John M. Basgen

Prevention and Reversal of Diabetic Nephropathy in db/db Mice Treated with Alagebrium (ALT-711)

Background: Alagebrium (ALT-711) has been shown to improve renal dysfunction in animal models of diabetes. Methods: To test its effects in diabetic nephropathy (DN), ALT-711 was administered (1 mg/kg daily i.p.) to 9-week-old female db/db mice (n = 15, group A1) for 3 weeks and to 3-month-old (n = 15, group A2), 7-month-old (n = 7, group A3), and 12-month-old (n = 5, group A4) female db/db mice for 12 weeks, while a similar number of diabetic and nondiabetic mice were used as controls. The ΕN-carboxymethyllysine (CML) levels in serum, urine, skin, and kidney tissue were measured by enzyme-linked immunosorbent assay. The renal morphometric parameters were assessed by electron and light microscopy. Results: By the 3rd week of treatment, the serum CML level decreased by 41%, and the urinary CML concentration increased by 138% from baseline, while the urinary albumin/creatinine ratio was lower (p < 0.05) in diabetic and nondiabetic group A1 mice. After 3 months of treatment, serum, skin, and kidney CML levels and urinary albumin/creatinine ratio were lower (p < 0.05) and the urinary CML levels higher (p < 0.05) in treated group A2, A3, and A4 animals compared with groups which received phosphate-buffered saline, with a similar pattern observed in nondiabetic mice. The renal morphological parameters characteristic of DN decreased in treated compared with untreated mice. Conclusion: Alagebrium may prevent, delay, and/or reverse established DN in db/db mice by reducing the systemic advanced glycation end product pools and facilitating the urinary excretion of advanced glycation end products.

Estimating Mean Glomerular Volume Using Two Arbitrary Parallel Sections

The most reliable method for estimation of mean glomerular volume (MGV), the disector/Cavalieri method, is technically demanding and time consuming. Other methods suffer either from a lack of precise correlation with the gold standard or from the need for a large number of glomeruli in the sample. Here, a new method (the 2-profile method) is described; it provides a reliable estimate of MGV by measuring the profile area of glomeruli in two arbitrary parallel sections. MGV was estimated in renal biopsies from 16 diabetic patients and 13 normal subjects using both the Cavalieri and the 2-profile methods. The range of individual glomerular volumes based on the Cavalieri measurements was 0.31 to 4.02 x10(6) micro m(3). There was a high correlation between the two methods for MGV (r = 0.97; P < 0.0001). However, the 2-profile method systematically overestimated MGV (P = 0.0005, paired t test). This overestimation was corrected by introducing a multiplication factor of 0.91, after which statistical criteria of interchangeability with the Cavalieri method were met. The optimal distance between two sections was determined as 20 micro m with a coefficient of variation of 7.4% in repeated measurements of MGV. On the basis of findings that values for MGV stabilize after ten glomeruli are measured by the disector/Cavalieri method, it was determined that the accuracy of MGV by the 2-profile method obtained by eight glomeruli was less than 7% different from ten in all cases. Thus, the 2-profile method is a practical alternative to the disector/Cavalieri method for estimating MGV, especially in small samples and blocks with limited residual tissue.

Preservation of mesangium and immunohistochemically defined antigens in glomerular basement membrane isolated by detergent extraction.

To define the characteristics of isolated glomerular basement membrane (GBM), immunohistochemical and morphometric analyses have been carried out on rat and human tissues. Site-specific arrays of antigens were identified in detergent-isolated GBM in a distribution similar to that observed in intact kidney. In the human, fibronectin, procollagen IV, and collagen V were observed along the internal aspect of GBM continuous with antigenic sites in the mesangium. Another array of antigens was identified in the GBM but not within the mesangium--Goodpastures antigen, bovine lens capsule type IV collagen, and amyloid P component. In addition, sites reactive with rabbit antiserum to laminin were present on both sides of the lamina densa as well as within the mesangial region. Actomyosin, a presumed mesangial cell antigen persisted in the mesangium of isolated GBM. Mesangial matrix was identified in detergent-isolated GBM in an amount equivalent to that present in intact glomeruli. Sonicated GBM contained the same antigens but it was not possible to quantitate the amount of mesangial material by immunofluorescence or morphometric analysis. The thickness of the lamina densa was greater in sonicated and detergent-treated rat GBM preparations than in native rat kidney. These studies demonstrated that isolated GBM is heterogeneous with respect to its antigenic constituents and in addition contains mesangial matrix, which is morphologically and immunohistochemically distinct from peripheral GBM.

Podocyte-Specific Deletion of Yes-Associated Protein Causes FSGS and Progressive Renal Failure

FSGS is the most common primary glomerular disease underlying ESRD in the United States and is increasing in incidence globally. FSGS results from podocyte injury, yet the mechanistic details of disease pathogenesis remain unclear. This has resulted in an unmet clinical need for cell-specific therapy in the treatment of FSGS and other proteinuric kidney diseases. We previously identified Yes-associated protein (YAP) as a prosurvival signaling molecule, the in vitro silencing of which increases podocyte susceptibility to apoptotic stimulus. YAP is a potent oncogene that is a prominent target for chemotherapeutic drug development. In this study, we tested the hypothesis that podocyte-specific deletion of Yap leads to proteinuric kidney disease through increased podocyte apoptosis. Yap was selectively silenced in podocytes using Cre-mediated recombination controlled by the podocin promoter. Yap silencing in podocytes resulted in podocyte apoptosis, podocyte depletion, proteinuria, and an increase in serum creatinine. Histologically, features characteristic of FSGS, including mesangial sclerosis, podocyte foot process effacement, tubular atrophy, interstitial fibrosis, and casts, were observed. In human primary FSGS, we noted reduced glomerular expression of YAP. Taken together, these results suggest a role for YAP as a physiologic antagonist of podocyte apoptosis, the signaling of which is essential for maintaining the integrity of the glomerular filtration barrier. These data suggest potential nephrotoxicity with strategies directed toward inhibition of YAP function. Further studies should evaluate the role of YAP in proteinuric glomerular disease pathogenesis and its potential utility as a therapeutic target.

A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models

Gaining a foothold on kidney disease? The leading cause of kidney disease worldwide is known as focal segmental glomerulosclerosis (FSGS). FSGS is associated with loss of podocytes, an unusual cell type critical for the kidneys blood filtration function. Podocytes form interdigitating foot processes that wrap around capillaries and prevent leakage of plasma proteins into urine (proteinuria). Zhou et al. suppressed proteinuria by preventing podocyte loss in two different rat models of kidney disease, using a compound that selectively inhibits the TRPC5 ion channel (see the Perspective by Chung and Shaw). In short-term studies, this compound had no detectable side effects. Thus, TRPC5 inhibitors may merit exploration as a therapy for progressive kidney disease. Science, this issue p. 1332; see also p. 1256 A drug inhibits kidney disease in rat models by preventing loss of podocytes, cells critical for blood filtration. Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases.

Using Stereologic Techniques for Podocyte Counting in the Mouse: Shifting the Paradigm

Background: The podocyte serves the important function of maintaining the glomerular filtration barrier, and many studies report a decrease in podocyte number relative to the development of proteinuric states. However, there is significant inconsistency in the number of podocytes counted, possibly due to different counting methods. We previously counted the three glomerular cell types in the mouse kidney and showed that the fractionator/disector method is a close approximation of the exhaustive count or the gold standard method. In this study, we compared the commonly used model-based approach with the design-based approach to count podocytes in the db/m and db/db mouse and illustrate that the design-based approach, which uses the fractionator/disector method, provides an accurate determination of podocyte number. Methods: In the design-based approach, toluidine blue-stained 1-µm-thick sections from glutaraldehyde perfusion-fixed kidneys were used (n = 15) with the fractionator/disector method. In the model-based approach, WT-1-immunolabeled podocyte nuclei in 3- to 4-µm-thick formalin-fixed paraffin-embedded sections of the same kidneys were counted with the Weibel-Gomez method. Glomerular volume was determined for each method. Results: We discovered that the fractionator/disector method counted 89 ± 10 podocytes compared to the Weibel-Gomez method, which counted 137 ± 38 podocytes and overestimated podocyte number by 54% (p < 0.05). In addition, glomerular volume (231 ± 52 × 103 vs. 192 ± 64 × 103 µm3) was significantly underestimated by 17% (p < 0.0002). Moreover, the model-based approach was more time consuming. Conclusion: We conclude that the fractionator/disector method offers an unbiased and efficient determination of podocyte counts.

Glomerular Structure in the Normal Human Kidney: Differences between Living and Cadaver Donors

Glomerular structure has been studied in tissues derived from normal living and cadaver kidney donors. Values obtained in such subjects were considered interchangeable and have been used to define the normal parameters when evaluating the effects of various renal diseases. The present study evaluated glomerular structure by light and electron microscopy in 83 living and 53 cadaver kidney donors. Glomerular basement membrane (GBM) width (356 +/- 52 versus 329 +/- 45 nm), glomerular volume (1.64 +/- 0.47 versus 1.33 +/- 0.39 x 10(6) microm(3)), and mesangial matrix volume per glomerulus (0.15 +/- 0.05 versus 0.12 +/- 0.04 x 10(6) microm(3)) were significantly greater in cadaver compared with living kidney donors, respectively. It is hypothesized that glomerular and extracellular matrix swelling is associated with the cadaver kidney preservation process. This study suggests that the normal values for glomerular structure should be derived only from living kidney donor tissues.

Dendrin Ablation Prolongs Life Span by Delaying Kidney Failure

Podocyte loss is central to the progression of proteinuric kidney diseases leading to end-stage kidney disease (ESKD), requiring renal replacement therapy, such as dialysis. Despite modern tools and techniques, the 5-year mortality of some patients requiring dialysis remains at about 70% to 80%. Thus, there is a great unmet need for podocyte-specific treatments aimed at preventing podocyte loss and the ensuing development of ESKD. Here, we show that ablation of the podocyte death-promoting protein dendrin delays the onset of ESKD, thereby expanding the life span of mice lacking the adapter protein CD2AP. Ablation of dendrin delays onset and severity of proteinuria and podocyte loss. In addition, dendrin ablation ameliorates mesangial volume expansion and up-regulation of mesangial fibronectin expression, which is mediated by a podocyte-secreted factor. In conclusion, onset of ESKD and death can be markedly delayed by blocking the function of dendrin.

Effect of angiotensin II on glomerular structure in streptozotocin-induced diabetic rats.

Background/Aims: The streptozotocin (STZ)-induced diabetic rat is a widely used animal model of human diabetic nephropathy. In this model, diabetic nephropathy progresses without significant elevation in blood pressure. Therefore, studies have examined the effect of hypertension in STZ spontaneously hypertensive rats (SHR). This study investigated angiotensin II (Ang II)-induced hypertension in diabetic nephropathy in the STZ-diabetic rat independent of deleterious genetic effects in SHR. Methods: Animals were divided as follows: nondiabetic controls (ND; n = 18); diabetic (STZ: 65 mg/kg; n = 16); Ang II-induced hypertensive ND (Ang II: 120 ng/kg/min; n = 9), and hypertensive diabetic rats (n = 18). Systolic blood pressure was measured by the tail-cuff method prior to STZ injection and then weekly. After 3 months, plasma creatinine, and 24-hour urine albumin and creatinine were measured and kidneys harvested for morphometry. Results: Ang II infusion increased systolic blood pressure in diabetic and ND rats. When combined with diabetes, Ang II increased albumin excretion rate (14-fold, p < 0.05), plasma creatinine (1.5-fold, p < 0.005) worsened creatinine clearance (37%, p < 0.002) and increased glomerular basement membrane width (1.2-fold, p < 0.0001). Conclusion: Ang II caused moderate hypertension and accelerated diabetic nephropathy and glomerular structural changes. The Ang II-infused STZ-diabetic rat is an excellent model to study the deleterious glomerular effects of hypertension on diabetes independent of genetic traits.

Quantitation of antigen in tissue by immunofluorescence image analysis

A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/micrograms) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.