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Glomerular mRNAS in human type 1 diabetes: Biochemical evidence for microalbuminuria as a manifestation of diabetic nephropathy
Abstract
In patients with type 1 diabetes, some consider microalbuminuria to be a predictor of diabetic nephropathy while others believe it is an early feature of diabetic nephropathy.
Levels of mRNAs that are of pathogenetic relevance in diabetic nephropathy were compared in glomeruli isolated from microalbuminuric and overtly proteinuric subjects and in control normoalbuminuric diabetic subjects and living renal transplant donors.
In subjects with microalbuminuria and overt proteinuria, glomerular mRNAs were virtually identical and approximately twofold higher for connective tissue growth factor (CTGF; P < 0.01) and collagen alpha2(IV) (P < 0.03) compared to living renal donors and normoalbuminuric patients. Glomerular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were not significantly different among the groups (P = 0.4). Weak but statistically significant correlations were noted between CTGF mRNA and albuminuria (assessed by rank), fractional mesangial surface area, and a composite renal biopsy index. Glomerular CTGF mRNA correlated inversely with creatinine clearance. Glomerular collagen alpha2(IV) mRNA levels correlated with albuminuria (by rank) and less strongly with fractional mesangial area.
To our knowledge, these data provide the first biochemical evidence demonstrating that the glomeruli of microalbuminuric patients and those with overt proteinuria do not differ significantly. The data support the concept that microalbuminuria is not "predictive" of diabetic nephropathy, but rather is an earlier point in the spectrum of diabetic nephropathy.
... CCN2, a matricellular protein, has been identified as a downstream target and is now established as a critical causal factor in many forms of fibrosis, including that of DN. 6,7 Furthermore, CCN2 is up-regulated early in human diabetic patients and in animal models of DN, 8,6 and increased urinary CCN2 levels predict and stage early DN. 9,10 Elevated CCN2 activity has been shown to be causal in glomerular and interstitial fibrosis, leading to reduced glomerular function, proteinuria, and, ultimately, diminished renal function. ...
... Although not commonly performed, when human biopsy samples have been studied at the time of microalbuminuria onset, early pathologic findings comparable with those described for the BTBR ob/ob model have been documented. 8 For the present studies, rhCCN3 was delivered by i.p. injection three times per week (Monday, Wednesday, and Friday), as shown in Figure 2. The treatment was stopped after 8 weeks (17 weeks of age) and 24 injections per mouse. The two doses chosen (0.604 and 6.04 mg/kg of rhCCN3) were based on the effective dose in vitro and the circulating dose in a normal mouse and were approximately 2 times (low dose) and 20 times (high dose) the circulating amount of endogenous CCN3. ...
Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any Food and Drug Administration–approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (nov) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col 1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).
... In the search for molecular linkers between haemodynamic dysregulation, the renin-angiotensin system and renal scarring, TGFβ expression was found to be upregulated in glomerular diseases including experimental glomerulonephritis [20] and experimental and human DN within the mesangium [21,22]. Increased glomerular expression of CCN2 has also been reported in a variety of human glomerulopathies, including IgA nephropathy, focal and segmental glomerulosclerosis as well as diabetic nephropathy [23][24][25]. Increased CCN2 expression has been documented in experimental models of diabetic glomerulosclerosis [26,27]; in vitro studies have shown that CCN2 is induced in HMCs by high glucose, AGEs, TGFβ and ROS [6,26,28]. In the streptozotocin (STZ) diabetic mouse model, upregulated expression of CCN2 has been found to be primarily contained to the podocyte in the immediate phases after induction of diabetes [29]; STZ mice that transgenically over-express CCN2 specifically in the podocyte developed more severe proteinuria and mesangial expansion than their control (non-transgenic) littermates [30]. ...
... In the kidney, CREB is reported as a positive and negative regulator of gene expression in disease pathophysiology. In tubular cells it transduces pro-survival signals after oxidant stress [22] and regulates hexosamine induced fibronectin synthesis in mesangial cells [23]. High extracellular glucose and TGFβ stimulate fibronectin production in mesangial cells by cAMP/CREB [24]; use of anti TGFβ antibodies has been reported to decrease CREB phosphorylation and hypertrophy in diabetic glomeruli [25]. ...
CCN2/CTGF is an established effector of TGFbeta driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFbeta leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function.
From a panel of genes regulated by TGFbeta and CCN2, we used co-inertia analysis to identify and then experimentally verify a subset of transcription factors, E2F1 and CREB, that regulate an expression fingerprint implicated in altered actin dynamics and cell hypertrophy. Importantly, actin related genes containing E2F1 and CREB binding sites, stratified by expression profile within the dataset. Further analysis of actin and cytoskeletal related genes from patients with diabetic nephropathy suggests recapitulation of this programme during the development of renal disease. The Rho family member Cdc42 was also found uniquely to be activated in cells treated with TGFbeta and CCN2; Cdc42 interacting genes were differentially regulated in diabetic nephropathy.
TGFbeta and CCN2 attenuate CREB and augment E2F1 transcriptional activation with the likely effect of altering actin cytoskeletal and cell growth/hypertrophic gene activity with implications for cell dysfunction in diabetic kidney disease. The cytoskeletal regulator Cdc42 may play a role in this signalling response.
... However, this seems unlikely when interpreted in the context of our findings that in experimental diabetes, alterations in available renal CCN2 are associated with the early development of glomerulosclerosis, and CCN2 in vitro stimulates ECM synthesis by renal cells [24]. Further, we recently determined by RT-PCR amplification of mRNA from renal biopsies that patients with type 1 diabetes have glomerular CCN2 transcripts that are elevated in the presence of microalbuminuria and overt proteinuria [25]. We found a positive correlation between glomerular CCN2 mRNA levels and albuminuria and fractional mesangial area [25]. ...
... Further, we recently determined by RT-PCR amplification of mRNA from renal biopsies that patients with type 1 diabetes have glomerular CCN2 transcripts that are elevated in the presence of microalbuminuria and overt proteinuria [25]. We found a positive correlation between glomerular CCN2 mRNA levels and albuminuria and fractional mesangial area [25]. Collectively, this supports the idea that the changes in urinary CCN2 levels observed in the present study reflect changes in synthesis occurring in the kidney. ...
It is currently impossible to reliably predict which diabetic patients will develop nephropathy and progress to kidney failure. Microalbuminuria, often regarded as a predictor of overt diabetic renal disease is, in fact, an indicator of established glomerular damage. We have shown that glomerular expression of the prosclerotic cytokine CCN2 (CTGF) is greatly up-regulated early in experimental and in human diabetes and mesangial cell exposure to CCN2 increases its production of extracellular matrix (ECM) molecules responsible for glomerulosclerosis. As an early marker, we therefore investigated the presence of CCN2 in urine and the relationship to diabetes and/or renal disease in an experimental model of diabetes and in a limited patient population.
Urine samples from (1) healthy rats, (2) rats made diabetic by streptozotocin (STZ), (3) healthy human volunteers, (4) diabetic patients with renal disease, and (5) diabetic patients without renal disease were examined by Western blotting and/or enzyme-linked immunosorbent assay (ELISA) for qualitative and quantitative analysis of the of CCN2.
Low levels of urinary CCN2 were present in healthy, control rats, but were increased approximately sevenfold overall in STZ-diabetic animals. CCN2 levels were the highest at week 3 of diabetes, then decreased with time, but remained significantly elevated over controls even after 32 weeks. Consistently low levels of urinary CCN2 were also detected in healthy volunteers (mean value, 7.1 CCN2/mg creatinine). However, levels were elevated approximately sixfold in the majority of diabetic patients with nephropathy. A small number of the diabetic patients not yet exhibiting evidence of renal involvement demonstrated CCN2 urinary levels that were ninefold greater than controls. The remaining normoalbuminuric diabetic patients demonstrated CCN2 levels indistinguishable from those of healthy volunteers. Analysis by Western blotting confirmed the identity of the urinary CCN2. A molecular species equivalent to full-length CCN2 (37/39 kD doublet) was present in healthy controls. In contrast, the nephropathic group demonstrated multiple CCN2 bands.
These findings support our hypothesis that CCN2 is up-regulated early in the evolution of glomerulosclerosis, including that of diabetes. We contend that urinary CCN2 may both stage nephropathy and predict those patients who are destined for progressive glomerulosclerosis and end-stage renal disease (ESRD). Cross-sectional and prospective studies of larger, well-defined diabetic patients groups will be required to prove this hypothesis, and are ongoing.
... antisense 5¢-GGTGTTTAAAGCCAATCGATT-3¢ ET-A R sense 5¢-TGGCCTTTTGATCACAATGACTTT-3¢ Bauer et al. (2002) antisense 5¢-TTTGATGTGGCATTGAGCATACAGGTT-3¢ CYR61 sense 5¢-ACTTCATGGTCCCAGTGCTC-3¢ Xie et al. (2001) antisense 5¢-AAATCCGGGTTTCTTTCACA-3¢ COL4A2 sense 5¢-TATTCCTTCCTCATGCACACGGCG-3¢ Adler et al. (2001) antisense 5¢-CCAATTTTTGGGTTGGCACC-3¢ CTGF sense 5¢-GAGTGGGTGTGTGACGAGCCCAAGG-3¢ Adler et al. (2001) antisense 5¢-ATGTCTCCGTACATCTTCCTGTAGT-3¢ a Ã-Actin sense 5¢-AAGGCCAACCGCGAGAAGAT-3¢ Sasaki et al. (2001) antisense 5¢-TCGGTGAGGATCTTCATGAG-3¢ (Figure 3) in addition to CYR61, CTGF and COL4A2 (Figure 4). For all ®ve of the genes, the gene expression microarray result was con®rmed. ...
... antisense 5¢-GGTGTTTAAAGCCAATCGATT-3¢ ET-A R sense 5¢-TGGCCTTTTGATCACAATGACTTT-3¢ Bauer et al. (2002) antisense 5¢-TTTGATGTGGCATTGAGCATACAGGTT-3¢ CYR61 sense 5¢-ACTTCATGGTCCCAGTGCTC-3¢ Xie et al. (2001) antisense 5¢-AAATCCGGGTTTCTTTCACA-3¢ COL4A2 sense 5¢-TATTCCTTCCTCATGCACACGGCG-3¢ Adler et al. (2001) antisense 5¢-CCAATTTTTGGGTTGGCACC-3¢ CTGF sense 5¢-GAGTGGGTGTGTGACGAGCCCAAGG-3¢ Adler et al. (2001) antisense 5¢-ATGTCTCCGTACATCTTCCTGTAGT-3¢ a Ã-Actin sense 5¢-AAGGCCAACCGCGAGAAGAT-3¢ Sasaki et al. (2001) antisense 5¢-TCGGTGAGGATCTTCATGAG-3¢ (Figure 3) in addition to CYR61, CTGF and COL4A2 (Figure 4). For all ®ve of the genes, the gene expression microarray result was con®rmed. ...
The aetiology of uterine fibroids remains unknown, despite causing significant gynaecological morbidity. Fibroids have a reduced microvascular density when compared with adjacent myometrial tissue. The aim of this study was to identify genes with differential expression between fibroid and adjacent normal myometrium, particularly genes with a role in angiogenesis. Total RNA was extracted from fibroid/myometrium pairs from 12 hysterectomy specimens, and used to perform 24 cDNA microarrays. There were 10,500 genes screened on each microarray for differential expression. Analysis of expression data was carried out using multiple t-tests, as well as a novel class prediction algorithm (GeneRaVE). The differential gene expression of selected genes was confirmed by quantitative 'real time' RT-PCR. Selected genes with a role in angiogenesis were further analysed for expression in isolated cell populations of endothelial cells (fibroid and myometrium) and smooth muscle cells (fibroid and myometrium), to see if their expression was confined to particular cell types. Twenty-five genes with differential gene expression between fibroid and myometrium were identified. Insulin-like growth factor-2, endothelin A receptor, connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61) and collagen 4alpha2 (COL4A2) were confirmed by RT-PCR. CTGF and CYR61, both angiogenesis promoters, were reduced in expression relative to myometrium. COL4A2, the precursor for an angiogenesis inhibitor, canstatin, was increased relative to myometrium. These three genes display an anti-angiogenic expression profile in fibroids relative to myometrium. These findings may explain the reduced microvascular density seen in fibroids relative to myometrium.
... Renal biopsy was performed in the standard manner 24 . Microdissection of the kidney biopsy samples were performed, as previously described 25 . Kidney tubules, with glomeruli removal through microdissection, was stored in RNAiso reagent at −70°C (Takara Bio Inc., Otsu, Shiga, Japan). ...
Kidney tubular cell death induced by transforming growth factor-β1 (TGF-β1) is known to contribute to diabetic nephropathy, a major complication of diabetes. Caspase-3-dependent apoptosis and caspase-1-dependent pyroptosis are also involved in tubular cell death under diabetic conditions. Recently, ferroptosis, an atypical form of iron-dependent cell death, was reported to cause kidney disease, including acute kidney injury. Ferroptosis is primed by lipid peroxide accumulation through the cystine/glutamate antiporter system X c ⁻ (xCT) and glutathione peroxidase 4 (GPX4)-dependent mechanisms. The aim of this study was to evaluate the role of ferroptosis in diabetes-induced tubular injury. TGF-β1-stimulated proximal tubular epithelial cells and diabetic mice models were used for in vitro and in vivo experiments, respectively. xCT and GPX4 expression, cell viability, glutathione concentration, and lipid peroxidation were quantified to indicate ferroptosis. The effect of ferroptosis inhibition was also assessed. In kidney biopsy samples from diabetic patients, xCT and GPX4 mRNA expression was decreased compared to nondiabetic samples. In TGF-β1-stimulated tubular cells, intracellular glutathione concentration was reduced and lipid peroxidation was enhanced, both of which are related to ferroptosis-related cell death. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, alleviated TGF-β1-induced ferroptosis. In diabetic mice, kidney mRNA and protein expressions of xCT and GPX4 were reduced compared to control. Kidney glutathione concentration was decreased, while lipid peroxidation was increased in these mice, and these changes were alleviated by Fer-1 treatment. Ferroptosis is involved in kidney tubular cell death under diabetic conditions. Ferroptosis inhibition could be a therapeutic option for diabetic nephropathy.
... Microalbuminuria is one of the earliest predictor of onset of diabetic nephropathy and its progression. 7 In renal diseases, diagnosis is based on the patient's clinical history, physical examination, laboratory tests, diagnostic imaging, scintigraphy, and histopathology. Early diabetic nephropathy is been identified by persistent microalbuminuria defined as an albumin excretion rate of 20-200 μg/min or 30-300 mg/24 h, or a spot urine albumin to creatinine ratio of 30-300 mg/g (3.5-3 5 mg/mmol) in males and 20-2 00 mg/g (2.5-2 5 mg/mmol) in females. ...
... To highlight seminal publications, CCN2 was originally shown to be induced in vitro in human renal mesangial cells in response to elevated extracellular D-glucose through a classical PKC dependent isoform and in a TGF-β1 dependent manner (Murphy et al. 1999). Subsequent work by others showed that circulating CCN2 is elevated in people with diabetic kidney disease, and in post-translationally modified forms in urine (Riser et al. 2003), (Gilbert et al. 2003), and also that higher circulating CCN2 (Nguyen et al. 2008) and CCN2 mRNA in human kidneys (Adler et al. 2001) each predict progressive diabetic nephropathy. We and many others studied the cellular mechanisms by which CCN2 may be up-regulated in diabetes, including in renal mesangial cells, podocytes, and tubular epithelial cells: factors downstream of glucose -advanced glycation-end products, protein kinase C isoforms, as well as saturated fatty acids, and reactive oxygen species, may each induce CCN2 (Twigg 2010), (van Nieuwenhoven et al. 2005). ...
Across the years the CCNs have been increasingly implicated in the development of obesity, diabetes and its complications. Evidence for this is currently derived from their dysregulation in key metabolic pathological states in humans, animal and in vitro models, and also pre-clinical effects of their bioactivities. CCN2 is the best studied in this disease process and the other CCNs are yet to be better defined. Key steps where CCNs may play a pathogenic metabolic role include: (i) obesity and insulin resistance, where CCN2 inhibits fat cell differentiation in vitro and CCN3 may induce obesity and insulin resistance; (ii) elevated blood glucose levels to diabetes mellitus onset, where CCN2 may contribute to pancreatic beta cell and islet function; and (iii) in diabetes complications, such as nephropathy, retinopathy, liver disease (NAFLD/NASH), CVD and diabetes with heart failure. In contrast, CCN1, CCN2 and possibly CCN3, may have a reparative role in wound healing in diabetes, and CCN2 in islet cell development. In terms of CCN2 regulation by a diabetes metabolic environment and related mechanisms, the author’s laboratory and others have progressively shown that advanced glycation-end products, protein kinase C isoforms, saturated fatty acids, reactive oxygen species and haemodynamic factors upregulate CCN2 in relevant cell and animal systems. Recent data has suggested that CCN2, CCN3 and CCN6 may affect energy homeostasis including in regulating glycolysis and mitochondrial function. This paper will address the current data implicating CCNs in diabetes and its complications, focusing on recent aspects with translational clinical relevance and future directions.
... In diabetic patients with glomerular endothelial dysfunction, the release of NO is impaired, which causes increased vasoconstriction, as evaluated by Doppler measurement. [22] The increased levels of microalbuminuria that were observed in our cirrhotic patients with diabetes may represent a marker of endothelial damage because it is known that in diabetic patients, the most likely initiation step is glomerular endothelial dysfunction, which leads to ultrastructural glomerular changes and eventually, to microalbuminuria [22][23][24]. Furthermore, Russo et al. have argued that glomerular damage is the primum movens of diabetic nephropathy. ...
Renal failure is a common complication of cirrhosis and is associated with poor prognosis. Several reports have demonstrated the clinical utility of renal resistive indices in the assessment of renal function in cirrhosis patients. It is unknown whether the occurrence of diabetes affects renal haemodynamic indices in patients with cirrhosis. Therefore, the aim of our study was to compare renal Doppler indices in cirrhotic patients with and without type 2 diabetes mellitus (T2DM) and in diabetics without cirrhosis, and to relate the Doppler parameters to albuminuria. We evaluated 89 consecutive patients with normal renal functioning, including 37 with cirrhosis and T2DM (CD-Group), 41 with cirrhosis without diabetes (C-Group) and 11 with diabetes without cirrhosis (D-Group). The kidney pulsatility index (PI) and resistance index (RI) were measured by Doppler ultrasound. Renal function was expressed as the estimated glomerular filtration rate (eGFR) using the modification of diet in renal disease (MDRD) formula. Microalbuminuria (µAlb) was also evaluated. No significant differences were observed with respect to age, the Child–Pugh class or the serum creatinine level. The eGFR was mildly reduced in the CD-Group compared with the C-Group and D-Group, and µAlb was present in 24.4 % of the patients in the CD-Group and in 9 % of those in the D-Group. The PI and RI were significantly increased in the CD-Group and D-Group compared with the C-Group. Both the PI and RI were significantly associated with µAlb independent of age and Child–Pugh class. The novel finding of this study was that T2DM potentially impairs renal haemodynamics in patients with cirrhosis.
... In fact, elevated CTGF mRNA levels were found in glomeruli of microalbuminuric and overt DN patients. In addition other study has added to this notion that CTGF levels are elevated in plasma of almost all type 1 diabetic patients with DN, and in a small minority of normoalbuminuric patients [48,49]. It was thus postulated that the nephropathy-associated increase of plasma CTGF reflects, at least in part, increased renal production in addition to the expected effect of its reduced renal clearance [50]. ...
Aim:
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease, and thus, the ability of antidiabetic drugs to ameliorate renal microvascular disease may be as important as their ability to control blood glucose. Therefore, we investigated the reno-protective effect of the antidiabetic drugs, Sitagliptin and Pioglitazone, versus combined Metformin/Enalapril in a rat model of type 2 diabetes.
Method:
Male Wistar rats were randomly assigned to be either normal control or diabetic. Induction of type 2 diabetes was done by intraperitoneal injection of\ low dose streptozotocin (35mg/kg) on top of 2 weeks of high fat diet. Hyperglycemic animals were divided into 4 groups: untreated diabetic, Sitagliptin (10mg/kg), Pioglitazone (10mg/kg) and Metformin/Enalapril (500, 10mg/kg, respectively) treated. After 6 weeks, fasting blood glucose, plasma insulin, β-cell function, insulin resistance, serum lipids, urea & creatinine, albuminurea, kidney weight, renal oxidative stress, plasma connective tissue growth factor (CTGF) and renal histopathology were assessed.
Key findings:
Sitagliptin decreased microalbuminurea, urea & creatinine, renal tropism, oxidative stress and CTGF to levels similar to Metformin/Enalapril group. It also preserved near normal renal histology. Although Pioglitazone treatment reduced urea, creatinine, renal tropism and oxidative stress, it did not improve renal pathological changes or significantly alter CTCF.
Significance:
Early Sitagliptin treatment in type 2 diabetes can equally ameliorate renal functions and structural changes as combined Metformin/Enalapril. Moreover Sitagliptin is a better renoprotective than Pioglitazone, probably due to its suppressor effect on CTGF, a key factor mediating diabetic renal injury.
... In our study, after 6 weeks of STZ-induced diabetes, there was a significant increase in expression of CTGF in rat renal cortex. Our data on expression of CTGF in renal cortex are in agreement with previously studies which reported that overexpression of CTGF at the mRNA or protein level has previously been demonstrated in vivo in experimental diabetic rats [25] and human kidney with DN [26], as well as in cultured high glucose stimulated HK-2 cells [27]. What's more, our results also revealed that 6-week administration of ELFPMF statistically significantly increased the expression of CTGF measured by ELISA. ...
Background
Extremely low frequency pulsed magnetic fields (ELFPMF) have been shown to induce Faraday currents and measurable effects on biological systems. A kind of very high frequency electromagnetic field was reported that it improved the symptoms of diabetic nephropathy (DN) which is a major complication of diabetes. However, few studies have examined the effects of ELFPMF DN at the present. The present study was designed to investigate the effects of ELFPMF on DN in streptozotocin (STZ)–induced type 1 diabetic rats.
Methods
Adult male SD rats were randomly divided into three weight-matched groups: Control (non-diabetic rats without DN), DN + ELFPMF (diabetic rats with DN exposed to ELFPMF, 8 h/days, 6 weeks) and DN (diabetic rats with DN exposed to sham ELFPMF). Renal morphology was examined by light and electron microscopy, vascular endothelial growth factor (VEGF)-A and connective tissue growth factor (CTGF) were measured by enzyme linked immune sorbent assay.
Results
After 6 weeks’ ELFPMF exposure, alterations of hyperglycemia and weight loss in STZ-treated rats with DN were not found, while both positive and negative effects of ELFPMF on the development of DN in diabetic rats were observed. The positive one was that ELFPMF exposure attenuated the pathological alterations in renal structure observed in STZ-treated rats with DN, which were demonstrated by slighter glomerular and tubule-interstitial lesions examined by light microscopy and slighter damage to glomerular basement membrane and podocyte foot processes examined by electron microscopy. And then, the negative one was that ELFPMF stimulation statistically significantly decreased renal expression of VEGF-A and statistically significantly increased renal expression of CTGF in diabetic rats with DN, which might partially aggravate the symptoms of DN.
Conclusion
Both positive and negative effects of ELFPMF on the development of DN in diabetic rats were observed. The positive effect induced by ELFPMF might play a dominant role in the procession of DN in diabetic rats, and it is suggested that the positive effect should be derived from the correction of pathogenic diabetes-induced mediators.
... In the urine from normal subjects is possible to find a low amount of albumin to a variable rate at different hours of the day and according to physical activity. The threshold value is 30 mg/die (5). The appearance of mycroalbuminuria in a diabetic patient sometime is concomitant with the onset of hypertension that critically increases the progression of albuminuria and the decline of glomerular filtration (6). ...
Diabetic nephropathy affects a subset of about 30% of patients with type 1 Diabetes Mellitus (DM); it also develops in a less defined percentage (20-30%) of patients with type 2, after a period of 15-20 years. It is usually divided into stages. The aim of this study is to assess the usefulness of duplex sonography with Doppler wave form analysis in the evaluation of early diabetic nephropathy, in order to detect patients at risk for irreversible renal disease. 262 patients (61 males, 201 females; age range: 48-81 years) with type 2 diabetes mellitus were studied; 100 healthy volunteers with no evidence of diabetes mellitus (74 females, 26 males; age range: 50-80 years) composed the control group. All of them underwent duplex Doppler sonography of the kidneys; a scanner with a 3.5 MHz transducer (Toshiba 270 SSA) was used, Pulsatily Index (P.I.) and Resistive Index (R.I.) of Doppler waveform were obtained at the intrarenal arteries; the average value of 3 bilateral measurements was taken. Doppler sonography was done by the same authors without knowledge of the patient group (case or control). Both indexes (PI. and R.I.) resulted to be higher in patients with DM compared to controls in patients with microalbuminuria: PI. = 1.49 +/- 0.34 vs. 1.07 +/- 0.06, p<0.05; R.I. = 0.79 +/- 0.15 vs 0.60 +/- 0.03, p<0.05. Even if our data have to be confirmed by further studies, they suggest that duplex Doppler sonography may be a useful complementary test in the evaluation of diabetic nephropathy, especially in the early stages, in order to identify more patients at risk of developing diabetic nephropathy.
... Identifying the factors that contribute to the pathogenesis of DN is a critical step towards halting its progression. Connective tissue growth factor (CTGF; CCN2) is a matricellular protein involved in modulation of the extracellular environment and plays a role in the development and progression of diabetic complications [2][3][4][5][6][7][8][9][10][11]. In DN, increased renal CTGF expression has been described both in glomeruli and in the tubulointerstitium [4]. ...
Connective tissue growth factor (CTGF; CCN2) plays a role in the development of diabetic nephropathy (DN). Urinary CTGF (uCTGF) is elevated in DN patients and has been proposed as a biomarker for disease progression, but it is unknown which pathophysiological factors contribute to elevated uCTGF. We studied renal handling of CTGF by infusion of recombinant CTGF in diabetic mice. In addition, uCTGF was measured in type 1 DN patients and compared with glomerular and tubular dysfunction and damage markers. In diabetic mice, uCTGF was increased and fractional excretion (FE) of recombinant CTGF was substantially elevated indicating reduced tubular reabsorption. FE of recombinant CTGF correlated with excretion of endogenous CTGF. CTGF mRNA was mainly localized in glomeruli and medullary tubules. Comparison of FE of endogenous and recombinant CTGF indicated that 60% of uCTGF had a direct renal source, while 40% originated from plasma CTGF. In DN patients, uCTGF was independently associated with markers of proximal and distal tubular dysfunction and damage. In conclusion, uCTGF in DN is elevated as a result of both increased local production and reduced reabsorption due to tubular dysfunction. We submit that uCTGF is a biomarker reflecting both glomerular and tubulointerstitial hallmarks of diabetic kidney disease.
... Several studies have reported increased CTGF expression in renal disorders associated with kidney fibrosis such as diabetic nephropathy [2,4,5]. Glomerular podocytes, mesangial cells and proximal tubular epithelial cells have been shown to express elevated levels of CTGF in human and experimental models of diabetic nephropathy67891011. Antisense oligonucleotides targeting CTGF in mouse models of type 1 and type 2 diabetes, for example, attenuated progression of nephropathy [11], while CTGF overexpression in podocytes of mice worsened proteinuria and mesangial expan- sion [12] . ...
... CCN2 is involved in diabetes-induced pathophysiological changes such as extracellular matrix synthesis, cell migration, and epithelial-to-mesenchymal transition [137,138]. Furthermore, upregulation of CCN2 has also been demonstrated in human and experimental models of diabetic nephropathy [139][140][141]. Inhibition of CCN2 signaling preserved the structure and function of the kidney in diabetic mice [142]. ...
Diabetic nephropathy is a major complication of diabetes mellitus and the leading cause of end-stage renal disease. Both hyperglycemia and hypertension (systemic and/or intraglomerular) are established causal factors for diabetic nephropathy. Nonetheless, there is growing evidence that activated innate immunity and inflammation are also contributing factors to the pathogenesis of diabetic nephropathy. This notion is based on increasing evidence indicating that both cytokines-chemokines and pro-fibrotic growth factors are important players in the progression of diabetic nephropathy, effectively accelerating and exacerbating inflammatory and fibrotic processes leading to end-stage renal disease. In this review, we focus on several predominant cytokines-chemokines as potential predictive markers for diabetic nephropathy. These cytokines-chemokines may also be helpful as biomarkers to monitor the progression of the disease and the impact of interventional modalities aimed at halting eventual manifestation of end-stage renal disease in diabetic patients.
... Several studies have reported increased CTGF expression in renal disorders associated with kidney fibrosis such as diabetic nephropathy [2,4,5]. Glomerular podocytes, mesangial cells and proximal tubular epithelial cells have been shown to express elevated levels of CTGF in human and experimental models of diabetic nephropathy [6][7][8][9][10][11]. Antisense oligonucleotides targeting CTGF in mouse models of type 1 and type 2 diabetes, for example, attenuated progression of nephropathy [11], while CTGF overexpression in podocytes of mice worsened proteinuria and mesangial expansion [12]. ...
... The ECM expansion has been proposed to be due to a combination of increased ECM production (Mason and Wahab 2003) and a reduction in ECM breakdown (Nakamura et al. 1994), (McLennan et al. 1994). CTGF/CCN-2 is a potent inducer of ECM in fibroblasts and it is elevated in the diabetic kidney in human diabetes (Adler et al. 2001), and in animal models (Murphy et al. 1999), (Riser et al. 2000 ). CTGF/CCN- 2 is induced by high glucose conditions in human renal mesangial cells (Murphy et al. 1999), and consequentially as CCN-2 can regulate ECM induction, as well as proteases and their inhibitors (Wang et al. 2003), it may mediate ECM expansion and fibrosis in diabetic nephropathy through both induction of ECM formation and inhibition of ECM degradation (McLennan et al. 2004), (Twigg 2011). ...
CCN-2, also known as connective tissue growth factor (CCN-2/CTGF) is a cysteine rich, extracellular matrix protein that acts as a pro-fibrotic cytokine in tissues in many diseases, including in diabetic nephropathy. We have published that soluble advanced glycation end products (AGEs), that are present in increased amounts in diabetes, induce CCN-2. However in vivo AGEs are known to be heavily tissue bound and whether matrix bound AGEs regulate CCN-2 has not been investigated. In this study we determined in human renal mesangial cells if CCN-2 is induced by matrix associated AGEs and if CCN-2 may then secondarily mediate effects of matrix AGEs on extracellular matrix expansion. Data generated show that CCN-2 mRNA and protein expression are induced by matrix bound AGEs, and in contrast, this was not the case for TGF-β1 mRNA regulation. Using CCN-2 adenoviral anti-sense it was found that CCN-2 mediated the up-regulation of fibronectin and the tissue inhibitor of matrix metalloproteinase, TIMP-1, that was caused by matrix bound AGEs. In conclusion, CCN-2 is induced by non-enzymatically glycated matrix and it mediates downstream fibronectin and TIMP-1 increases, thus through this mechanism potentially contributing to ECM accumulation in the renal glomerulus in diabetes.
... Patients with type 1 DM (overt nephropathy (n ¼ 5), microalbuminuria (n ¼ 5), normoalbuminuria (n ¼ 3), and live renal donors (n ¼ 6)) had renal biopsies, total RNA was extracted from cortex, cDNA was synthesized, stored at À80 1C, 18,19 and used for the first time herein. ...
We sought to find a urinary biomarker for chronic kidney disease and tested hematopoietic growth factor inducible neurokinin-1 (HGFIN, also known as Gpnmb/Osteoactivin) as it was found to be a kidney injury biomarker in microarray studies. Here, we studied whether HGFIN is a marker of kidney disease progression. Its increase in kidney disease was confirmed by real-time PCR after 5/6 nephrectomy, in streptozotocin-induced diabetes, and in patients with chronic kidney disease. In the remnant kidney, HGFIN mRNA increased over time reflecting lesion chronicity. HGFIN was identified in the infarct portion of the remnant kidney in infiltrating hematopoietic interstitial cells, and in distal nephron tubules of the viable remnant kidney expressed de novo with increasing time. In vitro, it localized to cytoplasmic vesicles and cell membranes. Epithelial cells lining distal tubules and sloughed luminal tubule cells of patients expressed HGFIN protein. The urine HGFIN-to-creatinine ratio increased over time after 5/6 nephrectomy; increased in patients with proteinuric and polycystic kidney disease; and remained detectable in urine after prolonged freezer storage. The urine HGFIN-to-creatinine ratio compared favorably with the urine neutrophil gelatinase-associated lipocalin (NGAL)-to-creatinine ratio (both measured by commercial enzyme-linked immunosorbent assays (ELISAs)), and correlated strongly with proteinuria, but weakly with estimated glomerular filtration rate and serum creatinine. Thus, HGFIN may be a biomarker of progressive kidney disease.
... In type 1 diabetes, plasma and urine CTGF levels correlate with the level of albuminuria and the stage of progressive renal insufficiency (31)(32)(33)(34), and the plasma CTGF level is an independent predictor of vascular disease as assessed by intimal medial thickness (35) and of mortality and progression to ESRD (36). In renal biopsy specimens from patients with diabetes, elevated levels of CTGF mRNA are associated with chronic tubulointerstitial damage, albuminuria, and progression of renal insufficiency (37)(38)(39). ...
This report summarizes the first phase 1 trial treating patients with microalbuminuric diabetic kidney disease (DKD) using FG-3019, a human monoclonal antibody to connective tissue growth factor (CTGF). CTGF is critically involved in processes of progressive fibrosis, including DKD. This phase 1, open-label, dose-escalation trial evaluated safety, pharmacokinetics, and possible therapeutic effects of FG-3019 on albuminuria, proteinuria, and tubular proteins.
Microalbuminuric subjects (n = 24) with type 2 (79%) or type 1 (21%) diabetes received 3 or 10 mg/kg FG-3019 dosed intravenously every 14 days for four doses. Albuminuria and safety follow-up were to days 62 and 365, respectively.
No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, suggesting a saturable elimination pathway. Although this study was not designed for efficacy testing, it was notable that urinary albumin/creatinine ratio (ACR) decreased significantly from mean pretreatment ACR of 48 mg/g to mean post-treatment (day 56) ACR of 20 mg/g (P = 0.027) without evidence for a dose-response relationship.
Treatment of microalbuminuric DKD subjects using FG-3019 was well tolerated and associated with a decrease in albuminuria. The data demonstrate a saturable pathway for drug elimination, minimal infusion adverse events, and no significant drug-attributable adverse effects over the year of follow-up. Changes in albuminuria were promising but require validation in a prospective, randomized, blinded study.
... The increased TGF-β 1 expression is likely to have resulted in the observed increased collagen IV (α1) mRNA expression. This may have important consequences for renal function, as an upregulation of collagen IV expression has previously been shown to predict the progression from normoalbuminuria to microalbuminuria in diabetic nephropathy (Adler et al. 2001). ...
In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross-fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted-on-Restricted offspring were born small but were of similar weight to Control-on-Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted-on-Restricted offspring at 6 months without glomerular hypertrophy. Cross-fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control-on-Control and Restricted-on-Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control-on-Control and Restricted-on-Restricted females at 18 months. Restricted-on-Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor-beta(1), collagen IV (alpha1) and matrix matelloproteinase-9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.
... Bar 5 25 mm. that in both human and experimental diabetic nephropathy, glomerular CTGF is strongly upregulated and that plasma CTGF, as well as urine CTGF, might serve as a relevant biomarker in the progression of human diabetic nephropathy (Ito et al. 1998;Riser et al. 2000;Adler et al. 2001Adler et al. ,2002Wahab et al. 2001;Nguyen et al. 2006Nguyen et al. ,2008bRoestenberg et al. 2006;Guha et al. 2007;Yokoi et al. 2008). Exogenous recombinant human CTGF reduced pSmad1/5/8 level in the renal cortex, indicating that CTGF level significantly affects renal BMP signaling activity (Nguyen et al. 2008a). ...
Diabetic nephropathy is characterized by decreased expression of bone morphogenetic protein-7 (BMP-7) and decreased podocyte number and differentiation. Extracellular antagonists such as connective tissue growth factor (CTGF; CCN-2) and sclerostin domain-containing-1 (SOSTDC1; USAG-1) are important determinants of BMP signaling activity in glomeruli. We studied BMP signaling activity in glomeruli from diabetic patients and non-diabetic individuals and from control and diabetic CTGF(+/+) and CTGF(+/-) mice. BMP signaling activity was visualized by phosphorylated Smad1, -5, and -8 (pSmad1/5/8) immunostaining, and related to expression of CTGF, SOSTDC1, and the podocyte differentiation markers WT1, synaptopodin, and nephrin. In control and diabetic glomeruli, pSmad1/5/8 was mainly localized in podocytes, but both number of positive cells and staining intensity were decreased in diabetes. Nephrin and synaptopodin were decreased in diabetic glomeruli. Decrease of pSmad1/5/8 was only partially explained by decrease in podocyte number. SOSTDC1 and CTGF were expressed exclusively in podocytes. In diabetic glomeruli, SOSTDC1 decreased in parallel with podocyte number, whereas CTGF was strongly increased. In diabetic CTGF(+/-) mice, pSmad1/5/8 was preserved, compared with diabetic CTGF(+/+) mice. In conclusion, in human diabetic nephropathy, BMP signaling activity is diminished, together with reduction of podocyte markers. This might relate to concomitant overexpression of CTGF but not SOSTDC1.
... Only low levels of glomerular CTGF are found in normal human glomeruli, but both mRNA and protein levels of the factor increase during the early (incipient) stage of DN and continue to do so as the disease progresses. Both glomerular podocytes and mesangial cells express CTGF in DN (Adler et al. 2001, Wahab et al. 2005a. Similarly, glomerular CTGF mRNA and protein levels increase in murine models of DN, initially in podocytes and later in parietal epithelia (lining the Bowman's capsule) and mesangial cells (Roestenberg at al. 2006). ...
Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of matricellular proteins. Its expression is induced by a number of factors including TGF-beta. It has been associated with fibrosis in various tissues including the kidney. Diabetic nephropathy (DN) develops in about 30% of patients with diabetes and is characterized by thickening of renal basement membranes, fibrosis in the glomerulus (glomerulosclerosis), tubular atrophy and interstitial fibrosis, all of which compromise kidney function. This review examines changes in CTGF expression in the kidney in DN, the effects they have on glomerular mesangial and podocyte cells and the tubulointerstitium, and how these contribute to driving fibrotic changes in the disease. CTGF can bind to several other growth factors modifying their function. CTGF is also able to interact with receptors on cells, including integrins, tyrosine receptor kinase A (TrkA), low density lipoprotein receptor-related protein (LRP) and heparan sulphate proteoglycans. These interactions, the intracellular signalling pathways they activate, and the cellular responses evoked are reviewed. CTGF also induces the expression of chemokines which themselves have pharmacological actions on cells. CTGF may prompt some responses by acting through several different mechanisms, possibly simultaneously. For example, CTGF is often described as an effector of TGF-beta. It can promote TGF-beta signalling by binding directly to the growth factor, promoting its interaction with the TGF-beta receptor; by triggering intracellular signalling on binding the TrkA receptor, which leads to the transcriptional repression of Smad7, an inhibitor of the TGF-beta signalling pathway; and by binding to BMP-7 whose own signalling pathway opposing TGF-beta is inhibited, leading to enhanced TGF-beta signalling.
... Our studies demonstrate a correlation between elevated levels of renal TGF-ß1 and changes in renal lysosomal activity as measured by the Russo/Osicka/Brammar/Candido/Jerums/ Comper degree of degradation of excreted albumin and dextran sulfate. These findings would concur with recent studies by Adler et al. [41] that demonstrate that intact albuminuria in diabetic nephropathy is correlated with increases in glomerular mRNA for connective tissue growth factor that is produced after cellular stimulation with TGF-ß. ...
Recent studies show that albuminuria may be the result of changes in post-glomerular cellular uptake and processing of albumin. This study aims to determine whether this processing is disrupted in diabetes and/or hypertension.
Diabetes (d) was induced using streptozotocin in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) and studied after 8, 16 and 24 weeks of disease. Intact albumin excretion was determined by radioimmunoassay. Total albumin was determined by [(14)C]albumin. Lysosomal activity was determined by dextran sulfate desulfation. Renal TGF-beta1 and transforming growth factor-beta1 inducible gene-h3 mRNA (betaig-h3) expression was determined by real time RT-PCR.
SHR-c rats exhibited an increase in intact albuminuria without significant change in total albumin excretion (intact plus albumin-derived peptides). For WKY-d rats, intact albuminuria developed initially, followed by an increase in total albumin excretion primarily in the form of albumin peptides (peptiduria). SHR-d rats exhibited both increases in peptiduria and intact albuminuria. There was no increase in glomerular permeability at 24 weeks for polydisperse [(3)H]Ficoll in all groups. Increased renal TGF-beta1 and betaig-h3 expression was correlated with a decrease in dextran sulfate desulfation and increased intact albuminuria independent of peptiduria.
Increased albumin excretion in hypertension and/or diabetes is manifested in different forms independent of glomerular permeability.
... Largely on the basis of experimental data and immunochemical analyses of biopsy samples, multiple genes have been proposed as potential candidates in the evaluation of prognosis. Those can be divided into ECM-related genes directly implicated in accumulation of sclerotic material or genes implicated in ECM degradation, cytokines/growth factors, apoptosis-related molecules, and components of the aldosterone-reninangiotensin system (6,11,16,19,20). ...
... Elevated CTGF levels in glomeruli of NOD mice appear to correlate with the duration of diabetes (89). Elevated CTGF expression was also detected in human DN (89, 162,163). In vitro studies have shown that CTGF is induced in renal cells by both high glucose and AGE (87,89,160), as well as ROS (164), which they generate. ...
Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstitium. This review summarizes the main changes in protein composition of the glomerular mesangium and basement membrane and the evidence that, in the mesangium, these are initiated by changes in glucose metabolism and the formation of advanced glycation end products. Both processes generate reactive oxygen species (ROS). The review includes discussion of how ROS may activate intracellular signaling pathways leading to the activation of redox-sensitive transcription factors. This in turn leads to change in the expression of genes encoding extracellular matrix proteins and the protease systems responsible for their turnover.
Fibrosis is defined as an excess of extracellular matrix (ECM) components, or a failure in degradation of existing ECM components. The ECM provides structural support for cells, comprising principally collagen, and other proteins such as elastin, glycoproteins, and integrins. Hyperglycemia, as a result of diabetes, can cause stimulation of signaling pathways, which lead to either of these outcomes, resulting in fibrosis. Many organs are affected in diabetes; principally the skin, causing chronic wounds and peripheral neuropathy, but also commonly the kidneys and large vessels. The pathogenesis of microvascular changes, which lead to these end-organ complications, includes under- or overexpression of growth factors, namely, transforming growth factor-β (TGF-β). Other fibrotic mechanisms include the renin angiotensin aldosterone system (RAAS), and formation and interaction of free radicals via oxidative stress, and advanced glycation end products (AGEs). Monitoring progression of fibrosis along with treatment is organ specific, utilizing biomarkers, which target the growth factors and signaling molecules involved as well as key ECM components in that organ; and while specific therapeutic targets have been identified in affected organs, ultimately maintaining good glycemic control prevents progression of fibrosis and further complications.
Diabetic kindney disease affects up to one third of patients withtype 1 and type 2 diabetes and is anow the commonest single cause of end-stage renal failure in the western world. The cumulative incidence of diabetic nephropathy continues to increase as type 2 diabetes becomes more prevalent. Managing increasing numbers of patients requiring renal replacement therapy is a major challenge for health services throughout the world. Diagnosis is usually made upon clinical grounds and is based on the detection of albuminuria. If the diagnosis is in doubt renal biopsy should be considered. The major clinical phases of diabetic kidney disease are microalbuminuria, proteniuria or overt nephropathy, progressive renal impairment, and end-stagerenal failure. Not all patients progress to end-stage renal afailure because of an associated excess cardiac mortality. Major risk factors for diabetic kidney disease are poor glycemic control and hypertension. Genetic factors also appear to play an important role in determining an individuals predisposition, but the gene(s) responsible have not yet been unequivocallly identified. Treatment is based upon primary prevention with good glycaemic control and treatment of hypertension. Secondary prevention with antihypertensive therapy, particularly with agents acting on the renin--angiotensin system, can slow the rate of progression of diabetic nephropathy and delay the onset of end-stage renal failure. Coexisting cardiovascular risk factors should also be aggressively managed. Patients with advanced diabetic kidney disease often have multiple problems that require the input of a large number of health care professionals.
Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) in the United States, accounting for nearly 40% of incident ESRD (1). Diabetes mellitus (DM) is also an independent and strong risk factor for ESRD ascribed to causes other than diabetes (2), such as hypertension, pyelonephritis, and other forms of glomerulopathies that can lead to chronic renal disease. Here we focus mainly on diabetic nephropathy as a major microvascular complication of both type 1 and type 2 diabetes.
The aetiology of uterine fibroids remains unknown, despite causing significant gynaecological morbidity. Fibroids have a reduced microvascular density when compared with adjacent myometrial tissue. The aim of this study was to identify genes with differential expression between fibroid and adjacent normal myometrium, particularly genes with a role in angiogenesis. Total RNA was extracted from fibroid/myometrium pairs from 12 hysterectomy specimens, and used to perform 24 cDNA microarrays. There were 10 500 genes screened on each microarray for differential expression. Analysis of expression data was carried out using multiple t-tests, as well as a novel class prediction algorithm (GeneRaVE‘). The differential gene expression of selected genes was confirmed by quantitative ‘real time’ RT‐PCR. Selected genes with a role in angiogenesis were further analysed for expression in isolated cell populations of endothelial cells (fibroid and myometrium) and smooth muscle cells (fibroid and myometrium), to see if their expression was confined to particular cell types. Twenty-five genes with differential gene expression between fibroid and myometrium were identified. Insulin-like growth factor-2, endothelin A receptor, connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61) and collagen 4a2 (COL4A2) were confirmed by RT‐PCR. CTGF and CYR61, both angiogenesis promoters, were reduced in expression relative to myometrium. COL4A2, the precursor for an angiogenesis inhibitor, canstatin, was increased relative to myometrium. These three genes display an anti-angiogenic expression profile in fibroids relative to myometrium. These findings may explain the reduced microvascular density seen in fibroids relative to myometrium.
Apart from clinical, histological and biochemical indices, genomics are now being employed to unravel the pathogenetic mechanisms in the disease progression of IgA nephritis (IgAN). The results of angiotensin converting enzyme (ACE) gene polymorphism have been controversial. Those patients with the DD genotype seem to have a poorer prognosis. However, with high dose angiotensin receptor blocker (ARB) therapy, the ACE gene polymorphism status of a patient may no longer be a matter for concern as those with the DD genotype would also respond favourably to high dose ARB therapy. Association studies with gene sequencing and haplotypes have suggested that multiple genes are involved in the pathogenesis of IgAN. Some workers have reported a synergistic effect in the combined analysis of AGT-M235T and ACE I/D polymorphism. With the use of deoxyribo nucleic acid (DNA) microarray, tens of thousands of gene expressions genome-wide can be examined together simultaneously. A locus of familial IgAN has been described with strong evidence of linkage to IgAN1 on chromosome 6q22-23. Two other loci were reported at 4q26-31 and 17q12-22. DNA microarray techniques could also help in the identification of specific pathogenic genes that are up- or down-regulated and this may allow genome wide analyses of these genes and their role in the pathogenesis and progression of IgAN. Recently, using genome-wide association studies (GWAS) more loci for disease susceptibility for IgAN have been identified at 17p13, 8p23, 22q12, 1q32 and 6p21.
Background/Aims: Recent studies show that albuminuria may be the result of changes in post-glomerular cellular uptake and processing of albumin. This study aims to determine whether this processing is disrupted in diabetes and/or hypertension. Methods: Diabetes (d) was induced using streptozotocin in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) and studied after 8, 16 and 24 weeks of disease. Intact albumin excretion was determined by radioimmunoassay. Total albumin was determined by [14C]albumin. Lysosomal activity was determined by dextran sulfate desulfation. Renal TGF-β1 and transforming growth factor-β1 inducible gene-h3 mRNA (βig-h3) expression was determined by real time RT-PCR. Results: SHR-c rats exhibited an increase in intact albuminuria without significant change in total albumin excretion (intact plus albumin-derived peptides). For WKY-d rats, intact albuminuria developed initially, followed by an increase in total albumin excretion primarily in the form of albumin peptides (peptiduria). SHR-d rats exhibited both increases in peptiduria and intact albuminuria. There was no increase in glomerular permeability at 24 weeks for polydisperse [3H]Ficoll in all groups. Increased renal TGF-β1 and βig-h3 expression was correlated with a decrease in dextran sulfate desulfation and increased intact albuminuria independent of peptiduria. Conclusion: Increased albumin excretion in hypertension and/or diabetes is manifested in different forms independent of glomerular permeability.
Renal failure is a common complication of cirrhosis and is associated with poor prognosis. Several reports have demonstrated the clinical utility of renal resistive indices in the assessment of renal function in cirrhosis patients. It is unknown whether the occurrence of diabetes affects renal haemodynamic indices in patients with cirrhosis. Therefore, the aim of our study was to compare renal Doppler indices in cirrhotic patients with and without type 2 diabetes mellitus (T2DM) and in diabetics without cirrhosis, and to relate the Doppler parameters to albuminuria. We evaluated 89 consecutive patients with normal renal functioning, including 37 with cirrhosis and T2DM (CD-Group), 41 with cirrhosis without diabetes (C-Group) and 11 with diabetes without cirrhosis (D-Group). The kidney pulsatility index (PI) and resistance index (RI) were measured by Doppler ultrasound. Renal function was expressed as the estimated glomerular filtration rate (eGFR) using the modification of diet in renal disease (MDRD) formula. Microalbuminuria (µAlb) was also evaluated. No significant differences were observed with respect to age, the Child-Pugh class or the serum creatinine level. The eGFR was mildly reduced in the CD-Group compared with the C-Group and D-Group, and µAlb was present in 24.4 % of the patients in the CD-Group and in 9 % of those in the D-Group. The PI and RI were significantly increased in the CD-Group and D-Group compared with the C-Group. Both the PI and RI were significantly associated with µAlb independent of age and Child-Pugh class. The novel finding of this study was that T2DM potentially impairs renal haemodynamics in patients with cirrhosis.
Type 1 diabetes can be diagnosed at any age, but dinical course, genetic, and environmental determinants appear to be heterogenous by age. The common pathway begins with preclinical beta-cell autoimmunity with progressive defect of insulin secretion, followed by onset of hyperglycemia, transient usually partial remission, and finally complete insulinopenia associated with acute and chronic complications and premature death. Current research effort is focused on identification of the genetic and environmental determinants of this process and the ways they interact.
Diabetes mellitus contributes greatly to morbidity, mortality, and overall health care costs. In major part, these outcomes derive from the high incidence of progressive kidney dysfunction in patients with diabetes making diabetic nephropathy a leading cause of end-stage renal disease. A better understanding of the molecular mechanism involved and of the early dysfunctions observed in the diabetic kidney may permit the development of new strategies to prevent diabetic nephropathy. Here we review the pathophysiological changes that occur in the kidney in response to hyperglycemia, including the cellular responses to high glucose and the responses in vascular, glomerular, podocyte, and tubular function. The molecular basis, characteristics, and consequences of the unique growth phenotypes observed in the diabetic kidney, including glomerular structures and tubular segments, are outlined. We delineate mechanisms of early diabetic glomerular hyperfiltration including primary vascular events as well as the primary role of tubular growth, hyperreabsorption, and tubuloglomerular communication as part of a "tubulocentric" concept of early diabetic kidney function. The latter also explains the "salt paradox" of the early diabetic kidney, that is, a unique and inverse relationship between glomerular filtration rate and dietary salt intake. The mechanisms and consequences of the intrarenal activation of the renin-angiotensin system and of diabetes-induced tubular glycogen accumulation are discussed. Moreover, we aim to link the changes that occur early in the diabetic kidney including the growth phenotype, oxidative stress, hypoxia, and formation of advanced glycation end products to mechanisms involved in progressive kidney disease. © 2011 American Physiological Society. Compr Physiol 1:1175-1232, 2011.
Diabetic nephropathy (DN) is a common complication of diabetes types 1 and 2. One of the hallmarks of DN is the development of mesangial expansion, which occurs through accumulation of extracellular matrix (ECM) components. Altered local gene expression of humoral factors (eg, transforming growth factor-b, connective tissue growth factor, and platelet-derived growth factor) can lead to increased production of ECM components (eg, fibronectin and collagen IV) or decreased degradation through matrix metalloproteinases (eg, MMP-1, MMP-2). In recent years, new techniques for examination of gene expression have been developed. Because of their large scale and high-throughput character, it is now possible to examine differential gene expression in a large number of samples. This paper provides an overview of techniques used and results obtained in studies of DN. Newly developed concepts of how altered gene expression may affect histomorphologic features or clinical symptoms are also discussed.
The prevalence of diabetes has dramatically increased worldwide due to the vast increase in the obesity rate. Diabetic nephropathy is one of the major complications of type 1 and type 2 diabetes and it is currently the leading cause of end-stage renal disease. Hyperglycemia is the driving force for the development of diabetic nephropathy. It is well known that hyperglycemia increases the production of free radicals resulting in oxidative stress. While increases in oxidative stress have been shown to contribute to the development and progression of diabetic nephropathy, the mechanisms by which this occurs are still being investigated. Historically, diabetes was not thought to be an immune disease; however, there is increasing evidence supporting a role for inflammation in type 1 and type 2 diabetes. Inflammatory cells, cytokines, and profibrotic growth factors including transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), connective tissue growth factor (CTGF), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-18 (IL-18), and cell adhesion molecules (CAMs) have all been implicated in the pathogenesis of diabetic nephropathy via increased vascular inflammation and fibrosis. The stimulus for the increase in inflammation in diabetes is still under investigation; however, reactive oxygen species are a primary candidate. Thus, targeting oxidative stress-inflammatory cytokine signaling could improve therapeutic options for diabetic nephropathy. The current review will focus on understanding the relationship between oxidative stress and inflammatory cytokines in diabetic nephropathy to help elucidate the question of which comes first in the progression of diabetic nephropathy, oxidative stress, or inflammation.
Proefschrift Universiteit Leiden. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands. Auteursnaam op omslag: Hans Baelde.
Diabetes mellitus is characterized by a lack of insulin causing elevated blood glucose, often with associated insulin resistance. Over time, especially in genetically susceptible individuals, such chronic hyperglycemia can cause tissue injury. One pathological response to tissue injury is the development of fibrosis, which involves predominant extracellular matrix (ECM) accumulation. The main factors that regulate ECM in diabetes are thought to be pro-sclerotic cytokines and protease/anti-protease systems. This review will examine the key markers and regulators of tissue fibrosis in diabetes and whether their levels in biological fluids may have clinical utility.
Glomerulosclerosis is a severe complication of many immunologically-mediated kidney diseases, eventually resulting in loss of renal function. In chronic graft-versus-host disease (GvHD) in mice, a model for human lupus nephritis, the end-stage sclerotic lesions were previously shown to contain large amounts of fibronectin (FN). This study investigated a domain-specific accumulation process of circulating plasma FN (pFN) in sclerotic lesions.
GvHD mice were injected with FITC-conjugated pFN or pFN-fragments, with or without heparin pre-incubation. pFN fragments were generated by digestion of pFN by cathepsin D, after which the fragments were separated on a heparin affinity column. Thus, two batches of fragments were obtained with either low or high affinity for heparin.
FN accumulation was accompanied by an up-regulated expression of integrin alpha5beta1, the FN receptor, in the periphery of sclerotic lesions. pFN-FITC injected into GvHD mice was trapped in sclerotic glomeruli within 24 hours. Both heparin and non-anti-coagulant heparin blocked the accumulation of pFN-FITC, indicating that the protective effect of heparin in the trapping of FN is independent of its anticoagulant properties, and probably results from preventing direct binding of FN in the sclerotic lesions. To investigate whether FN binds in the glomerulus via the heparin-binding regions, pFN fragments were generated and injected into GvHD mice. Whereas the fraction with high affinity for heparin did not accumulate in the sclerotic glomeruli, the fraction with low affinity for heparin did. Partial sequencing of the isolated peptides showed that in the glomerulus fibronectin does not bind via the heparin II binding region.
We hypothesize that the protective effect of heparin treatment may be the result of steric hindrance of the specific binding sites, that is, the I1-5 and/or III1 self-assembly sites of FN.
mRNAs of pathogenetic importance in the development of diabetic nephropathy were measured in subjects with type 1 diabetes to determine whether these might be used to predict progression from normoalbuminuria to microalbuminuria. We proposed that conversion from normoalbuminuria to microalbuminuria would be most likely in subjects whose connective tissue growth factor (CTGF) and collagen mRNAs were above the 95% confidence interval (CI) for live renal donors and within the 95% CI for subjects with abnormal albuminuria.
Glomerular CTGF, collagen alpha2(IV), and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured in microdissected glomeruli from living renal donors (n = 10), and subjects with normoalbuminuria (n = 12), microalbuminuria (n = 5), and overt proteinuria (n = 6).
After 44 +/- 2 months of follow-up, one subject converted from normoalbuminuria to microalbuminuria. Although the data are limited, progression from normoalbuminuria to microalbuminuria occurred in the only normoalbuminuric subject whose mRNA levels were above the live renal donors' 95% CI for CTGF and collagen alpha2(IV) and within the 95% CI of subjects with abnormal albuminuria. No clinical or histopathologic finding distinguished the progressor from the nonprogressors at the time of biopsy.
This case report provides proof-of-principle that a panel of glomerular mRNA markers chosen because of their pathogenetic relevance may be useful adjuncts to albuminuria and histology in predicting clinical stability or clinical progression in diabetic nephropathy.
Damage to the kidney generally elicits tissue repair mechanisms, but these processes themselves conversely may result in the progression of chronic renal disease. In a majority of patients chronic renal insufficiency progresses to a common histological end point, marked by the presence of a vast amount of scar tissue, that is, glomerulosclerosis and interstitial fibrosis. These lesions are the result of an excessive production of extracellular matrix (ECM) components. Studies on RNA expression in experimental kidney disease have shown that renal mRNA levels for ECM components and cytokines can function as prognostic tools. This suggests that mRNA levels potentially predict outcome and reaction to therapy in patients with renal diseases. Timely detection of molecular alterations could allow early therapeutic intervention that slows down or even prevents the development of sclerotic and fibrotic lesions. This review first provides a short introduction on mechanisms of initiation and progression of renal disease. Molecular techniques are available to identify renal RNA sequences potentially involved in disease progression. We discuss several molecular techniques that are being used in kidney research for quantitation and detection of mRNA. This is followed by a brief overview of investigation in experimental renal diseases, which reveal that alterations in tissue ECM mRNA levels precede histological damage and can function as predictors of clinical outcome. In particular, studies in human kidney biopsies that evaluate the prognostic value of mRNA levels with respect to renal function are examined, paying special attention to the pitfalls that potentially are encountered when interpreting the results of such studies. Then, we elaborate on ways of optimal exploitation of mRNA quantification as a prognostic tool. The potential and limitations of microarray technology in the search for genes specifically involved in progression of renal disease are reviewed, including RNA expression profiling and large-scale DNA mutation screening. Finally, the future utilities of microarray in nephrology and renal pathology are discussed.
The aim of this study was to determine whether aminoguanidine (AG), an inhibitor of advanced glycation, prevents expression of the profibrotic cytokine, connective tissue growth factor (CTGF), as well as accumulation of the previously reported CTGF-dependent matrix protein, fibronectin, in a model of experimental diabetic nephropathy. Diabetic animals were randomly allocated into groups receiving 32 wk of AG or vehicle. Diabetic rats showed increases in CTGF mRNA and protein expression as well as in advanced glycation end-product (AGE) and fibronectin immunostaining, compared with nondiabetic rats. In the diabetic kidney, the increase in CTGF gene and protein expression as well as expression of the extracellular matrix protein fibronectin were prevented by AG. To further explore the relationship between AGEs and mesangial CTGF and fibronectin production, cultured human mesangial cells were exposed in vitro to soluble AGE-BSA and carboxymethyl lysine-BSA, and this led to induction of both CTGF and fibronectin. On the basis of our in vitro findings in mesangial cells linking AGEs to CTGF expression, the known prosclerotic effects of CTGF, and the ability of AG to attenuate mesangial expansion, it is postulated that the antifibrotic effects of AG in this animal model may be partially mediated by CTGF.
Renal disease in older diabetic patients is costly in terms of morbidity, mortality and expenditure. Therefore, prevention and treatment of diabetic nephropathy has become a prominent goal in the treatment of patients with diabetes mellitus. Preventive treatment should begin no later than at the stage of microalbuminuria, and regular screening for microalbuminuria is recommended for all patients with diabetes, irrespective of age.
Improved metabolic control has been demonstrated to lower urinary albumin excretion. Target glycosylated haemoglobin levels should be below 7%, or 1% above the upper limit of normal of non-diabetic subjects. The use of an intensified treatment regimen is recommended. Insulin therapy has no adverse effects on renal indexes.
To preserve renal function in older diabetic patients, blood pressure should be kept at or below 130/80mm Hg. Treatment with ACE inhibitors or angiotensin II receptor antagonists (angiotensin II receptor blockers; ARBs) is superior to other pharmacological therapy, and should be initiated as first-line treatment. Most of the calcium channel antagonists have been found to increase or to have no effect on microalbuminuria despite blood pressure reduction. Moreover, there is substantial controversy as to whether they may be associated with increased cardiovascular morbidity. Non-dihydropyridine derivatives and calcium channel antagonists, such as nitrendipine, may be nephroprotective and have favourable effects on patients outcomes.
A renoprotective action of diuretics may be confined to indapamide. Although β-adrenoreceptor blockers are effective antihypertensive agents, they may not adequately preserve kidney function in older diabetic patients. However, as add-on treatment to ACE inhibitors or ARBs, they are particularly beneficial in nephropathic patients at risk of cardiovascular disease or with arrhythmias, in whom they may prove life-saving.
Much current epidemiologic research aims to find the factors that trigger the development of breast cancer in women who are genetically predisposed to it. In this issue of the Journal, Hamilton and Mack1 add to that rapidly growing literature some intriguing observations from a study of female twins. They find evidence that hormonal exposures at puberty may play an exaggerated part in some women who are likely to be at high genetic risk for breast cancer, even though the responsible genes are unknown. On the basis of a multifaceted examination of different risk factors in various groups of twins, they . . .
This article has no abstract; the first 100 words appear below.
Diabetes mellitus causes a dramatic increase in mortality, particularly from cardiovascular causes: the survival rate among persons with either type 1 or type 2 diabetes and no history of coronary heart disease was reported to be as poor as that of survivors of myocardial infarction.¹ Although accounting for only approximately 10 percent of all cases of diabetes, type 1 diabetes has been investigated far more completely than other forms. Trials concerning the importance of glycemic control²,³ or the response of nephropathy to intervention with antihypertensive agents⁴,⁵ have shown, however, that with certain limitations, the results of interventional studies . . .
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Microalbuminuria and hypertension with Over the past decade, there has been considerable focus on the concept of microalbuminuria, not only because it predicts renal disease in type 1 and type 2 diabetes, but also because it relates to premature mortality in the diabetic and in the general population. More importantly, intervention at this stage is now possible with the perspective of preserving glomerular filtration rate (GFR) and ameliorating cardiovascular disease and ensuing strong end-points. INITIAL STUDIES: The concept of microalbuminuria was introduced about 20 years ago and since then there has been a multitude of studies and papers on this subject using the original definition, but not always, in the US. Before that time it was suggested, mainly from the US, that diabetic renal disease was an untreatable relentlessly progressive condition.
Genetic studies:
There is an overwhelming number of studies on genetics and diabetes and also covering the genetics of diabetic complications including nephropathy. However, so far the results are extremely disappointing. Patients at risk cannot be identified and genetic analyses are of no value as a guide to treatment. The notion that the development of complications is controlled mainly by a special genetic pattern is increasingly doubtful. In genetic studies, it is rather phenotypic well-accepted risk factors that dominate. STRUCTURAL BASIS OF MICROALBUMINURIA: Patients with microalbuminuria have significant abnormalities in the kidney, including glomeruli. This is quite clear in patients with type 1 diabetes, but is also seen in type 2 diabetes, where on the other hand, other risk factors such as hypertension and dyslipidaemia also seem to be of importance, including loss of autoregulation. Renal biopsies are generally not indicated in the management of diabetic patients. MICROALBUMINURIA AND EARLY MORTALITY: It is quite clear that microalbuminuria predicts early mortality both in type 1 and type 2 diabetes. The association to other risk factors may partly explain this--but this does not account for the whole picture. Endothelial dysfunction as well as inflammatory and arteriosclerotic abnormalities in blood vessels may be a relevant hypothesis that needs to be further explored along with other possibilities. CLINICAL COURSE AND ASSOCIATED ABNORMALITIES: The risk factor for progression in normoalbuminuric patients to microalbuminuria is higher than normal albumin excretion (strongest factor), poor glycaemic control, elevated blood pressure, and to some extent smoking. The clinical course of microalbuminuria is usually progressive, but with the more effective intervention now available we encounter that the so-called natural history (without intervention) is increasingly difficult to study. Microalbuminuria is clearly associated with a number of abnormalities, almost in all organs, but GFR is generally well preserved in spite of more advanced structural lesions. Therefore, microalbuminuria is an important marker for more pronounced diabetic vascular disease in general as well as for nephropathy. Regression to normoalbuminuria only rarely occurs during standard unchanged nonintensive treatment. TREATMENT STRATEGIES: The best possible glycaemic control is important in preventing and ameliorating the course of normo- and micro-albuminuria. Another major treatment strategy, especially in microalbuminuric patients, is antihypertensive treatment including inhibition of the renal angiotensin aldosterone system. Numerous new studies are available, both in type 1 and type 2 diabetes, documenting that not only microalbuminuria but also renal and cardiovascular complications in these patient are also far better controlled by early detection and treatment. Therefore, screening for microalbuminuria should be a strategy in all diabetes management followed by effective intervention as outlined in this paper.
Connective tissue growth factor (CTGF) is a peptide secreted by cultured endothelial cells and fibroblasts when stimulated by transforming growth factor-beta (TGF-beta), and is overexpressed during fibrotic processes in coronary arteries and in skin. To determine whether CTGF is implicated in the pathogenesis of diabetic glomerulosclerosis, cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli were examined from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to recombinant human CTGF significantly increased fibronectin and collagen type I production. Furthermore, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36 to 38 kD) into the media. However, sodium heparin treatment resulted in a greater than fourfold increase in media-associated CTGF, suggesting that the majority of CTGF produced was cell- or matrix-bound. Exposure of MC to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in diabetic glomerulosclerosis, markedly induced the expression of CTGF transcripts, while recombinant human CTGF was able to autoinduce its own expression. TGF-, and high glucose, but not mechanical strain, stimulated the concomitant secretion of CTGF protein, the former also inducing abundant quantities of a small molecular weight form of CTGF (18 kD) containing the heparin-binding domain. The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta-neutralizing antibody blocked this stimulation. In vivo studies using quantitative reverse transcription-PCR demonstrated that although CTGF transcripts were low in the glomeruli of control mice, expression was increased 28-fold after approximately 3.5 mo of diabetes. This change occurred early in the course of diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (twofold) observed in whole kidney cortices indicated that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis, acting downstream of TGF-beta.
The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane.
We examined type IV collagen distribution and density in human diabetic kidneys by quantitative immunogold electron microscopy. We studied normal kidney transplant donors and "slow-track" and "fast-track" insulin dependent diabetic (IDDM) patients. The "slow-track" patients had IDDM for > or = 20 years and mesangial volume fraction (VvMes/glom) of < or = 0.32. The "fast-track" patients had IDDM for < or = 20 years and VvMes/glom > or = 0.37. Renal biopsies were embedded in Lowicryl, reacted with polyclonal anti-type IV collagen (in the distribution of the classical alpha 1(IV) and alpha 2(IV) collagen chains) and monoclonal anti-alpha 4(IV) collagen chain antibody followed by gold conjugated secondary antibody. We found, by morphometric techniques, a decrease in the immunogold densities of anti-type IV collagen in the subendothelial zone of the GBM in the "fast-track" IDDM patients. There was a trend towards a decrease in mesangial matrix (MM) particle density in the "fast-track" (P = 0.07) but not in the "slow-track" patients. However, because of the marked increase in MM in the "fast-track" patients, the per glomerulus estimated quantity of these antigens in MM was increased. In contrast, the density of alpha 4(IV) collagen chain was increased in the epithelial zone of the GBM in the "fast-track" IDDM patients. It is not known whether these changes in glomerular type IV collagen represent markers of advanced diabetic lesions or whether these changes might be detected earlier in diabetic patients destined for the later development of serious lesions.
Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)
Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation.
The steady-state levels of mRNA encoding for the alpha 1(IV) collagen chain, laminin B1 and B2 chains, basement membrane HSPG, and alpha 1(I) and alpha 1(III) collagen chains were examined in rat glomeruli at 4, 12, and 24 wk after injection of STZ. The mRNA levels for the alpha 1(IV) collagen chain, laminin B1 and B2 chains, and alpha 1(I) and alpha 1(III) collagen chains increased significantly with age in the STZ-induced diabetic rats before morphological thickening of basement membrane occurred. In contrast, the mRNA levels for HSPG decreased markedly 4 wk after STZ injection and then increased with age compared with those for control rats. The mRNA levels for these ECM components showed a continuous decline with age in controls. Treating the diabetic rats with insulin for 4 wk ameliorated the abnormally regulated ECM gene expression in the glomeruli. These data suggest that the abnormal regulation of ECM gene expression in the glomeruli may contribute to the expansion of mesangial matrix and basement membrane thickening in diabetic rats, and that hyperglycemia may play a role in the abnormal ECM gene expression.
We have undertaken an ultrastructural immunogold investigation of the distribution of type IV collagen and heparan sulphate proteoglycan (HSPG) in glomeruli from the kidneys of one normal control and three patients with diabetes mellitus and proteinuria. The sample included both diffuse and nodular diabetic glomerulosclerosis. In the control and diabetic kidneys, the type IV collagen was present predominantly on the endothelial aspect of the glomerular basement membrane (GBM), and by contrast the HSPG was found mainly on the epithelial side. In the mesangium in both control and diabetic glomeruli, type IV collagen was found predominantly in the central regions, while HSPG was mostly restricted to the region beneath the epithelial cells. Consequently, where there is a marked increase in mesangial matrix with nodule formation in diabetics there is a corresponding increase in the amount of type IV collagen but not of HSPG. Although the three diabetic patients were proteinuric, the HSPG was not decreased in the thickened GBMs.
The changes in glomerular extracellular matrices components in diabetic nephropathy were investigated. Indirect immunofluorescence staining, using polyclonal antibodies to heparan sulfate proteoglycan (HS-PG), laminin, type IV collagen, and fibronectin was carried out on renal specimens obtained by needle biopsy. Immunofluorescence intensity and distribution were observed. HS-PG and laminin decreased in the capillary walls; on the other hand, type IV collagen and fibronectin tended to increase in the mesangial area. HS-PG and laminin decreased in inverse proportion to sclerosis grades and proteinuria. These changes seemed to play an important role in progression of diabetic glomerulosclerosis.
We examined steady-state levels of mRNA encoding type IV collagen, B1 chain of laminin, and the basement membrane heparan sulfate proteoglycan in the kidney cortex of a mouse model (KKAy) of non-insulin-dependent diabetes. mRNAs encoding laminin B1 and the proteoglycan were unchanged in kidneys taken from diabetic mice with demonstrable basement membrane thickening. mRNA levels for type IV collagen, in contrast, were significantly elevated (2-fold) in diabetic mice concurrent with but not preceding morphologically thickened basement membranes. There was a negative correlation between a ratio of proteoglycan/type IV collagen and levels of albuminuria in the diabetic mice. No correlation was noted with laminin. We also examined the effects of inhibiting the synthesis of thromboxane, a potent vasoconstrictor, on the steady-state levels of type IV collagen in the diabetic mice. Inhibition of thromboxane stopped the progression of albuminuria and prevented an increase in type IV collagen mRNA levels. We conclude that basement membrane thickening in diabetes, a hallmark of diabetic nephropathy, is partly a consequence of an unbalanced increase in the production of type IV collagen. The relative decrease in proteoglycan production may contribute to chronic albuminuria.
Since several studies have suggested that a slight increase in urinary albumin excretion (microalbuminuria) is predictive of nephropathy in patients with diabetes mellitus, we studied the relation of albumin excretion to renal structure in patients with insulin-dependent (Type I) diabetes. Renal biopsy specimens were evaluated with light- and electron-microscopical morphometric techniques in 48 patients who had had diabetes for 5 to 40 years and who excreted less than 200 mg of urinary albumin per 24 hours. Patients in Group I (n = 26) had normal urinary albumin excretion, creatinine clearance, and blood pressure; those in Group II (n = 10) had increased urinary albumin excretion but normal creatinine clearance and blood pressure; those in Group III (n = 12) had increased urinary albumin excretion and hypertension, decreased creatinine clearance, or both. Glomerular structure varied similarly, ranging from normal to abnormal in Groups I and II, but was consistently abnormal in Group III. The thickness of the glomerular basement membrane, the fractional volume of the mesangium, and the mesangial volume per glomerulus in Group III exceeded the corresponding values in the other groups significantly. Thus, microalbuminuria, when present with hypertension, decreased creatinine clearance, or both, indicates established abnormalities of glomerular structure. Normal albumin excretion, or microalbuminuria without these other functional abnormalities, does not accurately predict the severity of the underlying glomerular lesions in patients with Type I diabetes.
The immunohistopathology of the intrinsic basement membrane-associated antigens were examined in diabetic nephropathy. In early and moderate stages of disease there was polyantigenic expansion of all the intrinsic components of mesangium, glomerular basement membrane (GBM), and tubular basement membrane (TBM) assessed by polyclonal antisera to collagen types IV and V, laminin, and by monoclonal antibodies to type IV collagen and fibronectin and to four other intrinsic components of normal renal extracellular matrices (MBM10, 11, 12, and 15). In the mesangium the first intrinsic antigens to increase were fibronectin and type V collagen. In late stages of disease, there was a diminution in the mesangium of all of these antigens with the exception of type V collagen, which persisted. Additionally, antigens appeared in the mesangium, recognized by MBM11 and MBM15, which are normally present in fetal but not adult mesangial regions. Similarly, in the GBM in late stages of disease, there was a decrease in all of the antigens, except for a persistence of the antigen recognized by MBM15. However, in TBM all of the antigens assessed increased in early, moderate, and severe disease. These studies document the complexity of polyantigenic alterations in the development of diabetic nephropathy.
Patients with Type 1 (insulin-dependent) diabetes without proteinuria were studied to define those patients who will later develop persistent proteinuria (more than 0.5 g protein/24 h). Two investigations were performed; 71 patients were studied longitudinally for 6 years and another 227 patients were studied cross-sectionally. All were less than 50 years of age and had developed diabetes before the age of 40 years. At entry into the study they had no proteinuria (Albustix method), had normal blood pressure and urinary albumin excretion rates less than 200 micrograms/min (normal less than or equal to 20 micrograms/min). The best predictor of persistent proteinuria or an albumin excretion rate greater than 200 micrograms/min was the initial urinary albumin excretion rate. During the longitudinal study, seven patients with an urinary albumin excretion rate of more than 70 micrograms/min at the start of the study developed persistent proteinuria or an albumin excretion rate greater than 200 micrograms/min. In contrast, only three out of the remaining 64 patients with urinary albumin excretion rate less than or equal to 70 micrograms/min developed urinary albumin excretion rate greater than 200 micrograms/min. Patients with an urinary albumin excretion rate greater than 70 micrograms/min are thus at risk of developing diabetic nephropathy. We designate this stage of renal involvement incipient nephropathy. Patients with incipient nephropathy were further characterized in the cross-sectional study. Compared with normoalbuminuric patients, patients with incipient nephropathy had increased systolic and diastolic blood pressure, but normal serum creatinine. The glomerular filtration rate was higher than normal in patients with incipient nephropathy though not different from that of normoalbuminuric patients.
We studied whether microalbuminuria (urinary albumin excretion rates of 15 to 150 micrograms per minute) would predict the development of increased proteinuria in Type I diabetes. We also studied the influence of glomerular filtration rate, renal blood flow, and blood pressure on the later development of proteinuria. Forty-four patients who had had Type I diabetes for at least seven years and who had albumin excretion rates below 150 micrograms per minute were studied from 1969 to 1976, and 43 were restudied in 1983. Of the 14 who initially had albumin excretion rates at or above 15 micrograms per minute, 12 had clinically detectable proteinuria (over 500 mg of protein per 24 hours) or an albumin excretion rate above 150 micrograms per minute at the later examination. Of the 29 who initially had albumin excretion rates below 15 micrograms per minute, none had clinically detectable proteinuria at the later examination, although four had microalbuminuria. Those whose condition progressed to clinically overt proteinuria had elevated glomerular filtration rates and higher blood pressures at the initial examination than did those in whom proteinuria did not develop. Renal blood flow was not elevated in these patients. We conclude that microalbuminuria predicts the development of diabetic nephropathy and that elevated glomerular filtration rates and increased blood pressure may also contribute to this progression.
In an attempt to detect patients at high risk of developing diabetic nephropathy, a longitudinal study of urinary albumin excretion rate (radial immunodiffusion) was carried out in 15 female and 8 male long-term insulin-dependent diabetics without proteinuria (negative Albustix test).
Five females and 3 males had an elevated urinary albumin excretion at the time of screening, mean 115 ± 26 ( sd ) mg/24 h. Our upper normal range for urinary albumin excretion is ≤ 40 mg/24 h. The 5 patients with the highest albumin excretion subsequently developed persistent albuminuria, 132 → 1007 mg/24 h, P < 0.05, elevated serum creatinine, 83 → 128 μmol/l, P < 0.05, and raised blood pressure, 135/86 → 163/112 mmHg, P < 0.05. One patient developed intermittent albuminuria (positive Albustix test), while the variables in the remaining 2 patients were about the same during the 6 years observation period. Fifteen patients had a normal urinary albumin excretion, mean 17 ± 9 ( sd ) mg/24 h, at the time of the screening. Intermittent and persistent albuminuria developed in 2 patients, while albumin excretion, serum creatinine, and blood pressure were nearly unchanged in the remaining 13 patients after 6 years.
Our longitudinal study indicates that early detection of patients at high and low risk of developing persistent proteinuria i.e. diabetic nephropathy, is possible by using a sensitive method for measuring urinary albumin excretion.
Although microalbuminuria is known to foretell the later development of overt proteinuria in patients with insulin-dependent diabetes mellitus (IDDM), different investigators have reported different levels of albuminuria as being predictive. However, whether different levels of albuminuria reflect differences in glomerular structure is not well known. In this study, we divided a cohort of 66 nonproteinuric long-standing (duration 20 +/- 7 years) IDDM patients, who had both renal functional and structural studies performed, into four groups according to their urinary albumin excretion rate (AER). The several different levels of microalbuminuria previously reported to be predictive served to demarcate these groups: group I, AER < or = 22 mg/24 h (upper limit for normal in our laboratory) (33 patients); group II, AER 23-45 mg/24 h (11 patients); group III, AER 46-100 mg/24 h (13 patients); and group IV, AER 101-220 mg/24 h (9 patients). Creatinine clearance was similar in groups I, II, and III but was lower in group IV. Systemic hypertension was present in five patients in group I, one in group II, seven in group III, and five in group IV. Mean values for glomerular basement membrane (GBM) width and volume fraction of the mesangium [Vv(mes/glom)] were greater in all groups than in a group of 52 age-matched normal kidney donors (P < 0.0001). Also, filtration surface density [Sv(PGBM)], inversely related to Vv(mes/glom) (r = 0.61, P < 0.0001), was reduced in all diabetic groups compared with the normal group (P < 0.0001). Structural measures were identical in group I and II.(ABSTRACT TRUNCATED AT 250 WORDS)
Nodular intercapillary glomerulosclerosis is the most typical lesion of diabetic nephropathy (DN) and is characterized by increased extracellular matrix (ECM) and amorphous masses of mesangial matrix. The local exaggeration of these deposits results in the formation of the typical diabetic nodule. To clarify the composition of the ECM of sclerotic lesions in DN, we investigated the distribution of type III and type IV collagens and their mRNAs by immunohistochemistry and in situ hybridization, respectively. In normal renal tissues, there was no intraglomerular immunostaining for type III collagen, while strongly positive staining was found in the extraglomerular interstitium. Positive immunostaining for type IV collagen was also present in the mesangium, glomerular basement membrane (GBM), Bowman's capsule, and the vascular pole of the normal glomerulus. In DN, the nodular lesions were negative for type III collagen and strongly positive for type IV collagen. On the other hand, in the late stage of global sclerosis, both type III and type IV collagens were diffusely present in the sclerotic matrix. To determine the origins of these type III and type IV collagens in the sclerotic matrix, in situ hybridization was performed, utilizing thymine-thymine (T-T) dimerized synthetic oligonucleotides complementary to either pro alpha 1(III) chain or pro alpha 1(IV) chain mRNAs as probes. The signals were detected by enzyme immunohistochemistry using an anti-T-T antibody. Intraglomerular cells (glomerular epithelial and mesangial cells) containing type III collagen mRNA were found in DN with sclerotic lesions, but not in normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)
FACTORS IN RENAL DISEASE: The factors that affect the development of renal disease are the same as those that influence its progression, that is, glycaemic control, blood pressure and albumin excretion rate. EFFECTS OF TREATMENT: Many of these factors are modifiable by treatment and their modification appears to delay the evolution of diabetic nephropathy. Of primary importance is the treatment and reduction of albuminuria, microalbuminuria in particular.
An accurate assessment of which patient with glomerular disease will progress to end-stage renal failure would be an important addition to establishing prognosis and evaluating therapeutic strategies. We previously found the development of glomerular scarring in animal models was preceded by an increase in glomerular type IV collagen mRNAs and that the level of scarring predicted the rate of progression. The purpose of this study was to determine whether these findings apply to human glomerular diseases using microdissected glomeruli and assessment of mRNA by competitive PCR. After showing that the levels of type IV collagen mRNAs were elevated in sclerotic glomeruli isolated from nephrectomies, we undertook this preliminary cross-sectional study of type IV collagen subchain mRNAs in renal biopsies in two of the leading causes of glomerulosclerosis, diabetic nephropathy, and membranous glomerulopathy. We found that glomerular type IV collagen mRNA levels were altered in disease-specific ways. The relative levels of the individual alpha-chains of type IV collagen depended on the anatomic site of the glomerular lesions. The alpha 2 type IV/alpha 3 type IV collagen mRNA ratio was high in diabetes mellitus, but not in membranous glomerulopathy. These data, coupled with those obtained from experimental animals, suggest that a dysregulation of basement collagen synthesis underlies progressive glomerular scarring. If these conclusions are verified in prospective studies it will be feasible to assess the risk of developing progressive glomerulosclerosis in the individual patient and to quantitatively assess therapeutic responses in a timely manner.
The earliest manifestations of type I diabetic nephropathy include mesangial matrix expansion, basement membrane thickening, and renal hypertrophy. Transforming growth factor (TGF)-beta, a potent inducer of matrix protein synthesis, is a prime candidate to mediate the glomerular changes observed in diabetes. However, the temporal expression of TGF-beta and matrix proteins during the early stage of diabetic nephropathy has not been clearly defined. Using in situ hybridization and immunohistochemistry, we determined the expression of TGF-beta and type IV collagen mRNAs and proteins in glomeruli and interstitium of diabetic rats 3, 7, and 14 days after streptozotocin (STZ) administration. There was a marked increase in the expression of TGF-beta and alpha1(IV) procollagen mRNAs in glomerular and tubulointerstitial cells as early as 3 days after induction of diabetes, an effect that persisted for 14 days. A concomitant increase in TGF-beta and type IV collagen proteins was also observed at each time point. Insulin treatment substantially inhibited the increased expression of TGF-beta and collagen type IV mRNAs and proteins. We conclude that TGF-beta is increased in glomeruli during the early phase of rapid renal growth in diabetes. These findings suggest that TGF-beta may be a key factor involved in the pathogenesis of basement membrane thickening and extracellular matrix accumulation. Inhibition of TGF-beta and type IV collagen expression by insulin treatment suggests that they may be useful structural markers for determining the efficacy of therapeutic intervention during early diabetic nephropathy.
As transforming growth factor-beta1 (TGF-beta1) is implicated in the pathogenesis of glomerulosclerosis, the aim of the study was to demonstrate if levels of glomerular TGF-beta1 mRNA in renal biopsies correlated with glomerulosclerosis. Glomeruli were collected by microdissection from renal biopsies in patients with membranous nephropathy, lupus nephritis, diabetic nephropathy, minimal change disease and IgA nephropathy presented by proteinuria when serum creatinine was <3 mg%. Glomerular mRNAs were reverse transcribed and TGF-beta1, alpha2(IV) collagen, beta-actin cDNA quantitated by competitive polymerase chain reaction (PCR). By semiquantitative electron microscopy, a 3.5-fold increase of glomerular TGF-beta1/beta-actin mRNA ratio in the moderate sclerotic group (n = 23, p < 0.01) and a 1.5-fold increase in the mild sclerotic group (n = 22, p < 0.05) were observed when compared to the minimal sclerotic group (n = 12). A concordant increase of glomerular alpha2(IV) collagen mRNA was found with 2.2- and 1.3-fold in moderate and mild sclerotic groups, respectively. The TGF-beta1/beta-actin mRNA ratios were highest in membranous nephropathy (466.4 +/- 133.4, n = 11), followed by lupus nephritis (394.9 +/- 94.8, n = 12) and diabetic nephropathy (333.2 +/- 97.6, n = 10). Patients with minimal change disease(233.1 +/- 54.1, n = 15)and IgA nephropathy(185.3 +/- 39.6, n = 9) had low levels. The degree of glomerulosclerosis in each group followed the TGF-beta1/beta-actin mRNA ratios indicating that the level is the major determinant ofglomerulosclerosis but not the disease entities. Glomerular TGF-beta1/beta-actin mRNA ratio did not correlate with clinical parameters such as the urinary protein excretion and creatinine clearance. These results suggest that glomerular TGF-beta1/beta-actin mRNA ratio may be used as a marker of glomerulosclerosis in renal biopsy to reflect the local sclerotic process.
Type IV collagen is a constituent of mesangial matrix and is increased in amount in many forms of glomerular injury.
We performed renal biopsies in patients who (1) were donating a kidney to a relative (LRD, N = 6), (2) had diabetic glomerulopathy with or without nephrosclerosis (DM, N = 6), or (3) had diabetic glomerulopathy with a superimposed glomerular lesion (DM+, N = 5). Glomerular collagen alpha2(IV) and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured, and the former correlated with clinical and morphological data to assess its usefulness in reflecting glomerular injury.
Collagen alpha2(IV) mRNA levels were lowest in LRD (2.9 +/- 0.6 attomol/glomerulus), higher in DM (5.9 +/- 1.6, P = 0.05), and highest in DM+ (12.7 +/- 2.8 attm/glomerulus, P < 0.05 vs. LRD and vs. DM). Control GAPDH mRNA levels were not significantly different (P > 0.05). Levels of proteinuria, serum creatinine, and glomerular size did not correlate with collagen alpha2(IV) mRNA levels. The fractional mesangial area and the fractional mesangial area occupied by type IV collagen were higher in both diabetic groups than in LRD (P < 10-6), but the intensity of type IV collagen staining in the diabetic patients was significantly less than that seen in the LRD (P < 0.01). In DM+ patients, extramesangial type IV collagen was present. Fractional mesangial area and glomerular collagen alpha2(IV) mRNA levels correlated (r = 0.45, P < 0.05).
These data are consistent with a view of diabetic nephropathy as a lesion of increased alpha2 type IV collagen transcription, increased total amount of collagen present, but decreased mesangial density relative to other matrix molecules. These data further demonstrate that glomerular injury superimposed on diabetic nephropathy contributes to additional structural damage by inducing increased synthesis of type IV collagen at extramesangial sites.
Diabetic glomeruloscle-rosis-immunogold ultrastructural studies on the glomerular distri-Reprint requests to Sharon Adler, M.D. ,Division of Nephrology bution of type IV collagen and heparan sulphate proteoglycan. and Hypertension, Harbor-UCLA Medical Center, 1124 W. Carson St
- Moss J I Shore
- Spiro
Woodrow D, Moss J, Shore I, Spiro RG: Diabetic glomeruloscle-rosis-immunogold ultrastructural studies on the glomerular distri-Reprint requests to Sharon Adler, M.D.,Division of Nephrology bution of type IV collagen and heparan sulphate proteoglycan. and Hypertension, Harbor-UCLA Medical Center, 1124 W. Carson St., J Pathol 167:49–58, 1992
Glomerular transforming E-mail: sadler@rei.edu growth factor-beta1 mRNA as a marker of glomerulosclerosis-application in renal biopsies
- Yang Cw S Hsueh
- Wu
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Center General Clinical Research Center (M01RR00425). It was pre-
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Our data do not rule out the possibility that other 13 Glomerular distribution of type factors not assessed in these analyses contribute to the IV collagen in diabetes by high resolution quantitative immunochemistry
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and Hypertension, Harbor-UCLA Medical Center, 1124 W. Carson St.,
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phropathy. J Pathol 174:131-138, 1994
Nerlich A, Schleicher E: Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions
actual diabetic nephropathy.
16. Nerlich A, Schleicher E: Immunohistochemical localization of
extracellular matrix components in human diabetic glomerular lesions. Am J Pathol 139:889-899, 1991
Changes in glomerular exsented in part in abstract form at the meeting of the American Society tracellular matrices components in diabetic nephropathy
- S Ikeda
- H Makino
- T Haramoto
Center General Clinical Research Center (M01RR00425). It was pre-18. Ikeda S, Makino H, Haramoto T, et al: Changes in glomerular exsented in part in abstract form at the meeting of the American Society
tracellular matrices components in diabetic nephropathy. J Diaof Nephrology in Toronto, October 2000. The authors thank Dr. Jacob
betes Compl 5:186-188, 1991
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