Idiopathic Minimal Lesion Nephrotic Syndrome Research Articles

T regulatory cell function in idiopathic minimal lesion nephrotic syndrome

The purpose of this study was to test the hypothesis that, in idiopathic minimal lesion nephrotic syndrome (IMLNS), the T regulatory (T reg) cell suppressor mechanism is deficient, thereby enhancing cytokine release by T effector cells. Twenty-one patients with IMLNS, eight healthy controls and two patients with nephrotic syndrome and membranoproliferative glomerulonephritis were studied. The percentage of T reg cells was similar in the healthy controls and in patients with IMLNS in relapse or in remission. Thymidine incorporation in autologous T effector cells, as well as expression of the regulatory cytokine interleukin (IL)-10, was significantly reduced in patients in relapse when compared with patients in remission and healthy subjects. IL-2 expression was also reduced in patients in relapse but did not achieve statistical significance. In a different set of experiments, T cells, from subjects with IMLNS in remission, when stimulated with antiCD3-antiCD28 antibodies, secreted increased levels of cytokines. No such increase in cytokines was observed when cells from healthy controls were stimulated with same mitogen. The impaired T reg cell function observed in these patients may have pathogenic and therapeutic implications, because it could explain the persistence of the proposed pathogenic cytokines observed in the patients with IMLNS.

A case of unfulfilled expectations. Cytokines in idiopathic minimal lesion nephrotic syndrome

Idiopathic minimal lesion nephrotic syndrome (IMLNS) was proposed to be a disorder of T-cell dysfunction by Shalhoub in 1974. The mechanisms by which T-cells increase glomerular permeability have remained elusive (and unproven). There is evidence that IMLNS may be due to a circulating factor released from activated T-cells. In recent years, efforts have been made to identify this pathogenetic cytokine as well as to understand the mechanism(s) for the increased release of this factor. This review attempts to critically analyze the available published data. Using different methodologies, investigators have focused on the production of cytokines in patients with IMLNS during relapse and remission. This has resulted in a plethora of data without definitive conclusions. The pathogenetic cytokine has not been identified, and it is questionable whether there is a Th2 dominance in IMLNS. The review of the available data illustrates the difficulties encountered when one is studying the cytokine secretory pattern in patients with IMLNS. Differences in patient population, type of cells studies, sample preservation, and methodology used to measure cytokines are some of the factors that could account for the disparity of observed results.

Proteinuria and Fusion of Podocyte Foot Processes in Rats after Infusion of Cytokine from Patients with Idiopathic Minimal Lesion Nephrotic Syndrome

Background/Aims: We report on the isolation of a factor secreted by peripheral blood mononuclear cells from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse and its effect on proteinuria and podocyte morphology in the rat. Methods: Peripheral blood mononuclear cells from patients with IMLNS (in relapse and in remission) and patients with focal segmental glomerulosclerosis were cultured for 72 h. Supernatants from 20 × 10⁶ cultured cells were separated by liquid chromatography into three fractions according to markers (bovine serum albumin, β-amylase, and apoferritin). Each supernatant fraction was infused into rats for 5 days using an osmotic pump. Proteinuria, 24-hour albumin excretion or albumin/creatinine ratio in a 24-hour urine collection, was measured daily starting 3 days prior to fraction infusion. Renal tissue was obtained for electron microscopy studies. The β-amylase fraction underwent electrophoresis using isoelectric focusing gel. Results: When protein excretion was compared prior to and during supernatant fraction infusion, a significant increase in proteinuria was observed only when β-amylase fraction from IMLNS patients in relapse was infused (p < 0.05). Protein electrophoresis of the β-amylase fraction showed a single band at pH 6.0 only in samples from IMLNS patients in relapse. The band was composed of two proteins, β-amylase and a 100-kDa glycoprotein. Fusion of foot processes was observed only when the β-amylase fraction from IMLNS patients in relapse was infused. Conclusions: The infusion of the β-amylase fraction containing a 100-kDa glycoprotein from IMLNS patients in relapse induced proteinuria and effacement of foot processes in the rat. This protein may play a role in the pathogenesis of IMLNS.

Cytokine mRNA Profile in Lipoid Nephrosis: Evidence for Increased IL-8 mRNA Stability

Background/Aims: Proteinuria in idiopathic minimal lesion nephrotic syndrome (IMLNS) is presumed to be due to the effect of circulating factors on glomerular permeability to plasma proteins. This study examines the expression of messenger ribonucleic acid (mRNA) for cytokines thought to mediate glomerular inflammation during different stages of the nephrotic syndrome. Methods: Messenger RNA expression and stability from peripheral blood mononuclear cells of IMLNS patients in relapse and in remission, and age matched normal controls were measured using a ribonuclease protection assay (RPA). The spontaneous and Interleukin 2 (IL-2) stimulated mRNA expression were studied. Results: Spontaneous mRNA expression for Interleukin 8 (IL-8) from IMLNS patients in relapse was significantly increased when compared to IMLNS patients in remission and normal controls (p < 0.05). After 14 h of IL-2 stimulation, mRNA IL-8 levels expressed by IMLNS PBMC patients in remission were not different from those observed in normal controls. However, after 5 days of PBMC incubation, a significant increase in mRNA for IL-8 in IMLNS patients compared to controls was found (p < 0.01). Stability assay demonstrated that IL-8 mRNA transcript from the nephrotic patients remained higher than those from controls and showed a significantly prolonged life t_1/2 (p = 0.02). Conclusions: IL-8 mRNA expression is increased in IMLNS patients in relapse. Moreover, stability studies show that IL-8 mRNA life t_1/2 is prolonged due to altered post-transcriptional regulation. This finding may explain the elevated serum IL-8 levels observed in these patients during relapse and may have pathogenic significance since IL-8 has been shown to induce proteinuria in the experimental animal.

Circulating mediators of proteinuria in idiopathic minimal lesion nephrotic syndrome.

The pathogenesis of proteinuria in idiopathic minimal lesion nephrotic syndrome (IMLNS) remains to be elucidated. The most-accepted hypothesis is that the increased glomerular permeability to plasma proteins results from the effect of circulating factors on glomerular capillaries. This report critically reviews the current studies that have attempted to isolate and characterize this putative factor(s). Products released from hepatocyte or peripheral blood mononuclear cells or isolated by chromatography from serum or plasma have been tested in rats for their role in inducing proteinuria. These factors have been infused into the isolated kidney preparation or into the intact animal as a single venous injection, or continuously by pump for a period of 4 h to 7 days. Several of these isolated factors have been shown to induce proteinuria in rats. However, their exclusive pathogenetic role is questionable since none is always present in all IMLNS patients during relapse. Therefore, the increase in proteinuria in these patients may result from a single or a variety of factors as yet to be identified.

Cytokine from Peripheral Blood Mononuclear Cells (PBMC) from Idiopathic Minimal Lesion Nephrotic Syndrome (ILMNS) Patients in Relapse Induces Albuminuria and Fusion of Foot Processes in Vivo in the Rat

Cytokine from Peripheral Blood Mononuclear Cells (PBMC) from Idiopathic Minimal Lesion Nephrotic Syndrome (ILMNS) Patients in Relapse Induces Albuminuria and Fusion of Foot Processes in Vivo in the Rat

Anti-interleukin 8 antibody abolishes effects of lipoid nephrosis cytokine.

Supernatant factor from peripheral blood mononuclear cell (PBMC) cultures of idiopathic minimal lesion nephrotic syndrome (IMLNS) patients in relapse induces in vivo albuminuria and increases 35sulfate uptake by glomerular basement membrane (GBM) in rats. The purpose of this study was to evaluate the effect of anti-interleukin 8 (IL8) neutralizing antibody on the effects induced by the supernatant factor. Supernatant factor collected from six cultures of PBMC from IMLNS patients in relapse were combined and aliquotted into two samples. Anti-IL8 neutralizing antibody (750 ng) was added to one. Supernatant factor or supernatant factor and anti-IL8 antibody were infused for 5 days into the left renal artery of Wistar rats using an osmotic pump. On the last day of infusion, rats were injected with 35sulfate (1.0 mCi/200 g) intraperitoneally and killed after 8 h. Glomeruli were isolated and GBM obtained. There was a significant increase in 35sulfate uptake of the infused kidney (169 +/- 52 cpm/mg dry glomerular weight, mean +/- SEM) compared with the uptake seen in the contralateral kidney (116 +/- 41, P < 0.05) when the supernatant factor was infused alone. No significant differences in 35sulfate incorporation were seen between infused kidney (173 +/- 5) and contralateral kidney (190 +/- 49) when supernatant factor and anti-IL8 antibody were administered. A significant increase in albuminuria was seen on the last day of infusion (0.43 +/- 0.11 albumin/ creatinine ratio, mean +/- SEM) compared with the ratio prior to infusion of the supernatant factor alone (0.18 +/- 0.03, P <0.05). No significant differences in urinary albumin/creatinine ratios prior to and on the 5th day of infusion were seen when the supernatant factor was administered with anti-IL8 antibody. Supernatant factor effects were abolished by the addition of anti-IL8 neutralizing antibody, suggesting that the described effects are mediated by IL8.

Failure to Down Regulate Interleukin 8 (IL8) mRNA in Patients with Idiopathic Minimal Lesion Nephrotic Syndrome (IMLNS) 1804

Failure to Down Regulate Interleukin 8 (IL8) mRNA in Patients with Idiopathic Minimal Lesion Nephrotic Syndrome (IMLNS) 1804

The effect of lymphocytes from renal transplant patients on glomerular basement membrane sulfate uptake

We have studied the effect of peripheral blood mononuclear cells (PBMC) from renal transplant patients on 35sulfate uptake by rat glomerular basement membrane (GBM). Nine patients were included in the study; six were studied during an episode of acute allograft rejection and seven while not undergoing acute rejection. The sulfate uptake index, calculated as the ratio between uptake by GBM from rat glomeruli cocultured with PBMC and 35sulfate incorporation by GBM from glomeruli cultured alone, was significantly higher when glomeruli were cocultured with PBMC from patients undergoing an acute rejection (3.26 +/- 1.18, mean +/- SEM) than it when glomeruli were cocultured with PBMC from nonrejecting transplant patients not showing proteinuria (0.81 +/- 0.11) (P = 0.0053). After reversing the acute allograft rejection with resolution of proteinuria, the sulfate uptake index returned to normal in four of five patients. The fifth patient had persistent nephrotic syndrome and his sulfate uptake index remained elevated. These findings are similar to those observed in idiopathic minimal lesion nephrotic syndrome patients in relapse. Because the GBM sulfated compounds may play a role in glomerular permeability to plasma proteins, by acting on these compounds PBMC may be a common mechanism for proteinuria.

IL-8 production by peripheral blood mononuclear cells in nephrotic patients

We studied the interleukin 8 (IL-8) gene expression by peripheral blood mononuclear cells (PBMC) and the IL-8 serum concentration in patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) and other glomerulopathies. PBMC from eight of the nine (IMLNS) patients in relapse demonstrated the presence of IL-8 mRNA. All three IMLNS patients in remission (P = 0.0026 when compared to patients in relapse) and the two patients with nephrotic syndrome with other glomerulopathies failed to elicit an IL-8 mRNA response. Eleven of the 12 IMLNS patients in relapse showed IL-8 serum concentration above the level of detection. Only one of the seven patients in remission had detectable serum levels of IL-8 (P = 0.0033 when compared to levels from IMLNS patients in relapse). IL-8 serum levels were not detectable in three patients with nephrotic syndrome and other glomerulopathies. Supernatants of PBMC cultures from IMLNS patients in relapse increased the 35sulfate uptake by rat GBM. This effect was abolished by the addition of anti-IL-8 neutralizing antibody to the culture media and reproduced by the addition to the media of IL-8 in concentrations found in the serum of IMLNS patients in relapse. Finally, the effect of IL-8 on the 35sulfate turnover of the glomerular basement membrane (GBM) sulfated compounds was evaluated in vitro. A significant decrease in the percentage of residual 35sulfate incorporated in the GBM (41 +/- 5, mean +/- SEM) was observed in cultures treated with IL-8 as compared to those that were not treated with IL-8 (58 +/- 8, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of lymphokine from nephrotic peripheral blood mononuclear cells on catabolism of rat glomerular basement membrane sulfated compounds.

We have previously shown a significant increase in sulfate-35 uptake in rat glomerular basement membrane (GBM) when glomeruli were cultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse. In the present study, we have isolated the lymphokine mediating the augmented sulfate-35 incorporation and evaluated its effect on the catabolism of the GBM sulfated compounds. Supernatants from IMLNS PBMC cultures of 13 patients in relapse and 10 in remission were fractionated using gel filtration chromatography. There was a significant increase in rat GBM sulfate-35 uptake when glomeruli were cultured in carbonic anhydrase fraction from patients in relapse (12.9 +/- 3.2; cpm/microgram GBM protein, mean +/- SEM) as compared to glomeruli cultured in the same fraction from patients in remission (8.2 +/- 2.5: cpm/microgram GBM protein; p < 0.05). The catabolism of the GBM sulfated compounds was determined by studying the washout of the sulfate-35 macromolecules after equilibration in sulfated isotope-free medium for 12 h. There was a significant decrease in residual sulfate-35 in rat GBM when glomeruli were cultured in a 29-kD fraction from patients in relapse (7.0 +/- 2.5; cpm/micrograms GBM protein, mean +/- SEM) as compared to glomeruli cultured in the same fraction from patients in remission (31.8 +/- 1.6; p < 0.005). No significant differences in sulfate-35 incorporation were seen when other fractions from patients in relapse and in remission were compared. These studies suggest that the lymphokine secreted by PBMC from IMLNS patients in relapse increases the catabolism of the GBM sulfated compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prednisone on nephrotic peripheral blood mononuclear cell mediated increase in 35sulfate uptake in rat glomerular basement membrane.

We have previously described a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal-lesion nephrotic syndrome (IMLNS) in relapse, but not with PBMC of IMLNS patients in remission. In the present study we examined the effect of prednisone therapy on the PBMC-mediated increase in 35sulfate GBM uptake. The GBM 35sulfate uptake after rat glomeruli were cocultured with PBMC from 11 IMLNS patients in relapse (geometric mean 437 cpm/mg dry glomerular weight) was significantly higher than the incorporation observed in glomeruli cultured alone (geometric mean 229 cpm/mg dry glomerular weight; p less than 0.01). However, no significant differences in 35sulfate uptake were seen between glomeruli cultured alone and glomeruli cocultured with PBMC from IMLNS patients when PBMC were obtained from the 11 patients on treatment with prednisone (2 mg/kg/day) or the same patients in remission and off prednisone therapy. Prednisone therapy abolished the PBMC-mediated increased 35sulfate uptake by rat GBM. GBM sulfated compounds seem to play a role in glomerular permeability. The temporal relationship between inhibition of GBM sulfate incorporation by prednisone and resolution of the proteinuria support the hypothesis that PBMC from IMLNS patients in relapse could secrete a lymphokine which by altering the metabolism of the GBM sulfated compounds may subsequently increase glomerular permeability to plasma proteins.

Effect of concanavalin A on nephrotic peripheral blood mononuclear cells mediated increased 35sulfate uptake in rat glomerular basement membrane.

We have previously shown a significant increase in 35sulfate uptake in rat glomerular basement membrane (GBM) when glomeruli were cocultured with peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse, but an uptake not different than normal controls if glomeruli were incubated with PBMC of patients in remission. In the present study we examined 35sulfate uptake by GBM after PBMC from 12 IMLNS patients in remission were stimulated with Concanavalin A (Con A) (10 micrograms/ml of culture media). There was a significant increase in 35sulfate GBM uptake when glomeruli were cocultured with Con A-stimulated IMLNS PBMC (geometric mean), 331 cpm/mg dry glomerular weight) as compared to glomeruli cocultured with IMLNS PBMC (geometric mean, 200) (p = 0.048); glomeruli alone stimulated with Con A (geometric mean, 182) (p = 0.008) or glomeruli alone (geometric mean, 146) (p = 0.002). No significant differences were seen between the groups when glomeruli were cocultured with PBMC from 12 normal adults. These data show that Con A stimulated PBMC from IMLNS patients in remission alter the sulfate metabolism of rat GBM. The stimulation of PBMC with Con A reproduces the increase in 35sulfate uptake observed when glomeruli are cocultured with PBMC from IMLNS in relapse. Sulfated compounds in the GBM may play a role in glomerular permeability. Since stimulated nephrotic PBMC alter the metabolism of GBM sulfated compounds, these findings may have pathogenic significance.

Effect of supernatants from nephrotic peripheral blood mononuclear cells on 35sulfate incorporation in rat glomerular basement membrane.

In previous research, we showed that when peripheral blood mononuclear cells (PBMC) from patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) in relapse were cocultured with rat glomeruli, there was an increased glomerular basement membrane (GBM) uptake of 35sulfate. This study was done to determine whether the increased uptake was due to substances secreted into the nephrotic PBMC culture supernatants. 35sulfate uptake in rat GBM was significantly higher when glomeruli were cocultured with PBMC from 12 IMLNS patients in relapse (geometric mean [GM] = 513 cpm/mg) than when simultaneous assays were done using either PBMC from eight control subjects (GM = 158) (p less than 0.05) or glomeruli incubated without PBMC (GM = 275 cpm/mg) (p less than 0.01). 35sulfate uptake did not increase when glomeruli were cocultured with PBMCs from 11 MLNS patients in remission. Rat GBM 35sulfate uptake was significantly higher when glomeruli were incubated in the supernatants of the PBMC cultures from 16 IMLNS patients in relapse (GM = 234 cpm/mg) than it was when glomeruli were cultured in the supernatants from normal control PBMC (GM = 141 cpm/mg; p less than 0.002) or from glomeruli cultured alone (GM = 141 cpm/mg) (p less than 0.04). Supernatants from PBMC cultures of 11 IMLNS patients in remission did not increase rat GBM 35sulfate uptake. These data suggest that PBMC from IMLNS patients in relapse secrete a factor(s) released into supernatants that increases the 35sulfate rat GBM uptake. Since sulfated compounds in the GBM play a role in glomerular permeability, this finding may have pathogenic significance.

EFFECT OF LYMPHOCYTES FROM NEPHROTIC PATIENTS ON UPTAKE OF 35S BY GLOMERULAR BASEMENT MEMBRANE

Patients with idiopathic minimal lesion nephrotic syndrome (IMLNS) have an increase in glomerular permeability to proteins due to the loss of fixed negative charges in the glomerular basement membrane (GBM). Heparan sulfate is the main compound carrying the anionic sites; SO4 in this molecule represents an important source of the negative charges.We have studied the effect of peripheral blood lymphocytes (PBL) from nephrotic patients on the incorporation of 35S in rat GBM. 35S uptake increased when glomeruli were incubated with lymphocytes from 10 patients with IMLNS in relapse compared to simultaneous assays on 5 controls (2295 ± 634 cpm/mg glomerular weight, mean ± SEM, and 962 ± 248 cpm/mg, respectively) (P<0.02).35S incorporation did not increase when glomeruli were incubated with PBL from seven patients with other glomerulopathies (1016 ± 503 cpm/mg) as compared to controls (1283 ± 642 cpm/mg) or from eight IMLNS patients in remission (822 ± 435 cpm/mg) as compared to controls (1475 ± 608 cpm/mg). No significant differences in 35S uptake were seen between glomerular cultures with and without control PBL.These data suggest a role for PBL in the pathogenesis of IMLNS, since the increased 35S incorporation may be the result of increase catabolism of proteoglycans in the GBM. This may subsequently lead to decrease in anionic sites and increase in glomerular permeability.

Indomethacin in the treatment of idiopathic minimal lesion nephrotic syndrome

Ten patients with idiopathic minimal lesion nephrotic syndrome were treated with indomethacin. Nine patients received the drug for five weeks and one patient for nine weeks. Indomethacin was administered in a dose of 1 mg/kg/day during the first week and 2 mg/kg/day, thereafter. At the end of the study, nine patients remained edematous and all had a urinary protein excretion greater than 50 mg/kg/day, within the nephrotic range. Indomethacin did not change the course of the disease and its use in the treatment of IMLNS is not advised.