Hemicentin 1 influences podocyte dynamic changes in glomerular diseases.

Different complex mechanisms control the morphology of podocyte foot processes and their interactions with the underlying basement membrane. Injuries to this system often cause glomerular dysfunction and albuminuria. The present study aimed at identifying early markers of glomerular damage in diabetic nephropathy. For this purpose, we performed a microarray analysis on kidneys of 3-wk-old peroxisome proliferator-activated receptor-γ (PPARγ)-null and AZIP/F1 mice, which are two models of diabetic nephropathy due to lipodystrophy. This was followed by functional annotation of the enriched clusters of genes. One of the significant changes in the early stages of glomerular damage was the increase of hemicentin 1 (HMCN1). Its expression and distribution were then studied by real-time PCR and immunofluorescence in various models of glomerular damage and on podocyte cell cultures. HMCN1 progressively increased in the glomeruli of diabetic mice, according to disease severity, as well as in puromycin aminonucleoside (PA)-treated rats. Studies on murine and human podocytes showed an increased HMCN1 deposition upon different pathological stimuli, such as hyperglycemia, transforming growth factor-ß (TGF-ß), and PA. In vitro silencing studies showed that HMCN1 mediated the rearrangements of podocyte cytoskeleton induced by TGF-ß. Finally, we demonstrated an increased expression of HMCN1 in the kidneys of patients with proteinuric nephropathies. In summary, our studies identified HMCN1 as a new molecule involved in the dynamic changes of podocyte foot processes. Its increased expression associated with podocyte dysfunction points to HMCN1 as a possible marker for the early glomerular damage occurring in different proteinuric nephropathies.

Small molecule membrane transporters in the mammalian podocyte: a pathogenic and therapeutic target.

The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

Extracorporeal photochemotherapy for the treatment of glomerulopathies with associated nephrotic syndrome.

INTRODUCTION: Patients with glomerulopathies associated with nephrotic syndrome have few effective treatment options. Here we report on the use of extracorporeal photochemotherapy (ECP) in 6 patients in whom glomerulopathy failed to respond to pharmacologic therapy. ECP is a promising immunomodulatory therapy associated with few side effects, that has been successfully used in the treatment of other immune-mediated conditions such as solid organ transplant rejection and graft-versus-host disease. METHODS: In this study, patients underwent 12 months of ECP. In all patients, progressive improvement in proteinuria was noted during the follow-up period, and total cholesterol and triglyceride levels also returned to near-normal values. RESULTS: In the 4 patients who had adequate renal function at study initiation, improvement of renal function was observed, as indicated by improved glomerular filtration rates (GFRs) and decreased proteinuria to creatininuria (Pru/Cru) ratios. In contrast, renal function progressively worsened in the 2 patients with inadequate renal function at study initiation. CONCLUSIONS: The results suggest that ECP is a feasible treatment for patients with glomerulopathies associated with nephrotic syndrome who have adequate baseline renal function. Additional clinical evaluation will have to be performed to better define the patient population in which ECP is most effective.

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.

[EBV-ASSOCIATED PNEUMONIA IN PATIENT WITH GRANULOMATOSIS WITH POLYANGIITIS (GPA) IN IMMUNOSOPPRESSIVE THERAPY TREATED WITH ACICLOVIR].

Granulomatosis polyangiitis (GPA) is an ANCA-related vasculitis (AAV) whose clinical manifestations mainly concern the respiratory tract (upper and lower) and the kidney. The treatment of GPA (as well as other AAV) includes the use of immunosuppressive drugs with numerous side effects; the most frequent complications are infectious and neoplastic. GPA frequently relapses. Epstein Barr Virus (EBV) is a ubiquitous virus; it is estimated that about 90% of the world's population has BEEN EXPOSED TO with this pathogen and has subsequently developed a latent infection. Under certain conditions including immunosuppression EBV may reactivate. We report the clinical case of a 67-year-old woman who presented with GPA involving the upper respiratory tract and renal failure with the need for hemodialysis treatment. The fourth month of induction therapy with cyclophosphamide and methylprednisone she presented with dyspnea and respiratory failure. After excluding pulmonary embolism and heart failure, a series of investigations including high resolution tomography and fibroscopy with broncoalveolar lavage (BAL) were performed which excluded recurrence of pulmonary vasculitis including alveolar haemorrhage A BAL demonstrated EBV-DNA. On this basis EBV pneumonia was diagnosed, and antiviral therapy with acyclovir was begun, followed by clinical and radiological improvement. In patients with GPA treated with immunosuppressive drugs pulmonary involvement may not only be due to the underlying vasculitis, but also to opportunistic agents, which must always be considered.

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 alphavbeta3. 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.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

The renal phenotype of allopurinol-treated HPRT-deficient mouse.

Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 µg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems to be directly involved in promoting lipid accumulation and subsequent phenotype changes of tubular cells, with activation of inflammation and fibrosis.

Rapid and cost-effective xenograft hepatocellular carcinoma model in Zebrafish for drug testing.

We developed a novel, rapid and cost-effective Zebrafish xenograft model of hepatocellular carcinoma (HCC) for drug screening in the disease. Following injection into the yolk sack of Zebrafish larvae of the human HCC cell line JHH6 stained by a vital dye, tumor mass growth was followed by fluorescence microscopy and by human Ki67 quantification. Tumor induced neo-angiogenesis was evaluated by alkaline phosphatase staining of the vessels, by using the Tg(fli1:EGFP)y1 strain of Zebrafish and by the quantification of the zebrafish vascular endothelial growth factor and of its receptor. We show that it is feasible to micro-inject JHH6 in Zebrafish larvae, that injected cells can grow for different days and that this induces a marked neo-angiogenesis. Finally, we show that our model allows testing the effects of anti-HCC drugs such as Bortezomib. Compared to more complex HCC mouse models, our model is far less expensive, faster to set up and does not need immunosuppressant treatment. Finally, the model makes use of JHH6, an aggressive form of HCC cell line never tested before in Zebrafish. In conclusion, the possibility to test anti HCC/neo-angiogenesis drugs makes our JHH6 model useful to select therapeutic molecules for a highly vascularized tumor such as HCC.

A nanoporous surface is essential for glomerular podocyte differentiation in three-dimensional culture.

Although it is well recognized that cell-matrix interactions are based on both molecular and geometrical characteristics, the relationship between specific cell types and the three-dimensional morphology of the surface to which they are attached is poorly understood. This is particularly true for glomerular podocytes - the gatekeepers of glomerular filtration - which completely enwrap the glomerular basement membrane with their primary and secondary ramifications. Nanotechnologies produce biocompatible materials which offer the possibility to build substrates which differ only by topology in order to mimic the spatial organization of diverse basement membranes. With this in mind, we produced and utilized rough and porous surfaces obtained from silicon to analyze the behavior of two diverse ramified cells: glomerular podocytes and a neuronal cell line used as a control. Proper differentiation and development of ramifications of both cell types was largely influenced by topographical characteristics. Confirming previous data, the neuronal cell line acquired features of maturation on rough nanosurfaces. In contrast, podocytes developed and matured preferentially on nanoporous surfaces provided with grooves, as shown by the organization of the actin cytoskeleton stress fibers and the proper development of vinculin-positive focal adhesions. On the basis of these findings, we suggest that in vitro studies regarding podocyte attachment to the glomerular basement membrane should take into account the geometrical properties of the surface on which the tests are conducted because physiological cellular activity depends on the three-dimensional microenvironment.

Glomerular permeability defect in hypertension is dependent on renin angiotensin system activation.

BACKGROUND: The aim of the present study was to compare glomerular permeability alterations associated with experimental hypertension models known to have different effects on the circulating renin-angiotensin system (RAS). METHODS: Five groups, 10 animals each, were studied. One group served as a nonhypertensive control. The other four groups of hypertensive animals were composed of spontaneously hypertensive rats, deoxycorticosterone acetate hypertensive rats, Goldblatt two-kidney, one-clip rats, and a group of Wistar rats infused with angiotensin II (200 ng/kg/min). Tail-cuff sphygmomanometric systolic blood pressure (BP), albumin permeability determined in isolated glomeruli exposed to oncotic gradients (P(alb)), glomerular filtration rate (GFR, iopamidol method), plasma renin activity (PRA), and albuminuria were evaluated. RESULTS: Alterations in P(alb) and albumin excretion rate were more evident in the experimental models with an activation of the RAS despite similar levels of systolic BP and GFR. A positive correlation was found between P(alb) and albuminuria (r = 0.51; P < .001) and between systolic BP and albuminuria (r = 0.37; P < .01). No relation was found between systolic BP and P(alb). CONCLUSIONS: The present study indicates that the activation of the RAS plays a significant role in the development of glomerular albumin permeability defects in hypertensive models and may contribute to the mechanisms that lead to target organ damage in hypertension.

Direct effect of plasma permeability factors from patients with idiopatic FSGS on nephrin and podocin expression in human podocytes.

The presence of circulating plasma factors (PF) altering renal permeability to proteins has been previously described in patients with focal segmental glomerulosclerosis (FSGS). Since these patients show reduced nephrin and podocin expression at renal biopsy, we evaluated the effect of serum and PF from patients with FSGS on nephrin and podocin expression in human podocytes. We studied 7 sera from patients with steroid-resistant FSGS, 3 from patients with nephrotic syndrome caused by non-immune disease, and 6 from healthy subjects. PF was prepared from plasmapheresis eluates of 2 patients with post-transplant recurrence of FSGS. Purification procedure was based on protein A Sepharose chromatography and differential precipitation in ammonium sulphate. Nephrin and podocin expression was semi-quantitatively evaluated by immunofluorescence. We found that serum and PF from FSGS patients rapidly induced redistribution and loss of nephrin in podocytes. This effect was associated with cytoskeleton redistribution and inhibited by cytochalasin B and sodium azide. On the contrary, podocin expression was unchanged after incubation with serum and PF from FSGS patients for short periods, but markedly reduced at 24 h. Our results demonstrate that serum and PF from FSGS patients may directly affect nephrin and podocin in human podocytes, thus providing new insights into the mechanisms causing proteinuria in FSGS.

Warfarin-related nephropathy: possible role for the warfarin pharmacogenetic profile.

Warfarin-related nephropathy (WRN) is a renal complication of warfarin treatment associated with over-anticoagulation. We describe a case of a 73-year-old man affected by chronic kidney disease, essential hypertension and atrial fibrillation treated with warfarin. The patient presented a rapid course of kidney failure after many episodes of over-anticoagulation, and renal biopsy demonstrated WRN. Interestingly, the patient's warfarin pharmacogenetic profile showed that he was warfarin sensitive. This is the first report describing the presence of gene polymorphisms affecting warfarin metabolism in a subject with a biopsy-proven WRN. The patient was treated with corticosteroids obtaining a partial clinical response.

Podocyte developmental defects caused by adriamycin in zebrafish embryos and larvae: a novel model of glomerular damage.

The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an in vivo model. In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 µM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 µM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury.

Role of components of frailty on quality of life in dialysis patients: a cross-sectional study.

BACKGROUND: Many people on dialysis suffer a variety of conditions that can affect frailty (the condition or quality of being frail), such as comorbidities, disabilities, dependence, malnutrition, cognitive impairment and poor social conditions. Frailty is suspected to affect quality of life (QoL). OBJECTIVES: The study aimed to evaluate the effect of the different components of frailty on the QoL of people on dialysis. METHODS: We enrolled 203 out of 233 prevalent patients on dialysis in the Trieste area of Italy. We applied the Short-Form 36 (SF-36) questionnaire, Activities of Daily Living, Instrumental Activities of Daily Living, Subjective Global Assessment scales and Karnofsky Index. In addition we analysed their social conditions. RESULTS: Dependence, malnutrition and disability had a negative role on QoL. Living with family and good social-economic conditions were significantly related to a better QoL. CONCLUSIONS: Dependence, malnutrition, disability, poor social and economic conditions have a significant effect on life quality. The role of comorbidities appears to be less important. Screening of patients, nutritional and functional rehabilitation and prevention of social isolation appear to be indispensable in guaranteeing a satisfactory life quality.

Podocyte expression of membrane transporters involved in puromycin aminonucleoside-mediated injury.

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA). We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family. Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration. In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.

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.

Transitions of serum albumin in patients with glomerulosclerosis 'in vivo' characterization by electrophoretic titration curves.

HSA functions as a physiological transporter of solutes and small molecules that induce structural transitions 'in vitro'. Analysis of these transitions requires prior purification of HSA that could introduce bias due to conformational changes. We utilized electrophoretic titration curves to describe a neutral to acid (N-A) transition of HSA directly in sera of seven patients with active focal segmental glomerulosclerosis (FSGS). The divergent electrophoretic profile of HSA was characterized by a shift in the range of pHs between 4.5 and 7.5 with an average variation of free electrophoretic mobility corresponding to loss of 1 positive charge in the pKa protonation range of histidyl residues and should involve domain I of HSA. 'In-gel' determination by maleimide-PEO2-biotin of free SH 34 of domain I showed inaccessibility of the dye at this site in pathological HSA and alkylation with the same complex induced N-A transition in normal HSA. Potential binders of free imidazoles such as Ca++ and/or of SH 34 such as NO were excluded on the basis of direct titration and studies on binding stimulation. This is the first report describing a transition of HSA directly 'in vivo', and the utilization of electrophoretic titration curves was critical to this purpose. This transition appears to be specific to FSGS and is unrelated to the nephrotic syndrome, Ca++ and NO binding. Spectroscopic analysis will elucidate the structural implication.

Nephrotic urine prevents increased rat glomerular albumin permeability induced by serum from the same patient with idiopathic nephrotic syndrome.

BACKGROUND: The putative humoral mediator thought to be involved in the pathogenesis of idiopathic nephrotic syndrome has not yet been identified. However, components exist in normal serum that block the permeability activity of FSGS serum in vitro. The potential of FSGS serum to increase glomerular albumin permeability may result from an imbalance between permeability factors and naturally occurring inhibitors. We hypothesized that this imbalance may be favoured by loss of inhibitory factors in nephrotic urine. METHODS: The study population consisted of seven patients with biopsy-proven FSGS, one with IgM nephropathy, and three with idiopathic nephrotic syndrome without biopsy, from whom frozen serum and dialysed and lyophilized urine samples were available. Glomerular albumin permeability (P(alb)) was determined from the change in glomerular volume induced by applying oncotic gradients across the basement membrane of normal isolated rat glomeruli pre-incubated with patient serum, normal control serum, patient serum mixed with an equal volume of urine from the same patient, or patient serum mixed with normal urine. Serum and urine apolipoproteins J and E were measured by dot-blot, utilizing peroxidase-labelled antibodies. The urinary capacity to scavenge oxygen radicals was determined after exposure of isolated glomeruli to superoxide generated by xanthine and xanthine oxidase. RESULTS: The mean P(alb) of the patients was markedly elevated at 0.74+/-0.08. The addition of urine from the same patient significantly reduced P(alb) (mean 0.15+/-0.23) in all but one of the patients with FSGS. Normal urine had no inhibitory effect in the 10 patients in which it was tested (mean 0.71+/-0.09). Serum apo J was slightly decreased and serum apo E was slightly increased compared with controls. Urine levels of both lipoproteins were significantly decreased compared with controls. Urine from FSGS patients effectively neutralized superoxide, whereas normal urine did not. CONCLUSIONS: Nephrotic urine but not normal urine contains components that block increased albumin permeability in isolated rat glomeruli induced by serum from patients with the idiopathic nephrotic syndrome. The inhibitory function of these components, which appear not to include apolipoproteins J and E, may involve scavenging of superoxide as a final common pathway. Loss in the urine from the serum of naturally occurring inhibitors in the initial stages of the disease may propagate proteinuria and glomerular injury.

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

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. Only the definitive characterization and quantification in vivo of the different molecules that play a role in FSGS may furnish adequate answer.