Change In Glomerular Volume Research Articles

Changes in Glomerular Volume, Sclerosis, and Ischemia at 5 Years after Kidney Transplantation: Incidence and Correlation with Late Graft Failure.

Glomerular volume, ischemic glomeruli, and global glomerulosclerosis are not consistently assessed on kidney transplant biopsies. The authors evaluated morphometric measures of glomerular volume, the percentage of global glomerulosclerosis, and the percentage of ischemic glomeruli and assessed changes in these measures over time to determine whether such changes predict late allograft failure. All three features increased from transplant to five-year biopsy. Kidneys with smaller glomeruli at 5 years had more global glomerulosclerosis and a higher percentage of ischemic-appearing glomeruli. Smaller glomeruli and increasing percentages of global glomerulosclerosis and ischemic glomeruli at 5 years predicted allograft failure. Only increased percentage of ischemic glomeruli predicted allograft failure at 5 years independent of all Banff scores. Glomerular changes reflect pathologic processes that predicted allograft loss; measuring them quantitatively might enhance the current Banff system and provide biomarkers for intervention trials. Histology can provide insight into the biology of renal allograft loss. However, studies are lacking that use quantitative morphometry to simultaneously assess changes in mean glomerular volume and in the percentages of globally sclerosed glomeruli (GSG) and ischemic-appearing glomeruli in surveillance biopsies over time to determine whether such changes are correlated with late graft failure. We used digital scans of surveillance biopsies (at implantation and at 1 and 5 years after transplantation) to morphometrically quantify glomerular volume and the percentages of GSG and ischemic-appearing glomeruli in a cohort of 835 kidney transplants. Cox proportional hazards models assessed the risk of allograft failure with these three glomerular features. From implantation to 5 years, mean glomerular volume increased by nearly 30% (from 2.8×10 6 to 3.6×10 6 µm 3 ), mean percentage of GSG increased from 3.2% to 13.2%, and mean percentage of ischemic-appearing glomeruli increased from 0.8% to 9.5%. Higher percentages of GSG and ischemic-appearing glomeruli at 5-year biopsy predicted allograft loss. The three glomerular features at 5-year biopsy were related; the percentage of GSG and the percentage of ischemic glomeruli were positively correlated, and both were inversely correlated to glomerular volume. At 5 years, only 5.3% of biopsies had ≥40% ischemic glomeruli, but 45% of these grafts failed (versus 11.6% for <40% ischemic glomeruli). Higher Banff scores were more common with increasing percentages of GSG and ischemia, but at 5 years, only the percentage of ischemic glomeruli added to predictive models adjusted for Banff scores. Glomerular changes reflect important pathologic processes that predict graft loss. Measuring glomerular changes quantitatively on surveillance biopsies, especially the proportion of ischemic-appearing glomeruli, may enhance the current Banff system and be a useful surrogate end point for clinical intervention trials. This article contains a podcast at.

EET Analogs and the Dual‐Inhibition of sEH/COX‐2 for the Treatment of Focal Segmental Glomerular Sclerosis

Focal segmental glomerulosclerosis (FSGS) is the single most common form of kidney disease. This devastating disorder can lead to end stage renal disease (ESRD), a terminal diagnosis in the absence of a kidney transplant. The progression of FSGS is defined by sclerotic damage to the glomerulus, the filtering unit of blood in the kidney. Epoxyeicosatrienoic acids (EETs) are a family of arachidonic acid metabolites important to glomerular function. EETs are metabolized by enzymes such as soluble epoxide hydrolase (sEH) and cyclooxygenase (COX), and we have developed a novel dual‐ligand drug (PTUPB) that inhibits both sEH and COX‐2. We hypothesize that EET analogs and the dual‐inhibition of sEH and COX‐2 will mitigate glomerular damage caused by FSGS. The long‐term goal of this research is to better understand the role of 8,9‐EET and its metabolites on glomerular function so that we can develop new therapies to treat FSGS and other nephropathies.MethodsGlomerular Permeability Assay: Glomeruli were pre‐incubated in 5% albumin (50kDa dialyzed BSA), followed by a bath exchange to 1% albumin. Under normal conditions the glomeruli are non‐permeable to albumin, and as a result, when the 5% albumin bath is exchanged for 1% albumin, water is taken up into the intra‐glomerular space due to the rapid change in oncotic pressure. Glomerular permeability was tested using our non‐hydrolyzable 8,9‐EET analog (1μM), our non‐hydrolyzable 14,15‐EET analog (EET‐A, 1μM) or PTUPB (1μM) with isolated SD rat glomeruli in the presence or absence of angiotensin II (AngII, 10μM), a known glomerular permeabilizing agent. 3D‐Confocal imaging (72μm z‐depth, 27‐layer z‐stack) was utilized to measure the relative changes in glomerular volume.ResultsGlomerular permeability to albumin (Palb) is derived from the albumin reflection coefficient (σalb), where a value of 1 indicates high permeability to albumin, and 0 indicates low permeability (relative to control). A baseline response to a 5% to 1% albumin exchange resulted in very low albumin permeability (Palb = −0.21 ± 0.21, n=6). Conversely, the presence of AngII resulted high albumin permeability (Palb = 1.21 ± 0.12, n=15). Our 8,9‐EET analog, in the presence of AngII, mitigated the effects of AngII‐induced permeability (Palb = 0.13 ± 0.18, n=7), as did our dual sEH/COX2 inhibitor, PTUPB, under the same Ang‐II stimulation (Palb = 0.71% ± 0.35, n=7), but not the 14,15‐EET analog (Palb = 0.99% ± 0.21, n=4) . All values are mean ± SEM.ConclusionFSGS is complex multi‐model disease, and glomerular damage/dysfunction are at the core of this condition. The ability of our 8,9‐EET analog and PUTPB to decrease AngII‐associated glomerular permeability suggest and important role for EETs and their metabolism. Future work will investigate not only the effects of 8,9‐EET analogs and PTUPB on glomerular health, but also the consequences of EET metabolites and other EET‐analogs, on both glomerular and mesangial function using cell and animal models.Support or Funding InformationR01 DK103616‐01 (John D. Imig). Dr. Ralph and Marian Falk Medical Research Trust ‐ Phase II (John D. Imig)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic kidney cortical damage is associated with baseline kidney function and albuminuria in patients managed with radical nephrectomy for kidney tumours

This study evaluated the relationship between histological markers of chronic kidney damage in patients undergoing radical nephrectomy for kidney tumours and preoperative kidney function, degree of albuminuria, and changes in glomerular volume. A schema to grade chronic kidney damage could be used to identify patients at risk of developing CKD following nephrectomy. Non-neoplastic cortical tissue was sourced from 150 patients undergoing radical nephrectomy for suspected kidney cancer. This tissue was evaluated for indicators of chronic damage, specifically: glomerulosclerosis, arteriosclerosis, interstitial fibrosis, and tubular atrophy. Glomerular volume was determined using the Weibel and Gomez method. Associations between these parameters and both estimated glomerular filtration rate (eGFR) and albumin-creatinine ratio (ACR) were determined using either a Mann-Whitney U-test or a Kruskal-Wallis ANOVA. Associations between both eGFR and ACR and glomerular volume were assessed using linear regression. eGFR was inversely associated with the degree of glomerulosclerosis (p < 0.001), vascular narrowing (p= 0.002), tubular atrophy (p < 0.001), and interstitial fibrosis (p < 0.001). ACR was associated only with the degree of interstitial fibrosis (p= 0.02) and tubular atrophy (p= 0.02). Glomerular volume was greater for males, diabetics, hypertensive patients, and patients with a greater degree of interstitial fibrosis. Glomerular volume was positively associated with ACR. A schema to grade chronic damage was developed. The proposed schema is associated with baseline clinical indices of kidney function and damage. Longitudinal validation is necessary to determine the prognostic utility of this schema.

Change in glomerular volume and its clinicopathological impact after kidney transplantation.

Both immunological and non-immunological etiologies affect graft function after kidney transplantation, including acute rejection, calcineurin inhibitor toxicity, and a recurrence of glomerulonephritis. Glomerular enlargement or glomerular sclerosis due to glomerular hyperfiltration related to increased renal blood flow is another cause. Although the glomerular volume in baseline biopsies predicts late allograft function, the relationship between allograft function and the annual changes in glomerular volume after kidney transplantation are unclear. We investigated changes in glomerular volume after kidney transplantation and their clinicopathological relationship. We enrolled 23 patients with stable kidney function without an episode of rejection or any complication resulting in a functional decrease in the graft. We measured glomerular volume (GV) using the Weibel-Gomez method and glomerular density (GD) using 0,1 h biopsy samples as baseline controls and 1 yr biopsy samples and investigated the association between the changes in them and clinical parameters, including graft function, proteinuria, and renal hemodynamic markers, including effective renal plasma flow (ERPF) and filtration fraction (FF). The ERPF was calculated from a 99mTc-mercaptoacetyltriglycine (MAG3) renogram. The GV and ERPF increased significantly 1 yr after kidney transplantation. In contrast, proteinuria decreased significantly and Δproteinuria (1 yr - 1 month after transplantation) was correlated with ΔGV (P < 0.05, rs = -0.467). Glomerular enlargement 1 yr after transplantation may be related to improved proteinuria. It is possible that glomerular enlargement serves as a renal adaptation after kidney transplantation.

Deficiency in Mitochondrial Complex I Activity Due toNdufs6Gene Trap Insertion Induces Renal Disease

Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied. Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C. We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease. These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.

Relationship Among Total Kidney Volume, Renal Function and Age

We measured kidney volume using software and investigated the relationship between kidney volume and renal function. Age, gender, height, body weight, body mass index, body surface area and serum creatinine were recorded for 539 normal individuals. A tissue segmentation tool program was used to measure kidney volume from computerized tomography images. The glomerular filtration rate was calculated using the Cockcroft-Gault equation and an abbreviated modification of diet in renal disease equation. We looked for correlations of renal parenchymal volume with age and anthropometric measurements. We also tested for a correlation between kidney volume and renal function using the glomerular filtration rate according to the Cockcroft-Gault and modification of diet in renal disease equations. Mean kidney volume in all participants was 261.3 ± 58.1 ml. Mean volume in men was approximately 14 ml greater than in women (266.1 vs 251.8 ml, p = 0.004). Kidney volume correlated significantly with height (r = 0.344, p <0.001), body weight (r = 0.343, p <0.001), body mass index (r = 0.177, p <0.001), body surface area (r = 0.371, p <0.001) and age (r = -0.418, p <0.001). Kidney volume also correlated with the glomerular filtration rate according to the Cockcroft-Gault and modification of diet in renal disease equations (p <0.001, r = 0.615 and p <0.001, r = 0.432, respectively). Kidney volume and the glomerular filtration rate decreased in parallel with increasing age. Kidney volume correlates well with renal function and anthropometric measurements. Knowledge of these relationships will be valuable in clinical urology and nephrology.

Endothelin receptor A-specific stimulation of glomerular inflammation and injury in a streptozotocin-induced rat model of diabetes

Activation of endothelin receptor-A (ET(A)) increases glomerular permeability to albumin (P(alb)) and elevates pro-inflammatory markers in hyperglycaemic rats. Male Sprague-Dawley rats were given streptozotocin (n = 32) or saline (sham; n = 32). Half of the animals in each group received the ET(A)-selective antagonist, ABT-627 (atrasentan; orally), beginning immediately after hyperglycaemia was confirmed. Glomeruli were isolated by sieving techniques and P(alb) determined from the change in glomerular volume induced by oncotic gradients of albumin. Glomerular nephrin levels were assessed by immunofluorescence, whereas urinary nephrin was measured by immunoassay. At 3 and 6 weeks after streptozotocin injection, proteinuria was significantly increased compared with sham controls and significantly reduced by ABT-627 treatment. P(alb) was also increased at 3 and 6 weeks post-streptozotocin. ABT-627 had no effect on P(alb) or protein excretion in sham control rats. In glomeruli isolated from hyperglycaemic rats, incubation with BQ-123, a selective ET(A) antagonist, reduced P(alb), whereas BQ-788, a selective endothelin receptor-B antagonist had no effect (n = 6 rats per group, 5-8 glomeruli per rat). Glomerular and plasma content of soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 were significantly increased 6 weeks after streptozotocin (ELISA). ABT-627 attenuated these increases. After 6 weeks of hyperglycaemia, glomerular nephrin content was decreased with a concurrent increase in urinary nephrin excretion. ABT-627 prevented glomerular nephrin loss in hyperglycaemic rats (n = 5-8 rats per group; eight groups). These observations support the hypothesis that endothelin-1, via the ET(A) receptor, directly increases P(alb), possibly via nephrin loss, as well as early inflammation in the hyperglycaemic rat.

Endothelin-1 Increases Glomerular Permeability and Inflammation Independent of Blood Pressure in the Rat

Endothelin (ET) 1 is a potent vasoactive peptide implicated in the pathogenesis of hypertension and renal disease. The aim of the current study was to test the hypotheses that ET-1 increases albumin permeability of glomeruli isolated from normal rats and that chronic ET-1 infusion will increase glomerular permeability and inflammation independent of blood pressure. Glomerular permeability to albumin was determined from the change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients. Incubation of glomeruli taken from normal rats with ET-1 at a concentration that did not produce direct glomerular contraction (1 nmol/L) significantly increased glomerular permeability to albumin, reaching a maximum after 4 hours. Chronic ET-1 infusion for 2 weeks in Sprague-Dawley rats significantly increased glomerular permeability to albumin and nephrin excretion rate, effects that were attenuated in rats given an ET(A) receptor antagonist (ABT-627, 5 mg/kg per day). Urinary protein and albumin excretion and mean arterial pressure (telemetry) were not changed by ET-1 infusion. Acute incubation of glomeruli isolated from ET-1-infused rats with the selective ET(A) antagonist significantly reduced glomerular permeability to albumin, an effect not observed with acute treatment with a selective ET(B) antagonist. Chronic ET-1 infusion increased glomerular and plasma soluble intercellular adhesion molecule 1 and monocyte chemoattractant protein 1 and elevated the number of macrophages and lymphocytes in renal cortices (ED-1 and CD3-positive staining, respectively). These effects were all attenuated in rats given an ET(A) selective antagonist. These data support the hypothesis that ET-1 directly increases glomerular permeability to albumin and renal inflammation via ET(A) receptor activation independent of changes in arterial pressure.

Free Radical Scavenging Decreases Endothelin‐1 (ET‐1) Excretion and Glomerular Permeability During Diabetes

Hyperglycemia induces renal oxidative stress and renal ET‐1 production. In the present study, we hypothesized that diabetes‐induced oxidative stress activates renal ET‐1 production and/or glomerular permeability. Hyperglycemia was induced in 250–275 g male Sprague‐Dawley rats by a single i.v. dose of streptozotocin (65 mg/kg). The free radical scavenger, tempol (1 mM), was provided to half of the hyperglycemic rats in the drinking water and the other half received vehicle. After 3 weeks, urine, plasma, and renal tissue were collected. Tempol treatment decreased urinary excretion of thiobarbituric acid reactive substances (3959 ± 329.1 nmol/day vs. 6706 ± 608.9 nmol/day, N=8, P&lt;0.001), as well as ameliorated glomerular superoxide production as monitored by dihydroethidium staining. Tempol treatment diminished urinary ET‐1 excretion (16.4 ± 1.5 pg/day vs. 30.6 ± 2.6 pg/day, N=8, P&lt;0.001). Inner medullary prepro‐ET‐1 was attenuated with tempol treatment (0.7±0.12 vs. 1.1±0.12, N=8, P&lt;0.05), although tempol treatment did not change cortical or outer medullary preproET‐1 levels. Glomerular permeability, Palb, (change in glomerular volume induced by exposing isolated glomeruli to oncotic gradients of albumin) was attenuated with tempol treatment (0.42±0.17 vs. 0.80±0.04, N=4, P&lt;0.05). Proteinuria and creatinine clearance were similar in both groups of rats. In conclusion, the free radical scavenger, tempol, attenuated the increase in renal oxidative stress, ET‐1 excretion, and glomerular permeability associated with the early phase of diabetes.

ETA receptor dependent increases in glomerular permeability in experimentally induced diabetic rats

A glomerular permeability defect occurs early in type 1 diabetes and precedes the onset of albuminuria and renal morphological changes. The aim of the current study was to test the hypothesis that the ETA receptor contributes to increased glomerular albumin permeability (Palb) in a model of type 1 diabetes. Male SD rats (250‐275 g) were given streptozotocin (diabetic, D; i.v., 65 mg/kg) or saline (S). Half of each group received the ETA‐selective antagonist, ABT‐627 (ABT), in drinking water at 5 mg/kg/d (n=6 in all groups). Treatment was begun immediately after confirming plasma glucose &gt;300 mg/dl. Six weeks later, glomeruli were isolated by sieving techniques and Palb determined from the change in glomerular volume induced by exposing glomeruli to oncotic gradients of albumin. Palb was increased in D (0.91 ±0.09 vs. S, 0.05 ±0.01, P&lt;0.01); ABT significantly reduced Palb when compared with D (D+ABT, 0.40 ±0.10, P&lt;0.05). ABT had no effect on Palb in S rats. Acute in vitro incubation of glomeruli isolated from D with ABT (10−6 M, 15 min at 37°C), significantly reduced Palb (0.89±0.11 to 0.41±0.09, P&lt;0.05) while selective ETB blockade, A192621 (10−3 to 10−7 M), had no effect. In conclusion, these data support the hypothesis that ETA but not ETB receptors play an important role in maintaining elevated Palb in type 1 diabetes. Mechanisms responsible for additional changes not affected by ABT remain to be determined.

Applicability of the glomerular size distribution coefficient in assessing human glomerular volume: the Weibel and Gomez method revisited

Changes in glomerular volume (V(glom)) play an important role in the initiation and progression of various glomerulopathies. Estimation of V(glom) in the normal kidney provides baseline values for studies of glomerular hypertrophy in disease. The traditional model-based method of Weibel and Gomez is widely applied to estimate V(glom) in clinical biopsy specimens. Assumptions of glomerular size distribution and shape required by this method are potential sources of bias that have not been verified. We evaluated the applicability of the glomerular size distribution coefficient in estimating V(glom) in human kidneys. V(glom) of 720 non-sclerotic glomeruli in histologically normal kidneys of 24 males (20-69 years) was estimated by the unbiased disector/Cavalieri approach. Accurate glomerular diameters were calculated from Cavalieri estimates of V(glom) assuming glomerular sphericity. The coefficients of variation (CV) of glomerular diameters were compared with the corresponding values of the size distribution coefficient predicted by the Weibel and Gomez method. Mean (SD) glomerular diameter was 201 (28) mm (range 110-276 mm). The CV of glomerular diameter within each kidney ranged from 4.9 to 14.6%. Corresponding glomerular size distribution coefficients predicted by the formula of Weibel and Gomez ranged from 1.00 to just 1.03. The value of the size distribution coefficient required by the Weibel and Gomez technique when estimating V(glom) in normal human kidneys is remarkably constant. This is despite large variations in V(glom). Future studies should examine the extent of bias introduced by the glomerular shape assumptions of this method.

Chymase increases glomerular albumin permeability via protease-activated receptor-2

Increased infiltration of the kidney by mast cells is associated with proteinuria, and interstitial fibrosis in various renal diseases. Mast cells produce serine proteases including tryptase and chymase (MCC) that act via protease-activated receptors (PARs) to induce synthesis of fibrogenic cytokines by renal cells. In the present study, we investigated direct effect of MCC and role of PARs on glomerular albumin permeability (P(alb)). Isolated rat glomeruli were incubated with MCC (0.1, 1, 10, and 100 ng/ml) for 5-30 min in presence or absence of PAR-1 and PAR-2 blocking antibodies. P(alb) was determined from the change in glomerular volume in response to an albumin oncotic gradient. The effect of direct activation of PARs on P(alb) was verified by incubating glomeruli with synthetic hexapeptide known to activate PAR-1 and PAR-2. MCC increased P(alb) of isolated rat glomeruli in a dose- and time-dependent manner. Blocking PAR-2 prevented MCC-mediated increase in P(alb). RT-PCR analysis of glomerular RNA demonstrated the presence of constitutively expressed PAR-1, -2, and -3 and low levels of PAR-4. In addition, direct activation of PAR-2 by hexapeptide SLIGKV increased P(alb) comparable to MCC, whereas PAR-1 activation by TFLLRN had no effect on P(alb). Our results document that MCC induces increase in P(alb) and that this effect is mediated through PAR-2. MCC may also play a role in renal scarring. We propose that inhibiting MCC activity or blocking the activation of PAR-2 may provide new targets for therapy in renal diseases.

Nitric oxide preserves the glomerular protein permeability barrier by antagonizing superoxide

The interaction of nitric oxide with superoxide (O2-) is a major O2- scavenging mechanism that can minimize O2 (-)-mediated oxidative stress. Glomeruli produce both nitric oxide and O2- and generation of both radicals is increased in various forms of glomerular disease. O2- increases glomerular capillary permeability to albumin (P(alb)). The present studies tested the hypothesis that nitric oxide opposes this effect, thereby preserving the glomerular protein permeability barrier. P(alb) was determined in isolated rat glomeruli by measuring the change in glomerular volume in response to an experimental oncotic gradient. Changes in P(alb) in response to O2- generated by tumor necrosis factor-alpha (TNF-alpha) or xanthine/xanthine oxidase (X/XO) was assessed under conditions of nitric oxide depletion and repletion. Incubation of rat glomeruli with the nitric oxide synthase (NOS) inhibitor L-N(G)-monomethyl arginine (L-NMMA) increased P(alb.) This effect was reversed by the nitric oxide donor diethylenetriamine NONOate (DETA-NONOate) and by the superoxide dismutase (SOD) mimetic Tempol. O2- generated after incubation with TNF-alpha or X/XO increased P(alb). This effect was blocked by DETA-NONOate. We demonstrate that nitric oxide protects the glomerular filtration barrier from injury caused by O2- and suggest that inhibition of nitric oxide synthesis could enhance O2(-)-mediated oxidative injury under pathologic conditions.

Superoxide Dismutase Mimetic Preserves the Glomerular Capillary Permeability Barrier to Protein

Overproduction of superoxide (O2*) occurs in glomerular disease and may overwhelm the capacity of superoxide dismutase (SOD), thereby intensifying oxidant injury by O2* and related radical species that disrupt the glomerular capillary permeability barrier to protein. We examined the efficacy of the SOD mimetic tempol in preserving glomerular permeability to protein using 1) a rat model of glomerular immune injury induced by an antiglomerular basement membrane antibody (anti-GBM), and 2) isolated rat glomeruli in which injury was induced by the cytokine tumor necrosis factor-alpha (TNFalpha). To induce glomerular immune injury, rats received anti-GBM using a protocol that results in prominent infiltration of glomeruli by macrophages and in which macrophage-derived TNFalpha has been shown to mediate albuminuria. To increase glomerular capillary permeability to albumin (P(alb)) ex vivo, isolated glomeruli were incubated with TNFalpha at concentrations (0.5-4.0 microg/ml) known to stimulate O2* production. Increments in P(alb) were detected by measuring changes in glomerular volume in response to an applied oncotic gradient. Significant increases in the urine excretion of albumin and F(2alpha)-isoprostane were observed in rats with glomerular immune injury without a significant change in systolic blood pressure. Tempol treatment significantly reduced urine isoprostane and albumin excretion. In isolated glomeruli, TNFalpha increased P(alb) and tempol abrogated this effect, both in a dose-dependent manner. These observations indicate that SOD mimetics can preserve the glomerular permeability barrier to protein under conditions of oxidative stress from O2* production.

The effect of proteinase inhibitors on glomerular albumin permeability induced in vitro by serum from patients with idiopathic focal segmental glomerulosclerosis.

The putative circulating factor responsible for the glomerular permeability alterations induced in vitro by serum from patients affected by focal segmental glomerulosclerosis (FSGS) remains unidentified. We have observed that a serine proteinase isolated from patient serum increases albumin permeability in isolated glomeruli. The objective of the present study was to determine the effect of various proteinase inhibitors on glomerular albumin permeability (P(alb)) in isolated glomeruli incubated with FSGS serum. The study population consisted of 12 FSGS patients (eight males; mean age: 21+/-10 years) previously shown to have elevated serum albumin permeability activity. P(alb) was determined by measuring the change in glomerular volume induced by applying oncotic gradients to isolated healthy rat glomeruli treated with patient serum in comparison to control serum. Solutions of seven different proteinase inhibitors (0.5 mg/ml) were added to the incubation media with the sera (1:1 vol/vol): serine proteinase inhibitors (PMSF, leupeptin, aprotinin, gabexate mesylate), the cysteine proteinase inhibitor E-64, the metalloproteinase inhibitor EDTA and the aspartate proteinase inhibitor pepstatin. Sera from the same patients were also tested with the addition to the incubation media of quinaprilat, an inhibitor of the metalloproteinase angiotensin-converting enzyme. Mean P(alb) of the sera was 0.86+/-0.11, with the addition of PMSF 0.41+/-0.09, leupeptin 0.30+/-0.17, aprotinin 0.09+/-0.14, gabexate mesylate 0.27+/-0.25, E-64 0.81+/-0.09, EDTA 0.68+/-0.10 or pepstatin 0.76+/-0.11. The mean P(alb) of the sera combined with quinaprilat was reduced to 0.34+/-0.35. Thus, only the serine proteinase inhibitors consistently blocked the increased P(alb) induced by the FSGS sera. In the cascade of events that lead to the initiation of glomerular fibrosis in FSGS, the putative glomerular permeability factor associated with FSGS may require a serine proteinase to effect its activity.

Sera from patients with collapsing focal segmental glomerulosclerosis increase albumin permeability of isolated glomeruli

The collapsing variant of focal segment glomerulosclerosis (FSGS) is characterized by heavy proteinuria and rapid progression to renal failure. Its cause is not known. We have characterized a substance in the circulation of patients with classic FSGS that increases in vitro permeability of glomeruli to albumin (P alb) and causes proteinuria when injected into rats. Inclusion of normal serum prevents the increase in P alb caused by this FSGS factor. We investigated the effect of sera from patients with collapsing FSGS on P alb, as well as the effect of inclusion of normal serum. Isolated glomeruli were incubated with serum from each of 11 patients with collapsing FSGS (1:50 dilution) or with patient serum and an equal volume of pooled normal serum. P alb was determined on the basis of changes in glomerular volume in response to an oncotic gradient. Sera from 10 of the 11 patients with collapsing FSGS increased P alb of isolated glomeruli to a value of 0.5 or greater. In each of the 5 cases tested, inclusion of normal serum abolished the increase in P alb. Sera of patients with collapsing FSGS increased glomerular P alb. Our finding that the increase in P alb is abolished by normal serum suggests that the substance and its mechanism of action are similar or identical to the FSGS factor we have isolated from the plasma of patients with recurrent FSGS. The presence of a circulating factor in collapsing FSGS has implications for prognosis and treatment in primary and recurrent collapsing FSGS.

An inhibitor of advanced glycation end product formation reducesNϵ-(carboxymethyl)lysine accumulation in glomeruli of diabetic rats

Background: An inhibitor of advanced glycation, OPB-9195, retards the progression of nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus. The aim of this study is to evaluate histologically the role of Nϵ-(carboxymethyl)lysine (CML) in the development of diabetic nephropathy and investigate whether inhibition of CML accumulation by OPB-9195 is associated directly with the prevention of glomerular lesions in OLETF rats. Methods: Kidneys of OLETF and Long-Evans Tokushima Otsuka rats were obtained at ages 7, 20, 50, and 68 weeks after collecting their blood and urine samples. OPB-9195 had been administered to the rats from age 24 weeks to the end of the experiments. CML in kidneys was detected by using a monoclonal antibody against CML according to an indirect immunofluorescence technique. CML-positive glomerular area was measured using NIH Image software (Research Services Branch of NIMH, Bethesda, MD). Hyalinized and/or sclerotic areas in glomeruli and mesangial and glomerular volume were measured using a point-counting technique. Results: CML-positive area in glomeruli correlated closely not only with urinary albumin excretion (r = 0.912; P = 0.001), but also with volumes of mesangium and hyalinized and/or sclerotic lesions (r = 0.859; P = 0.0019 and r = 0.833; P = 0.0027, respectively). Treatment with OPB-9195 reduced CML-positive area and prevented the increase in mesangial volume, with no significant change in glomerular volume at age 68 weeks. The volume of hyalinized and/or sclerotic lesions also decreased by treatment with OPB-9195 in three of four rats at age 68 weeks. Conclusion: CML is a major advanced glycation end product contributing to the development of diabetic nephropathy, and inhibition of its accumulation by OPB-9195 results in amelioration of glomerular lesions in OLETF rats. Am J Kidney Dis 41(S1):S68-S71. © 2003 by the National Kidney Foundation, Inc.

Twenty-one years of developmental nephrology: the kidney then and now.

Developmental renal physiology was guided for a long time by the notion that the "immature" kidney is characterized by glomerular-tubular imbalance, with glomerular preponderance. In accordance with this concept, the filtering capacity of the developing kidney exceeds the ability of the renal tubules to handle the filtrate, resulting in the urinary loss of substances such as amino acids and bicarbonate. Estimates of age-related changes in glomerular volume, based on measurements of glomerular diameter, and of proximal tubular volume, based on measurements of tubular length and tubular diameter, appeared to support this contention. The experimental approach that led to these conclusions was based on the assumptions that the increase in glomerular size is distributed evenly between the vascular and nonvascular elements and that tubular volume increases pari passu with the luminal surface area. Both assumptions were proved wrong. Moreover, micropuncture studies performed in guinea pigs and rats revealed that the proportionality between glomerular filtration and proximal reabsorption of fluid (i.e., glomerulo-tubular balance) is maintained throughout development. Subsequent studies showed that several transport mechanisms function quite adequately from the first days of extrauterine life. Sodium is avidly reabsorbed in distal nephron segments, under the stimulus of the high levels of aldosterone present during infancy. The increase in the secretion of potassium that would occur in the adult under these circumstances is mitigated by the low expression of potassium channels. In the case of phosphate, there is enhanced reabsorption, particularly at the level of the proximal tubule, due in part to a growth-specific NaPi type II transporter. These different adaptive mechanisms converge towards the maintenance of a positive external balance for substances that are essential to growth. The emergence of cellular and molecular biology has also encouraged research in the field of renal morphology. The genes that control mesenchymal-epithelial interactions and the signaling factors that mediate their effect have been described. Progress is being made in the identification of genes involved in certain forms of renal malformations. Novel methods of investigation, such as DNA arrays, are likely to lead to an even fuller, dynamic portrayal of gene expression during nephrogenesis. Characterizing the functional correlates of these genes will require investigators who are not only proficient in molecular biology, but who are also masters of physiological methods.

Apolipoproteins prevent glomerular albumin permeability induced in vitro by serum from patients with focal segmental glomerulosclerosis.

Glomerular albumin permeability alterations can be induced in vitro by serum from patients with end-stage renal disease caused by primary focal segmental glomerulosclerosis (FSGS). It was hypothesized that inhibitory substances may be present in normal serum, which may prevent the permeability alterations in isolated glomeruli, and the present study sought to isolate and characterize these factors. Albumin permeability was determined from the change in glomerular volume induced by applying oncotic gradients across the basement membrane of healthy isolated rat glomeruli preincubated with FSGS serum and normal serum fractionated using standard techniques. Fractions of normal serum with inhibitory activity obtained by a multistep chromatographic procedure underwent two-dimensional electrophoresis and staining. Approximately 50 protein spots were recovered, renatured, and tested for antipermeability activity. Five of these proteins demonstrated consistent inhibitory activity, and desorption ionization and mass spectrometry proved them to be components of high-density lipoprotein: apolipoproteins (apo) E(2) and E(4), high-molecular-weight J and L, and a 28-kD fragment of A-IV. Polyclonal antibodies to apo E or apo J added to the whole normal serum restored the permeability activity of the FSGS serum in the bioassay. Commercially available apo E and apo J also demonstrated antipermeability activity when added to FSGS serum. Cyanogen bromide digestion of apo A-IV produced fragments that inhibited the permeability activity of the FSGS serum, whereas the intact protein did not. Thus, components of high-density lipoprotein are capable of preventing glomerular albumin permeability induced by serum from patients with FSGS in an in vitro system. The specificity and mechanism of the inhibition remain to be determined; the alteration of normal inhibitory activity in vivo may be a component in the pathophysiology of FSGS.

F-actin fiber distribution in glomerular cells: Structural and functional implications

Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture. Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy. The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli. Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.