No abstract

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.

Abstract.A plasma factor displaying permeability activity in vitro and possibly determining proteinuria has been hypothesized in idiopathic focal segmental glomerulosclerosis (FSGS). In vitro permeability activity (P alb ) was determined in sera of five patients with autosomal recessive steroid-resistant nephrotic syndrome (NPHS2), an inherited condition indistinguishable from idiopathic FSGS on clinical grounds, but in which proteinuria is determined by homozygous mutations of podocin, a key component of the glomerular podocyte. All patients had presented intractable proteinuria with nephrotic syndrome; four developed renal failure and received a renal allograft. For comparison, sera from 31 children with nephrotic syndrome were tested. Pretransplant P alb was high in all cases (mean 0.81 Ϯ 0.06), equivalent to levels observed in idiopathic FSGS. Overall, P alb did not correlate with proteinuria. The posttransplant outcome was complicated in two patients by recurrence of proteinuria after 10 and 300 d, respectively, that responded to plasmapheresis plus cyclophosphamide. P alb levels were high at the time of the recurrence episodes and steadily decreased after plasmapheresis, to reach normal levels in the absence of proteinuria after the seventh cycle. In an attempt to explain high P alb in these patients, putative inhibitors of the permeability activity were studied. Coincubation of serum with homologous nephrotic urine reduced P alb to 0, whereas normal urine did not determine any change, which suggests loss of inhibitory substances in nephrotic urine. The urinary levels of the serum P alb inhibitors apo J and apo E were negligible in all cases, thus suggesting that other urinary inhibitors were responsible for the neutralizing effect. These data indicate that P alb is high in NPHS2, probably resulting from loss of inhibitors in urine. Lack of correlation of P alb with proteinuria suggests a selective loss of inhibitors. As in idiopathic FSGS, proteinuria may also recur after renal transplantation in NPHS2 patients, and post-transplant proteinuria is associated with high P alb . The relationship between elevated P alb and proteinuria in NPHS2 remains to be determined.

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.

The efficacy and safety of nonionic dimeric contrast media in subjects with impaired renal function is largely unknown. The present study was aimed at determining the risk of tubular nephrotoxicity in patients with mild to moderate renal insufficiency who underwent intravenous urography (IVU) with the nonionic dimeric contrast agent iodixanol (Visipaque, Nycomed Imaging, Oslo, Norway). In a double-blind protocol 64 patients (55 males; mean age 68.3 years) with serum creatinine between 135 and 265 micromol/l who were to undergo IVU were randomized to receive iodixanol (a nonionic dimer) or iopromide (a nonionic monomer), 600 mg I/kg b. w. Renal function was evaluated before and 1 h, 6 h, 24 h, 48 h and 7 days after IVU with analysis of serum creatinine, urinary enzymes alanylaminopeptidase and N-acetyl-beta-glucosaminidase, and urinary microproteins alpha-1-microglobulin and albumin. Renal function remained stable in both contrast medium groups during the follow-up period. No statistically significant differences were observed between the monomer and the dimer in terms of urinary enzyme and microprotein excretion or serum creatinine. Transient radiocontrast-induced nephropathy developed in 1 patient who had received iodixanol. The administration of the nonionic dimeric contrast medium iodixanol, or of the nonionic monomer iopromide, entailed a low nephrotoxic potential in patients with mild to moderate renal insufficiency undergoing excretory urography.

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.

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, gamma 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.

Neither the course of enzyme excretion nor the incidence of acute renal failure following radiocontrast administration were affected by single doses of calcium antagonists. AAP levels were lower in general in subjects taking the 20-mg dose of nitrendipine. This study also indicates that a single low or normal dose of nitrendipine per os is not effective prophylaxis before radiocontrast administration. The designs of future studies investigating the "nephroprotective' effect of calcium-channel antagonists per os should incorporate (1) the use of repeated doses to saturate hepatic metabolic pathways, and (2) the control of confounding variables in the measurement of urinary enzymes.