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ABSTRACT: Recent studies suggested that miRNAs are involved in the development of the pathogenesis of HIV-associated nephropathy (HIVAN). Rapamycin, a widely used mTOR inhibitor, has been demonstrated to slow down the progression of HIVAN. However, the role of miRNA in the regulation of these processes has not been investigated so far. In the current study, we have used a microarray-based approach in combination with real-time PCR to profile the miRNA expression patterns in rapamycin-treated HIVAN mice (Tg26). Our results demonstrated that 19 miRNAs belonging to 13 different families expressed differentially in renal tissues of rapamycin-receiving Tg26 mice when compared to Tg26 mice-receiving saline only. The patterns of miRNAs expression in rapamycin-receiving Tg26 mice took a reverse turn. These miRNAs were classified into 8 functional categories. In in vitro studies, we examined the expression of specific miRNAs in HIV-1 transduced human podocytes (HIV/HPs). HIV/HPs displayed attenuation of expression of miR-99a, -100a, -199a and miR-200, whereas, rapamycin inhibited this effect of HIV. These findings suggest that rapamycin-mediated up-regulation of specific miRNAs could contribute to amelioration of renal lesions in HIVAN mice.
Experimental Cell Research 04/2013; · 3.58 Impact Factor
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ABSTRACT: Alterations in podocyte actin cytoskeleton have been implicated in the development of proteinuric kidney diseases. In the present study, we evaluated the effect of HIV on podocyte actin cytoskeleton and mechanism involved. We hypothesized that HIV may be compromising actin cytoskeleton via down regulation of human podocytes (CIDHPs) vitamin D receptor (VDR). HIV-transduced podocytes (HIV/CIDHPs) not only displayed down regulation of VDR but also showed activation of renin angiotensin system (RAS) in the form of enhanced expression of renin and increased production of Ang II. Moreover, CIDHPs lacking VDR displayed enhanced Ang II production and treatment of HIV/CIDHPs with EB1089 (vitamin D3, VD) attenuated Ang II production. HIV/CIDHPs as well as Ang II-treated CIDHPs exhibited enhanced expression of of cathepsin (CTS) L. Additionally, losartan (an Ang II type I receptor blocker) inhibited both HIV- and Ang II induced podocyte cathepsin L expression. Furthermore, VD down regulated HIV-induced podocyte CTSL expression. Both losartan and free radical scavengers attenuated HIV and Ang II-induced podocyte reactive oxygen species (ROS) generation. HIV also led to cytosolic CTSL accumulation through enhancement of podocyte lysosomal membrane permeabilization (LMP); on the other hand, VD, losartan, and superoxide dismutase (SOD) attenuated HIV-induced enhanced podocyte cytosolic CTSL accumulation. Morphologic evaluation of HIV/CIDHPs revealed sparse actin filaments and attenuated expression of dynamin. Interestingly, podocytes lacking CTSL displayed enhanced dynamin expression and HIV/CIDHP expressing CTSL exhibited down regulation of dynamin. These findings indicate that HIV-induced down regulation of podocyte VDR and associated RAS activation and cytosolic CTSL accumulation compromised actin cytoskeleton.
AJP Renal Physiology 03/2013; · 4.42 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S and H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.
PLoS ONE 01/2013; 8(2):e55852. · 4.09 Impact Factor
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Xiqian Lan,
Partab Rai,
Nirupama Chandel,
Kang Cheng,
Rivka Lederman,
Moin A Saleem, Peter W Mathieson,
Mohammad Husain,
John T Crosson,
Kalpna Gupta,
Ashwani Malhotra,
Pravin C Singhal
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ABSTRACT: Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.
PLoS ONE 01/2013; 8(3):e55748. · 4.09 Impact Factor
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Himanshu Vashistha,
Pravin C Singhal,
Ashwani Malhotra,
Mohammad Husain, Peter W Mathieson,
Moin A Saleem,
Cyril Kuriakose,
Surya Seshan,
Anna Wilk,
Luis Delvalle,
Francesca Peruzzi,
Marco Giorgio,
Pier Giuseppe Pelicci,
Oliver Smithies,
Hyung-Suk Kim,
Masao Kakoki,
Krzysztof Reiss,
Leonard G Meggs
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ABSTRACT: Candidate genes have been identified that confer increased risk for diabetic glomerulosclerosis (DG). Mice heterozygous for the Akita (Ins2(+/C96Y)) diabetogenic mutation with a second mutation introduced at the bradykinin 2 receptor (B2R(-/-)) locus, express a disease phenotype that approximates human DG. Src homology 2 domain transforming protein 1 (p66) controls mitochondrial metabolism and cellular responses to oxidative stress, aging and apoptosis. We generated p66-null Akita mice to test whether inactivating mutations at the p66 locus will rescue kidneys of Akita mice from disease-causing mutations at the Ins2 and B2R loci. Here we show null mutations at the p66 and B2R loci interact with the Akita (Ins2(+/C96Y)) mutation, independently and in combination, inducing divergent phenotypes in the kidney. The B2R(-/-) mutation induces detrimental phenotypes, as judged by increased systemic and renal levels of oxidative stress, histology and urine albumin excretion, whereas the p66-null mutation confers a powerful protection phenotype. To elucidate the mechanism(s) of the protection phenotype, we turned to our in vitro system. Experiments with cultured podocytes revealed previously unrecognized crosstalk between p66 and the redox sensitive transcription factor p53 that controls hyperglycemia-induced ROS metabolism, transcription of p53 target genes (angiotensinogen, angiotensin II type-1 receptor; bax) angiotensin II generation and apoptosis. RNA-interference targeting p66 inhibits all of the above. Finally, protein levels of p53 target genes were upregulated in kidneys of Akita mice, but unchanged in p66-null Akita mice. Taken together, p66 is a potential molecular target for therapeutic intervention in DG.
AJP Renal Physiology 09/2012; · 4.42 Impact Factor
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Pratab Rai,
Andrei Plagov,
Dileep Kumar,
Shresh Pathak,
Kamesh R Ayasolla,
Amrita Chawla, Peter W Mathieson,
Moin A Saleem,
Mohammad Husain,
Ashwani Malhotra,
Pravin C Singhal
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ABSTRACT: HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5mg/kg/day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.
Experimental and Molecular Pathology 09/2012; · 2.42 Impact Factor
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Raymond Tan,
Hitesh Patni,
Pranai Tandon,
Liming Luan,
Bipin Sharma,
Divya Salhan,
Moin A Saleem, Peter W Mathieson,
Ashwani Malhotra,
Mohammad Husain,
Poornima Upadhya,
Pravin C Singhal
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ABSTRACT: The HIV-1 accessory protein Nef is considered to play an important role in the development of a podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering the podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef-actin and Nef-zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had a decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projections (filopodia). Nef/CIHP displayed an enhanced cortical F-actin score index (P<0.001) and thus indicated a reorganization of F-actin in the cortical regions. Microarray analysis showed that Nef enhanced the expression of Rac1, syndecan-4, Rif, and CDC42 and attenuated the expression of syndecan-3 and syntenin. In addition, Nef/CIHPs displayed a diminished sphingomyelinase (ASMase) activity. Functionally, Nef/CIHPs displayed diminished attachment and enhanced detachment to their substrate. These findings indicate that Nef interaction with actin compromises the podocyte cytoskeleton integrity.
Experimental and Molecular Pathology 06/2012; · 2.42 Impact Factor
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ABSTRACT: Laminar shear stress (LSS), induced by flowing blood, plays a key role in determining vascular health by modulating endothelial behaviour and vascular tone. In systemic endothelium many of the beneficial effects of chronic LSS are mediated through the transcription factor Kruppel-like factor 2 (KLF2), but little is known regarding the role of chronic LSS in the renal glomerulus. We demonstrate that exposure of glomerular endothelial cells to chronic (>24h) LSS of 10 dyn/cm(2) increases phosphorylation of extra-cellular signal-related kinase 5 (ERK5) and increases expression of KLF2, leading to increased expression of the downstream molecules endothelial nitric oxide synthase (eNOS), thrombomodulin, endothelin-1 and nitric oxide. However, the proportion of eNOS which was phosphorylated at serine 1117 and threonine 495 residues was decreased. We demonstrated dependence of these effects on the ERK5 pathway by using the inhibitor UO126. We found high levels of KLF2 expression in human glomeruli confirming the relevance of our in vitro observations and, as KLF2 is specifically induced by chronic LSS, suggesting the physiological importance of shear stress in the glomerulus. Conditioned medium from glomerular endothelial cells under chronic LSS decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein. The latter effect was more pronounced using a novel insert-based direct co-culture system in which endothelial cells were exposed to chronic LSS. These data provide the first direct evidence of glomerular endothelial cell to podocyte cross-talk.
The international journal of biochemistry & cell biology 06/2012; 44(9):1482-90. · 4.89 Impact Factor
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S A Asgeirsdóttir,
C van Solingen,
N F Kurniati,
P J Zwiers,
P Heeringa,
M van Meurs,
S C Satchell,
M A Saleem, P W Mathieson,
B Banas,
J A A M Kamps,
T J Rabelink,
A J van Zonneveld,
G Molema
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ABSTRACT: Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.
AJP Renal Physiology 03/2012; 302(12):F1630-9. · 4.42 Impact Factor
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Katsuyuki Tanabe,
Miguel A Lanaspa,
Wataru Kitagawa,
Christopher J Rivard,
Makoto Miyazaki,
Jelena Klawitter,
George F Schreiner,
Moin A Saleem, Peter W Mathieson,
Hirofumi Makino,
Richard J Johnson,
Takahiko Nakagawa
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ABSTRACT: Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.
AJP Renal Physiology 02/2012; 302(9):F1151-60. · 4.42 Impact Factor
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Lubka T Roumenina,
Marie Frimat,
Elizabeth C Miller,
Francois Provot,
Marie-Agnes Dragon-Durey,
Pauline Bordereau,
Sylvain Bigot,
Christophe Hue,
Simon C Satchell, Peter W Mathieson,
Christiane Mousson,
Christian Noel,
Catherine Sautes-Fridman,
Lise Halbwachs-Mecarelli,
John P Atkinson,
Arnaud Lionet,
Veronique Fremeaux-Bacchi
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ABSTRACT: Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.
Blood 01/2012; 119(18):4182-91. · 9.90 Impact Factor
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ABSTRACT: Prominent vasculopathy in Fabry disease patients is caused by excessive intracellular accumulation of globotriaosylceramide (GL-3) throughout the vascular endothelial cells causing progressive cerebrovascular, cardiac and renal impairments. The vascular lesions lead to myocardial ischemia, atherogenesis, stroke, aneurysm, thrombosis, and nephropathy. Hence, injury to the endothelial cells in the kidney is a key mechanism in human glomerular disease and endothelial cell repair is an important therapeutic target. We investigated the mechanism of uptake of α-galactosidase A (α-Gal A) in renal endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and (125)I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α-Gal A binds to human M6PR with very high affinity, but M6PR also binds to sortilin in a way that prevents α-Gal A binding to sortilin. Taken together, our data provide evidence that sortilin is a new α-Gal A receptor expressed in renal endothelial cells and that this receptor together with the M6PR is able to internalize circulating α-Gal A during enzyme replacement therapy in patients with Fabry disease.
PLoS ONE 01/2012; 7(6):e39975. · 4.09 Impact Factor
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Nan Hu,
Johanna Westra,
Abraham Rutgers,
Berber Doornbos-Van der Meer,
Minke G Huitema,
Coen A Stegeman,
Wayel H Abdulahad,
Simon C Satchell, Peter W Mathieson,
Peter Heeringa,
Cees G M Kallenberg
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ABSTRACT: In anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.
Membrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.
Expression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.
Expression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.
Arthritis research & therapy 12/2011; 13(6):R201. · 4.27 Impact Factor
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ABSTRACT: Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFβ1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN.
Laboratory Investigation 09/2011; 92(1):32-45. · 3.64 Impact Factor
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ABSTRACT: Background/Aims: Proteinuria is associated with cardiovascular and chronic kidney disease. Microparticles (MPs) are bioactive vesicles shed from activated cells and also linked to cardiovascular disease. MP-like structures have been identified in the glomerular basement membrane, urinary space and between the glomerular basement membrane and the podocyte. We hypothesised that circulating MPs may provide a link between vascular injury and kidney diseases by inducing podocyte phenotypic alterations, thus propagating glomerular dysfunction and proteinuria. Methods:Human umbilical vein endothelial cells and U937 monocytes were stimulated with TNF-α to produce MPs. These MPs were confirmed by electron microscopy, and added to differentiated podocyte monolayers to determine effects on podocyte albumin endocytosis and the production of soluble mediators. Results:Monocyte and endothelial MPs upregulated podocyte production of pro-inflammatory mediators monocyte chemoattractant protein-1 (p < 0.001) and interleukin-6 (p < 0.001). Only monocyte MPs upregulated podocyte secretion of VEGF (p < 0.001), known to regulate glomerular permeability. Endothelial MPs decreased podocyte albumin endocytosis by 13% compared to control cells (p < 0.01). Conclusion:MPs alter endocytic functions of podocytes and induce secretion of pro-inflammatory cytokines, potentially leading to glomerular inflammation in vivo and the development of proteinuria. This study identifies a potential pathophysiological role for circulating MPs in the kidney through effects on the podocyte.
Nephron Experimental Nephrology 08/2011; 119(3):e58-e66. · 1.86 Impact Factor
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ABSTRACT: Various studies indicate a role for both anti-nucleosome and anti-C1q autoantibodies in glomerulonephritis in patients with systemic lupus erythematosus. However, a causal relationship between these autoantibodies and the development of lupus nephritis has not been fully established. Since injury of the endothelium is a major target in lupus nephritis we assessed the interaction of C1q and nucleosomes with glomerular endothelial cells in vitro in the presence or absence of autoantibodies against these antigens. We demonstrate a direct and dose-dependent binding of both nucleosomes and C1q to immortalized human glomerular endothelial cells (GEnC) in vitro, which in part is mediated by cell surface heparan sulfate. We demonstrate that nucleosomes and C1q serve as targets for monoclonal and polyclonal antibodies as well as for anti-nuclear autoantibodies from patients with systemic lupus erythematosus. An additive effect of anti-C1q autoantibodies on anti-nucleosome mediated complement activation was observed. Furthermore, we showed that the activation of complement on glomerular endothelial cells is mediated by the classical pathway since the deposition of C3 on GEnC is abrogated by MgEGTA and does not occur in C1q-depleted serum. Taken together, our studies demonstrate a direct binding of both nucleosomes and C1q to glomerular endothelial cells in vitro. The subsequent binding of autoantibodies against nucleosomes in patients with systemic lupus erythematosus is potentially pathogenic and autoantibodies against C1q seem to have an additional effect.
Molecular Immunology 08/2011; 49(1-2):75-83. · 2.90 Impact Factor
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ABSTRACT: Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.
AJP Renal Physiology 07/2011; 301(4):F733-42. · 4.42 Impact Factor
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ABSTRACT: Transforming growth factor-β1 (TGF-β1) and the macrophage inhibitory factor receptor CD74 link the metabolic disorder with tissue injury in diabetic nephropathy. Fabry disease is an X-linked lysosomal glycosphingolipid storage disorder resulting from a deficient activity of α-galactosidase A that leads to proteinuric renal injury. However, the link between the metabolic abnormality and renal injury is poorly characterized. Globotriaosylsphingosine (lyso-Gb3) was recently identified as a bioactive molecule accumulating in Fabry disease. We hypothesized that lyso-Gb3 could modulate the release of secondary mediators of injury in glomerular podocytes and that recently described nephroprotective actions of vitamin D receptor activation in diabetic nephropathy may apply to lyso-Gb3.
Real time RT-PCR, ELISA and Western blot were used to study the biological activity of lyso-Gb3 in cultured human podocytes and potential modulation by vitamin D receptor activation.
In human podocytes, lyso-Gb3 dose and time dependently increased the expression of TGF-β1, extracellular matrix proteins (fibronectin and type IV collagen) and CD74. TGF-β1 mediated lyso-Gb3 effects on extracellular matrix production. Vitamin D receptor activation with paricalcitol or calcitriol prevented the increase in TGF-β1, CD74 and extracellular matrix induced by lyso-Gb3.
Lyso-Gb3 may have a role in glomerular injury in Fabry disease by promoting the release of secondary mediators of glomerular injury common to diabetic nephropathy. These effects are prevented by paricalcitol, raising the issue of vitamin D receptor activation as potential adjunctive therapy in Fabry nephropathy.
Nephrology Dialysis Transplantation 06/2011; 26(6):1797-802. · 3.40 Impact Factor
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Michiko Shimada,
Takuji Ishimoto,
Pui Y Lee,
Miguel A Lanaspa,
Christopher J Rivard,
Carlos A Roncal-Jimenez,
David T Wymer,
Hideaki Yamabe, Peter W Mathieson,
Moin A Saleem,
Eduardo H Garin,
Richard J Johnson
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ABSTRACT: Recent studies suggest that CD80 (also known as B7.1) is expressed on podocytes in minimal-change disease (MCD) and may have a role in mediating proteinuria. CD80 expression is known to be induced by Toll-like receptor (TLR) ligands in dendritic cells. We therefore evaluated the ability of TLR to induce CD80 in human cultured podocytes.
Conditionally immortalized human podocytes were evaluated for TLR expression. Based on high expression of TLR3, we evaluated the effect of polyinosinic-polycytidylic acid (polyIC), a TLR3 ligand, to induce CD80 expression in vitro.
TLR1-6 and 9 messenger RNA (mRNA) were expressed in podocytes. Among TLR ligands 1-9, CD80 mRNA expression was significantly induced by polyIC and lipopolysaccharide (TLR4 ligand) with the greatest stimulation by polyIC (6.8 ± 0.7 times at 6 h, P < 0.001 versus control). PolyIC induced increased expression of Cathepsin L, decreased synaptopodin expression and resulted in actin reorganization which suggested a similar injury pattern as observed with lipopolyssaccharide. PolyIC induced type I and type II interferon signaling, nuclear factor kappa B (NF-κB) activation and the induction of CD80 expression. Knockdown of CD80 protected against actin reorganization and reduced synaptopodin expression in response to polyIC. Dexamethasone, a corticosteroid commonly used to treat MCD, also blocked both basal and polyIC-stimulated CD80 expression, as did inhibition of NF-κB.
Activation of TLR3 on cultured human podocytes induces CD80 expression and phenotypic change via an NF-κB-dependent mechanism and is partially blocked by dexamethasone. These studies provide a mechanism by which viral infections may cause proteinuria.
Nephrology Dialysis Transplantation 05/2011; 27(1):81-9. · 3.40 Impact Factor
