Preemptive plasmapheresis and recurrence of FSGS in high-risk renal transplant recipients.

Recurrent focal segmental glomerulosclerosis (FSGS) following transplantation is ascribed to the presence of a circulating FSGS permeability factor (FSPF). Plasmapheresis (PP) can induce remission of proteinuria in recurrent FSGS. This study addressed the efficacy of pre-transplant PP in decreasing the incidence of recurrence in high-risk patients. Ten patients at high-risk for FSGS recurrence because of rapid progression to renal failure (n = 4) or prior transplant recurrence of FSGS (n = 6) underwent a course of 8 PP treatments in the peri-operative period. Recurrences were identified by proteinuria >3 g/day and confirmed by biopsy. Seven patients, including all 4 with first grafts and 3 of 6 with prior recurrence, were free of recurrence at follow-up (238-1258 days). Final serum creatinine in 8 patients with functioning kidneys averaged 1.53 mg/dL. FSGS recurred within 3 months in 3 patients, each of whom had lost prior transplants to recurrent FSGS. Two of these progressed to end-stage renal disease (ESRD) and the third has significant renal dysfunction. Based on inclusion criteria, recurrence rates of 60% were expected if no treatment was given. Therefore, PP may decrease the incidence of recurrent FSGS in high-risk patients. Definitive conclusions regarding optimal management can only be drawn from larger, randomized, controlled studies.

Early detection of radiation-induced glomerular injury by albumin permeability assay.

Renal irradiation leads predictably to glomerular vascular injury, cell lysis, matrix accumulation, sclerosis and loss of renal function. The immediate effects of renal irradiation that may be associated with glomerular pathology and proteinuria are not clear in the human disease or its rat model. We hypothesized that radiation-induced injury causes immediate and subtle alterations in glomerular physiology independent of the neurohumoral and hemodynamic regulatory mechanisms. We employed a sensitive in vitro functional assay of glomerular albumin permeability (P(alb)) to demonstrate radiation-induced damage to the glomerular filtration barrier immediately after total-body irradiation of rats. In blinded experiments, control rats were sham-treated, and experimental rats received 9.5 Gy X rays. Rats were killed humanely at 1 h to 9 weeks after irradiation and glomeruli were isolated. In parallel experiments, glomeruli were isolated from normal rats and irradiated in vitro. The change in glomerular capillary permeability due to an experimental oncotic gradient was determined using videomicroscopy and P(alb) was calculated. Results show that in vivo or in vitro irradiation of glomeruli caused an increased P(alb) at 1 h. Increased P(alb) was observed up to 3 weeks after irradiation. Glomeruli from mice irradiated with 9.5 or 19.0 Gy X rays did not show increased P(alb) at 1 h postirradiation. We conclude that glomerular protein permeability of irradiated rats increases in a dose-dependent manner immediately after irradiation and that it appears to be independent of hemodynamic or systemic influences.

Components of normal serum block the focal segmental glomerulosclerosis factor activity in vitro.

BACKGROUND: Sera from some patients with focal segmental glomerulosclerosis (FSGS) increase glomerular albumin permeability (P(alb)) in vitro. The hypothesis that a component of normal serum can protect the glomerular permeability barrier was tested using sera from FSGS patients, normal individuals, and several mammalian and avian species. METHODS: In most experiments, isolated rat glomeruli were incubated in medium containing FSGS serum known to increase P(alb) in vitro, normal serum, or both active FSGS and normal serum. In other experiments, fractions of normal serum and serum from other vertebrate species were incubated with active FSGS serum. P(alb) was calculated from glomerular capillary expansion in response to an oncotic gradient. To enrich the blocking activity, normal pooled human plasma was subjected to various biochemical manipulations. RESULTS: Normal human serum prevented the increase in P(alb) (active FSGS sera, 0.77 +/- 0.12; active FSGS sera:normal serum, 1:1 mix, 0.06 +/- 0.30, P < 0.001). Protection diminished as the concentration of normal serum was decreased. Specific fractions of human serum, including human albumin and immunoglobulin fractions, were not protective. Blocking activity was present in 80% ammonium sulfate precipitate and certain fractions from size-exclusion chromatography of normal pooled human plasma. Normal serum from each of the vertebrate species tested also prevented the increase in P(alb). Preincubation with normal serum was protective during subsequent incubation with FSGS serum, but normal serum was not protective after preincubation with FSGS serum. CONCLUSIONS: We conclude that a factor or factors in normal serum block the permeability effect of active FSGS sera. This phenomenon may account for variability in proteinuria among patients with FSGS and may explain inconsistent proteinuria following injection of FSGS sera into experimental animals. Characterization of the protective substance(s) and the mechanism by which the increase in permeability is blocked may provide insight into the pathogenesis of FSGS.

"The FSGS factor:" enrichment and in vivo effect of activity from focal segmental glomerulosclerosis plasma.

A circulating causative factor has been postulated in focal segmental glomerulosclerosis (FSGS). It has been shown that serum or plasma from some FSGS increases glomerular albumin permeability (Palb) in vitro. Palb greater than 0.5 (i.e., FS activity) is associated with recurrence after transplantation. Specimens from 15 FSGS patients were studied to document the presence of a permeability factor, to isolate this factor, to characterize its biochemical properties, and to show its effect in vivo. Total lipids were extracted by chloroform/methanol (2: 1); FS activity was absent from total lipid extract. Chylomicrons and lipoproteins were removed from the plasma with dextran sulfate, followed by sequential precipitation of proteins at 50 and 70% ammonium sulfate saturation. FS activity was retained in the 70% ammonium sulfate supernatant and exhibited a 100-fold purification. FS activity was lost after heating at 100 degrees C for 10 min or after protease digestion. Under nondenaturing conditions, electrophoresis of the FSGS 70% supernatant showed a prominent low molecular weight band that was not evident in the 70% supernatant from normal plasma. Dialysis and centrifugation-based membrane ultrafiltration of the FSGS factor indicated a molecular size between 30 and 50 kD. Injection of the 70% FSGS supernatant into rats caused a threefold increase in urine protein in collections from 6 to 24 h after injection. No increase in proteinuria occurred in rats injected with 70% supernatant from normal individuals. It is concluded that the FSGS factor is a low molecular weight protein with the potential to increase Palb in vitro and to cause proteinuria in vivo.

Documentation of angiotensin II receptors in glomerular epithelial cells.

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

TNF-alpha increases albumin permeability of isolated rat glomeruli through the generation of superoxide.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that plays a central role in inflammation. Glomerular levels of TNF-alpha are elevated in human and experimental glomerulonephritis. Glomerular cells produce and respond to TNF-alpha. One of the mechanisms by which these cells respond to TNF-alpha is through generation of reactive oxygen species. In this study, the effect of TNF-alpha on albumin permeability (P(albumin)) of isolated rat glomeruli and the possible mechanism of this effect were examined. Isolated rat glomeruli were incubated with TNF-alpha (0.4 ng/ml), TNF-alpha with anti-TNF-alpha antibodies, and TNF-alpha with the reactive oxygen species scavengers superoxide dismutase, catalase, DMSO, or dimethylthiourea for 12 min at 37 degrees C, and P(albumin) was calculated. TNF-alpha increased P(albumin) of isolated glomeruli compared with control (0.70 +/- 0.02, n = 25 versus 0.00 +/- 0.05, n = 26), and this effect was abrogated by anti-TNF-alpha antibodies (-0.18 +/- 0.05, n = 23). Superoxide dismutase abolished the increase in P(albumin) (-0.04 +/- 0.11, n = 23), whereas catalase (0.73 +/- 0.08, n = 30), DMSO (0.64 +/- 0.03, n = 10), or dimethylthiourea (0.51 +/- 0.08, n = 10) did not alter the effect of TNF-alpha. These results indicate that TNF-alpha increased P(albumin+)++ of isolated glomeruli and that the mediator of the increased P(albumin) is superoxide. It is concluded that TNF-alpha derived from glomerular or extraglomerular sources can increase glomerular P(albumin) through generation of superoxide and may lead to proteinuria.

Inhibitory effect of Tripterygium wilfordii multiglycoside on increased glomerular albumin permeability in vitro.

BACKGROUND: Tripterygium wilfordii Hook F is a medicinal plant used for the treatment of glomerulonephritis in China. We studied the effect of Tripterygium wilfordii multiglycoside (TWG) on glomerular albumin permeability (Palbumin) in vitro. METHODS: Isolated rat glomeruli were incubated with protamine (600 micrograms/ml) for 30 min, or with human recombinant tumour necrosis factor (TNF-alpha 0.4 ng/ml), superoxide (10 units/ml), or serum from a focal segmental glomerular sclerosis (FSGS) patient for 10 min at 37 degrees C. TWG, 1 mg/ml, was added in parallel tubes to study the effect on Palbumin. Control glomeruli were incubated under identical conditions. The albumin reflection coefficient (sigma albumin) was calculated from the change in glomerular volume in response to an applied oncotic gradient. Convectional permeability (Palbumin) was calculated as (1 - sigma albumin). RESULTS: Compared with controls, protamine increased the Palbumin of glomeruli (0.83 +/- 0.05, n = 25, vs 0.18 +/- 0.03, n = 20); pretreatment with TWG blocked this effect (0.13 +/- 0.04, n = 25). TNF-alpha also increased the Palbumin (0.79 +/- 0.04, n = 24 vs 0.04 +/- 0.07, n = 19); preincubation with TWG blocked this effect (0.03 +/- 0.09, n = 24). Palbumin of glomeruli incubated with xanthine and xanthine oxidase, resulting in the production of superoxide, also increased as compared to controls (0.85 +/- 0.04, n = 15 vs 0.08 +/- 0.05, n = 14); TWG blocked this effect as well (0.21 +/- 0.08, n = 14). FSGS serum also increased Palbumin of glomeruli significantly (0.88 +/- 0.02, n = 49 vs 0.00 +/- 0.02, n = 49); preincubation with TWG blocked this effect (0.05 +/- 0.07, n = 30). TWG by itself had no effect on Palbumin (0.19 +/- 0.10, n = 15). CONCLUSIONS: Our results show that TWG blocks protamine, TNF-alpha, superoxide, and FSGS serum-mediated increase in glomerular albumin permeability in vitro. We conclude that reduction of proteinuria by Tripterygium wilfordii multiglycoside in various kinds of glomerular diseases in vivo might be due to protection of the glomerular filtration barrier.

Direct effects of platelet-activating factor on glomerular capillary permeability.

Platelet-activating factor (PAF) is an important mediator of injury in acute renal failure and glomerulonephritis. Intrarenal infusion of PAF reduces glomerular filtration rate and renal plasma flow and increases glomerular permselectivity via its renal hemodynamic and/or immunologic effects. Direct effects of PAF on glomerular capillary permeability are not known. We studied the direct effects of PAF on mesangial contraction (a measure of filtration area), glomerular capillary hydraulic conductivity (L[p]) and capillary albumin permeability (P[albumin]). Glomeruli were isolated from Sprague-Dawley rats and incubated with or without various concentrations of PAF (10[-9], 10[-7] and 10[-5] M) for up to 5 h at 37 degrees C. Mesangial contraction (percent change in glomerular volume) was assessed from the gradual decrease in volume of glomeruli during 20 min of incubation with PAF. L(p) was calculated from the rate of change in glomerular volume during the 0.1 s of capillary expansion in response to a transcapillary oncotic gradient. P(albumin) was calculated from a change in relative volume of glomeruli in response to an oncotic gradient. Mesangial contraction was maximal after 20 min of incubation and was concentration dependent (5.2+/-0.9, 7.9+/-1.0 and 10.0+/-1.0%, respectively, with PAF 10(-9), 10(-7) and 10(-5) M). Incubation of glomeruli with PAF 10(-7) M for 60 min at 37 degrees C caused a significant decrease in L(p) (2.25+/-0.30 vs. control 3.12+/-0.28 microl x min(-1) x mm Hg(-1) x cm(-1), n = 5). P(albumin) of glomeruli incubated with PAF was unchanged up to 2 h but increased significantly with the highest concentration of PAF (10(-5) M) after 3 h of incubation (0.60+/-0.18, n=15, vs. control 0.00+/-0.08, n = 20), whereas lower concentrations of PAF (10[-7] or 10[-9] M) required at least 5 h of incubation with glomeruli to cause a significant increase in P(albumin) (0.45+/-0.09 and 0.48+/-0.07, respectively, n=15, vs. control 0.00+/-0.08, n=15). We conclude that PAF has multiple direct effects on glomerular functions, which are time dependent and may contribute to the altered capillary permeability in vivo.

Cyclosporine protects glomeruli from FSGS factor via an increase in glomerular cAMP.

Cyclosporine (CsA) administration to patients with recurrent focal segmental glomerulosclerosis (FSGS) after transplantation results in remission of proteinuria. We have shown that sera from patients with recurrent FSGS can increase the glomerular albumin permeability (Palbumin) and that increase in glomerular cAMP levels can alter the permeability characteristics of glomeruli in vitro. The purpose of this study was to determine if the increased glomerular levels of cAMP were related to the protective effects of CsA on an increase in Palbumin by FSGS sera. Glomeruli from Sprague-Dawley rats following intraperitoneal administration of CsA (25 mg/kg/day), cremophore (25 mg/kg/day), or saline for 5 days were incubated with 1:50 dilution of serum from three FSGS patients or with pooled normal human serum prior to calculation of Palbumin. Glomerular cAMP was measured by radioimmunoassay. Glomerular ultrastructural changes were assessed by transmission electron microscopy (TEM). Serum from three FSGS patients markedly increased Palbumin of glomeruli from saline or cremophore treated rats (saline, 0.68+/-0.08; 0.72+/-0.07; 0.70+/-0.07; and cremophore, 0.79+/-0.05; 0.81+/-0.02; 0.79+/-0.01; n=25 glomeruli in each group). In contrast Palbumin of glomeruli from CsA treated rats was not increased by any of the three FSGS sera tested (0.03+/-0.02; 0.04+/-0.05; 0.02+/-0.07, n=25 glomeruli in each group). Glomerular cAMP (pmol/mg of protein) increased 5 fold in CsA treated rats (328+/-26; 5 rats) compared with cremophore or saline treated rats (87+/-24 and 65+/-23, P<0.01; 5 rats in each group). The glomerular basement membrane appeared to be thickened and the lamina densa had an irregular appearance after treatment with CsA. No ultrastructural changes of glomerular epithelial or endothelial cells were evident. We conclude that CsA may have a direct protective effect on the glomerular filtration barrier in FSGS. We postulate that increased levels of glomerular cAMP by CsA may play an important role in protecting the glomerular Palbumin effect of the FSGS factor and may contribute to remission of proteinuria in FSGS patients.

Cyclosporine in glomerular disease.

Cyclosporine was introduced in 1981 as an immunosuppressive agent in renal transplantation. Its use was soon extended to the treatment of various glomerular disorders. In light of its known immunomodulating effects, the use of cyclosporine has been most prominent in those glomerular diseases thought to have an immune basis. The most careful studies of cyclosporine in glomerular diseases have been performed in the pediatric population with idiopathic nephrotic syndrome (i.e., minimal change disease and focal segmental glomerulosclerosis), although data are accumulating regarding efficacy and safety in adults with idiopathic nephrotic syndrome. In patients who are steroid-dependent, cyclosporine therapy can induce complete or partial remission in a significant proportion of cases; success rates in patients with steroid-resistant nephrotic syndrome are less encouraging. Treatment with cyclosporine allows for dose reduction or elimination of corticosteroids, and the consequent salutary effect on growth in the child and glucose and bone metabolism in all patients. Studies that suggest a potential benefit of cyclosporine in recurrent nephrotic syndrome in renal allografts and in other glomerular diseases are also discussed.

Circulating factor associated with increased glomerular permeability to albumin in recurrent focal segmental glomerulosclerosis.

BACKGROUND: Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence. METHODS: To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin. Of the 56 patients who had undergone renal transplantation, 33 had recurrences. Sixty-four patients, many of whom had undergone transplantation, were being treated with dialysis. Thirty-one patients with other renal diseases and nine normal subjects were also studied. RESULTS: The 33 patients with recurrent focal segmental glomerulosclerosis after transplantation had a higher mean (+/-SE) value for permeability to albumin (0.47+/-0.06) than the normal subjects (0.06+/-0.07) or the patients who did not have recurrences (0.14+/-0.06). After plasmapheresis in six patients with recurrences, the permeability was reduced (from 0.79+/-0.06 to 0.10+/-0.05, P = 0.008), and proteinuria was significantly decreased. Patients with corticosteroid-sensitive nephrotic syndrome or with membranous nephropathy after transplantation had low levels of serum activity. The circulating factor bound to protein A and hydrophobic-interaction columns and had an apparent molecular mass of about 50 kd. CONCLUSIONS: A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury.