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 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)     

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.     

Heparanase activity is dysregulated in children with steroid-sensitive nephrotic syndrome

BACKGROUND: Immune cells express heparanase, an endoglycosidase, able to degrade heparan sulfate glycosaminoglycan (HSGAG) in the glomerular capillary wall (GCW) and potentially induce proteinuria. The aim of this study was to determine whether dysregulated heparanase expression is associated with the heavy proteinuria of childhood steroid-sensitive nephrotic syndrome (SSNS). METHODS: Plasma and urinary heparanase activity and peripheral blood mononuclear cell (PBMC) mRNA heparanase levels [real-time polymerase chain reaction (PCR)] were measured in children with SSNS in relapse and remission. Plasma and urinary heparanase activity was determined in adult patients with nephrotic syndrome and in age- and gender-matched controls. RESULTS: Plasma heparanase activity was reduced in SSNS with relapse (811.2 units) compared to remission (1147.96 units) (P= 0.003) and control subjects (1390.51 units) (P < 0.001). In adult nephrotic syndrome, plasma heparanase activity was significantly lower in patients compared to controls. However, there was no difference between remission and relapse states. In children, urinary heparanase activity/urinary creatinine ratio was highest in SSNS relapse (14.26 units/mg) compared with remission (7.43 units/mg) (P= 0.016) and controls (2.29) (P < 0.001). However, PBMC heparanase mRNA expression was not different between these three groups. In adult nephrotic syndrome, urinary heparanase activity/urinary creatinine levels were lower in both remission and relapse compared to controls and there was no difference between remission and relapse states. CONCLUSION: In childhood SSNS, there is a qualitative and quantitative difference in urinary heparanase activity expression that is not paralleled in adult nephrotic syndrome. These data suggest that dysregulated heparanase expression may play a significant role in the pathogenesis of SSNS, possibly through an abnormality in post-translational control of latent heparanase activation.     

Interleukin-8 and tumor necrosis factor-alpha are increased in minimal change disease but do not alter albumin permeability

AIMS: Minimal change disease (MCD) is the most common primary nephrotic syndrome in children. Some suggested that interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) are involved in the pathogenesis of MCD. This study was done to see changes of plasma and urinary IL-8, TNF-alpha, and their effects on determination of permeability of glomerular basement membrane (BM) contributed by heparan sulfate proteoglycan (HSPG). METHODS: Study patients consisted of 19 biopsy-proven MCD children aged 2-15 years old. Both plasma, urinary IL-8 and TNF-alpha were measured. Employing the Millicell system, IL-8 and TNF-alpha were screened for the permeability factors. We examined whether IL-8 and TNF-alpha regulated BM HSPG gene expression and HS synthesis in the glomerular epithelial cells (GECs). RESULTS: Urinary IL-8 during relapse was significantly increased when compared with that of during remission or controls (13,996 +/- 2,811 vs. 2,941 +/- 373, 5,331 +/- 640 ng/mg.cr) (p < 0.05). Urinary TNF-alpha during relapse was also significantly increased (364.4 +/- 51.2 vs. 155.3 +/- 20.8, 36.0 +/- 4.5 ng/mg.cr) (p < 0.05). Plasma IL-8 during relapse was significantly increased compared to that during remission(1.19 +/- 0.62 vs. 0.51 +/- 0.42 ng/ml) (p < 0.05). However, the negative results were obtained in the permeability assay using the Millicell system. No difference was seen in BM HSPG gene expression and HS synthesis in the GECs. CONCLUSION: Therefore, it seems that both IL-8 and TNF-alpha may not play a disease-specific role in the pathogenesis of MCD.     

Effect of lipoid nephrosis cytokine on glomerular sulfated compounds and albuminuria

Supernatants from peripheral blood mononuclear cells cultures of 30 idiopathic, minimal lesion, nephrotic syndrome (IMLNS) patients in relapse and the same patients in remission were fractionated by gel filtration chromatography. Fractions eluting with carbonic anhydrase (29 kilodaltons) were infused for 5 days at the rate of 10 l/h into the left renal artery of Wistar rats using an Alzet osmotic pump. On the last day of infusion, rats were injected with³⁵sulfate (1.0 mCi/200 g) intraperitoneally and killed after 8 h. Glomeruli were isolated and glomerular basement membrane (GBM) obtained. There was a significant increase in³⁵sulfate uptake by GBM of the infused kidney (302±92 cpm/mg dry glomerular weight, mean±SEM) compared with the uptake seen in the contralateral kidney (157±36,P<0.01) when the fraction from IMLNS patients in relapse was infused. No significant differences in³⁵sulfate incorporation were seen between infused kidney (166±41) and contralateral kidney (172±64) when the same fraction from patients in remission was administered. A significant increase in albuminuria was seen on the last day of infusion (14.2±1.0 mg/24 h, mean±SEM) when supernatant factor from IMLNS patients in relapse was used. No significant differences in urinary albumin excretion prior to and after infusion were seen when the same fraction from IMLNS patients in remission was administered. The in vivo infusion of supernatant factor from IMLNS patients in relapse increased the³⁵sulfate uptake by GBM and augmented albuminuria, suggesting that the factor may have pathogenic significance in the proteinuria of IMLNS.     

T-cell subsets,interleukin-2 receptor expression and production of interleukin-2 in minimal change nephrotic syndrome

This study was designed to investigate T-lymphocyte subsets interleukin-2 receptor (IL-2R) expression and IL-2 production in minimal change nephrotic syndrome (MCNS). Peripheral blood T-lymphocytes and IL-2R expression were analysed using fluorescein isothiocyanatelabelled CD3, CD4, CD8 and CD25 monoclonal antibodies with flow cytometry. IL-2 production was determined by enzyme immunoassay. Ten children with MCNS in relapse and in remission were evaluated. Thirteen healthy children served as controls. The patients in relapse demonstrated a moderate decrease in the total absolute lymphocyte counts and CD8(+) T-lymphocytes compared with controls (P<0.05) and had a greatly increased IL-2R expression in frashly isolated, unstimulated peripheral lymphocytes compared with patients in remission and controls. While this was not statistically significant, IL-2R expression on cultured lymphocytes stimulated with phytohaemagglutinin was significantly elevated in relapse compared with those in remission and controls (P<0.05). IL-2 production did not correlate well with IL-2R expression and there was no significant difference between the groups. Our results suggest that T-cell subset changes and high IL-2R expression on peripheral lymphocytes may indicate the presence of stimulated T-cell populations in MCNS which could contribute to the immunopathogenesis.     

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.     

Interleukin-12 and interferon-γ production in childhood idiopathic nephrotic syndrome

Cellular immune disturbances, and T lymphocyte function in particular, have been previously implicated in idiopathic nephrotic syndrome (INS) of childhood. There are different patterns of cytokine expression in various forms of glomerulonephritis, which suggests that local production of these peptides plays an important role in the pathogenesis and progression of glomerulonephritis. To investigate T-cell and monocyte/macrophage cytokine production in INS, interleukin-12 (IL-12) and interferon-γ (IFN-γ) production by peripheral blood mononuclear cells (PBMC) of 11 children with steroid-sensitive nephrotic syndrome (SSNS), 9 with focal segmental glomerulosclerosis (FSGS), and 17 healthy controls was determined. Children with SSNS were studied in relapse, during corticosteroid treatment, and in stable remission, off corticosteroid treatment. IL-12 was not detected in serum, urine, and in supernatants of unstimulated PBMC. IL-12 production by concanavalin A (Con A)-stimulated PBMC of children with SSNS and FSGS was not different from controls. IFN-γ production by Con A-stimulated PBMC was decreased in children with relapsing SSNS, both in relapse and and during corticosteroid treatment. However, in stable remission it was similar to controls. Markedly decreased IFN-γ production (P<0.001) was observed by pokeweed mitogen-stimulated PBMC of relapsing SSNS patients and moderately decreased production by PBMC of FSGS patients. This study has established a decreased production of IFN-γ by PBMC of relapsing SSNS and FSGS patients, but does not allow differentiation between these two different conditions. IL-12 did not have a pathogenic role in either SSNS or FSGS. Received May 24, 1997; received in revised form March 19, 1998; accepted March 23, 1998     

Serum from minimal change patients in relapse increases CD80 expression in cultured podocytes

Background

Minimal change disease (MCD) is the most common cause of nephrotic syndrome in children and is associated with the expression of CD80 in podocytes and the increased excretion of CD80 in urine. We hypothesized that serum from patients with MCD might stimulate CD80 expression in cultured podocytes.

Methods

Sera and peripheral blood mononuclear cells (PBMCs) were collected from subjects with MCD in relapse and remission and from normal controls. Immortalized human podocytes were incubated with culture media containing patient sera or supernatants from patient and control PBMC cultures. CD80 expression was measured by quantitative PCR and western blot analysis.

Results

Sera collected from patients with MCD in relapse, but not in remission, significantly increased CD80 expression (mean ± standard deviation: 1.8?±?0.7 vs. 0.8?±?0.2; p?<?0.004) and CD80 protein secretion by podocytes (p?<?0.05 between relapse and normal controls). No such CD80 increase was observed when podocytes were incubated with supernatants of PBMC cultures from patients in relapse.

Conclusions

Sera from MCD patients in relapse, but not in remission, stimulated CD80 expression in cultured podocytes. Identifying this factor in sera could provide insights into the pathogenesis of this disorder. No role in CD80 expression by podocytes was found for cytokines released by PBMCs.     

Augmented interleukin-18 production by peripheral blood monocytes in patients with minimal-change nephrotic syndrome

BACKGROUND/AIM: The etiology of minimal-change nephrotic syndrome (MCNS) is poorly understood. It has been proposed that cell-mediated immunity and T-cell activation are key features of this glomerular disease. Interleukin (IL)-18, a novel interferon-gamma-stimulating factor, may act as an important effector molecule involved in various immune responses. To our knowledge, very little is known about the involvement of IL-18 in NCNS. The aim here was to define further the involvement of IL-18 in MCNS. METHODS: To understand the role of this cytokine, in vitro IL-18 levels were analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA) method in 16 patients with MCNS who were either in a stable or active condition. The disease controls included 16 patients with IgA nephropathy (IgAN). The IL-18 levels were compared with values in healthy controls. RESULTS: Significantly increased spontaneous and lipopolysaccharide (LPS)-stimulated production of IL-18 was detected in peripheral blood monocyte (PBM) cultures of MCNS patients with the nephrotic syndrome (NS) as compared with those of normal controls. Moreover, when individual MCNS patients were followed through their clinical illness, IL-18 levels were increased during the active phase and normalized as the patients went into remission. The amounts of IL-18 are significantly correlated with the levels of vascular permeability factor (VPF) in MCNS patients. CONCLUSIONS: Thus, in MCNS patients, the level of IL-18 was increased and this increase was related to the activity of this disease. The data provide circumstantial evidence for a role of IL-18 in MCNS.     

Transcriptional and post-transcriptional alterations of IkappaBalpha in active minimal-change nephrotic syndrome.

Minimal-change nephrotic syndrome (MCNS) is a renal disease characterized by heavy glomerular proteinuria and increased production of cytokines by immune cells. Because of the central role of nuclear factor-kappaB (NF-kappaB) in the regulation of cytokine expression, its activity during the relapse and remission phases of steroid-sensitive MCNS was analyzed. During relapse, nuclear extracts from peripheral blood mononuclear cells displayed high levels of NF-kappaB DNA-binding activity, consisting primarily of p50/RelA (p65) complexes. NF-kappaB p65 and IkappaBalpha proteins were barely detected or not detected in cytosolic fractions during relapse, in contrast to remission. The lack of expression of IkappaBalpha protein was associated with downregulation of IkappaBalpha mRNA and increases in the levels of the mRNA encoding the proteasome alpha2 subunit proteolytic pathway. In addition, inhibition of proteasome activity induced cytosolic accumulation of phosphorylated IkappaBalpha and significant reductions in the NF-kappaB binding activity in nuclear extracts from peripheral blood mononuclear cells from patients experiencing relapses. These results suggest that alterations in the NF-kappaB/IkappaBalpha regulatory feedback loop may contribute to the immunologic abnormalities that occur in steroidsensitive MCNS.     

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. Received: 1 September 1999 / Revised: 4 November 1999 / Accepted: 4 November 1999     

T cell CD3 receptor zeta (TCRζ)-chain expression in children with idiopathic nephrotic syndrome

Children with idiopathic nephrotic syndrome (INS) have an increased risk of developing life-threatening infections. Several studies have demonstrated functional abnormalities in the T lymphocytes of patients with nephrotic syndrome. Although T cells are activated in INS during relapse, as indicated by an increased expression of interleukin (IL)-2 receptor, these cells have a decreased ability to proliferate. The T-cell receptor (TCR) plays an important role in signal transduction and T cell activation, with the TCR-zeta (TCRζ) chain being a key element in early signaling. We measured the expression of the TCRζ chain in patients with INS (steroid resistant and steroid sensitive) during relapse and remission by flow cytometry and by PCR ELISA. The results showed a significant decrease in the expression of the TCRζ chain at both the protein and mRNA level in INS patients during relapse as compared with normal controls (p < 0.05). In contrast, when patients with INS achieved remission, the expression of TCRζ normalized and was similar to that expressed in normal controls. Therefore, a decreased expression of the TCRζ chain may explain the abnormal function of T cells in patients with INS, and it may also contribute to the increased risk for infections seen in these patients.     

T-lymphocyte subsets in nephrotic syndrome.

T-lymphocyte subsets when measured in steroid responsive nephrotic syndrome (SRNS) have demonstrated significant variance from normal values. T-cell subsets were studied by using two-color flow cytometric analysis in 32 children (9.2 +/- 5 years of age) with SRNS. The children were divided into four groups: a) SRNS in acute relapse, on prednisone; b) SRNS in acute relapse, off prednisone; c) SRNS in long-term remission, off prednisone (nephrotic controls); d) patients in remission on long-term prednisone therapy; and e) 15 age-matched normal controls. Children suffering an acute relapse of SRNS showed an increase in Leu2a+/DR+ (CD8) activated lymphocytes (P less than 0.05), a decrease in Leu4a+ total T-lymphocytes (P = 0.01) and a decrease in Leu3a+ (CD4) helper T-cells (P less than 0.05) when compared to normal controls and nephrotic controls. Though some subset changes may represent a prednisone effect and the functional role of these lymphocytes in the disease process is unknown, this study provides additional evidence to support a role for abnormal T-cell subsets in the etiology of SRNS.