No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Effects of Insulin and High Glucose on Mobilization of Slo1 BKCa Channels in Podocytes
Abstract
Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Podocytes are affected in the earliest stages of diabetic nephropathy and insulin signaling to podocytes is essential for normal glomerular function. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) encoded by the Slo1 gene are expressed in podocytes in a complex with multiple glomerular slit diaphragm proteins including nephrin, TRPC6 channels, and several different actin-binding proteins. Here we show that insulin increases cell surface expression of podocyte BK(Ca) channels, which is accompanied by a corresponding increase in the density of current flowing through these channels. Insulin stimulation of BK(Ca) channels was detectable in 15 min and required activation of both Erk and Akt signaling cascades. Exposure to high glucose (36.1 mM) for 24 h caused a marked reduction in the steady-state surface expression of BK(Ca) channels as well as of the slit diaphragm signaling molecule nephrin. High glucose treatment also abolished the stimulatory effects of insulin on BK(Ca) current density, although insulin continued to increase phosphorylation of Erk and Akt under those conditions. Therefore, in contrast to most other cell types, high glucose abrogates the effects of insulin in podocytes at relatively distal steps in its signaling pathway. Insulin stimulation of BK(Ca) channels in podocytes may prepare podocytes to adapt to changes in pressure gradients that occur during postprandial hyperfiltration.
... In neurons, this process can be regulated by multiple growth and differentiation factors (335), including factors that require Akt signaling (336). Through an analogous process, exposure of mouse podocytes to physiological concentrations of insulin markedly increases the steady-state surface expression of functional KCa1.1 channels within minutes (328,337). Insulin also induces a rapid increase in the surface expression of TRPC6 channels in podocytes (338,339) and it is tempting to hypothesize that some or all of the KCa1.1 channels in podocytes traffic to the cell surface as a complex with TRPC6 (325). On the other hand, there are conditions where TRPC6 and KCa1.1 trafficking is uncoupled. ...
... For example, exposing podocytes to high glucose or H2O2 results in a rapid increase in the steady-state surface expression of TRPC6 (338,340,341), and the effects of many physiological signals on podocyte TRPC6 are mediated by localized production of ROS through activation of NADPH oxidases (311-313, 342, 343). By contrast, exposure to high glucose or H2O2 inhibits surface expression of KCa1.1 in podocytes and can even abrogate the stimulatory effects of insulin (337). The effects of insulin on KCa1.1 in podocytes appear to follow a standard transduction pathway that entails activation of Akt, Erk, and PI3-kinase (337) along with the activation and possible dimerization of PKG type 1 subunit (328). ...
... There is evidence that insulin increases glomerular protein permeability through TRPC6-dependent activation of PKGIα signaling (439) and cytoskeleton reorganization (440). Insulin also increases mobilization of KCa1.1 channels in podocytes, although in part through different transduction mechanisms than are used for mobilization of TRPC6 (328,337). An enhancement of KCa1.1 could serve to enhance Ca 2+ efflux triggered by activation of TRPC6 by helping to maintain an adequate driving force to allow for efficient Ca 2+ permeation (225). ...
An essential step in renal function entails the formation of an ultrafiltrate that is delivered to the renal tubules for subsequent processing. This process, known as glomerular filtration, is controlled by intrinsic regulatory systems and by paracrine, neuronal, and endocrine signals that converge onto glomerular cells. In addition, the characteristics of glomerular fluid flow, such as the glomerular filtration rate and the glomerular filtration fraction, play an important role in determining blood flow to the rest of the kidney. Consequently, disease processes that initially affect glomeruli are the most likely to lead to end-stage kidney failure. The cells that compromise the glomerular filter, especially podocytes and mesangial cells, express many different types of ion channels that regulate intrinsic aspects of cell function and celllular responses to the local environment, such as changes in glomerular capillary pressure. Dysregulation of glomerular ion channels, such as changes in TRPC6, can lead to devastating glomerular diseases, and a number of channels, including TRPC6, TRPC5, and various ionotropic receptors, are promising targets for drug development. This review discusses glomerular structure and glomerular disease processes. It also describes the types of plasma membrane ion channels that have been identified in glomerular cells, the physiological and pathophysiological contexts in which they operate, and the pathways by which they are regulated and dysregulated. The contributions of these channels to glomerular disease processes, such as focal and segmental glomerulosclerosis (FSGS) and diabetic nephropathy, as well as the development of drugs that target these channels are also discussed.
... To date, some podocyte proteins functionally related to the foot process cytoskeleton, such as nephrin, Nep1, the nephrin-like protein, and transient receptor potential cation channels, such as TRPC6, have been found to interact with BK channels directly and regulate the physiology and pathology of podocytes (Kim et al., 2008(Kim et al., , 2009a(Kim et al., ,b, 2010. Abnormal expression of BK channels on the podocyte cell surface in some pathological conditions causes the destruction of cell actin cytoskeleton integrity, indicating that BK channels are essential for modulating the podocyte cytoskeleton (Kim and Dryer, 2011;Piwkowska et al., 2015). However, BK channels could promote Ca 2+ influx through TRPC6, which maintains activation of podocyte BK channels (Kim et al., 2009a). ...
... However, the relationships between mTORC complexes and BK channels have not been studied. In addition, Eun Young Kim's group investigated insulin modulation of BK channel activity associated with activation of Erk/MAPK and PI3K/Akt signaling pathways in podocytes (Kim and Dryer, 2011). Insulin, as a growth factor, can activate the PI3K/Akt signaling pathway, leading to activation of mTORC1 and mTORC2 (Saxton and Sabatini, 2017). ...
... Insulin, as a growth factor, can activate the PI3K/Akt signaling pathway, leading to activation of mTORC1 and mTORC2 (Saxton and Sabatini, 2017). LY294002, a PI3K inhibitor, can inhibit the insulin-induced increase in BK surface expression and macroscopic BK currents without altering the total BK protein expression levels (Kim and Dryer, 2011). However, whether mTOR complexes regulate BK channels in podocytes is unclear. ...
Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
... Recent studies showed that insulin stimulates glucose uptake by increasing the cell surface expression of glucose transporters (9,10). More recently, we showed that insulin also increases surface expression of BK Ca channels in podocytes (29). ...
... ally immortalized mouse podocyte cell line (MPC-5 cells). After differentiation for 2 wk at 37°C in our laboratory, these cells express the podocyte markers nephrin, Neph1, and synaptopodin (27,28,30) and they show physiological responses to insulin (29). We initially examined the effect of insulin on the steady-state surface expression of TRPC6 by means of cell surface biotinylation assays. ...
... Changes in transmural pressure gradients are thought to require mechanical compensation by glomerular cells to maintain the integrity of the filtration barrier (33,34). We propose that insulin signaling, by increasing surface expression of TRPC6 channels as reported here, and BK Ca channels as we reported previously (29), may prepare podocytes to carry out this compensation by allowing for increased Ca 2ϩ influx. Podocytes contract in response to increased Ca 2ϩ influx (45), and this could contribute necessary stability to the glomerular capillary wall-either by helping to maintain the integrity of the slit diaphragm or by preventing podocytes from detaching from the glomerular basement membrane. ...
Insulin receptors in podocytes are essential for normal kidney function. Here, we show that insulin evokes a rapid increase in the surface expression of canonical transient receptor potential-6 channel (TRPC6) channels in cultured podocytes, but caused a decrease in surface expression of TRPC5. These effects are accompanied by a marked increase in outwardly rectifying cationic currents that can be blocked by 10 μM SKF96365 or 100 μM La(3+). Application of oleoyl-2-acetyl-sn-glycerol (OAG) also increased SKF96365- and La(3+)-sensitive cationic currents in podocytes. Importantly, current responses to a combination of OAG and insulin were the same amplitude as those evoked by either agent applied alone. This occlusion effect suggests that OAG and insulin are targeting the same population of channels. In addition, shRNA knockdown of TRPC6 markedly reduced cationic currents stimulated by insulin. The effects of insulin on TRPC6 were mimicked by treating podocytes with H(2)O(2). Insulin treatment rapidly increased the generation of H(2)O(2) in podocytes, and it increased the surface expression of the NADPH oxidase NOX4 in cultured podocytes. Basal and insulin-stimulated surface expression of TRPC6 were reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases and other flavin-dependent enzymes, by siRNA knockdown of NOX4, and by manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, a membrane-permeable mimetic of superoxide dismutase and catalase. These observations suggest that insulin increases generation of ROS in part through activation of NADPH oxidases, and that this step contributes to modulation of podocyte TRPC6 channels.
... Insulin resistance is one of the main causes of podocyte injury in DN [3]. Physiologically, in response to insulin stimulation, podocytes activate insulin receptor and the downstream phosphoinositide 3-kinase (PI3K)/Akt cascades to maintain podocyte viability and GFB integrity [4,5]. On the contrary, insulin resistance increases the risk of renal dysfunction in DN. ...
... (C) Real-time PCR analysis of the mRNA levels of nephrin, collagen I and α-SMA in podocytes, which were treated with different concentrations of acetate (3, 5, 10, 20 and 40 mmol/L) under the HG condition for 24h (mean ± SD, * P < 0.05 vs. Ctrl, n = 3). (D) Western blotting analysis of the protein levels of nephrin, collagen I and α-SMA in podocytes, which were treated with different concentrations of acetate(3,5,10,20 and 40 mmol/L) under the HG condition for 24h (mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Ctrl, n = 3). (E) Western blotting analysis of the protein level of nephrin, collagen I and α-SMA in podocytes (mean ± SD, * P < 0.05 vs. HG+ siRNA control, # P < 0.05 vs. HG + acetate + siRNA control, n = 3). ...
Rationale: Albuminuria is an early clinical feature in the progression of diabetic nephropathy (DN). Podocyte insulin resistance is a main cause of podocyte injury, playing crucial roles by contributing to albuminuria in early DN. G protein-coupled receptor 43 (GPR43) is a metabolite sensor modulating the cell signalling pathways to maintain metabolic homeostasis. However, the roles of GPR43 in podocyte insulin resistance and its potential mechanisms in the development of DN are unclear.
Methods: The experiments were conducted by using kidney tissues from biopsied DN patients, streptozotocin (STZ) induced diabetic mice with or without global GPR43 gene knockout, diabetic rats treated with broad-spectrum oral antibiotics or fecal microbiota transplantation, and cell culture model of podocytes. Renal pathological injuries were evaluated by periodic acid-schiff staining and transmission electron microscopy. The expression of GPR43 with other podocyte insulin resistance related molecules was checked by immunofluorescent staining, real-time PCR, and Western blotting. Serum acetate level was examined by gas chromatographic analysis. The distribution of gut microbiota was measured by 16S ribosomal DNA sequencing with faeces.
Results: Our results demonstrated that GPR43 expression was increased in kidney samples of DN patients, diabetic animal models, and high glucose-stimulated podocytes. Interestingly, deletion of GPR43 alleviated albuminuria and renal injury in diabetic mice. Pharmacological inhibition and knockdown of GPR43 expression in podocytes increased insulin-induced Akt phosphorylation through the restoration of adenosine 5'-monophosphate-activated protein kinase α (AMPKα) activity. This effect was associated with the suppression of AMPKα activity through post-transcriptional phosphorylation via the protein kinase C-phospholipase C (PKC-PLC) pathway. Antibiotic treatment-mediated gut microbiota depletion, and faecal microbiota transplantation from the healthy donor controls substantially improved podocyte insulin sensitivity and attenuated glomerular injury in diabetic rats accompanied by the downregulation of the GPR43 expression and a decrease in the level of serum acetate.
Conclusion: These findings suggested that dysbiosis of gut microbiota-modulated GPR43 activation contributed to albuminuria in DN, which could be mediated by podocyte insulin resistance through the inhibition of AMPKα activity.
... The importance of intact podocyte insulin responses for glomerular function was subsequently highlighted in podocyte-specific IR knock-out mice, which develop features of DKD, despite normal blood glucose levels (3). Insulin has since been shown to modulate several downstream responses in podocytes including changes in mitochondrial function (22), autophagy (23), ER stress (24), VEGF-A secretion (25), actin dynamics (26), contractility (27), albumin permeability (27)(28)(29), and calcium mobilization (28,30). Current knowledge of insulinstimulated responses in podocytes is summarized in Figure 1 (2, 33). ...
... The mammalian target of rapamycin complex 2 (mTORC2) is responsible for Akt phosphorylation at Ser473. Akt can also activate mTORC1; (B) Insulin-stimulated contractility is regulated by calcium mobilization, via co-ordinated action of BK channels and TRPC6, which are regulated by Akt/p44/42 MAPK signaling (29) and increased ROS production (28), respectively. Insulin-stimulated dimerization of PKGIα, which may also involve TRPC6 (30), also contributes toward podocyte contractility (27); (C) Insulin-stimulated glucose-uptake in podocytes (4) is dependent on the expression and function of IRS-2 (31), Akt and nephrin (26,32). ...
Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease worldwide, occuring in approximately one-third of diabetic patients. One of the earliest hallmarks of DKD is albuminuria, often occurring following disruptions to the glomerular filtration barrier. Podocytes are highly specialized cells with a central role in filtration barrier maintenance; hence, podocyte dysfunction is a major cause of albuminuria in many settings, including DKD. Numerous studies over the last decade have highlighted the importance of intact podocyte insulin responses in the maintenance of podocyte function. This review summarizes our current perspectives on podocyte insulin signaling, highlighting evidence to support the notion that dysregulated podocyte insulin responses contribute toward podocyte damage, particularly during the pathogenesis of DKD.
... Moreover, in mouse podocytes, specific deletion of the gene encoding the insulin receptor causes a loss of podocyte foot processes [30]. In another study, insulin stimulated the surface expression of BK ca channel pore-forming subunits (Slo1) [29,31]. These channels require nephrin for steadystate surface expression [32]. ...
... Actin reorganization results in changes in the podocyte structure [28,30]. Other groups have suggested a role of insulin in the control of podocyte contractility, which may contribute to glomerular permeability [14,31]. ...
Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner.
... Growing evidences have suggested that several hormones (Figure 1A), such as angiotensin II and insulin, as well as pathological environment regulating BK expression and function playing important roles in podocyte injury (Kim and Dryer, 2011;Gao et al., 2015;Piwkowska et al., 2015). Angiotensin II (Ang II) which could induce the oxidative stress and podocyte death not only inhibits the current amplitude of Podocyte BK, but also facilitates the BK activation (Gao et al., 2015). ...
... Insulin increases cell surface expression of podocyte BK channels, with accompanied by a corresponding increase in the current density, via ERK (extracellular signal-regulated kinase) and AKT (PKB, protein kinase B) signaling cascades. While, high glucose treatment decreases the number of functional surface BK channels and nephrin as well as abolishes the stimulatory effects of insulin on BK (Kim and Dryer, 2011). Podocyte BK is also considered as a key player mediating insulin-increased filtration barrier permeability along with PKGI-dependent transepithelial albumin flux through participating in the disruption of the actin cytoskeleton induced by insulin. ...
Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca2+, possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus.
... The direct effect of insulin on podocyte vascular endothelial growth factor production [50] supports a role of insulin as an important endocrine modulator of podocyte function. Furthermore, insulin may control podocyte depolarization and contractility [51,52]. Although podocytes are not excitable cells, such contractility may be regulated by calcium ion influx via the coordinated action of large conductance Ca þ -activated K þ channels and the cation channel, transient receptor potential cation channel 6 [51,52]. ...
... Furthermore, insulin may control podocyte depolarization and contractility [51,52]. Although podocytes are not excitable cells, such contractility may be regulated by calcium ion influx via the coordinated action of large conductance Ca þ -activated K þ channels and the cation channel, transient receptor potential cation channel 6 [51,52]. Recent study in support of such mechanism shows that high concentrations of insulin increase albumin permeability of both isolated rat glomeruli and podocyte monolayers in the culture [53] and that insulin induces oxidative stress in podocytes. ...
Several key elements of the insulin signaling cascade contribute to podocyte function and survival. While it was initially thought that the consequences of altered insulin signaling to podocyte function was strictly related to altered glucose uptake, it has become clear that upstream signaling events involved in cell survival, lipid metabolism or nutrient sensing and modulated by insulin are strong independent contributors to podocyte function.
Akt2, the major isoform of Akt activated following cellular insulin stimulation, protects against the progression of renal disease in nephron-deficient mice, and podocyte-specific deletion of Akt2 results in a more rapid progression of experimental glomerular disease. In diabetes, podocyte mammalian target of rapamycin activation clearly contributes to podocyte injury and regulated autophagy. Furthermore, podocyte-specific glucose transporter type 4 (GLUT4) deficiency protects podocytes by preventing mammalian target of rapamycin signaling independently of glucose uptake. Finally, intracellular lipids have been recently recognized as major modulators of podocyte insulin signaling and as a new therapeutic target.
The identification of new contributors to podocyte insulin signaling is of extreme translational value as it may lead to new drug development strategies for diabetic kidney disease, as well as for other proteinuric kidney diseases.
... Other groups have suggested a role of insulin in the control of podocyte contractility, which may contribute to glomerular permeability [18,19]. Although podocytes are not excitable cells, it is suggested that such contractility is regulated by calcium ion influx via the coordinated action of largeconductance Ca 2+ -activated K + (BK) channels and the cation channel, TRPC6 [20,21]. ...
... Although podocytes are not excitable cells, it is suggested that such contractility is regulated by calcium ion influx via the coordinated action of largeconductance Ca 2+ -activated K + (BK) channels and the cation channel, TRPC6 [20,21]. Insulin has been shown not only to increase BK channel activity (via PI3K/Akt and ERK MAPK signalling pathways) [18], but to increase the surface expression of TRPC6 [19]. TRPC6 mobilization in response to insulin was demonstrated to be via a mechanism involving reactive oxygen species (ROS) production (specifically H 2 O 2 ), and NADPH oxidase 4 (Nox4), the catalytic subunit of NADPH oxidase [19]. ...
It is becoming increasingly clear that the insulin responses of a number of different cell types within the kidney are important
in the maintenance of normal renal function. This review summarizes our current understanding of renal insulin signalling,
with specific focus on the podocyte, presenting recent evidence that suggests these responses are altered in systemic insulin-resistant
states and chronic kidney disease via a number of different mechanisms.
... 48 However, high glucose (36.1 mM) treatment for 24 h was found to significantly reduce the expression of nephrin, thus affecting the expression of BK Ca channels and partially eliminating the stimulatory effect of insulin on the current density of BK Ca channels. 49 BK Ca channels can interact with Ca 2þ -mediated classical transient receptor potential canonical 6 channels in Potassium channel dysfunction in DKD podocytes, leading to Ca 2þ influx through these channels. This phenomenon may lead to the activation of BK Ca channels and prevent membrane depolarization, thus maintaining the driving force of Ca 2þ influx through these channels. ...
Diabetic kidney disease is a leading cause of end-stage renal disease, making it a global public health concern. The molecular mechanisms underlying diabetic kidney disease have not been elucidated due to its complex pathogenesis. Thus, exploring these mechanisms from new perspectives is the current focus of research concerning diabetic kidney disease. Ion channels are important proteins that maintain the physiological functions of cells and organs. Among ion channels, potassium channels stand out, because they are the most common and important channels on eukaryotic cell surfaces and function as the basis for cell excitability. Certain potassium channel abnormalities have been found to be closely related to diabetic kidney disease progression and genetic susceptibility, such as KATP, KCa, Kir, and KV. In this review, we summarized the roles of different types of potassium channels in the occurrence and development of diabetic kidney disease to discuss whether the development of DKD is due to potassium channel dysfunction and present new ideas for the treatment of DKD.
... Podocytes have an essential role in filtration barrier maintenance [145]. Some studies have highlighted the role of the insulin signaling in regulation of podocyte depolarization and contractility [146,147], and regulation of filtration barrier permeability [148]. In this regard, genetic mutation of the IR may lead to development of DN. ...
Insulin resistance, the most important characteristic of the type 2 diabetes mellitus (T2DM), is mostly caused by impairment in the insulin receptor (IR) signal transduction pathway. Protein tyrosine phosphatase 1B (PTP1B), one of the main negative regulators of the IR signaling pathway, is broadly expressed in various cells and tissues. PTP1B decreases the phosphorylation of the IR resulting in insulin resistance in various tissues. The evidence for the physiological role of PTP1B in regulation of metabolic pathways came from whole-body PTP1B-knockout mice. Whole-body and tissue-specific PTP1B-knockout mice showed improvement in adiposity, insulin resistance, and glucose tolerance. In addition, the key role of PTP1B in the pathogenesis of T2DM and its complications was further investigated in mice models of PTP1B deficient/overexpression. In recent years, targeting PTP1B using PTP1B inhibitors is being considered an attractive target to treat T2DM. PTP1B inhibitors improve the sensitivity of the insulin receptor and have the ability to cure insulin resistance-related diseases. We herein summarized the biological functions of PTP1B in different tissues in vivo and in vitro. We also describe the effectiveness of potent PTP1B inhibitors as pharmaceutical agents to treat T2DM.
... The function, structure and survival of podocytes are under the control of insulin stimulation [92]. The podocytes have high expression of insulin receptors [93], and insulin can control podocyte contractility which is related to glomerular permeability [94,95]. Podocytes isolated from mice model of kidney dysfunction were found to be unresponsive to insulin in terms of glucose metabolism [91]. ...
: Low birth weight (LBW) is associated with diseases in adulthood. The birthweight attributed risk is independent of confounding such as gestational age, sex of the newborn but also social factors. The birthweight attributed risk for diseases in later life holds for the whole spectrum of birthweight. This raises the question what pathophysiological principle is actually behind the association. In this review, we provide evidence that LBW is a surrogate of insulin resistance. Insulin resistance has been identified as a key factor leading to type 2 diabetes, cardiovascular disease as well as kidney diseases. We first provide evidence linking LBW to insulin resistance during intrauterine life. This might be caused by both genetic (genetic variations of genes controlling glucose homeostasis) and/or environmental factors (due to alterations of macronutrition and micronutrition of the mother during pregnancy, but also effects of paternal nutrition prior to conception) leading via epigenetic modifications to early life insulin resistance and alterations of intrauterine growth, as insulin is a growth factor in early life. LBW is rather a surrogate of insulin resistance in early life - either due to inborn genetic or environmental reasons - rather than a player on its own.
... This interaction allows Ca 2+ influx through TRPC6 to cause BK Ca activation, which by reducing the tendency for depolarization, could allow TRPC6 to function as an efficient and sustained source of Ca 2+ influx. These dynamics might explain why insulin increases the surface expression of both channels in podocytes [54,157,158,196,197]. In addition, podocytes also express a sodium-calcium exchange protein, which could also allow for increased Ca 2+ influx following TRPC6 activation, even if the channel is primarily conducting Na + [198]. ...
Mutations in the gene encoding canonical transient receptor potential-6 (TRPC6) channels result in severe nephrotic syndromes that typically lead to end-stage renal disease. Many but not all of these mutations result in a gain in the function of the resulting channel protein. Since those observations were first made, substantial work has supported the hypothesis that TRPC6 channels can also contribute to progression of acquired (non-genetic) glomerular diseases, including primary and secondary FSGS, glomerulosclerosis during autoimmune glomerulonephritis, and possibly in type-1 diabetes. Their regulation has been extensively studied, especially in podocytes, but also in mesangial cells and other cell types present in the kidney. More recent evidence has implicated TRPC6 in renal fibrosis and tubulointerstitial disease caused by urinary obstruction. Consequently TRPC6 is being extensively investigated as a target for drug discovery. Other TRPC family members are present in kidney. TRPC6 can form a functional heteromultimer with TRPC3, and it has been suggested that TRPC5 may also play a role in glomerular disease progression, although the evidence on this is contradictory. Here we review literature on the expression and regulation of TRPC6, TRPC3 and TRPC5 in various cell types of the vertebrate kidney, the evidence that these channels are dysregulated in disease models, and research showing that knock-out or pharmacological inhibition of these channels can reduce the severity of kidney disease. We also summarize several areas that remain controversial, and some of the large gaps of knowledge concerning the fundamental role of these proteins in regulation of renal function.
... These properties, when combined with detection methods, could offer new prospects to accomplish new glucose sensors [33,34]. In pursuit of this, the manipulation of the chemical integrity of hydrogels via new fabrication strategies allowed to report the swelling of hydrogels as a function of blood's glucose concentration [35]. This could become possible by incorporating the glucose-responsive hydrogel into a micro-or nanostructured holographic thin films [36], crystalline colloidal arrays [37], Fabry-Perot cavities [38], fluorescent dyes [39], quantum dots [40] and field effect transistor [41]. ...
Phenylboronic acids (PBAs) have gained considerable interest in recent years due to their recognition for diol-containing molecules such as glucose. Their response to the elevated glucose concentrations can be reported by measuring the change in the size or optical properties of polymers-bearing PBAs. In this context, fast response and good mechanical properties are crucial factors for constructing glucose-responsive sensors. Toward this goal, we have synthesized glucose-responsive nanostructured gels (NSGs) using 3-acrylamidophenylboronic acid and N-isopropylacrylamide. Herein, activated nanogels with controllable size were prepared and used as nano-cross-linkers. The prepared NSGs showed glucose-responsiveness in a remarkable concentration-dependent manner, exhibited high elasticity upon compression and slicing and resisted high level of deformation such as bending, twisting and stretching.
... BK channels in the podocytes are responsive to insulin and glucose and interact with TRPC channels and nephrin, both of which are implicated in DKD. Thus it is feasible that the malfunction of these channels is involved in the progression of DKD (Kim & Dryer, 2011). ...
Type 1 and 2 diabetes mellitus are major medical epidemics affecting millions of patients worldwide. Diabetes mellitus is the leading cause of diabetic kidney disease (DKD), which is the most common cause of end-stage renal disease (ESRD). DKD is associated with significant changes in renal hemodynamics and electrolyte transport. Alterations in renal ion transport triggered by pathophysiological conditions in diabetes can exacerbate hypertension, accelerate renal injury, and are integral to the development of DKD. Renal ion transporters and electrolyte homeostasis play a fundamental role in functional changes and injury to the kidney during DKD. With the large number of ion transporters involved in DKD, understanding the roles of individual transporters as well as the complex cascades through which they interact is essential in the development of effective treatments for patients suffering from this disease. This chapter aims to gather current knowledge of the major renal ion transporters with altered expression and activity under diabetic conditions, and provide a comprehensive overview of their interactions and collective functions in DKD.
... Actin reorganization leads to changes in podocyte structure, and insulin receptor stimulation causes the retraction of podocyte processes [5]. Some groups have suggested that insulin plays a role in the control of podocyte contractility, which may contribute to glomerular permeability [6][7][8][9]. We recently demonstrated that insulin remodels the actin cytoskeleton and increases the albumin permeability of both isolated rat glomeruli and podocytes; we further showed that the underlying mechanism is calcium-dependent [10,11]. ...
Background/Aims: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes. Methods: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-3H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA). Results: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway. Conclusion: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes.
... BK Ca channels, encoded by the Slo1 gene, play an important role in regulating the physiological membrane potential [36]. Moreover, many evidences suggested that some hormones, such as Ang II and insulin, as well as pathological environment could regulate the expression and function of BK Ca and play important roles in podocyte injury [37,38]. It is reported that Ang II could induce the increase of oxidative stress and podocyte death and inhibit the currents of BK Ca in podocytes with facilitating the BK Ca activation [38]. ...
... Further, we recently showed that reductions in neuronal calcium levels and calcium-mediated potentials were not seen in the ZDF rat even following a 7-week period of sustained peripheral hyperglycemia and hyperinsulinemia [29]. Additionally, activation of the PI3K/ mTOR/AKT pathway was shown to rapidly increase synaptic protein levels within minutes [67], while other pathways, such as MEK/ERK, a pathway which has nuclear targets, have been implicated in modulating the expression of calcium-sensitive channels [68]. It follows that targeting nuclear factors would be slower and likely longer-lasting compared to pathways involved with acute IR activation. ...
Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.
... As nephrin expression is reduced with progression of diabetic nephropathy and albuminuria, insulin signaling is expected to be reduced and, thus, to negatively affect the filtration barrier. Insulin´s action on podocytes also includes up-regulation of the ion channels TRPC (transient receptor potential cation channel, subfamily C) and BKCa channels 129,130 . In podocytes isolated from insulin-resistant obese db/db mice insulin failed to phosphorylate PKB and this was associated with reduced viability of the cells 131 . ...
Insulin resistance is a systemic disorder that affects many organs and insulin-regulated pathways. The disorder is characterized by a reduced action of insulin despite increased insulin concentrations (hyperinsulinaemia). The effects of insulin on the kidney and vasculature differ in part from the effects on classical insulin target organs. Insulin causes vasodilation by enhancing endothelial nitric oxide production through activation of the phosphatidylinositol 3-kinase pathway. In insulin-resistant states, this pathway is impaired and the mitogen-activated protein kinase pathway stimulates vasoconstriction. The action of insulin on perivascular fat tissue and the subsequent effects on the vascular wall are not fully understood, but the hepatokine fetuin-A, which is released by fatty liver, might promote the proinflammatory effects of perivascular fat. The strong association of salt-sensitive arterial hypertension with insulin resistance indicates an involvement of the kidney in the insulin resistance syndrome. The insulin receptor is expressed on renal tubular cells and podocytes and insulin signalling has important roles in podocyte viability and tubular function. Renal sodium transport is preserved in insulin resistance and contributes to the salt-sensitivity of blood pressure in hyperinsulinaemia. Therapeutically, renal and vascular insulin resistance can be improved by an integrated holistic approach aimed at restoring overall insulin sensitivity and improving insulin signalling.
... In primary cultures, podocytes have the highest IR and IRS1 expression levels compared with endothelial cells and mesangial cells [10]. Insulin was suggested to play a role in the regulation of podocyte contractility, which may contribute to glomerular permeability [91,92]. Podocyte-specific IR knockout mice develop albuminuria, the effacement of podocyte foot process, and podocyte apoptosis. ...
Insulin resistance has been characterized as attenuation of insulin sensitivity at target organs and tissues, such as muscle and fat tissues and the liver. The insulin signaling cascade is divided into major pathways such as the PI3K/Akt pathway and the MAPK/MEK pathway. In insulin resistance, however, these pathways are not equally impaired. For example, in the liver, inhibition of gluconeogenesis by the insulin receptor substrate (IRS) 2 pathway is impaired, while lipogenesis by the IRS1 pathway is preserved, thus causing hyperglycemia and hyperlipidemia. It has been recently suggested that selective impairment of insulin signaling cascades in insulin resistance also occurs in the kidney. In the renal proximal tubule, insulin signaling via IRS1 is inhibited, while insulin signaling via IRS2 is preserved. Insulin signaling via IRS2 continues to stimulate sodium reabsorption in the proximal tubule and causes sodium retention, edema, and hypertension. IRS1 signaling deficiency in the proximal tubule may impair IRS1-mediated inhibition of gluconeogenesis, which could induce hyperglycemia by preserving glucose production. In the glomerulus, the impairment of IRS1 signaling deteriorates the structure and function of podocyte and endothelial cells, possibly causing diabetic nephropathy. This paper mainly describes selective insulin resistance in the kidney, focusing on the proximal tubule.
... These studies pointed to some involved insulin signaling pathways in podocytes, e.g. the activation of the mitogen-activated protein kinases and phosphatidylinositol-4, 5-bisphosphate 3-kinase pathways [8]. Other groups showed that insulin increases the expression and mobilization of large-conductance Ca(2+)-activated K(+) channels as well as transient receptor potential canonical (TRPC6) channels in podocytes [9,10]. Using biotinylation assays, Kim et al. showed that insulin increased surface expression of TRPC6 [10]. ...
Background/aims:
Insulin signaling to podocytes is relevant for the function of the glomerulus. Now, we tested the hypothesis that insulin increases the surface expression of canonical transient receptor potential canonical type 6 (TRPC6) channels in podocytes by a calcineurin-dependent pathway.
Methods:
We used quantitative RT-PCR, immunoblotting, immunofluorescence and fluorescence spectrophotometry in cultured podocytes. Activation of Nuclear Factor of Activated T-cells (NFATc1) was measured using a specific calorimetric assay.
Results:
Insulin increased the expression of TRPC6 transcripts and protein in podocytes. Insulin increased TRPC6 transcripts in a time and dose-dependent manner. The insulin-induced elevation of TRPC6 transcripts was blocked in the presence of tacrolimus, cyclosporine A, and NFAT-inhibitor (each p < 0.01 by ANOVA and Bonferroni's multiple comparison test). Transcripts of NOX4, another target gene of the calcineurin-NFAT pathway, were affected in a similar way. Immunoblotting showed that the administration of 100 nmol/L insulin increased TRPC6-proteins 2-fold within 48 hours. Insulin increased the activity of NFATc1 in nuclear extracts (p < 0.001) whereas tacrolimus, cyclosporine A, and NFAT-inhibitor blocked that insulin effect (p < 0.001; two way ANOVA). Immunofluorescence showed that insulin increased TRPC6-expression on the cell surface. Fluorescence-spectrophotometry and manganese quench experiments indicated that the increased TRPC6-expression after insulin administration was accompanied by an elevated transplasmamembrane cation influx. Insulin-stimulated surface expression of TRPC6 as well as transplasmamembrane cation influx could be reduced by pretreatment with tacrolimus.
Conclusion:
Insulin increases the expression of TRPC6 channels in podocytes by activation of the calcineurin-dependent pathway.
... Podocytes, a major component of the glomerular filtration barrier, seem to have the highest levels of insulin receptors compared with endothelial and mesangial cells (58). In fact, insulin may control podocyte contractility that contributes to glomerular permeability (43,44). Insulin may also regulate GFR through local renal vasodilation, which can be blocked by indomethacin (18) and augmented by activation of endothelial NO synthase (36), implicating prostaglandins and NO, respectively, in the effect of insulin. ...
... Moreover, in mice, specific deletion of the gene encoding the insulin receptor in podocytes caused loss of podocyte foot processes [10]. Other groups have suggested that insulin played a role in controlling podocyte contractility, which may contribute to glomerular permeability [11] [12]. Recently, we demonstrated that insulin increased albumin permeability in both isolated rat glomeruli and podocytes. ...
Podocytes play a fundamental role in regulating glomerular permeability to albumin. This mechanism is disrupted in the course of diabetes. Both insulin and high glucose concentrations enhance the permeability of podocytes to albumin by stimulating oxygen free radical production, primarily by NAD(P)H oxidase-4 (NOX4), and by activating protein kinase G, isoform Iα (PKGIα). However, no study has investigated the combined effects of insulin and high glucose concentration. Here, we investigated the effects of applying insulin (INS, 300 nM) and high glucose (HG, 30 mM), both separately and combined, for 5 days, on cultured rat podocyte permeability to albumin. We measured podocyte permeability with a transmembrane albumin flux assay. We measured NOX4 and PKGIα mRNA expression with real-time PCR. We used Western blots to evaluate protein expression levels of NOX4, PKGIα, the myosin-binding subunit of myosin phosphatase 1, and myosin light chain. We found that INS and HG had a synergistic effect on podocyte permeability to albumin, and this synergy was not dependent on NOX4 or PKGIα. These results suggested that the combined action of INS and HG may exacerbate glomerular dysfunction in diabetes.
Copyright © 2015. Published by Elsevier Inc.
... Insulin can rapidly signal to the podocyte 13 after a meal to trigger several potentially beneficial homeostatic responses. These include the rapid absorption of glucose through translocation of glucose transporters to the plasma membrane of the cell, remodeling of its actin cytoskeleton, 13 and incorporation of potassium 14 and calcium channels 15 into the plasma membrane. Together, these responses allow the cell a readily accessible energy source (glucose) and the ability to contract and remodel the cytoskeleton. ...
Alterations and injury to glomerular podocytes play a key role in the initiation and progression of diabetic kidney disease (DKD). Multiple factors in diabetes cause abnormalities in podocyte signaling that lead to podocyte foot process effacement, hypertrophy, detachment, loss, and death. Alterations in insulin action and mammalian target of rapamycin activation have been well documented to lead to pathology. Reduced insulin action directly leads to albuminuria, increased glomerular matrix accumulation, thickening of the glomerular basement membrane, podocyte apoptosis, and glomerulosclerosis. In addition, podocytes generate factors that alter signaling in other glomerular cells. Prominent among these is vascular endothelial growth factor-A, which maintains glomerular endothelium viability but causes endothelial cell pathology when generated at too high a level. Finally, circulating vascular factors (eg, activated protein C) have a profound effect on podocyte stability and survival. This cytoprotective factor is critical for podocyte health, and its deficiency promotes podocyte injury and apoptosis. Thus, the podocyte sits in the center of a network of paracrine and hormonal signaling systems that in health keep the podocyte adaptable and viable, but in diabetes they can lead to pathologic changes, detachment, and death.
... This is in contrast to some of the other cellular effects that insulin has on the podocyte, which have been shown to occur rapidly, within minutes. These include glucose uptake [via glucose transporter translocation (5)], actin remodeling (33), and translocation of ion channels into the plasma membrane (16,17). ...
Podocytes are critically important for maintaining integrity of the glomerular filtration barrier and preventing albuminuria. Recently it has become clear that to achieve this they need to be insulin sensitive, and produce an optimal amount of vascular endothelial growth factor-A (VEGF-A). In other tissues insulin has been shown to regulate VEGF-A release but this has not been previously examined in the podocyte. Using an in-vitro and in-vivo approach we now show that insulin regulates VEGF-A in the podocyte in both mouse and man via the insulin receptor (IR). Insulin directly increases VEGF-A messenger RNA levels and protein production in conditionally immortalized wild-type human and murine podocytes. Furthermore, when podocytes are rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the IR) or in vivo (using transgenic podocyte specific IR knockout mice) podocyte VEGF-A production is impaired. Importantly, in vivo this occurs prior to the development of any podocyte damage due to podocyte insulin resistance. Modulation of VEGF-A by insulin in the podocyte may be another important factor in the development of glomerular disease associated with conditions in which insulin signaling to the podocyte is deranged.
... BK Ca channels contain binding sites for Ca 2+ on a large cytosolic domain. Binding of Ca 2+ to these sites allows for the voltage-dependent gating of BK Ca channels at physiological membrane potentials (Kim and Dryer 2011). Moreover, they were shown to interact directly with Ca 2+ -permeable TRPC6 channels in differentiated cells of a podocyte cell line (Kim et al. 2009a). ...
The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Podocytes are of keen interests because of their key roles in kidney development and disease. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are important ion channels located in podocytes and play the essential role in regulating calcium homeostasis cell signaling. In this research, we studied the undergoing developmental changes of BK(Ca) channels and their contribution to functional maturation of podocytes. Our results showed that the distribution of BK(Ca) channels changed with the maturity of differentiation in a conditionally immortalized mouse podocyte cell line. Additionally, the increase of BK(Ca) channel protein expression was detected by immunofluorescence staining with confocal microscopy in podocytes, which was consistent with the increase in the current density of BK(Ca) channels examined by whole-cell patch-clamp technique. Our results suggested that the developmental changes of BK(Ca) channels may help podocytes adapt to changes in pressure gradients occurring in physiological conditions. Those findings may have implications for understanding the physiology and development of kidney and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in podocytes.
... This elegant study went on to show that the effects of insulin on TRPC6 were mediated through the production of ROS (reactive oxygen species) via activation of NADPH oxidases [100]. The same group have also shown that, in addition to modulating TRPC channels in podocytes, insulin also rapidly causes Ca 2 + -regulated K + channels to locate to the plasma membrane of this cell [101]. Collectively these findings suggest that, when insulin stimulates the podocyte, as occurs after a meal, it causes the podocyte to rapidly take up a readily usable energy source, glucose, remodel its actin cytoskeleton and contract, which is facilitated by ionic flux into the cell. ...
Ninety-one years ago insulin was discovered, which was one of the most important medical discoveries in the past century, transforming the lives of millions of diabetic patients. Initially insulin was considered only important for rapid control of blood glucose by its action on a restricted number of tissues; however, it has now become clear that this hormone controls an array of cellular processes in many different tissues. The present review will focus on the role of insulin in the kidney in health and disease.
... The obesity-related factor(s) that caused the changes in BK Ca  1 -expression and BK Ca function are difficult to identify. Of the factors measured (leptin, insulin, and blood glucose), insulin has been shown to increase BK Ca expression and function in the plasma membrane of mouse podocytes, whereas high concentrations of glucose had the opposite effect (38). In contrast with the current findings, BK Ca function was inhibited in cerebral arteries from insulin-resistant rats (19). ...
Membranous nephropathy (MN) is a chronic kidney disease and a precursor to end-stage kidney disease. In this study, we evaluated the potential protective effects of acidic and neutral Stigma maydis polysaccharides (ASMP and NSMP, respectively) on cationized bovine serum albumin-induced MN in mice. Both polysaccharides (SMPs) provided effective protection from kidney injury by decreasing daily proteinuria, kidney dysfunction, and hyperlipidemia and minimizing structural changes and immune complex expression. Furthermore, SMPs improved intestinal barrier damage by increasing the expression of tight junction proteins in the intestinal tissue. They also maintained the integrity of the glomerular filtration barrier by promoting slit diaphragm proteins expression and PI3K/AKT signaling. However, ASMP offered better protection against podocyte injury than NSMP. The use of natural polysaccharides could thus be a new protective measure against podocyte injury and perhaps be utilized for the development of functional foods to protect against MN.
Introduction
A bidirectional association exists between insulin resistance (IR) and chronic kidney disease (CKD) in Type 2 Diabetes Mellitus (T2DM). Baseline measures of IR are predictive of CKD progression, and uraemia in progressive CKD is itself, in turn, associated with a worsening of IR. Pre-clinical research reveals that intrinsic IR in glomerular podocytes and the renal tubule may serve as a pathogenic driver of CKD in T2DM.
Areas covered
The present manuscript takes as its point of departure, the recently identified prognostic utility of severe insulin resistance as a predictor of CKD in T2DM. Findings from a series of studies describing the association of IR with pathological alterations in both established, and less commonly assessed dynamic measures of renal impairment are discussed. Drawing upon the pre-clinical mechanistic evidence base, the cellular and molecular basis of intrinsic renal IR as a promoter of CKD is considered.
Expert opinion
Measurement of insulin sensitivity may add value to profiling of renal risk in T2DM. Rational selection of therapeutic strategies targeting the enhancement of insulin sensitivity merits special attention regarding the personalised management of CKD in insulin resistance predominant T2DM.
Background: Despite multifactorial interventions, the prevalence of diabetic kidney disease (DKD) is reaching epidemic proportions worldwide. Loss of podocytes, the cells forming the glomerular filtration barrier, occurs early in the development of DKD and is associated with the presence of albuminuria. Summary: Recent studies have implicated circulating hormones, adipokines and lipids in the development and progression of DKD. Key Message: Although the endocrine effects of these factors are widely recognized, less is known about their direct effects on podocytes and the resulting implications for DKD.
Insulin plays a major role in regulating glucose homeostasis in podocytes. Protein kinase G type Iα (PKGIα) plays an important role in regulating glucose uptake in these cells. Rac1 signaling plays an essential role in the reorganization of the actin cytoskeleton and is also essential for insulin‐stimulated glucose transport. The experiments were conducted using primary rat podocytes. We performed western blot analysis, evaluated small GTPases activity assays, measured radioactive glucose uptake, and performed immunofluorescence imaging to analyze the role of PKGIα‐Rac1 signaling in regulating podocyte function. We also utilized a small‐interfering RNA‐mediated approach to determine the role of PKGIα and Rac1 in regulating glucose uptake in podocytes. The present study investigated the influence of the PKGI pathway on the insulin‐dependent regulation of activity and cellular localization of small guanosine triphosphatases in podocytes. We found that the PKGIα‐dependent activation of Rac1 signaling induced activation of the PAK/cofilin pathway and increased insulin‐mediated glucose uptake in podocytes. The downregulation of PKGIα or Rac1 expression abolished this effect. Rac1 silencing prevented actin remodeling and GLUT4 translocation close to the cell membrane. These data provide evidence that PKGIα‐dependent activation of the Rac1 signaling pathways is a novel regulator of insulin‐mediated glucose uptake in cultured rat podocytes.
Purpose of review:
Deregulation of protecting factor signaling actions in podocytes has emerged as an alternative pathway of podocyte injury mechanisms. Here, we review recent knowledge that highlighted how podocyte protecting factors are modulated by protein phosphatases.
Recent findings:
Protein tyrosine kinases and phosphatases participate in many, if not all, aspects of cellular function by turning on or off multiple signaling cascades and podocytes are no exception. Modulation of tyrosine residue phosphorylation of podocyte factors such as nephrin, vascular endothelial growth factor, insulin receptors and substrates has been shown to promote podocyte damage and cell death that contributed to multiple glomerular diseases. Protein phosphatase activity can cause either an increase [Src homology 2 domain-containing phosphatase 2 (SHP-2)] or a decrease [Protein tyrosine phosphatase1B (PTP1B), SHP-1 and SH2 domain-containing 5'-inositol phosphatase 2 (SHIP2)] in nephrin tyrosine phosphorylation depending on which podocyte injury model was used. Insulin resistance is closely linked to the development and progression of renal disease. Expression of PTP1B, SHP-1, phosphatase and tensin homolog and SHIP2 are potential mechanisms of podocytes insulin resistance in diabetic kidney disease.
Summary:
Tight regulation of protein phosphatases is critical to maintain cell homeostasis and may offer new perceptive targets to restore protecting factor actions in order to prevent podocyte dysfunction and glomerular diseases.
The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including insulin resistance (IR), dyslipidemia and hypertension, which may also foster development of chronic kidney disease. The mechanisms of MetS-induced kidney disease are not fully understood. The purpose of this review is to summarize recent discoveries regarding the impact of MetS on the kidney, particularly on the renal microvasculature and cellular mitochondria. Fundamental manifestations of MetS include insulin resistance (IR) and adipose tissue expansion, the latter promoting chronic inflammation and oxidative stress that exacerbate IR. Those in turn can elicit various kidney injurious events through endothelial dysfunction, activation of the renin-angiotensin-aldosterone system, and adipokine imbalance. IR and inflammation are also major contributors to microvascular remodeling and podocyte injury. Hence, these events may result in hypertension, albuminuria, and parenchymal damage. In addition, dyslipidemia and excessive nutrient availability may impair mitochondrial function and thereby promote progression of kidney cell damage. Elucidation of the link between MetS and kidney injury may help develop preventative measures and possibly novel therapeutic targets to alleviate and avert development of renal manifestations.
KCNMA1 is a pore-forming α-subunit of the large conductance Ca2+- and voltage-activated K+ channels, referred to as BK channels, which play key roles in various physiological functions. However, the role of KCNMA1 in mature adipocytes remains unclear. In this study, we reveal that kcnma1 expression is downregulated in white adipose tissue of mice fed a high fat-diet and in hypertrophied adipocytes. Furthermore, inhibition of kcnma1 expression or treatment with a BK channel blocker attenuated insulin-induced Akt phosphorylation in mature adipocytes. These results strongly indicate that KCNMA1 contributes to the regulation of insulin signalling in mature adipocytes. This article is protected by copyright. All rights reserved.
Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGI?) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGI? activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGI? and PKGI-dependent signaling pathways in podocytes. This article is protected by copyright. All rights reserved
Diabetes mellitus (DM) is the major cause of end-stage renal disease (ESRD) globally, and novel treatments are urgently needed. Current therapeutic approaches for diabetic nephropathy (DN) are focussing on blood pressure control with inhibitors of the renin-angiotensin-aldosterone system, on glycaemic and lipid control, and life-style changes. In this review, we highlight new molecular insights aiding our understanding of the initiation and progression of DN, including glomerular insulin resistance, dysregulation of cellular substrate utilisation, podocyte-endothelial communication, and inhibition of tubular sodium coupled glucose reabsorption. We believe that these mechanisms offer new therapeutic targets that can be exploited to develop important renoprotective treatments for DN over the next decade.
Podocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca(2+)-activated K(+) channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability. Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression was examined in podocytes with Western blotting and immunofluorescence. The BKCa-PKGIα interaction was assessed with co-immunoprecipitation. RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory β subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA. These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes.
Copyright © 2015. Published by Elsevier B.V.
Chronic kidney disease (CKD) has become a serious public health problem because of its associated morbidity, premature mortality and attendant healthcare costs. The rising number of persons with CKD is linked with ageing population structure and an increased prevalence of diabetes, hypertension and obesity. There is an inherited risk associated with developing CKD as evidenced by familial clustering and differing prevalence rates across ethnic groups. Earlier studies to determine the inherited risk factors for CKD rarely identified genetic variants that were robustly replicated. However, improvements in genotyping technologies and analytical methods are now helping to identify promising genetic loci aided by international collaboration and multi-consortia efforts. More recently, epigenetic modifications have been proposed to play a role in both the inherited susceptibility to CKD and, importantly, to explain how the environment dynamically interacts with the genome to alter an individual's disease risk. Genome-wide, epigenome-wide and whole transcriptome studies have been performed and optimal approaches for integrative analysis are being developed. This review summarises recent research and the current status of genetic and epigenetic risk factors influencing CKD using population-based information.
Systemic insulin resistance is becoming more prevalent in the young due to modern lifestyles predisposing to the metabolic syndrome and obesity. There is also evidence that there are critical insulin-resistant phases for the developing child, including puberty, and that renal disease per se causes systemic insulin resistance. This review considers the factors that render children insulin resistant, as well as the accumulating evidence that the kidney is an insulin-responsive organ and could be affected by insulin resistance.
Diabetes is increasing at daunting rates worldwide, and approximately 40% of affected individuals will develop kidney complications. Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease, and there are significant healthcare costs providing appropriate renal replacement therapies to affected individuals. For several decades, investigators have sought to discover inherited risk factors and biomarkers for DKD. In recent years, advances in high-throughput laboratory techniques and computational analyses, coupled with the establishment of multicenter consortia, have helped to identify genetic loci that are replicated across multiple populations. Several genome-wide association studies (GWAS) have been conducted for DKD with further meta-analysis of GWAS and comprehensive "single gene" meta-analyses now published. Despite these efforts, much of the inherited predisposition to DKD remains unexplained. Meta-analyses and integrated-omics pathway studies are being used to help elucidate underlying genetic risks. Epigenetic phenomena are increasingly recognized as important drivers of disease risk, and several epigenome-wide association studies have now been completed. This review describes key findings and ongoing genetic and epigenetic initiatives for DKD.
Slit diaphragm and podocyte damage is crucial in proteinuria pathogenesis in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and induced podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP.
Podocytes are key cells in the glomerular filtration barrier with a major role in the development of diabetic nephropathy. Podocytes are insulin-sensitive cells and have a functionally active local renin-angiotensin system. The presence and activity of angiotensin-converting enzyme 2 (ACE2), the main role of which is cleaving pro-fibrotic and pro-inflammatory angiotensin-II into angiotensin-(1-7), has been demonstrated in podocytes. Conditionally immortalized mouse podocytes were cultured with insulin in the presence and absence of albumin. We found that insulin increases ACE2 gene and protein expression, by real-time PCR and western blot respectively, and enzymatic activity within the podocyte and these increases were maintained over time. Furthermore, insulin favored an "anti-angiotensin-II" regarding ACE/ACE2 gene expression balance and decreased fibronectin gene expression as a marker of fibrosis in the podocytes, all studied by real-time PCR. Likewise insulin incubation seemed to protect podocyte from cell death, studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. However, all these effects disappeared in the presence of albumin, which may mimic albuminuria, a main feature in DN pathophysiology. Our results suggest that modulation of renin-angiotensin system balance, fibrosis and apoptosis by insulin in the podocyte may be an important factor in preventing the development and progression of diabetic kidney disease but the presence of albuminuria seems to block these beneficial effects.
Extracellular ATP may contribute to Ca(2+) signaling in podocytes during tubuloglomerular feedback (TGF) and possibly as a result of local tissue damage. TRPC6 channels are Ca(2+)-permeable cationic channels that have been implicated in the pathophysiology of podocyte diseases. Here we show using whole-cell recordings that ATP evokes robust activation of TRPC6 channels in mouse podocyte cell lines and in rat podocytes attached to glomerular capillaries in ex vivo glomerular explants. The ED50 for ATP is around 10 μM, is maximal at 100 μM, and currents were blocked by the P2 antagonist suramin. In terms of maximal currents that can be evoked, ATP is the strongest activator of podocyte TRPC6 that we have characterized to date. Smaller currents were observed in response to ADP, UTP, and UDP. ATP-evoked currents in podocytes were abolished by TRPC6 knockdown and by pretreatment with 10 μM SKF-96365 or 50 μM La(3+). ATP effects were also abolished by inhibiting G protein signaling and by the PLC/PLA2 inhibitor D-609. ATP effects on TRPC6 were also suppressed by knockdown of the slit diaphragm scaffolding protein podocin, and also by tempol, a membrane-permeable quencher of reactive oxygen species. Modulation of podocyte TRPC6 channels, especially in foot processes, could provide a mechanism for regulation of glomerular function by extracellular nucleotides, possibly leading to changes in permeation through slit diaphragms. These results raise the possibility that sustained ATP signaling could contribute to foot process effacement, Ca(2+)-dependent changes in gene expression, and/or detachment of podocytes.
Podocyte injury may contribute to the pathogenesis of diabetic nephropathy (DN), but the underlying mechanism of hyperglycemia induced podocyte damage is not fully understood. The Ras GTPase-activating-like protein IQGAP1 is associated to the slit diaphragm proteins and the actin cytoskeleton in podocyte. Here, we studied IQGAP1 expression alterations in human DN biopsies and extracellular signal-regulated kinase (ERK)-dependent pathways of IQGAP1 expression in podocyte under high glucose (HG) media. In vivo, analysis of renal biopsies from patients with DN revealed a significant reduction in IQGAP1 expression compared to controls. In vitro, IQGAP1 mRNA and protein expression were observed to decline under HG media at 48 h. But phosphorylation of ERK1/2 was activated under HG media at 24 h and 48 h. However, HG-induced downregulation of IQGAP1 protein was attenuated by specific ERK1/2 activation inhibitor PD98059. Taken together, these results highlight the importance of IQGAP1 in DN, and suggest that IQGAP1 expression in podocyte under HG media is modulated by the ERK1/2 pathway, which may lead to the future development of therapies targeting IQGAP1 dysfunction in podocytes in DN.
To explore the effects of leflunomide active metabolite A771726 on high glucose-induced podocyte cytoskeleton and its possible signaling pathway.
The conditionally immortal human glomeroular podocytes were divided into normal glucose (NG), mannitol (MA), high glucose (HG), high glucose with PDTC (pyrrolidine dithiocarbamate, a NF-κBp65 inhibitor) and high glucose with active leflunomide metabolite A771726 groups. Western blot was used to measure the ratio of p-NF-κBp65 to total NF-κBp65. And the protein and mRNA expressions of NF-κBp65, TRPC6 and nephrin were detected by Western blot and reverse transcription polymerase chain reaction (PCR). Immunofluorescence staining was used to detect the changes in the skeleton of podocyte.
(1) Podocytes with high glucose could activate the NF-κBp65 signaling pathway. There was a significant increase of p-NF-κBp65 protein at 60 min versus 0 min (1.20 ± 0.04 vs 0.79 ± 0.02, P < 0.01). Little activation of the pathways was observed in groups NG and MA. The up-regulated protein expression of p-NF-κBp65 induced with high glucose was significantly inhibited by PDTC and A771726 (both P < 0.05). The difference of NF-κBp65 mRNA expression was not statistically significant between the groups (all P > 0.05). (2) High glucose-induced podocyte activated the NF-κBp65 signaling path. Its downstream TRPC6 mRNA and protein expression significantly increased than NG while nephrin became down-regulated more than NG. PDTC and A771726 inhibited the high expression of TRPC6 while the expression of nephrin was elevated (all P < 0.05). (3) Immunofluorescent assay of high glucose-induced podocyte cytoskeleton showed disorderly F-actin and a disappearance of tensile fiber after 72 h.
Active leflunomide metabolite A771726 may protect podocytes through blocking the high glucose-induced signaling pathway of NF-κBp65.
Diabetic nephropathy (DN) represents a major public health cost. Tight glycemic and blood pressure control can dramatically slow, but not stop, the progression of the disease, and a large number of patients progress toward end-stage renal disease despite currently available interventions. An early and key event in the development of DN is loss of podocyte function (or glomerular visceral epithelial cells) from the kidney glomerulus, where they contribute to the integrity of the glomerular filtration barrier. Recent evidence suggests that podocytes can be the direct target of circulating hormones, lipids, and adipokines that are affected in diabetes. We review the clinical and experimental evidence implicating novel endocrine and metabolic pathways in the pathogenesis of podocyte dysfunction and the development of DN.
Stability and leakiness: Opposing challenges to the glomerulus. The complex architecture of the glomerular tuft is stabilized by several mechanisms. The basic system consists of the GBM and the mesangium maintaining the branching pattern of the capillary network. Superimposed are the podocytes, which appear to take effect by two mechanisms. First, podocytes contribute to the stabilization of the capillary folding pattern by supporting the angles between neighboring capillaries. Second, podocyte foot processes fixed to the outer aspect of the GBM probably function as contractile patches counteracting the elastic distension of the GBM. Simultaneously, the pattern of foot process interdigitation underlies the elaboration of a filtration slit and is thus pivotal for the high hydraulic permeability and the specifity of the glomerular filter. The loss of this pattern—commonly termed "foot process effacement" or "foot process fusion"—is frequently found in pathological situations and results in a decrease in permeability and impairment in specifity. On the other hand, foot process effacement is associated with prominent hypertrophy of the contractile apparatus of podocytes, suggesting an increased ability to generate forces counteracting capillary expansion. Thus, foot process effacement appears as an adaptive change in podocyte phenotype giving priority to the support function of podocytes for the prize of reducing the specific permeability.
Diabetic nephropathy is a major complication of diabetes leading to thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial expansion, and overt renal disease. The pathophysiologic mechanisms of diabetic nephropathy remain poorly understood. Nephrin is a recently found podocyte protein crucial for the interpodocyte slit membrane structure and maintenance of an intact filtration barrier. Here we have assessed the role of nephrin in two widely used animal models of diabetes, the streptozotocin model of the rat and the nonobese diabetic mouse. In both models, the expression levels of nephrin-specific mRNA as determined by real-time quantitative polymerase chain reaction increased up to two-fold during several weeks of follow-up. Immunohistochemical stainings revealed nephrin also more centrally within the glomerular tuft along with its preferential site in podocytes. Interestingly, as detected by immunoblotting, nephrin protein was also found in the urine of streptozotocin-induced rats. We conclude that nephrin is connected to the early changes of diabetic nephropathy and thus may contribute to the loss of glomerular filtration function.
The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1- and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.
Mechanosensitive large-conductance Ca(2+)-activated K(+) channels encoded by the Slo1 gene (BK(Ca) channels) are expressed in podocytes. Here we show that BK(Ca) channels reciprocally coimmunoprecipitate with synaptopodin (Synpo) in mouse glomeruli, in mouse podocytes, and in a heterologous expression system (HEK293T cells) in which these proteins are transiently expressed. Synpo and Slo1 colocalize along the surface of the glomerular basement membrane in mouse glomeruli. Synpo interacts with BK(Ca) channels at COOH-terminal domains that overlap with an actin-binding domain on the channel molecule that is necessary for trafficking of BK(Ca) channels to the cell surface. Moreover, addition of exogenous beta-actin to mouse podocyte lysates reduces BK(Ca)-Synpo interactions. Coexpression of Synpo increases steady-state surface expression of BK(Ca) channels in HEK293T cells. However, Synpo does not affect the stability of cell surface BK(Ca) channels, suggesting a primary effect on the rate of forward trafficking, and Synpo coexpression does not affect BK(Ca) gating. Conversely, stable knockdown of Synpo expression in mouse podocyte cell lines reduces steady-state surface expression of BK(Ca) channels but does not affect total expression of BK(Ca) channels or their gating. The effects of Synpo on surface expression of BK(Ca) are blocked by inhibition of Rho signaling in HEK293T cells and in podocytes. Functional cell surface BK(Ca) channels in podocytes are also reduced by sustained (2 h) but not acute (15 min) depolymerization of actin with cytochalasin D. Synpo may regulate BK(Ca) channels through its effects on actin dynamics and by modulating interactions between BK(Ca) channels and regulatory proteins of the podocyte slit diaphragm.
Multiphoton excitation fluorescence microscopy is a powerful noninvasive imaging technique for the deep optical sectioning of living tissues. Its application in several intact tissues is a significant advance in our understanding of organ function, including renal pathophysiological mechanisms. The glomerulus, the filtering unit in the kidney, is one good example of a relatively inaccessible and complex structure, with cell types that are otherwise difficult to study at high resolution in their native environment. In this article, we address the application, advantages, and limitations of this imaging technology for the study of the glomerular filtration barrier and the controversy it recently generated regarding the glomerular filtration of macromolecules. More advanced and accurate multiphoton determinations of the glomerular sieving coefficient that are presented here dismiss previous claims on the filtration of nephrotic levels of albumin. The sieving coefficient of 70-kD dextran was found to be around 0.001. Using a model of focal segmental glomerulosclerosis, increased filtration barrier permeability is restricted only to areas of podocyte damage, consistent with the generally accepted role of podocytes and the glomerular origin of albuminuria. Time-lapse imaging provides new details and important in vivo confirmation of the dynamics of podocyte movement, shedding, replacement, and the role of the parietal epithelial cells and Bowman's capsule in the pathology of glomerulosclerosis.
Microalbuminuria is an early lesion during the development of diabetic nephropathy. The loss of high molecular weight proteins in the urine is usually associated with decreased expression of slit diaphragm proteins. Nephrin, is the major component of the glomerular slit diaphragm and loss of nephrin has been well described in rodent models of experimental diabetes as well as in human diabetic nephropathy.
In this manuscript we analyzed the role of PKC-alpha (PKCalpha) on endocytosis of nephrin in podocytes. We found that treatment of diabetic mice with a PKCalpha-inhibitor (GO6976) leads to preserved nephrin expression and reduced proteinuria. In vitro, we found that high glucose stimulation would induce PKCalpha protein expression in murine and human podocytes. We can demonstrate that PKCalpha mediates nephrin endocytosis in podocytes and that overexpression of PKCalpha leads to an augmented endocytosis response. After PKC-activation, we demonstrate an inducible association of PKCalpha, PICK1 and nephrin in podocytes. Moreover, we can demonstrate a strong induction of PKCalpha in podocytes of patients with diabetic nephropathy.
We therefore conclude that activation of PKCalpha is a pathomechanistic key event during the development of diabetic nephropathy. PKCalpha is involved in reduction of nephrin surface expression and therefore PKCalpha inhibition might be a novel target molecule for anti-proteinuric therapy.
The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.
The highly ordered, isoporous substructure of the glomerular slit diaphragm was revealed in rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde. The slit diaphragm was similar in both animal species and appeared as a continuous junctional band, 300-450 A wide, consistently present within all slits formed by the epithelial foot processes. The diaphragm exhibited a zipper-like substructure with alternating, periodic cross bridges extending from the podocyte plasma membranes to a central filament which ran parallel to and equidistant from the cell membranes. The dimensions and spacing of the cross bridges defined a uniform population of rectangular pores approximately 40 by 140 A in cross section and 70 A in length. The total area of the pores was calculated to be about 2-3% of the total surface area of the glomerular capillaries. Physiological data indicate that the glomerular filter functions as if it were an isoporous membrane which excludes proteins larger than serum albumin. The similarity between the dimensions of the pores in the slit diaphragm and estimates for the size and shape of serum albumin supports the conclusion from tracer experiments that the slit diaphragm may serve as the principal filtration barrier to plasma proteins in the kidney.
Rat-1 fibroblasts stably overexpressing high levels of human insulin receptor were used as a model system to study the effects of hyperglycemia on insulin receptor tyrosine kinase (IRK) activity and protein kinase C (PKC) translocation in parallel in the intact cell. Glucose (10-25 mM) induced a significant reduction of IRK activity (tyrosine phosphorylation of IR-beta-subunit and IR-substrate-1) within 10 min. This effect was paralleled by a rapid translocation of several PKC isoforms (cPKC alpha, nPKC delta, nPKC epsilon, nPKC zeta) to the plasma membrane within 1 min. Kinetics of IRK inhibition and PKC translocation are consistent with the idea that the glucose effect on IRK is mediated by PKC activation. This hypothesis is supported by further observations. Addition of the protein kinase C inhibitor H-7 can prevent the effect of glucose on IRK. Inhibition of IRK is also observed after stimulation of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which can substitute for a physiological activator of PKC. Glucose (25 mM) increases the 32P incorporation in serine residues of the beta-subunit of IRK. We conclude that high levels of glucose induce inhibition of IRK in vivo. There is indirect evidence that this effect is mediated by a glucose-induced PKC translocation/activation and serine phosphorylation of the insulin receptor.
A mouse model for congenital nephrotic syndrome (NPHS1) was generated by inactivating the nephrin gene (Nphs1) in embryonic stem cells by homologous recombination. The targeting construct contained the Escherichia coli lacZ gene as a reporter for the Nphs1 promoter. Mice homozygous for inactivated Nphs1 were born at an expected frequency of 25%. Although seemingly normal at birth, they immediately developed massive proteinuria and edema and died within 24 h. The kidneys of null mice exhibited enlarged Bowman's spaces, dilated tubuli, effacement of podocyte foot processes and absence of the slit diaphragm, essentially as found in human NPHS1 patients. In addition to expression in glomerular podocytes, the reporter gene was expressed in the brain and pancreas of (+/-) and (-/-) mice. In the brain, expression was localized to the ventricular zone of the fourth ventricle, the developing spinal cord, cerebellum, hippocampus and olfactory bulb. In the cerebellum, the expression was seen in radial glial cells. Neither anatomical nor morphological abnormalities were observed in the brains of null mice.
Proteinuria, reflecting increased glomerular permeability to macromolecules is a characteristic feature of diabetic nephropathy. Nephrin, a 1241-residue transmembrane protein is a key component of the podocyte slit pore membrane and a major contributor of the glomerular filtration barrier. We investigated the expression of nephrin in human kidney tissue from patients with diabetic nephropathy to elucidate its relationship with proteinuria and the effects of anti-proteinuric therapy with angiotensin converting enzyme inhibition.
Renal biopsies were examined from 14 patients with Type II (non-insulin-dependent) diabetes mellitus and proteinuria who had been randomised to receive treatment with the ACE inhibitor, perindopril (4 mg/day) or placebo for the preceding 2 years. These specimens were compared with control human tissue sections, obtained from areas of normal renal cortex following nephrectomy for malignancy. Proteinuria was measured, specimens were examined histologically for injury and the expression of nephrin messenger RNA was assessed by quantitative in situ hybridisation.
Glomeruli from placebo-treated patients with diabetic nephropathy, showed a 62% reduction in nephrin expression compared with control subjects (p=0.0003). In contrast, nephrin RNA in glomeruli from perindopril treated patients was similar to that in the non-diabetic control group. In both placebo and perindopril treated patients, a close inverse correlation was noted between the magnitude of nephrin gene expression and the degree of proteinuria (placebo: r=0.86, p=0.013, perindopril: r=0.91, p=0.004).
Modulation in nephrin expression is related to the extent of proteinuria in diabetic nephropathy. These changes define, at a molecular level alterations in the glomerulus that occur in relation to proteinuria in diabetes and the effects of anti-proteinuric treatment with ACE inhibition.
We studied the distribution of nephrin in renal biopsies from 17 patients with diabetes and nephrotic syndrome (7 type 1 and 10 type 2 diabetes), 6 patients with diabetes and microalbuminuria (1 type 1 and 5 type 2 diabetes), and 10 normal subjects. Nephrin expression was semiquantitatively evaluated by measuring immunofluorescence intensity by digital image analysis. We found an extensive reduction of nephrin staining in both type 1 (67 +/- 9%; P < 0.001) and type 2 (65 +/- 10%; P < 0.001) diabetic patients with diabetes and nephrotic syndrome when compared with control subjects. The pattern of staining shifted from punctate/linear distribution to granular. In patients with microalbuminuria, the staining pattern of nephrin also showed granular distribution and reduction intensity of 69% in the patient with type 1 diabetes and of 62 +/- 4% (P < 0.001) in the patients with type 2 diabetes. In vitro studies on human cultured podocytes demonstrated that glycated albumin and angiotensin II reduced nephrin expression. Glycated albumin inhibited nephrin synthesis through the engagement of receptor for advanced glycation end products, whereas angiotensin II acted on cytoskeleton redistribution, inducing the shedding of nephrin. This study indicates that the alteration in nephrin expression is an early event in proteinuric patients with diabetes and suggests that glycated albumin and angiotensin II contribute to nephrin downregulation.
Evidence is accumulating that, in addition to regulating peripheral energy metabolism, insulin is an important modulator of neuronal function. Indeed, high levels of insulin and insulin receptors are expressed in several brain regions including the hippocampus. We have shown previously that insulin inhibits aberrant synaptic activity in hippocampal neurons via activation of large conductance Ca(2+)-activated K+ (BK) channels. In this study, we have examined further the effects of insulin on native hippocampal and recombinant (hSlo) BK channels expressed in human embryonic kidney (HEK) 293 cells. Pipette or bath application of insulin evoked a rapid increase in hippocampal BK channel activity, an action caused by activation of insulin receptors because insulin-like growth factor 1 (IGF-1) failed to mimic insulin action. In parallel studies, insulin, applied via the pipette or bath, also activated hSlo channels expressed in HEK293 cells. Although phosphoinositide 3-kinase is a key component of insulin and IGF-1 receptor signaling pathways, activation of this lipid kinase does not underlie the effects of insulin because neither 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) nor wortmannin inhibited or reversed insulin action. However, specific inhibitors of mitogen-activated protein kinase (MAPK) activation, 2'-amino-3'-methoxyflavone (PD98059) or 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126), attenuated insulin action, indicating that a MAPK-dependent mechanism underlies this process. Furthermore, insulin activation of this pathway enhances BK channel activity by shifting the Ca(2+)-sensitivity such that BK channels are active at more hyperpolarized membrane potentials. Because postsynaptic BK channels are important regulators of neuronal hyperexcitability, insulin-induced activation of BK channels, via stimulation of a MAPK-dependent pathway, may be an important process for regulating hippocampal function under normal and pathological conditions.
The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.
Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.
ATP release from macula densa (MD) cells into the interstitium of the juxtaglomerular (JG) apparatus (JGA) is an integral component of the tubuloglomerular feedback (TGF) mechanism that controls the glomerular filtration rate. Because the cells of the JGA express a number of calcium-coupled purinergic receptors, these studies tested the hypothesis that TGF activation triggers a calcium wave that spreads from the MD toward distant cells of the JGA and glomerulus. Ratiometric calcium imaging of in vitro microperfused isolated JGA-glomerulus complex dissected from rabbits was performed with fluo-4/fura red and confocal fluorescence microscopy. Activation of TGF by increasing tubular flow rate at the MD rapidly produced a significant elevation in intracellular Ca(2+) concentration ([Ca(2+)](i)) in extraglomerular mesangial cells (by 187.6 +/- 45.1 nM) and JG renin granular cells (by 281.4 +/- 66.6 nM). Subsequently, cell-to-cell propagation of the calcium signal at a rate of 12.6 +/- 1.1 microm/s was observed upstream toward proximal segments of the afferent arteriole and adjacent glomeruli, as well as toward intraglomerular elements including the most distant podocytes (5.9 +/- 0.4 microm/s). The same calcium wave was observed in nonperfusing glomeruli, causing vasoconstriction and contractions of the glomerular tuft. Gap junction uncoupling, an ATP scavenger enzyme cocktail, and pharmacological inhibition of P(2) purinergic receptors, but not adenosine A(1) receptor blockade, abolished the changes in [Ca(2+)](i) and propagation of the calcium wave. These studies provided evidence that both gap junctional communication and extracellular ATP are integral components of the TGF calcium wave.
Albuminuria in diabetic nephropathy is due to endothelial dysfunction, a loss of negative charges in the basement membrane, and changes a of the slit-membrane diaphragm composition. We have recently shown that protein kinase C alpha (PKCalpha)-deficient mice are protected against the development of albuminuria under diabetic conditions. We here tested the hypothesis that PKCalpha mediates the hyperglycemia-induced downregulation of the slit-diaphragm protein nephrin. After 8 weeks of streptozotocin (STZ)-induced hyperglycemia the expression of glomerular nephrin was significantly reduced. In contrast, other slit-diaphragm proteins such as podocin and CD2AP were unaltered in diabetic state. In PKCalpha-/- mice, hyperglycemia-induced downregulation of nephrin was prevented. Podocin and CD2AP remained unchanged. In addition, the nephrin messenger RNA expression was also reduced in hyperglycemic wild-type mice but remained unaltered in PKCalpha-/- mice. We postulate that the underlying mechanism of the hyperglycemia-induced regulation of various proteins of the glomerular filtration barrier is a PKCalpha-dependent regulation of the Wilms' Tumor Suppressor (WT1) which previously has been shown to act as a direct transcription factor on the nephrin promoter. Our data suggest that PKCalpha activation may be an important intracellular signaling pathway in the regulation of nephrin expression and glomerular albumin permeability in the diabetic state.
Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.
Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the physiological properties of many cell types. The gating properties of BK(Ca) channels are Ca(2+)-, voltage- and stretch-sensitive, and stretch-sensitive gating of these channels requires interactions with actin microfilaments subjacent to the plasma membrane. Moreover, we have previously shown that trafficking of BK(Ca) channels to the plasma membrane is associated with processes that alter cytoskeletal dynamics. Here, we show that the Slo1 subunits of BK(Ca) channels contain a novel cytoplasmic actin-binding domain (ABD) close to the C terminus, considerably downstream from regions of the channel molecule that play a major role in determining channel-gating properties. Binding of actin to the ABD can occur in a binary mixture in the absence of other proteins. Coexpression of a small ABD-green fluorescent protein fusion protein that competes with full-length Slo1 channels for binding to actin markedly suppresses trafficking of full-length Slo1 channels to the plasma membrane. In addition, Slo1 channels containing deletions of the ABD that eliminate actin binding are retained in intracellular pools, and they are not expressed on the cell surface. At least one point mutation within the ABD (L1020A) reduces surface expression of Slo1 channels to approximately 25% of wild type, but it does not cause a marked effect on the gating of point mutant channels that reach the cell surface. These data suggest that Slo1-actin interactions are necessary for normal trafficking of BK(Ca) channels to the plasma membrane and that the mechanisms of this interaction may be different from those that underlie F-actin and stretch-sensitive gating.
Diabetic nephropathy (DN) is the leading cause of renal failure in the world. It is characterized by albuminuria and abnormal glomerular function and is considered a hyperglycemic "microvascular" complication of diabetes, implying a primary defect in the endothelium. However, we have previously shown that human podocytes have robust responses to insulin. To determine whether insulin signaling in podocytes affects glomerular function in vivo, we generated mice with specific deletion of the insulin receptor from their podocytes. These animals develop significant albuminuria together with histological features that recapitulate DN, but in a normoglycemic environment. Examination of "normal" insulin-responsive podocytes in vivo and in vitro demonstrates that insulin signals through the MAPK and PI3K pathways via the insulin receptor and directly remodels the actin cytoskeleton of this cell. Collectively, this work reveals the critical importance of podocyte insulin sensitivity for kidney function.
Loss or dysfunction of podocytes is a major cause of glomerular kidney disease. Several genetic forms of glomerular disease are caused by mutations in genes that encode structural elements of the slit diaphragm or the underlying cytoskeleton of podocyte foot processes. The recent discovery that gain-of-function mutations in Ca(2+)-permeable canonical transient receptor potential-6 channels (TRPC6) underlie a subset of familial forms of focal segmental glomerulosclerosis (FSGS) has focused attention on the basic cellular physiology of podocytes. Several recent studies have examined the role of Ca(2+) dynamics in normal podocyte function and their possible contributions to glomerular disease. This review summarizes the properties of TRPC6 and related channels, focusing on their permeation and gating properties, the nature of mutations associated with familial FSGS, and the role of TRPC channels in podocyte cell biology as well as in glomerular pathophysiology. TRPC6 interacts with several proteins in podocytes, including essential slit diaphragm proteins and mechanosensitive large-conductance Ca(2+)-activated K(+) channels. The signaling dynamics controlling ion channel function and localization in podocytes appear to be quite complex.
Elevated glomerular filtration rate (GFR) is a common observation in early diabetes mellitus and closely correlates with the progression of diabetic nephropathy. Hyperfiltration has been explained to be the result of a reduced load of sodium and chloride passing macula densa, secondarily to an increased proximal reabsorption of glucose and sodium by the sodium-glucose co-transporters. This results in an inactivation of the tubuloglomerular feedback (TGF), leading to a reduced afferent arteriolar vasoconstriction and subsequently an increase in GFR. This hypothesis has recently been questioned due to the observation that adenosine A(1)-receptor knockout mice, previously shown to lack a functional TGF mechanism, still display a pronounced hyperfiltration when diabetes is induced. Leyssac demonstrated in the 1960s (Acta Physiol Scand58, 1963:236) that GFR and proximal reabsorption can work independently of each other. Furthermore, by the use of micropuncture technique a reduced hydrostatic pressure in Bowman's space or in the proximal tubule of diabetic rats has been observed. A reduced pressure in Bowman's space will increase the pressure gradient over the filtration barrier and can contribute to the development of diabetic hyperfiltration. When inhibiting proximal reabsorption with a carbonic anhydrase inhibitor, GFR decreases and proximal tubular pressure increases. Measuring intratubular pressure allows a sufficient time resolution to reveal that net filtration pressure decreases before TGF is activated which highlights the importance of intratubular pressure as a regulator of GFR. Taken together, these results imply that the reduced intratubular pressure observed in diabetes might be crucial for the development of glomerular hyperfiltration.
Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1(VEDEC), Slo1(QEERL), and Slo1(EMVYR)) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca(2+)-dependent K(+) current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BK(Ca) currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons.
Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BK(Ca) channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BK(Ca) channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BK(Ca) channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BK(Ca) channels in the kidney and possibly in other tissues.
Large-conductance (BK(Ca) type) Ca(2+)-activated K(+) channels encoded by the Slo1 gene and various canonical transient receptor potential channels (TRPCs) are coexpressed in many cell types, including podocytes (visceral epithelial cells) of the renal glomerulus. In this study, we show by coimmunoprecipitation and GST pull-down assays that BK(Ca) channels can associate with endogenous TRPC3 and TRPC6 channels in differentiated cells of a podocyte cell line. Both types of TRPC channels colocalize with Slo1 in podocytes and in human embryonic kidney (HEK) 293T cells transiently coexpressing the TRPC channels with Slo1. In HEK293T cells, coexpression of TRPC6 increased surface expression of a Slo1 subunit splice variant (Slo1(VEDEC)) that is typically retained in intracellular compartments, as assessed by cell-surface biotinylation assays and confocal microscopy. Corresponding currents through BK(Ca) channels were also increased with TRPC6 coexpression, as assessed by whole-cell and excised inside-out patch recordings. By contrast, coexpression of TRPC3 had no effect on the surface expression of BK(Ca) channels in HEK293T cells or on the amplitudes of currents in whole cells or excised patches. In podocytes, small interfering RNA knockdown of endogenous TRPC6 reduced steady-state surface expression of endogenous Slo1 channels, but knockdown of TRPC3 had no effect. TRPC6, but not TRPC3 knockdown also reduced voltage-evoked outward current through podocyte BK(Ca) channels. These data indicate that TRPC6 and TRPC3 channels can bind to Slo1, and this colocalization may allow them to serve as a source of Ca(2+) for the activation of BK(Ca) channels. TRPC6 channels also play a role in the regulation of surface expression of a subset of podocyte BK(Ca) channels.
Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low-(5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [gamma-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effect on autophosphorylation (30 +/- 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 +/- 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 microM to 80 microM) for ATP of IRK from high-glucose-treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells after acute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
The nature of renal hemodynamic response to a large carbohydrate-rich meal and the associated renal excretory functions were examined. Seven normal subjects were studied after ingestion of 300 g of rice meal. Five healthy men served as a time control. After eating the test meal, the mean creatinine clearance began to rise and became significantly higher in the 2nd hour compared with the control (118 +/- 7 vs. 91 +/- 3 ml/min/1.73 m2; p less than 0.01). Urinary excretion rates of sodium and chloride after the meal were significantly higher compared with those in the premeal period. Blood glucose concentrations rose significantly following the meal. The postprandial plasma levels of amino acids did not vary significantly compared with the premeal values. These results demonstrate that rice meal ingestion increased glomerular filtration rate via a mechanism(s) different from the protein-induced glomerular hyperfiltration.
By immunoelectron microscopy the podocyte foot processes of the rat and human kidney have been shown to contain three major proteins of the contractile apparatus in muscle, i.e., actin, myosin, and the Z-line protein, alpha-actinin. Gel electrophoresis and immunoblot analysis of isolated glomeruli suggests that these proteins constitute an important part of the total glomerular protein contents. In the chicken kidney, the plasmalemmal portion of the foot processes that abuts the glomerular basement membrane was specifically labeled with antibodies against chicken gizzard vinculin and talin, two proteins thought to be important for the linkage of actin filaments to the lipid bilayer and to the receptor for fibronectin and laminin. Such a linkage may not only be important for the attachment of actin filaments to the plasma membrane, but could also be of functional significance for restricting the fibronectin-laminin receptor in its lateral diffusion in the plane of the lipid bilayer and to localize it at the basis of the podocytic foot processes. Assuming that actin, myosin, and alpha-actinin are arranged in a way that would allow the foot processes to generate contractile force this filament system might help the glomerular capillaries to resist the high intraluminal hydrostatic pressure as well as to actively modify the surface area for filtration. Vimentin and tubulin, the main protein subunits of intermediate filaments and microtubules, respectively, were confined to the podocyte cell body and the major processes but were virtually absent from the foot processes. This suggests that both proteins and their polymers are not important for the structure and function of the foot processes.
Food intake increases glomerular filtration and proteinuria in adult rats. That this postprandial hyperfiltration could be age dependent was investigated in 3-, 10-, 20-, and 30-mo-old rats. Glomerular filtration rate and protein excretion were measured in fed or 24 h fasted conscious animals. In the 3-mo-old rats food ingestion increased renal filtration by 45% from 1.17 +/- 0.08 to 1.73 +/- 0.11 ml.min-1.g kidney wt-1 (n = 6). As the animals became older, the differences between fed and fasted periods became smaller: in 30-mo-old rats glomerular filtration rate was 0.85 +/- 0.03 and 1.01 +/- 0.06 ml.min-1.g kidney wt-1 (n = 6) in fasted and fed conditions, respectively. Proteinuria, which was mainly albuminuria, increased slightly with age and was more markedly reduced by acute food restriction in the 30-mo-old than in the 3-mo-old rats. Because the renin-angiotensin system activity decreases with age, its role in postprandial hyperfiltration was assessed by measuring glomerular filtration in 3-mo-old animals whose angiotensin II converting-enzyme activity was chronically inhibited by daily administration of perindopril. In such experimental conditions there was no longer a difference in renal filtration between fed and fasted rats. These data indicate that 1) postprandial increase in glomerular filtration is to some extent related to the renin-angiotensin system activity; 2) short-term reduction of food intake reduces proteinuria even in senescent rats, although the feeding dependence of the glomerular filtration is blunted with age.
The effect of amino acid (AA) infusion on renal hemodynamics was examined in 19 healthy subjects. Thirteen subjects participated in the first protocol (normal protein intake) and six in the second protocol (low protein intake). The first protocol consisted of three studies: study 1 (n = 13), AA were infused over 3 h to increase plasma amino acid levels two- to threefold; study 2 (n = 7), AA were infused with somatostatin and peripheral replacement of insulin, glucagon, and growth hormone; study 3 (n = 6), somatostatin was infused with basal hormonal replacement as in study 2. During study 1, glomerular filtration rate (GFR) rose by 20% (from 107 +/- 5 to 128 +/- 4 ml . 1.73 m-2 . min-1, P less than 0.001). Renal plasma flow (RPF) increased by a similar percentage (599 +/- 35 to 704 +/- 33 ml . 1.73 m-2 . min-1, P less than 0.001). When somatostatin was infused with AA (study 2), neither GFR nor RPF changed from base line. Somatostatin infusion alone (study 3) had no effect on GFR or RPF. During protocol 2, six subjects received a low-protein diet (40 g/day) for 7 days and AA were infused as per study 1. Base-line GFR (104 +/- 5 to 96 +/- 4 ml . 1.73 m-2 . min-1 and RPF (593 +/- 32 to 507 +/- 23 ml . 1.73 m-2 . min-1) both decreased (P less than 0.02) after the low-protein diet.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of intravenous glucose infusion on glomerular filtration rate and renal plasma flow (constant infusion technique using 125I-iothalamate and 131I-hippuran) and on urinary excretion of albumin and beta-2-microglobulin were studied in ten normal subjects and seven metabolically well-controlled insulin-dependent diabetics. Following glucose infusion in normal subjects (n = 10) blood glucose increased from 4.7 +/- 0.1 to 10.9 +/- 0.4 mmol/l (SEM) (p less than or equal to 0.01). Glomerular filtration rate increased from 116 +/- 2 to 123 +/- 3 ml/mi x 1.73 m2 (p less than or equal to 0.01), while no change in renal plasma flow was seen - 552 +/- 11 versus 553 +/- 18 ml/min x 1.73 m2. Volume expansion with intravenous saline infusion in six of the normal subjects induced no changes in blood glucose or kidney function. In seven strictly controlled insulin-dependent diabetics, blood glucose values were raised from 4.6 +/- 0.4 to 16.0 +/- 0.6 mmol/l and clamped by means of an 'artificial beta cell'. Glomerular filtration rate increased in all patients, from 133 +/- 5 to 140 +/- 6 ml/min x 1.73 m2 (p less than or equal to 0.02), as did renal plasma flow from 576 +/- 26 to 623 +/- 38 ml/min x 1.73 m2 (p less than or equal to 0.02). Urinary albumin excretion remained unchanged in both normal subjects and diabetics. beta-2-microglobulin excretion rate increased significantly in the diabetics following glucose infusion, while no significant change was seen in the normal subjects. Our results show that hyperglycaemia per se contributes to the increased glomerular filtration rate and renal plasma flow in insulin-dependent diabetes.
In a previous study of the changes in glomerular structure in the isolated perfused kidney (IPK), perfusion at high pressures lead to an enlargement of the glomerular tuft and to the formation of giant capillaries. The present paper analyzes the morphological and dimensional changes of the peripheral glomerular capillary wall under these circumstances. The enlargement of glomerular capillaries at high pressure perfusion was accompanied by a considerable increase in the surface area of the glomerular basement membrane (GBM). The podocyte as well as the endothelial layer perfectly adapted to the acute challenge in covering increasing GBM area. The interdigitating foot process pattern showed up in an ideal arrangement. The capillary wall expansion was associated with a significant increase in total pericapillary slit area. Compared to the corresponding low pressure groups (65 mm Hg, without and with the application of vasodilators) the slit area increased in the high pressure groups (105 mm Hg, without and with vasodilator) by approximately 50 and 75%, respectively. This increase of the slit area was mainly due to an increase in slit length; the slit width remained fairly constant. These findings indicate that the pericapillary wall is distensible based on a distensibility of the GBM. We suggest that the contractile apparatus of podocyte foot processes regulates the expansion of the GBM.
To investigate the mechanism for the impairment of insulin receptor kinase activity induced by high glucose (HG) in Rat 1 fibroblasts that expressed human insulin receptors (HIRc), we measured protein tyrosine phosphatase (PTPase) activity in HG cells. Incubating HIRc cells for 4 days in 27 mM D-glucose (HG) stimulated cytosolic PTPase activities, but not particulate PTPase activity as determined by two methods using the dephosphorylation of insulin receptors. Furthermore, PTP1B, a major non-transmembrane PTPase in the cytosolic fraction, was increased in HG cells according to Western blots. These results indicate that desensitization of insulin receptor function by a high glucose condition is associated with the activation of PTPase activity.
Kidney biopsies from Pima Indians with type II diabetes were analyzed. Subjects were classified clinically as having early diabetes (n = 10), microalbuminuria (n = 17), normoalbuminuria, despite a duration of diabetes equal to that of the subjects with microalbuminuria (n = 12), or clinical nephropathy (n = 12). Subjects with microalbuminuria exhibited moderate increases in glomerular and mesangial volume when compared with those with early diabetes, but could not be distinguished from subjects who remained normoalbuminuric after an equal duration of diabetes. Subjects with clinical nephropathy exhibited global glomerular sclerosis and more prominent structural abnormalities in nonsclerosed glomeruli. Marked mesangial expansion was accompanied by a further increase in total glomerular volume. Glomerular capillary surface area remained stable, but the glomerular basement membrane thickness was increased and podocyte foot processes were broadened. Broadening of podocyte foot processes was associated with a reduction in the number of podocytes per glomerulus and an increase in the surface area covered by remaining podocytes. These findings suggest that podocyte loss contributes to the progression of diabetic nephropathy.
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.
In mammals, protein ingestion increases the glomerular filtration rate (GFR), an effect which has been incriminated as a risk factor in progression of renal disease. Some studies suggest that a postprandial increase in GFR is absent or mild with vegetable proteins compared to animal proteins. The objective of this experiment was to determine whether vegetable (soy) protein had different effects than animal protein on GFR in dogs with normal or reduced renal function. A trial was conducted in which GFR was measured in four dogs with normal kidney function and seven dogs with reduced renal mass before and after administering protein. Normal dogs were fed four protein sources (casein, soy meal, soy flakes and purified soy protein). Dogs with reduced renal mass were fed three protein sources (casein, purified soy protein and pork liver). All proteins significantly (P < 0.05) increased the GFR in both groups except for casein (P = 0.066) in normal dogs. Proteins did not differ significantly in the magnitude of the increase in GFR that was induced. This study indicates that soy proteins in dogs have the same effect on GFR as animal-source proteins, which is contrary to reports of effects in humans.
The number of cells in glomeruli has been a challenging measure, especially in human kidneys, with only a small amount of tissue obtained by biopsy. However, the number of cells and their function are important determinants of renal function in health and disease.
Modern morphometric techniques have now provided the means to determine the numerical density (Nv) and number (with a measure of glomerular volume) of endothelial cells, mesangial cells, and podocytes in plastic-embedded renal tissue biopsied from nondiabetic subjects (N = 36) and type 1 diabetic patients (N = 46) over an extended age range from childhood through late adult.
Nv values for all glomerular cells varied only slightly with age and did not change within the range of glomerular lesions of diabetes studied. Thus, the increase in glomerular volume during childhood to a steady level thereafter was the primary determinant of total glomerular cell number. The number of mesangial cells and endothelial cells increased with age, reflecting the increase in all cells, while the podocytes remained unchanged in number over all ages studied (10 to 69 years). Numbers of total glomerular cells, mesangial cells, and endothelial cells were not changed with diabetes, while podocytes were fewer in number in diabetic patients of all ages, with reduced podocyte numbers even in diabetes of short duration.
The essentially constant glomerular cell density in nondiabetic and diabetic subjects under different circumstances possibly indicates an underlying propensity for the glomerulus to regulate its architecture to maintain a constant number of cells per volume, no matter the size of the glomerulus or the severity of diabetic nephropathy studied in this set of patients. The reductions in podocyte numbers in both younger and older diabetic patients indicate a significant risk for functional abnormalities as diabetic nephropathy progresses. Moreover, these observations do not support the suggestion of marked increases in glomerular cell number (and especially mesangial cells) with the development and progression of diabetic nephropathy.
Changes in the intrinsic spike discharge properties in one neuronal population can alter the functions and even the formation of an entire neuronal network. Therefore it is important to understand the factors that regulate acquisition of a mature electrophysiological phenotype. Here we focus on large-conductance K(Ca) channels, which shape the pattern of repetitive discharge and which are therefore likely to play a role in the refinement of neural networks during development. In the parasympathetic ciliary ganglion of chick, the developmental expression of K(Ca) channels coincides with stages at which ciliary cells form synapses with target tissues. Moreover, K(Ca) expression requires formation of synapses with target tissues, and with afferent preganglionic inputs. The trophic effect of targets is mediated by TGFbeta1, whereas the effect of the preganglionic input is mediated by an isoform of beta-neuregulin-1. These trophic factors act synergistically, and this appears to be a normal feature of their actions in vivo. The acute effects of TGFbeta1 entail translocation of preexisting K(Ca) channels from intracellular stores to the plasma membrane. This requires activation of the signaling enzymes Ras, Erk MAP kinase and PI3 kinase. TGFbeta1 also causes a more sustained increase in K(Ca) channels (i.e. for up to 2 weeks) that requires synthesis of new channel proteins. Inductive regulation of K(Ca) expression is also observed in CNS cells that form more complex networks. In lumbar motoneurons, the largest changes in K(Ca) expression coincide with the elimination of synapses with hindlimb targets. Interactions with target tissues play a key role in regulation of motoneuron K(Ca) expression, and this trophic effect of target muscle is mediated by GDNF or a closely related factor. In addition, K(Ca) expression in motoneurons is dependent on ongoing electrical activity both in vivo and in vitro. This provides an additional mechanism for use-dependent refinement of neural networks during embryonic development.
Insulin is a key hormone regulating the control of metabolism and the maintenance of normoglycaemia and normolipidaemia. Insulin acts by binding to its cell surface receptor, thus activating the receptor’s intrinsic tyrosine kinase activity, resulting in receptor autophosphorylation and phosphorylation of several substrates. Tyrosine phosphorylated residues on the receptor itself and on subsequently bound receptor substrates provide docking sites for downstream signalling molecules, including adapters, protein serine/threonine kinases, phosphoinositide kinases and exchange factors. Collectively, those molecules orchestrate the numerous insulin-mediated physiological responses.
A clear picture is emerging of the way in which insulin elicits several intracellular signalling pathways to mediate its physiologic functions. A further challenge, being pursued by several laboratories, is to understand the molecular mechanisms that underlie insulin action at the peripheral level, deregulation of which ultimately leads to hyperglycaemia and Type 2 diabetes.
We review how circulating factors such as insulin itself, TNF-α, interleukins, fatty acids and glycation products influence insulin action through insulin signalling molecules themselves or through other pathways ultimately impinging on the insulin-signalling pathway. Understanding how the mechanism by which molecular insulin action is modulated by these factors will potentially provide new targets for pharmacological agents, to enable the control of altered glucose and lipid metabolism and diabetes.
Recent disclosure of podocyte proteins has unraveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we addressed the role of nephrin, CD2-associated protein (CD2AP), and podocin together with the integrity of the slit diaphragm in the pathogenesis of proteinuria of patients with diabetes and nephropathy.
Nephrin mRNA and protein expression were evaluated in parallel in adult diabetic patients by in situ hybridization and immunohistochemistry. For comparison, nondiabetic patients with minimal change nephrosis and normal control patients were evaluated. CD2AP and podocin expression by immunohistochemistry was also assessed. The filtration slit was analyzed by morphometry and transmission electron microscopy.
Extracellular nephrin mRNA and protein were markedly reduced in diabetic patients. No changes were found in patients with minimal change versus controls. CD2AP and podocin were comparable in all subjects. Ultrastructural analysis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a frequency of the filtration slits comparable to control patients.
Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.
Diseases of the glomerular filter of the kidney are a leading cause of end-stage renal failure. Recent studies have empha- sized the critical role of the slit diaphragm of podocytes for the size-selective filtration barrier of the kidney and revealed novel aspects of the mechanisms that lead to proteinuria, in both inherited and acquired diseases (1- 4). Several critical struc- tural protein components of the slit diaphragm have been identified. Recently, it has been speculated that these slit dia- phragm proteins, in addition to their structural functions, par- ticipate in common signaling pathways. This review focuses on what is known about signaling at the slit diaphragm. It provides a snapshot of our current understanding of the signaling prop- erties of slit diaphragm proteins and projects a framework for further studies necessary to delineate the function and dynam- ics of the slit diaphragm protein complex and the pathogenesis of nephrotic syndrome. Ultrafiltration of plasma in the renal glomeruli is a major function of the kidney. The glomerular filter through which the ultrafiltrate has to pass consists of three layers: the fenestrated endothelium, the intervening glomerular basement membrane, and the epithelial podocyte foot processes. This filtration bar- rier behaves as a size-selective sieve restricting the passage of macromolecules on the basis of their size, shape, and charge (5-7). Although the glomerular filter is a primary target of a large number of progressive disorders that lead to chronic renal insufficiency, until recently, little was known about the impor- tance of podocytes for establishing the size-selective filtration barrier of the kidney. The recent description of gene defects in hereditary nephrotic syndrome resulting in the loss of podocyte proteins has dramatically changed this situation. Together with data from various animal models, these studies have unraveled
Podocytes express many proteins characteristic of smooth muscle, such as actin and myosin. They also express receptors to several vasoactive agents, including acetylcholine and angiotensin II; these phenotypic properties suggest that podocytes are not static entities but may respond to physiologic stimuli. The electrophysiologic properties of a conditionally immortalized human podocyte cell line that expresses the specific podocyte proteins nephrin, podocin, and synaptopodin were examined by patch clamp. Channels that were highly K(+)-selective and had a conductance of 224 +/- 11.5 pS in symmetrical 150 mM K(+) solutions were identified. Channel activity was Ca(2+)- and voltage-dependent, being increased with an increase in Ca(2+) or depolarization, and inhibited by penitrem A. The conductance and voltage- and Ca(2+)-dependence suggest that this is the large-conductance calcium-activated K(+) channel, BK (KCNMA1)-this was supported by reverse transcription-PCR experiments that showed the presence of the BK encoding mRNA, along with expression of KCNMB subunit types 3 and 4. In sections of human glomeruli, immunocytochemistry revealed that BK co-localizes with the podocyte-specific protein nephrin, indicating that these channels are present in native human podocytes. In whole-cell experiments, penitrem A inhibited outward currents to the same extent as tetra-ethyl ammonium (TEA) but did not affect the membrane potential. Channel activity was also increased by applying suction to the patch pipette or by dilution of the bathing medium, indicating that these channels are stretch sensitive. Thus, these channels do not contribute to the resting membrane potential but are activated by a rise in intracellular Ca(2+), membrane depolarization, cell swelling, or membrane stretch. By implication, these results suggest that podocytes may be able to respond to changes in the glomerular capillary pressure and modulate the GFR.
Pathways to nephron loss starting from glomerular diseases—Insights from animal models.
Studies of glomerular diseases in animal models show that progression toward nephron loss starts with extracapillary lesions, whereby podocytes play the central role. If injuries remain bound within the endocapillary compartment, they will undergo recovery or be repaired by scaring.
Degenerative, inflammatory and dysregulative mechanisms leading to nephron loss are distinguished. In addition to several other unique features, the dysregulative mechanisms leading to collapsing glomerulopathy are particular in that glomeruli and tubules are affected in parallel. In contrast, in degenerative and inflammatory diseases, tubular injury is secondary to glomerular lesions. In both of the latter groups of diseases, the progression starts in the glomerulus with the loss of the separation between the tuft and Bowman's capsule by forming cell bridges (parietal cells and/or podocytes) between the glomerular and the parietal basement membranes. Cell bridges develop into tuft adhesions to Bowman's capsule, which initiate the formation of crescents, either by misdirected filtration (proteinaceous crescents) or by epithelial cell proliferation (cellular crescents). Crescents may spread over the entire circumference of the glomerulus and, via the glomerulotubular junction, may extend onto the tubule.
Two mechanisms concerning the transfer of a glomerular injury onto the tubulointerstitium are discussed: (1) direct encroachment of extracapillary lesions and (2) protein leakage into tubular urine, resulting in injury to the tubule and the interstitium. There is evidence that direct encroachment is the crucial mechanism.
Progression of chronic renal disease is underlain by a vicious cycle which passes on the damage from lost and/or damaged nephrons to so far healthy nephrons. Presently, two mechanisms are discussed: (1) the loss of nephrons leads to compensatory mechanisms in the remaining nephrons (glomerular hypertension, hyperfiltration, hypertrophy) which increase their vulnerability to any further challenge (overload hypothesis); and (2) a proteinuric glomerular disease leads, by some way or another, to tubulointerstitial inflammation and fibrosis, accounting for the further deterioration of renal function (fibrosis hypothesis). So far, no convincing evidence has been published that in primary glomerular diseases fibrosis is harmful to healthy nephrons.
The potential of glomerular injuries to regenerate or to be repaired by scaring is limited. The only option for extracapillary injuries with tuft adhesion is repair by formation of a segmental adherent scar (i.e., segmental glomerulosclerosis).
The trafficking of large-conductance Ca2+-activated K+ channels (K(Ca)) in chick ciliary ganglion neurons is regulated by growth factors. Here we show that a canonical p38 cascade inhibits K(Ca) trafficking in ciliary ganglion neurons. Two different p38 inhibitors (SB202190 or SB203580) or over-expression of dominant-negative forms of several components of the p38 cascade increased K(Ca) in ciliary neurons. Inhibition of protein synthesis or Golgi processing had no effect on this phenomenon, suggesting that p38 is acting at a distal step of the trafficking pathway. Depolymerization of filamentous actin (F-actin) increased functional expression of K(Ca), whereas stabilization of F-actin inhibited the effect of SB202190 on K(Ca) trafficking. SB202190 also caused an immunochemically detectable increase in K(Ca) on the plasma membrane. Inhibition of p38 decreased the extent of cortical F-actin in ciliary neurons. Macroscopic K(Ca) is suppressed by transforming growth factor (TGF) beta3. Application of TGFbeta3 increased the phosphorylation of p38 in ciliary neurons and increased cortical F-actin. Thus, the p38 signaling cascade endogenously suppresses development of functional K(Ca), in part by stabilizing an F-actin barrier that prevents plasma membrane insertion of functional channel complexes. This cascade also appears to mediate inhibitory effects of TGFbeta3 on the expression of K(Ca).
TRPC6 is thought to be a Ca2+-permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca2+ concentration ([Ca2+]i) when the cells were bathed in high-K+ buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca2+ permeability relative to monovalent cation permeability and/or that Ca2+ influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca2+ permeability, TRPC6 currents were examined in extracellular buffer in which Ca2+ was varied from 0.02 to 20 mm. The results were consistent with a pore-permeation model in which Ca2+ acts primarily as a blocking ion and contributes only a small percentage (∼4%) to whole-cell currents in the presence of extracellular Na+. Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca2+]i 50–100 nm when the membrane potential was clamped at between −50 and −80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca2+-activated K+ current), activation of TRPC6 will lead to a sustained increase in [Ca2+]i. Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca2+ response will be cell specific and dependent upon the complement of other channel types.
Podocytes form an epithelial layer on the outer aspect of the basement membrane of glomerular capillaries. The interdigitating pattern of podocyte foot processes (PFPs) generates a unique and extremely long cell-cell contact area - the filtration slit. Thus, the interdigitating PFPs are the morphological basis for the high hydraulic conductivity of the glomerular capillaries. Any disturbance in this interdigitating pattern results in a drop of glomerular filtration rate impairing renal function. PFPs are based on the actin cytoskeleton, consisting of a subplasmalemmal network and a central core of filament bundles. Besides giving PFPs their morphology, the actin cytoskeleton anchors cell-cell contact and cell-matrix proteins in podocytes. Several human genetic diseases as well as transgenic mouse models provide evidence for the crucial role of the actin cytoskeleton in podocytes. Varying flow rates of the filtrate, increased glomerular capillary pressure in glomerular hypertension, and varying activation states of contractile proteins in PFPs impose a mechanical load on the actin cytoskeleton, challenging the intricate arrangement of PFPs and podocyte adhesion. Here we review data about the actin cytoskeleton of podocytes and the response of podocytes to mechanical load. From these data possible mechanisms are emerging how the actin cytoskeleton may allow podocytes to adapt to states of increased mechanical load.
The terminally differentiated podocyte, also called glomerular visceral epithelial cell, are highly specialized cells. They function as a critical size and charge barrier to prevent proteinuria. Podocytes are injured in diabetic and non-diabetic renal diseases. The clinical signature of podocyte injury is proteinuria, with or without loss of renal function owing to glomerulosclerosis. There is an exciting and expanding literature showing that hereditary, congenital, or acquired abnormalities in the molecular anatomy of podocytes leads to proteinuria, and at times, glomerulosclerosis. The change in podocyte shape, called effacement, is not simply a passive process following injury, but is owing to a complex interplay of proteins that comprise the molecular anatomy of the different protein domains of podocytes. These will be discussed in this review. Recent studies have also highlighted that a reduction in podocyte number directly causes proteinuria and glomerulosclerosis. This is owing to several factors, including the relative inability for these cells to proliferate, detachment, and apoptosis. The mechanisms of these events are being elucidated, and are discussed in this review. It is the hope that by delineating the events following injury to podocytes, therapies might be developed to reduce the burden of proteinuric renal diseases.
Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform-like activity, in part by activating large-conductance Ca2+-activated potassium (BK) channels. Moreover, the mitogen-activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho-MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK-dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability.