Michele Carraro

Podocyte Glutamatergic Signaling Contributes to the Function of the Glomerular Filtration Barrier

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.

Characterization of plasma factors that alter the permeability to albumin within isolated glomeruli

Focal segmental glomerulosclerosis (FSGS) is responsible for intractable proteinuria and has become the leading cause of renal insufficiency in children. Protenuria in FSGS is probably due to the effect of one or more permeability plasma factors which increase the glomerular permeability to proteins. We fractioned serum from children with FSGS using two mixed chromatographic‐electrophoretic approaches and have purified ten proteins among several hundreds which maintained the original permeability activity after renaturation, utilizing an isolated rat glomeruli assay. Six proteins were successfully characterized by mass spectometry as fibulin, apolipoprotein J, vitronectin, albumin isoforms, γ chain fibrinogen and mannan‐binding lectin‐associated serine protease. Both procedures utilized for purification were based on affinity chromatography with Protein A‐Sepharose and ended with two‐dimensional electrophoresis, whereas the intermediate steps were different. Cross inhibition with zinc and aprotinin of purified factors and whole FSGS serum indicate strong homology. These are the first data demonstrating permeability activity for serum proteins, an observation with important implications in pathogenesis of proteinuria. Determination of the serum levels of each protein and a careful differentiation of FSGS from normal serum could provide the basis for clarifying the mechanism of proteinuria.

HIV-1 Tat reduces nephrin in human podocytes : a potential mechanism for enhanced glomerular permeability in HIV-associated nephropathy

Objective:To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Design:Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Methods:Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. Results:We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin αvβ3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. Conclusion:The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

Circulating anti‐actin and anti‐ATP synthase antibodies identify a sub‐set of patients with idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome (iNS) with resistance or dependence to steroids is a common disease in children but in spite of an increasing clinical impact its pathogenesis is unknown. We screened for the presence of circulating antibodies against glomerular (podocytes, mesangium) and tubular cells (tubular epithelia) a cohort of 60 children with iNS including 8 patients with a familial trait of iNS or with proven mutation of NPHS1‐NPHS2 and 12 with good sensitivity to steroids. Positive sera were found in 8 cases, all belonging to the category without familial trait/molecular defects. The targets of antibodies were characterized with Western blot and MALDI‐Mass utilizing β‐hexyl cell extracts separated with two‐dimensional electrophoresis. In all cases antibodies of the IgM class were directed against ATP synthase β chain alone (4 cases) or in combination with actin (3 cases); one child presented IgG against aldose reductase. The clinical picture was nephrotic syndrome with steroid resistance or dependence and variable cyclosporin sensitivity; 3 patients developed end stage renal failure. The basic pathology picture was focal segmental glomerulosclerosis (FSGS) in 4 cases and mesangial proliferative glomerulonephrites with deposition of IgM in 2. Overall, patients with circulating auto‐antibodies could not be readely differentiated on clinical grounds with the exception of 3 children who developed positivity for antinuclear antibodies during the follow‐up. Affinity‐purified IgM from one patient who underwent plasmapheresis for therapeutical pourposes (but not from a normal pool) induced proteinuria in Sprague‐Dawley rats and concomitant human IgM deposition within glomeruli. This is the first report of circulating anti‐actin/ATP synthase β chain antibodies in a subset of patients with iNS. Both pathological significance and clinical impact given by the presence of these antibodies and the relationship with other conditions such as lupus‐erythematosus, characterized by their presence, must be defined.

Albumin permeability in isolated glomeruli in incipient experimental diabetes mellitus

Aims/hypothesis. The pre-clinical phase of diabetic nephropathy is characterised by increased glomerular filtration rate and episodes of microalbuminuria. The cause of the microalbuminuria has been variably ascribed to alterations of the size or charge selective barriers of the glomerulus or both or as a consequence of the haemodynamic changes. Our aim was to investigate very early albumin permeability alterations in isolated glomeruli which were not subject to perfusion pressure.¶Methods. Isolated glomeruli were studied from 120 male Wistar rats, divided into three groups: streptozotocin-treated, streptozotocin-treated with insulin pellet implants, and controls. From each group ten animals were killed at 7, 14, 28, and 56 days after induction. Study variables included blood pressure, proteinuria, iopamidol clearance, albumin permeability and glomerular area. Subsequently, albumin permeability, proteinuria, and iopamidol clearance were determined in an additional group of 40 diabetic animals studied at 24, 72, 96, and 120 h after induction.¶Results. Albumin permeability increased steadily from induction in streptozotocin-treated animals, reaching a plateau at approximately 120 h. Glomerular filtration rate was shown to increase significantly at approximately 7 days and proteinuria correlated with it. Glomerular hypertrophy was observed both in streptozotocin-treated animals and in streptozotocin-treated rats with insulin pellet implants. Strict blood glucose control delayed the appearance of the permeability defect in isolated glomeruli and inhibited the increase in glomerular filtration in intact animals. It did not prevent glomerular hypertrophy.¶Conclusion/interpretation. An albumin permeability defect exists early in isolated non-perfused glomeruli from streptozotocin-treated rats and seems to be independent of glomerular filtration rate alterations. [Diabetologia (2000) 43: 235–241]

Glomerular albumin permeability as an in vitro model for characterizing the mechanism of focal glomerulosclerosis and predicting post-transplant recurrence

Abstract:  The putative mechanisms of proteinuria in idiopathic focal glomerulosclerosis and of its post‐transplant recurrence are discussed. It is proposed that a balance between circulating factors with permeability activity on glomeruli and putative inhibitors play a key role. The characterization of inductors is currently in progress; most inhibitors appear to be apolipoproteins (mainly apoJ and apo E) but we cannot exclude other substances. The goal is now to evaluate the concentration of both inducers and inhibitors of glomerular permeability in vivo. Permeability activity in plasma of patients with FSGS with and without recurrence of the disease may be evaluated by an in vitro functional essay with isolated glomeruli. Published data on permeability activity evaluated with this method in different proteinuric states gave, however, controversial results and this test cannot be readily considered of clear clinical utility.

Proteomics of Plasma and Urine in Primary Nephrotic Syndrome in Children

Primary nephrotic syndrome in children, especially the variant with segmental glomerulosclerosis, remains an unsolved clinical problem. In spite of some progress, its pathogenesis is still unknown and the therapy options are confined to gross immune modulation. Indirect evidence based on posttransplant recurrence of the disease suggested an implication of plasma factors, whose characterization remains in course. Besides historical candidates, research is now considering glyco- and lipoderivatives. Structural analysis of plasma and urinary proteins based on proteomics has recently shown an increased proteolysis of major components such as albumin and the implication of alpha 1-antitrypsin that represents the first-line defense against exogenous and endogenous substances with proteolytic activity. Albumin has also emerged as a major plasma antioxidant, and recent studies have demonstrated that in patients with active focal segmental glomerulosclerosis albumin undergoes massive and stable oxidation with sulfonation of Cys34, formation of an adduct with +48 Da molecular weight, changes of the net charge due to additional negative residues, and loss of free thiol group (SH) titration. Altogether, these data suggest that oxidative stress determines selective protein damages in focal segmental glomerulosclerosis patients with formation of new adducts and fragmentation of plasma proteins. Research should now address whether oxidation of podocyte proteins is important for the maintenance of renal selectivity and is involved in proteinuria.

Modulation of Incipient Glomerular Lesions in Experimental Diabetic Nephropathy by Hypotensive and Subhypotensive Dosages of an ACE Inhibitor

A glomerular permeability defect occurs early in the course of type 1 diabetes and precedes the onset of microalbuminuria and renal morphological changes. Recently, ACE inhibitors have been shown to prevent loss of glomerular membrane permselective function, but the mechanism of this nephroprotective effect is still being debated. The objective of the present study was to evaluate the effects of hypotensive and subhypotensive dosages of the ACE inhibitor quinapril ex vivo and of its active metabolite quinaprilat in vitro on the glomerular albumin permeability (P(alb)) defect in the early phases of experimental diabetes. For the ex vivo study, six groups of male Wistar rats were evaluated for 4 weeks. One group served as a nondiabetic control (C); the other five groups were rendered diabetic and included untreated diabetic rats (D) and diabetic rats receiving quinapril at the dosages of 5 (DQ1), 2.5 (DQ2), 1.25 (DQ3), and 0.625 (DQ4) mg. kg(-1). day(-1). Dosage-dependent effects of quinapril on systolic blood pressure and the glomerular filtration rate were observed. In contrast, control of P(alb) in isolated glomeruli exposed to oncotic gradients, proteinuria, and glomerular and tubular hypertrophy was obtained with subhypotensive dosages (DQ3 and DQ4 groups) of the ACE inhibitor. In the in vitro study, quinaprilat reduced P(alb) significantly in concentration ranges from 10(-6) to 10(-14) mol/l compared with results in control glomeruli. The effect on P(alb) may have occurred by mechanisms different from kidney ACE inhibitor. These study results indicated that ACE inhibitor treatment prevents the early onset of the P(alb) defect in experimental diabetes. This effect seemed to occur independently of systemic or glomerular hemodynamic changes and, at least partially, from kidney ACE inhibition.

Involvement of the Renin Angiotensin System in the Pathogenesis of Postexercise Proteinuria

Proteinuria after strenuous exercise is common in healthy subjects. The pathophysiologic mechanism of postexercise proteinuria (PEP) is not clear, although the phenomenon has long been known and many explanatory theories have been proposed. It is widely recognized that angiotensin II may increase filtration of protein through the glomerular membrane, and that its concentration in plasma increases during exercise. The aim of this study was to evaluate possible involvement of angiotensin II in the pathogenesis of PEP. Of 25 young volunteers who performed maximal aerobic exercise, eight showed PEP. The exercise was repeated after an interval of at least one week, now 90 minutes after administration of captopril (25 mg). Captopril did not affect the achieved work load of the maximal blood pressure and heart rate during the exercise, but PEP was not found. As it was possible to prevent PEP by administering an angiotensin-converting enzyme inhibitor, the study supports the theory that the renin angiotensin system is involved in the pathogenesis of PEP.

Inhibition of renal permeability towards albumin: A new function of apolipoproteins with possible pathogenetic relevance in focal glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a degenerative renal disease characterized by the accumulation of extracellular matrix and lipids within the glomerular tuft. It has been proposed that an abnormal renal permeabilization towards proteins induced by a putative plasma factor is, in some way, involved in the pathogenesis of the disease. In this paper, we measured the plasma permeability activity (Palb) in several sera of patients with FSGS and found a mean activity of 0.82 ± 0.03 which means a marked increase compared to a mean Palb of 0.16±0.03 in normal controls. Coincubation of FSGS and normal serum reduced the permeability activity within the normal range; normal serum added to the incubation medium after the glomeruli had already been exposed to the FSGS serum had no effect, suggesting the presence of inhibitory substances with a direct effect on a circulating substrate. Finally, the antipermeability activity was retained when heated to 60°C but not to 100°C. By serial fractionations of normal serum and reported activity measurements at each step, five natural occurring inhibitors of albumin permeabilization were purified and characterized by matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS), as components of apolipoproteins (apo) (apo E2 and E4, apo L, the high Mr  apo J and a 28 kDa fragment of apo A‐IV). Coincubation of each apolipoprotein with FSGS serum inhibited permeability, but only apo J and apo E2 and E4 were found to be crucial for the process. In conclusion, we have purified from normal serum five inhibitors of permeability induced by FSGS serum, all corresponding to apolipoproteins. An imbalance between permeability factors and apolipoproteins may play a pathogenetic role in FSGS.