Glomerular Protein Research Articles

Abstract 453: Angiogenesis Inhibition-Induced Hypertension and Nephrotoxicity: Not All Antihypertensives are the Same

Angiogenesis inhibition with the VEGF inhibitor sunitinib is an established anti-cancer therapy inducing hypertension and nephrotoxicity. Here we compared the effects of the endothelin antagonist (ET-R) macitentan (30 mg/kg/day, p.o; n=8), the calcium channel blocker (CCB) amlodipine (3 mg/kg/day, p.o; n=9), or the ACE-inhibitor captopril (3 or 12 mg/kg/day, s.c.; n=9) vs. no treatment (n=14) in sunitinib (26.7 mg/kg/day, p.o)-exposed hypertensive WKY rats. Treatment lasted 8 days; mean arterial pressure (MAP) was monitored telemetrically. At the end of the treatment period, 24-hour urine samples for protein and endothelin-1 (ET-1) measurements, and blood samples and kidneys for histological evaluation were collected. With sunitinib, MAP increased from 94.7±0.9 mmHg to 125.8±1.5 mmHg (Δ31.1±0.9 mmHg, p<0.001). Co-administration of macitentan (Δ12.3±1.5 mmHg, p<0.001) or amlodipine (Δ11.4±1.7 mmHg, p<0.001) attenuated the sunitinib-induced MAP rise, whereas low and high captopril doses did not (Δ28.1±2.0 and Δ27.2±1.1 mmHg). With sunitinib, serum creatinine increased from 8.0±2.7 to 29.9±4.6 μmol/l (p<0.01) and proteinuria from 7.5±1.3 to 33.3±4.8 mg/day, p<0.05. Although no agent could prevent the sunitinib-induced rise in creatinine, the induced proteinuria was attenuated by 62% (p<0.01) with macitentan and by 88% with low and 114% (p<0.001) with high dose of captopril, while proteinuria increased by 56% (p=NS) with amlodipine. With sunitinib, urinary ET-1 increased from 3.3±0.5 to 4.8±1.0 pg/day (p<0.05). Macitentan and captopril abolished this increase. Renal histology revealed extensive glomerular ischemia and endothelial cell swelling. Concomitant with the decrease in proteinuria, glomerular intra-epithelial protein deposition decreased with macitentan and captopril. In conclusion, ET-R antagonism and CCB effectively reduced the sunitinib-induced hypertension, whereas ACE-inhibition did not. Both ET-R antagonism and ACE inhibition diminished the sunitinib-induced proteinuria. Since ET-R antagonists are not yet available as anti-hypertensive agents, CCBs are preferred in angiogenesis inhibition-induced hypertension, while adding an ACE-inhibitor might be considered when proteinuria is also present.

Berberine ameliorates experimental diabetes-induced renal inflammation and fibronectin by inhibiting the activation of RhoA/ROCK signaling

The accumulation of glomerular extracellular matrix proteins, especially fibronectin (FN), is a critical pathological characteristic of diabetic renal fibrosis. Inflammation mediated by nuclear factor-κB (NF-κB) plays a critical role in the pathogenesis of diabetic nephropathy (DN). RhoA/ROCK signaling is responsible for FN accumulation and NF-κB activation. Berberine (BBR) treatment significantly inhibited renal inflammation and thus improved renal damage in diabetes. Here, we study whether BBR inhibits FN accumulation and NF-κB activation by inhibiting RhoA/ROCK signaling and the underlying mechanisms involved. Results showed that BBR effectively inhibited RhoA/ROCK signaling activation in diabetic rat kidneys and high glucose-induced glomerular mesangial cells (GMCs) and simultaneously down-regulated NF-κB activity, which was accompanied by reduced intercellular adhesionmolecule-1, transforming growth factor-beta 1 and FN overproduction. Furthermore, we observed that BBR abrogated high glucose-mediated reactive oxygen species generation in GMCs. BBR and N-acetylcysteine inhibited RhoA/ROCK signaling activation in high glucose-exposed GMCs. Collectively, our data suggest that the renoprotective effect of BBR on DN partly depends on RhoA/ROCK inhibition. The anti-oxidative stress effect of BBR is responsible for RhoA/ROCK inhibition in DN.

Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy

Glomerular matrix protein accumulation, mediated largely by resident mesangial cells (MCs), is central to the pathogenesis of diabetic nephropathy. We previously showed that caveolin (CAV)-1/caveolae mediate matrix upregulation by MCs in response to high glucose and TGFβ, two important pathogenic mediators of diabetic glomerular sclerosis. Here, we evaluated the in vivo role of CAV-1/caveolae in the development of diabetic nephropathy. Diabetes was induced in Cav1-knockout (KO) mice and their wild-type (WT) counterparts by streptozotocin injection. After 10 months, kidneys were evaluated for the development of nephropathy, including glomerular sclerosis and upregulation of matrix proteins. Parallel experiments assessing glucose-induced matrix upregulation were carried out in MCs isolated from KO mice. KO diabetic mice developed hyperglycaemia and renal hypertrophy, but were protected from developing albuminuria and glomerular sclerosis compared with WT mice. KO mice were significantly protected from the upregulation of glomerular collagen I, fibronectin, connective tissue growth factor (CTGF) and TGFβ. In vitro, glucose induced collagen I A1 promoter activation and collagen I, fibronectin and CTGF protein upregulation in WT but not KO MCs. Re-expression of Cav1 in KO cells restored this response. Cav1 deletion rendered significant protection from glomerular matrix accumulation and albuminuria in a mouse model of type 1 diabetes. These studies provide a foundation for the development of renal-targeted interference with CAV-1/caveolae as a novel approach to the treatment of diabetic nephropathy.

Angiotensin II Contributes to Diabetic Renal Dysfunction in Rodents and Humans via Notch1/Snail Pathway

In nondiabetic rat models of renal disease, angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. Herein, we wanted to explore the role of Ang II in diabetic nephropathy by a translational approach spanning from in vitro to in vivo rat and human studies, and to dissect the intracellular pathways involved. In isolated perfused rat kidneys and in cultured human podocytes, Ang II down-regulated nephrin expression via Notch1 activation and nuclear translocation of Snail. Hairy enhancer of split-1 was a Notch1-downstream gene effector that activated Snail in cultured podocytes. In vitro changes of the Snail/nephrin axis were similar to those in renal biopsy specimens of Zucker diabetic fatty rats and patients with advanced diabetic nephropathy, and were normalized by pharmacological inhibition of the renin-angiotensin system. Collectively, the present studies provide evidence that Ang II plays a relevant role in perpetuating glomerular injury in experimental and human diabetic nephropathy via persistent activation of Notch1 and Snail signaling in podocytes, eventually resulting in down-regulation of nephrin expression, the integrity of which is crucial for the glomerular filtration barrier.

Molecular signaling cascade of miRNAs in causing Diabetes Nephropathy.

Diabetic nephropathy (DN) is one of the major microvascular diseases and most common in diabetic patient, finally results in kidney failure. The main features of DN are basement membrane thickening, microalbuminuria, proteinuria, glomerular, mesangial hypertrophy and ECM protein accumulation. Recent discoveries have been shown that numerous pathways are activated during the development of DN in Diabetes mellitus. The small non-coding miRNA plays an important role in regulating the pathway which is involved in DN. In our study we consolidate different pathways which regulated by miRNAs in molecular signaling which results in causing DN. We embedded entire pathway in the form of regulatory network and we could able to understand that some of the miRNAs like miR-29 family, miR-377 and miR-25 would be able to control DN.

SREBP‐1 Mediates Angiotensin II‐induced TGFβ Upregulation in Kidney Mesangial Cells

Angiotensin II (Ang II) is a key determinant of the progression of kidney disease of diverse etiology. Mesangial cells (MC) contribute importantly to the accumulation of glomerular matrix proteins and hence glomerular sclerosis. We recently demonstrated a role for the transcription factor sterol regulatory element binding protein (SREBP‐1), best known for its regulation of lipid metabolism, in high glucose fibrotic responses in MC. Here we assessed whether it mediated Ang II profibrotic signaling. We found that Ang II induced generation of the mature transcription factor (mSREBP‐1) in a dose‐ and time‐dependent manner in primary MC. Transcriptional activation was confirmed. The chaperone SCAP and protease S1P, as well as signaling through the Ang II type 1 receptor and PI3K/Akt were required for SREBP‐1 activation. Endoplasmic reticulum (ER) stress was a central mediator of Ang II‐induced PI3K/Akt and SREBP‐1 activation. In uninephrectomized C57BL/6 mice, Ang II infusion for 1 week upregulated expression of glomerular SREBP‐1 and the ER stress chaperone GRP78. The profibrotic cytokine transforming growth factor beta (TGFβ) is a central mediator of glomerular sclerosis. Inhibition of either ER stress or SREBP‐1 in MC prevented Ang II‐induced TGFβ upregulation. SREBP‐1 is thus an important mediator of Ang II‐induced profibrotic signaling through its effects on TGFβ upregulation.

Celecoxib Offsets The Negative Renal Influences of Cyclosporine Via COX‐2/Endothelin ETB Receptor crosstalk

We tested the hypotheses that (i) celecoxib, a selective COX‐2 inhibitor, counterbalances renal derangements caused by cyclosporine A (CSA) in male rats, and (ii) the endothelin ETB receptor mediates the CSA‐celecoxib interactions. Ten‐day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea and creatinine) and inflammatory cytokines (renal transforming growth factor‐β and interleukin‐2). CSA also caused renal tubular atrophy and congestion in renal arterioles along with perivascular and interstitial fibrosis. These detrimental renal effects of CSA were dramatically reduced in rats treated concurrently with celecoxib (10 mg/kg/day). Moreover, immunohistochemical analyses showed that glomerular and tubular protein expressions of COX‐2 and ETB were reduced by CSA and restored to near‐control values in rats treated simultaneously with celecoxib. The adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days). The BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, facilitation of the interplay between COX‐2 and endothelin ETB receptors is the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in male rats.

601 SEVERE RENAL FUNCTIONAL DETERIORATION AND CARDIOVASCULAR DISEASE DEATH AFTER RADICAL NEPHRECTOMY IN PATIENTS WITH RENAL CELL CARCINOMA CAN BE PREDICTED USING A COMBINED ASSESSMENT OF HISTOPATHOLOGY AND FAS EXPRESSION IN SURGICALLY RESECTED NON-NEOPLASTIC SPECIMENS

You have accessJournal of UrologyKidney Cancer: Basic Research (IV)1 Apr 2013601 SEVERE RENAL FUNCTIONAL DETERIORATION AND CARDIOVASCULAR DISEASE DEATH AFTER RADICAL NEPHRECTOMY IN PATIENTS WITH RENAL CELL CARCINOMA CAN BE PREDICTED USING A COMBINED ASSESSMENT OF HISTOPATHOLOGY AND FAS EXPRESSION IN SURGICALLY RESECTED NON-NEOPLASTIC SPECIMENS Takehiro Sejima, Hideto Iwamoto, Toshihiko Masago, Shuichi Morizane, Akihisa Yao, Tadahiro Isoyama, and Atsushi Takenaka Takehiro SejimaTakehiro Sejima Yonago, Japan More articles by this author , Hideto IwamotoHideto Iwamoto Yonago, Japan More articles by this author , Toshihiko MasagoToshihiko Masago Yonago, Japan More articles by this author , Shuichi MorizaneShuichi Morizane Yonago, Japan More articles by this author , Akihisa YaoAkihisa Yao Yonago, Japan More articles by this author , Tadahiro IsoyamaTadahiro Isoyama Yonago, Japan More articles by this author , and Atsushi TakenakaAtsushi Takenaka Yonago, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.152AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The purpose of the study was to identify accurate indicators that predict adverse consequences after radical nephrectomy (RN) with regard to chronic kidney disease (CKD) progression and its related mortality. This study utilized surgical specimens and long-term follow up data from patients with renal cell carcinoma (RCC). Our methodology was used to speculate on the functional outcome of the remaining kidney using the results generated from the nephrectomized kidney. METHODS Histological analyses of global glomerulosclerosis (GS), arteriosclerosis (AS) and interstitial fibrosis (IF)/tubular atrophy (TA), as well as Fas mRNA quantification and immunohistochemistry, were performed on surgically resected non-neoplastic parenchyma from 100 patients with RCC. The estimated glomerular filtration rates (eGFR) were determined using the Modification of Diet in Renal Disease study equations. The prognoses, which included the detailed cause of death, were collected. Several factors associated with comorbid disease and the assessments of non-neoplastic specimens were also analyzed. RESULTS Median follow up period was 78.5 months (range 12.5-154.8). Mean (Standard deviation [SD]) 1 year post-operative percentage for eGFR decline was 30.2 (15.2). Severe renal functional deterioration (SRFD) was categorized as an approximate mean + SD value for the eGFR decline greater than 45 percent, after 1 year post-operation. The GS extent percentage (> 15) and glomerular Fas protein expression were independent predictors for SRFD. These two predictors and the SRFD indicated the significantly increased mortality due to cardiovascular disease (each P value < 0.05). CONCLUSIONS This is the first report to indicate the predictors of progressive CKD after RN using the combination assessment of histopathology and the expression of an apoptotic molecule in surgically resected non-neoplastic tissues. Our results suggest that Fas-induced glomerular cell apoptosis and the subsequent GS that occurs in cortical cells cause the functional deterioration in the remaining kidney, and ultimately the increase in mortality caused by cardiovascular disease. Multivariate logistic regression analysis of the six variables used to predict severe renal functional deterioration Variables Odds ratio (95% CI) P value Diabetes mellitus (yes vs. no) 5.793 (0.494–67.904) 0.169 Proteinuria (yes vs. no) 0.431 (0.041–4.572) 0.4851 Pre-operative stage 3 or 4 CKD (yes vs. no) 7.316 (0.443–120.856) 0.1643 Fas mRNA quantity ( >0.3 vs. <0.3 Fas / beta-actin ratio) 7.883 (0.633–98.122) 0.1085 Glomerular Fas staining (positive vs. negative) 26.82 (2.141–335.913) 0.0108 Percent of global glomerulosclerosis ( >15 vs. <15%) 13.139 (1.283–134.565) 0.03 © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e245-e246 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takehiro Sejima Yonago, Japan More articles by this author Hideto Iwamoto Yonago, Japan More articles by this author Toshihiko Masago Yonago, Japan More articles by this author Shuichi Morizane Yonago, Japan More articles by this author Akihisa Yao Yonago, Japan More articles by this author Tadahiro Isoyama Yonago, Japan More articles by this author Atsushi Takenaka Yonago, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Glomerular Endothelial Surface Layer Acts as a Barrier against Albumin Filtration

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of native ferritin [corrected] in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

Isolating glomeruli from mice: A practical approach for beginners

A modified procedure for Dynabead perfusion was developed to provide a practical methodology for obtaining large quantities of glomeruli from mice with a high purity. The glomeruli may be useful in exploring the mechanism behind glomerular diseases in conjunction with proteomics. The aim of the study was to save on costs and help researchers, particularly beginners, in the practical application of this method in their studies. Kidneys of C57BL/6 mice were perfused via two different techniques with Dynabeads. The purity and structures of the isolated glomeruli were investigated. The amounts of glomerular protein were measured and the costs of kidney and heart perfusions were compared. There was a 100% success rate at all stages involved in separating the glomeruli of mice via kidney perfusion. The isolated glomeruli remained intact and the purity was 96.7±1.2%. The average amounts of protein in the isolated glomeruli of 8- and 20-week-old mice were 45.6±13.4 and 55.8±17.0 μg, respectively. The cost of glomerular isolation via kidney perfusion was one-fortieth of the cost of isolation via heart perfusion. The described procedure is practical and has a high success rate. The isolated glomeruli of mice were intact and pure and a large quantity was obtained at a lower cost.

A Novel Domain Regulating Degradation of the Glomerular Slit Diaphragm Protein Podocin in Cell Culture Systems

Mutations in the gene NPHS2 are the most common cause of hereditary steroid-resistant nephrotic syndrome. Its gene product, the stomatin family member protein podocin represents a core component of the slit diaphragm, a unique structure that bridges the space between adjacent podocyte foot processes in the kidney glomerulus. Dislocation and misexpression of slit diaphragm components have been described in the pathogenesis of acquired and hereditary nephrotic syndrome. However, little is known about mechanisms regulating cellular trafficking and turnover of podocin. Here, we discover a three amino acids-comprising motif regulating intracellular localization of podocin in cell culture systems. Mutations of this motif led to markedly reduced degradation of podocin. These findings give novel insight into the molecular biology of the slit diaphragm protein podocin, enabling future research to establish the biological relevance of podocin turnover and localization.

Prognostic Value of NGAL Staining in Patients with IgA Nephropathy

Background: Renal tubulointerstitial injury plays an important role in disease progression of IgAN. Neutrophil gelatinase-associated lipocalin (NGAL) is a stress protein released by tubular cells. NGAL is a promising biomarker of acute kidney injury. There is a growing literature suggesting that NGAL is also a marker of chronic kidney disease and severity. Our aim was to evaluate the prognostic value of NGAL staining in patients with IgAN. Methods: This retrospective study included all consecutive patients who underwent a renal biopsy at our center between January 2005 and December 2009. Forty-five patients with IgA nephritis were enrolled, and renal biopsy specimens of 29 patients were evaluated. We evaluated baseline age, sex, hypertension, serum creatinine, glomerular filtration rate (GFR), urine protein, NGAL staining, glomerulosclerosis, interstitial fibrosis, and extracapillary proliferation. The primary endpoint of this study was doubling of baseline serum creatinine and/or the onset of ESRD in the course of the study. At the end of the follow-up, patients whose estimated GFR (eGFR) was ≤15 mL/min/1.73 m2 and/or baseline serum creatinine doubled, were defined as the progressor group. Results: Nineteen patients (65.5%) were NGAL positive and 10 patients (34.5%) were NGAL negative. Female gender and hypertension were associated with NGAL-positive staining. Urinary protein excretion and serum creatinine levels were more elevated in the NGAL-positive group, but the difference was not significant. We found NGAL-positive staining in major proportion in the progressor group (88.9%) than the non-progressor group (55%) (p = 0.076). Conclusion: NGAL staining can be a new histological marker in IgAN progression.

Changes of podocyte p130Cas in diabetic conditions

Proteinuria results from increased glomerular permeability and is associated with retraction and effacement of the highly specialized interdigitating podocyte foot processes. In podocytes (glomerular epithelial cells [GECs]), p130Cas localizes diffusely to the cytoplasm and connects focal adhesion proteins and kinases to the glomerular basement membrane and adapter proteins of slit diaphragm insertion sites. To investigate whether high-glucose (HG) and advanced glycosylation end products (AGEs) induce quantitative and distributional changes of podocyte p130Cas protein, a docking protein connecting F-actin fibers to the glomerular basement membrane and adapter proteins, we cultured rat GECs (r-GECs) and mouse GECs (m-GECs) under (i) normal glucose (5 mM = control), (ii) HG (30 mM), (iii) AGE-added or (iv) HG plus AGE-added conditions. We also prepared streptozotocin-induced diabetic renal tissues. We found that p130Cas stainings were located in peripheral cytoplasm of r-GECs and m-GECs as dots in linear alignment, partially colocalized with actin-binding proteins such as synaptopodin and alpha-actinin, and locally connected to the ends of actin filaments. In diabetic conditions, the intensities of p130Cas stainings were diminished in more pathological HG plus AGE-added condition of r-GECs and m-GECs and in chronic diabetic renal tissues. p130Cas protein was decreased significantly by HG, AGE and HG plus AGE in both cells compared with normal glucose or osmotic control conditions. p130Cas mRNA expression levels were also suppressed similarly in diabetic conditions. We suggest that diabetic conditions modulate the quantitative and distributional changes of podocyte p130Cas and therefore affect the filtration function of podocytes.

Short Communication Mutations in NPHS2 (podocin) in Mexican children with nephrotic syndrome who respond to standard steroid treatment

Human nephrotic syndrome has been related to mutations in glomerular proteins. Mutations in the NPHS2 gene that encodes podocin have been described as responsible for steroid-resistant nephrotic syndrome. It has been advised to test for NPHS2 mutations in parallel or before giving steroid treatment in nephrotic syndrome patients in order to avoid unnecessary therapy. We identified NPHS2 mutations in Mexican children with nephrotic syndrome. The study included 13 children with nephrotic syndrome and 2 healthy control individuals; 8 patients were steroid-resistant and 5 were steroid-sensitive. We analyzed the 3rd exon of NPHS2 by DNA sequencing. Podocin heterozygous missense mutations L139R and L142P were found; the former was found in both steroid-sensitive and steroid-resistant children, while the latter was found in a steroid-resistant child. We conclude that NPHS2 mutations should be investigated to help decide the course of treatment in nephrotic syndrome patients.

Bone Marrow-Derived Mononuclear Cells Promote Improvement in Glomerular Function in Rats with Early Diabetic Nephropathy

Background/Aims: Diabetic nephropathy is one of the main causes of end-stage renal disease. The present study investigated the effect of mononuclear cell (MC) therapy in rats subjected to diabetic nephropathy. Methods: Male Wistar rats were divided into control (CTRL), diabetic (DM), CTRL+MC and DM+MC groups. Diabetes was induced by a single injection of streptozotocin (45 mg/kg, i.p.) and, 4 weeks later, 2×10⁷ MCs were injected via the jugular vein. Results: The rats in the DM and DM+MC groups showed increased glycemia, glomerular filtration rate and glomerular tuff area versus control groups. The glomerular filtration rate and glomerular tuff area were normalized in the DM+MC group. No alterations were observed in the fractional excretion of electrolytes and proteinuria between the DM and DM+MC groups. TGF-β1 protein levels in the DM group were significantly increased versus control animals and normalized in the DM+MC group. An increase in ED1⁺/arginase I⁺ macrophages and IL-10 renal expression was observed in the DM+MC group versus DM group. Conclusions: Bone marrow-derived MC therapy was able to prevent glomerular alterations and TGF-β1 protein overexpression and modulated glomerular arginase I⁺ macrophage infiltration in rats subjected to early diabetic nephropathy.

Unchanged Distribution Density of Anionic Sites on the Glomerular Wall in Rats with Active Heymann Nephritis

In various kinds of glomerulonephritis, alteration of anionic charge on the glomerular basement membrane (GBM) and podocytes has been controversial for more than decade. To elucidate the relation between glomerular protein leakage and anionic sites on the glomerular wall, we examined the distribution of anionic sites on the GBM and podocytes of rats with active Heymann nephritis (AHN). Urinalysis for protein levels was conducted, and the kidneys were examined using electron microscopic cytochemistry for the assessment of anionic charge with two cationic probes. The anionic sites on podocytes were decreased in number in the AHN rats; however, the distributions of anionic sites on the GBM were similar in density to those seen in the control animals. From these results, we consider that the decrease in anionic charge density on podocytes might be attributable to protein leakage and that the charge barrier of the GBM is irrelevant to the protein leakage in AHN rats.

Novel carbocyclic nucleoside analogs suppress glomerular mesangial cells proliferation and matrix protein accumulation through ROS-dependent mechanism in the diabetic milieu

The synthesis of a series of novel 3,4-cis- and 3,4-trans-substituted carbocyclic nucleoside analogs from protected uracil and thymine is described. The key reaction in the followed synthetic protocols utilized the Mitsunobu reaction to couple 3,4-substituted cyclopentanols to 3N-benzoyl uracil or 3N-benzoyl thymine. These molecules were evaluated with regard to their ability to treat diabetic nephropathy. Our results show that two analogs significantly reduced high-glucose induced glomerular mesangial cells proliferation and matrix protein accumulation in vitro and, more interestingly, exhibited an anti-oxidative effect suggesting that the activity may be mediated through ROS-dependent mechanism.

Involvement of the Tubular ClC-Type Exchanger ClC-5 in Glomeruli of Human Proteinuric Nephropathies

Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.AimTo investigate whether ClC-5 is expressed in the glomerular compartment and whether it has a role in proteinuric nephropathies. ClC-5 expression was studied using Real-time PCR in manually- and laser-microdissected biopsies from patients with type 2 diabetes (n 37) and IgA nephropathy (n 10); in biopsies of membranous glomerulopathy (MG) (n 14) immunohistochemistry for ClC-5 (with morphometric analysis) and for WT1 was done. Controls: cortical tissue (n 23) obtained from unaffected parts of tumor-related nephrectomy specimens.ResultsClC-5 was expressed at glomerular level in all biopsies. Glomerular ClC-5 levels were significantly higher in diabetic nephropaty and MG at both mRNA and protein level (p<0.002; p<0.01). ClC-5 and WT1 double-staining analysis in MG showed that ClC-5 was localized in the podocytes. ClC-5 ultrastructural immunolocalization was demonstrated in podocytes foot processes. Our study is the first to demonstrate that ClC-5 is expressed in human podocytes. The ClC-5 overexpression found in biopsies of proteinuric patients suggests that proteinuria may play a part in its expression and that podocytes are likely to have a key role in albumin handling in proteinuric states.

Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: Exploration of proteome after removal of blood‐derived proteins

Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples. Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins. Two times PBS washes of frozen sections effectively eliminated blood-derived proteins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer generated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins. Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study.

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.