SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts.

BACKGROUND: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. METHODS: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. RESULTS: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. CONCLUSIONS: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

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.

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.

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli.

Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time-consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic- and paraffin-embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2-profile and Weibel-Gomez (WG) methods and a novel 3-profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10-glomerular sampling versus 20-glomerular sampling using the Cav method, while 5-glomerular sampling was less precise. In plastic tissue, 2- or 3-profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2- or 3-profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3-profile method offered incremental benefit to the 2-profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin-embedded versus plastic-embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3-profile method offers slightly improved concordance with reduced bias versus 2-profile. Our findings have implications for future studies using glomerular morphometry.

KIBRA upregulation increases susceptibility to podocyte injury and glomerular disease progression.

Despite recent progress in the identification of mediators of podocyte injury, mechanisms underlying podocyte loss remain poorly understood, and cell-specific therapy is lacking. We previously reported that kidney and brain expressed protein (KIBRA), encoded by WWC1, promotes podocyte injury in vitro through activation of the Hippo signaling pathway. KIBRA expression is increased in the glomeruli of patients with focal segmental glomerulosclerosis, and KIBRA depletion in vivo is protective against acute podocyte injury. Here, we tested the consequences of transgenic podocyte-specific WWC1 expression in immortalized human podocytes and in mice, and we explored the association between glomerular WWC1 expression and glomerular disease progression. We found that KIBRA overexpression in immortalized human podocytes promoted cytoplasmic localization of Yes-associated protein (YAP), induced actin cytoskeletal reorganization, and altered focal adhesion expression and morphology. WWC1-transgenic (KIBRA-overexpressing) mice were more susceptible to acute and chronic glomerular injury, with evidence of YAP inhibition in vivo. Of clinical relevance, glomerular WWC1 expression negatively correlated with renal survival among patients with primary glomerular diseases. These findings highlight the importance of KIBRA/YAP signaling to the regulation of podocyte structural integrity and identify KIBRA-mediated injury as a potential target for podocyte-specific therapy in glomerular disease.

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 × 10(3) vs. 192 ± 64 × 10(3) µm(3)) 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.

Podocyte number in the maturing rat kidney.

BACKGROUND/AIMS: The podocyte is an important cell for maintaining the normal structure and function of the glomerulus. In recent years much attention has been given to the number of podocytes in glomeruli. During this time there has been a debate as to whether podocytes can divide after the capillary-loop stage of development. The aim of this study was to use an unbiased counting method to determine if podocyte number increases after the capillary-loop stage of development. METHODS: The fractionator/disector method was used to count podocytes in glomeruli from rats aged 1 day, 5, 12, and 24 weeks. Glomerular volume was also measured with the unbiased Cavalieri principle and used to calculate the density of podocytes per glomerulus. RESULTS: The number of podocytes did not increase from the capillary-loop stage of glomerular development to 24 weeks of age. Glomerular volume increased 3.6-fold during this time, which resulted in a decrease of podocyte density as the rats aged. CONCLUSION: The study documents that the number of podocytes is stable after the capillary-loop stage of glomerular development. The data does not confirm but adds evidence that podocytes do not divide from the capillary-loop stage of glomerular development to 24 weeks of age in the normal rat.

Podocyte Foot Process Effacement Precedes Albuminuria and Glomerular Hypertrophy in CD2-Associated Protein Deficient Mice.

Background: Podocyte foot process effacement is a key histologic finding in proteinuric kidney disease. We previously showed that 3-week old CD2AP-deficient mice have significant proteinuria, glomerular hypertrophy and mesangial expansion. The goal of this study is to use morphometry to establish the temporal sequence of podocyte foot process effacement, glomerular volume expansion and albuminuria in Cd2ap -/- mice by measuring these parameters at the 2-week time point. Methods: Wild-type mice age 14 ± 1 days with the Cd2ap gene (WT, N = 5) and mice deficient for Cd2ap (Cd2ap KO, N = 5) were generated. Kidneys were harvested and fixed in 2.5% glutaraldehyde and processed for examination by light and electron microscopy. An average of 415.2 (range 268-716) grid points were counted for all the glomeruli, and quantification of glomerular volume from each kidney. Urine was collected the day prior to sacrifice for urine albumin-to-creatinine ratio (ACR) measurements. Results: There was no difference in albuminuria [median (range) mg/g] between WT [212.2 (177.6-388.4) mg/g] vs. Cd2ap KO mice [203.3 (164.7-910.2) mg/g], P = 0.89; or glomerular volume 68,307[10,931] vs. 66,844[13,022] µm3, p = 0.92. The volume densities of glomerular components of the podocyte, capillary lumen and mesangium were not different for the two groups, P = 0.14, 0.14 and 0.17 respectively. However, foot process width was increased in Cd2ap KO 1128[286] vs. WT [374 ± 42] nm, P = 0.02. Conclusion: Here we show that while 2-week old WT and Cd2ap KO mice have similar levels of albuminuria, glomerular and mesangial volume, Cd2ap KO mice have more extensive podocyte foot process effacement. The data suggests that podocyte injury is the initiating event leading to mesangial expansion and albuminuria in this model.

DACH1 protects podocytes from experimental diabetic injury and modulates PTIP-H3K4Me3 activity.

Dachshund homolog 1 (DACH1), a key cell-fate determinant, regulates transcription by DNA sequence-specific binding. We identified diminished Dach1 expression in a large-scale screen for mutations that convert injury-resistant podocytes into injury-susceptible podocytes. In diabetic kidney disease (DKD) patients, podocyte DACH1 expression levels are diminished, a condition that strongly correlates with poor clinical outcomes. Global Dach1 KO mice manifest renal hypoplasia and die perinatally. Podocyte-specific Dach1 KO mice, however, maintain normal glomerular architecture at baseline, but rapidly exhibit podocyte injury after diabetes onset. Furthermore, podocyte-specific augmentation of DACH1 expression in mice protects from DKD. Combined RNA sequencing and in silico promoter analysis reveal conversely overlapping glomerular transcriptomic signatures between podocyte-specific Dach1 and Pax transactivation-domain interacting protein (Ptip) KO mice, with upregulated genes possessing higher-than-expected numbers of promoter Dach1-binding sites. PTIP, an essential component of the activating histone H3 lysine 4 trimethylation (H3K4Me3) complex, interacts with DACH1 and is recruited by DACH1 to its promoter-binding sites. DACH1-PTIP recruitment represses transcription and reduces promoter H3K4Me3 levels. DACH1 knockdown in podocytes combined with hyperglycemia triggers target gene upregulation and increases promoter H3K4Me3. These findings reveal that in DKD, diminished DACH1 expression enhances podocyte injury vulnerability via epigenetic derepression of its target genes.

AMPK mediates regulation of glomerular volume and podocyte survival.

Herein, we report that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without focal segmental glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease (MCD), where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral and 5/6th nephrectomy models in Shroom3-KD mice. Knockdown mice exhibited less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying that Fyn was downstream of Shroom3. Using SHROOM3 or FYN knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMPK, a negative regulator of anabolism. AMPK activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMPK abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMPK activation. In agreement with this, treatment of glomerular injury models with AMPK activators restricted glomerular volume, podocytopenia, and progression to FSGS. Glomerular transcriptomes from MCD biopsies also showed significant enrichment of Fyn inactivation and Ampk activation versus FSGS glomeruli. In summary, we demonstrated the important role of AMPK in glomerular volume regulation and podocyte survival. Our data suggest that AMPK activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

Advanced pathological study for definite diagnosis of mitochondrial cardiomyopathy.

AIMS: Mitochondrial cardiomyopathy (MCM) is difficult to make a definite diagnosis because of various cardiovascular phenotypes and no diagnostic criteria in the pathology examination. We aim to add myocardial pathology to the diagnostic criteria for mitochondrial respiratory chain disorders. METHODS: Quantitative analysis of mitochondria using electron microscopy and immunohistopathological analysis with respiratory chain enzyme antibodies were performed in 11 patients with hypertrophic or restrictive cardiomyopathy who underwent endomyocardial biopsy for possible MCM . Respiratory chain enzymatic assay in biopsied myocardium and genetic studies were also performed in all the subjects to define MCM. RESULTS: Four patients were diagnosed with MCM according to the recent criteria of mitochondrial respiratory chain disorders. Using electron microscopy with quantitative analysis, the volume density of mitochondria within cardiac muscle cells was significantly increased in the MCM group compared with the non-MCM group (p=0.007). Immunohistopathological results were compatible with the result of the respiratory chain enzymatic assay. CONCLUSIONS: Pathological diagnosis of MCM could be confirmed by a quantitative study of electron microscopy and immunohistopathological analysis using the mitochondrial respiratory chain enzyme subunit antibody.

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

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.

Comparison of methods for counting cells in the mouse glomerulus.

BACKGROUND: Researchers have long been interested in counting the number of cells within the glomerulus. Investigators using different techniques have yielded conflicting results. The most direct method is to count the cells in serial sections from the entire glomerulus. This Exhaustive Count method is not usually practical. The disector/fractionator method counts the number of cells in a fraction of sections. The total is estimated from the product of the cell count and the reciprocal of the fraction. The Weibel-Gomez method determines the density of cells per glomerulus, then multiplies this density by glomerular volume to obtain cell number. In this study, we compared the disector/fractionator and Weibel-Gomez methods to identify a practical alternative for the time-consuming Exhaustive Count method. METHODS: Glomeruli from a normal mouse kidney were completely sectioned and images obtained. Appropriate images were used to count glomerular cell number using each method. RESULTS: The Exhaustive Count method yielded 213 +/- 22 (mean +/- SD) cells/glomerulus vs. the disector/fractionator average of 211 +/- 29 cells/glomerulus (p = 0.82). The Weibel-Gomez method average of 235 +/- 26 cells/glomerulus was statistically different from the Exhaustive Count method (p = 0.003). CONCLUSION: The Weibel-Gomez produced a 10% overestimation, whereas the disector/fractionator method was unbiased and thus a good substitute for the Exhaustive Count method.

Increased Expression of Beige/Brown Adipose Markers from Host and Breast Cancer Cells Influence Xenograft Formation in Mice.

UNLABELLED: The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1(+) or Myf5(+) cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue-related markers and contained cells that morphologically resembled brown adipocytes. IMPLICATIONS: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design.

Early chronic low-level lead exposure produces glomerular hypertrophy in young C57BL/6J mice.

Early chronic lead exposure continues to pose serious health risks for children, particularly those living in lower socioeconomic environments. This study examined effects on developing glomeruli in young C57BL/6J mice exposed to low (30 ppm), higher (330 ppm) or no lead via dams' drinking water from birth to sacrifice on post-natal day 28. Low-level lead exposed mice [BLL mean (SD); 3.19 (0.70) µg/dL] had an increase in glomerular volume but no change in podocyte number compared to control mice [0.03 (0.01) µg/dL]. Higher-level lead exposed mice [14.68 (2.74) µg/dL] had no change in either glomerular volume or podocyte number. The increase in glomerular volume was explained by increases in glomerular capillary and mesangial volumes with no change in podocyte volume. Early chronic lead exposure yielding very low blood lead levels alters glomerular development in pre-adolescent animals.

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.

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.

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.

An increase in the cell component of the cortical interstitium antedates interstitial fibrosis in type 1 diabetic patients.

BACKGROUND: Interstitial expansion is important in the progression of a variety of kidney diseases, including diabetic nephropathy (DN). However, the interstitial elements that constitute interstitial expansion in DN are unknown and are the subject of this report. METHODS: Interstitial composition was analyzed in 15 long-standing type 1 diabetic patients, 8 with mild ( congruent with 1.5 x normal) and 7 with moderate ( congruent with 2 x normal) increases in cortical interstitial fractional volume [Vv(Int/cortex]. The mild group was 29 +/- 5 (mean +/- SD) years old with diabetes duration of 17 +/- 5 years. The moderate group was older (41 +/- 7 years; P < 0.03), had longer diabetes duration (28 +/- 7 years; P = 0.002), lower creatinine clearance (90 +/- 14 mL/min/1.73 m2 vs. 109 +/- 18 mL/min/1.73 m2; P = 0.05) and used antihypertensive medications more frequently (0/8 vs. 4/7; P < 0.03) compared to the mild group. Age- and gender-matched normal controls (N = 9) also were studied. Interstitial composition was evaluated by morphometric analysis of electron microscopic (EM) micrographs systematically obtained without bias at high (x 7500) and low (x 1500) magnification. RESULTS: Mild interstitial expansion was associated with an congruent with 50% increase in fractional volume of interstitial cells (P < 0.001) and congruent with 70% increase in fractional volume of interstitial nuclei (P < 0.01). Numerical density of interstitial nuclei was normal in these patients, suggesting that the interstitial cells might be larger rather than simply more numerous. An increase over normal in the interstitial fractional volume of fibrillary collagen of congruent with 50% was seen only with moderate expansion (P < 0.001), when creatinine clearance was already decreased. Interstitial expansion was associated with a decrease in volume and surface of peritubular capillaries as well as with a reduction in surface ratio of capillaries to tubules. CONCLUSIONS: In contrast to early mesangial expansion where matrix accumulation plays a dominant role, mild interstitial expansion in long-standing type 1 diabetic patients is largely due to an increase in the cell component of the interstitium. Increased fractional volume of interstitial fibrillary collagen is only seen at later stages of the disease, when the glomerular filtration rate is already reduced. Different pathogenetic processes may be operative in early diabetic glomerular and interstitial diseases.

Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat.

BACKGROUND: Nonenzymatic reactions between sugars or lipids and protein and formation of advanced glycation and lipoxidation end products (AGE/ALEs) contribute to the chemical modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is implicated in the development of diabetic complications. In this study, we examined the effect of the AGE/ALE inhibitor pyridoxamine on chemical modification and cross-linking of collagen and development of renal disease in the streptozotocin-diabetic rat. METHODS: Diabetic rats were treated with pyridoxamine; parallel experiments were conducted with aminoguanidine, the prototype AGE inhibitor. Progression of renal disease was evaluated by measurements of albuminuria and plasma creatinine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ALEs in skin collagen by HPLC and GC-MS assays. RESULTS: Pyridoxamine significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia and plasma lactate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and cross-linking of skin collagen increased approximately twofold in diabetic versus control rats after seven months of diabetes. Pyridoxamine caused a significant (25 to 50%) decrease the AGE/ALEs, carboxymethyllysine and carboxyethyllysine, cross-linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. CONCLUSIONS: Pyridoxamine inhibits the progression of renal disease, and decreases hyperlipidemia and apparent redox imbalances in diabetic rats. Pyridoxamine and aminoguanidine had similar effects on parameters measured, supporting a mechanism of action involving AGE/ALE inhibition.