Two-color analysis of lymphocyte subpopulations in membranous nephropathy and minimal change nephrotic syndrome

Two-color flow cytometry was carried out to determine the correlation between cell mediated immunity and the development of the nephrotic stage in patients with membranous nephropathy (MN) and minimal change nephrotic syndrome (MCNS). In this study, lymphocyte subpopulations were measured by two-color flow cytometry using various monoclonal antibodies of the Leu series. Thirty patients with MN and 25 patients with MCNS were examined. Clinically, those patients were divided into four stages as follows: (1) untreated nephrotic stage, (2) prednisolone (PSL) treated nephrotic stage, (3) persistent proteinuria stage (incomplete remission, ICR), and (4) complete remission (CR). Pathologically, the patients with MN also divided into four stages I-IV, according to Churg's classification. The values of the Leu 3a/Leu 2a ratio in patients in the untreated nephrotic stage of MN and MCNS were significantly higher than those in the remission stage in both diseases (P less than 0.01, P less than 0.05, respectively). Two-color flow cytometry showed that the reduction of Leu2a positive cells was mainly due to a decrease of Leu 2a+15+ subsets (suppressor T cells) in the untreated nephrotic stage and relative increase of Leu 3a+8+ subsets (suppressor inducer T cells). There was no significant difference in these findings among the histopathological stages in patients with MN. Patients with MN and MCNS showed a significant elevation of Leu 2a+DR+ cells after the treatment of PSL. The abnormalities of suppressor T cells and suppressor inducer T cells in the peripheral blood appear to be correlated with clinical activities of the nephrotic syndrome due to MN or MCNS, but not to be related to the pathogenesis of either disease. It is postulated that PSL might stimulate Leu 2a positive cells and Leu 3a positive cells, and then increase the number of Leu 2a+15+ cells in the peripheral blood of patients with MN and MCNS.     

Rituximab treatment of idiopathic membranous nephropathy

Idiopathic membranous nephropathy is a common cause of nephrotic syndrome whose pathogenesis may involve B-cell functions. Rituximab is a monoclonal antibody that binds to the CD20 antigen on B cells thereby deleting them. We conducted an open-label pilot trial of rituximab treatment in 15 severely nephrotic patients with proteinuria refractory to angiotensin-converting enzyme inhibition and/or receptor blockade but with adequately controlled blood pressure. Rituximab was given 2 weeks apart and, at 6 months, patients who remained proteinuric but had recovered B-cell counts were given a second course of treatment. Proteinuria was significantly decreased by about half at 12 months. Of the 14 patients who completed follow-up, full remission was achieved in two and partial remission in six patients based upon the degree of proteinuria. Side effects were minor; however, we found no relationship between the response and number of B cells in the blood, CD20 cells in the kidney biopsy, degree of tubulointerstitial fibrosis, starting proteinuria or creatinine values. Rituximab appears effective in reducing proteinuria in some patients with idiopathic membranous nephropathy but prospective identification of responsive patients was not possible.     

Long-term outcome of patients with membranous nephropathy after complete remission of proteinuria

To assess the prognostic significance of complete remission in patients with idiopathic membranous nephropathy, 33 patients were followed for a median of 96 months after remission of proteinuria. All patients had had a histological diagnosis of membranous nephropathy and a nephrotic syndrome. Only patients with a complete remission lasting for at least six months and with a follow-up of at least four years after remission were considered. No relapse of proteinuria developed in 17 patients (51%), 7 patients had relapse of non-nephrotic proteinuria (21%) and 9 (27%) relapse of nephrotic proteinuria. However proteinuria disappeared again in some patients so that at follow-up 73% of patients are in complete remission, 21% have non-nephrotic proteinuria and only 6% have nephrotic syndrome. All patients maintained a normal plasma creatinine over the years. It is concluded that complete remission of proteinuria is a strong predictor of long-term favourable outcome in patients with idiopathic membranous nephropathy.     

Relationship between interstitial infiltrates and steroid responsiveness of proteinuria in membranous nephropathy

Mononuclear inflammatory cells were retrospectively analysedusing monoclonal antibodies in the interstitium and glomeruliof 16 renal biopsy specimens from patients with nephrotic syndromedue to idio-pathic membranous nephropathy (IMN). The aim ofthe study was to determine the composition of the infiltrateand to assess the ability to predict the response of proteinuriato corticosteroids. All patients had received prednisolone asa sole treatment. Nine patients had shown a complete or partialremission of proteinuria (group A) and seven did not respondat all (group B). Both groups were matched for age and degreeof proteinuria; also both groups had normal renal function atthe time of biopsy. Very few intraglomerular leukocytes, mostlymonocytes/ macrophages (MM) were found. The majority of interstitialcells were T lymphocytes and MM. CD4+ve T helper/inducer cellspredominated among the interstitial T cell population and Bcells were a minor component. No significant differences werefound between the two groups regarding the types of the intraglomerularcells. However, interstitial T-cells, CD4 + ve T helper/inducercells, CD8+ve T cytotoxic/suppressor cells and MM were significantlyhigher in group A than in group B. Also HLA-DR expressing interstitialcells were much in excess in group A. In addition patients with complete remission of proteinuriahad higher numbers of interstitial cells compared to those withpartial response. There was no correlation between the numbersof types of intraglomerular and interstitial cells and the degreeof proteinuria at presentation. Also no association was foundbetween intraglomerular or interstitial cell population andsubsequent relapse of proteinuria. In conclusion, interstitial but not intraglomerular mononuclearcells seem to determine the initial response of proteinuriato corticosteroids in patients with IMN.     

Decreased production and responsiveness of interleukin 2 in lymphocytes of patients with nephrotic syndrome

Production of and responsiveness to interleukin 2 (IL-2) were evaluated in lymphocytes from 18 patients with nephrotic syndrome. The IL-2 production of T cells, when stimulated with autologous non-T cells separated on a polystyrene resin column, was significantly decreased in patients with minimal-change nephrotic syndrome. No significant difference in the level of IL-2 production was noted between minimal-change nephrotic syndrome patients in the nephrotic stage and those in remission. The IL-2 production was also significantly decreased in lymphocytes from patients with membranous nephropathy. The responsiveness to IL-2 was inconstant among patients with nephrotic syndrome. These results indicate that the immune system of regulating IL-2 production is impaired in patients with nephrotic syndrome.     

A clinical study of immunological status in patients with gastric cancer: with special reference to T-cell subsets, interleukin-2 in the lymphocytes of peripheral blood

Peripheral blood lymphocytes of 63 patients with gastric cancer were studied by using different monoclonal antibodies and flow cytometry. Used monoclonal antibodies were OKT3 (total T cell), OKT4 (helper/inducer), OKT8 (suppressor/cytotoxic), Leu 7 and Leu 11 (NK/K cell). Interleukin-2 was measured by tritium-labelled thymidine CTLL assay on the supernatant of peripheral blood lymphocytes after 24 hours stimulation with phytohemagglutinin. Interleukin-2 receptor was also studied by using monoclonal antibody (for Tac antigen) and flow cytometry. The results were as follows: among peripheral blood lymphocytes; 1. the number of OKT3, OKT4 cells and the percentage of OKT4 cells decreased significantly with more advanced stage of cancer. 2. production of interleukin-2 also decreased with the progression of the cancer. 3. decreases in the OKT4/OKT8 ratio were found with cancer progression. 4. the percentage and the number of OKT8 cells increased. 5. the percentage and the number of Leu 11 cells and the number of Leu 7 cells were increased significantly in the stage III (moderately advanced cancer). These results suggested that the activated helper T cells decreased, the induction capability of cytotoxic T cells decreased and the suppressor T cells increased with the progression of cancer. Quantitative and qualitative change in T-cell subsets in advanced stage may be one factor responsible for immunosuppression.     

Two-color flow cytometric analysis of splenic lymphocyte subpopulations in patients with gastric cancer

Lymphocyte subpopulations of the spleen were assayed in 26 patients with gastric cancer and 5 patients with benign disease using two-color flow cytometric analysis. The ratio of Leu 2a+·Leu 15+ cells, or suppressor T cells, in the gastric cancer patients was about 6 per cent, being higher than that in the patients with benign disease (p<0.05). There were fewer Leu 7+·Leu 11– cells, or natural killer-NK-cells, in the gastric cancer patients in stage III or IV than in those with stages I or II (p<0.05). The ratio of Leu 3a+·Leu 8– cells, or helper T cells, in the stage IV patients accounted for about 15 per cent of the splenic lymphocytes, which was less than that seen in the patients in stages I or II (p<0.05). The ratio of Leu 2a+·Leu 15– cells, or cytotoxic T cells, was approximately twice that of suppressor T cells. The pre-operative administration of lentinan plus OK-432 increased the ratio of Leu 4+·HLA-DR+ cells, or activated T cells, and cytotoxic T cells (p<0.05 and p<0.01, respectively). The above results suggest that lymphocyte subpopulations in the spleen may have more immuno-suppressive potential in proportion with the stage of gastric cancer, but that this reduced immune state may be altered when lentinan and OK-432 are given to these patients.     

Measurement of the charge on red blood cells in patients with various glomerulopathies

Red blood cells have a negative charge on their surface which prevents Rouloau formation. This charge may reflect the charge on glomerular capillaries. Using alcian blue, we measured the negative charge on red blood cells in patients with IgA nephropathy (IgAN), membranous nephropathy (MN), minimal change nephropathy (MCN) and focal glomerular sclerosis (FGS), for which the diagnosis was determined by renal biopsy. The alcian blue values for the normal control, IgAN, MCN (nephrotic phase), MCN (remission) and MN were 155.3 +/- 12.3, 140.9 +/- 23.9, 101.7 +/- 18.4, 152.9 +/- 11.2 and 140.9 +/- 23.6 ng per 1 x 10(6) red blood cells, respectively. The charge was more significantly decreased in MCN during nephrotic phase than other renal diseases and the normal control. The charge in MCN was within the normal range on remission. When we studied the correlation between the charge on red blood cells and proteinuria during steroid therapy, the charge in MCN was found to increase with reduction of proteinuria, while there was no change in MN and FGS with reduction of proteinuria, i.e. it was usually within normal range. The reduced charge was associated with the nephrotic phase in MCN. The difference in the charge on the red blood cells between MCN and FGS during nephrotic phase seems to suggest a different etiology of proteinuria. Measurement of the charge on red blood cells could be an useful method for differentiating MCN from other renal diseases in the nephrotic phase.     

A case of langerhans’-cell histiocytosis with membranous nephropathy

A 26-year-old man with a history of Langerhans’-cell histiocytosis (LCH) of the bone presented with nephrotic-range proteinuria. Renal biopsy results showed changes characteristic of membranous nephropathy. During the current hospitalization, the patient had 2 episodes of pulmonary embolism. LCH at this time was documented in the lymph nodes. The patient was treated with repeated courses of vinblastine and high doses of corticosteroids to achieve remission of the basic disease and the renal involvement. After 2 years, complete remission of both the lymphadenopathy and the nephrotic syndrome was achieved. The association of membranous nephropathy with LCH might be attributable to an underlying abnormality in the immune system, a paraneoplastic manifestation, or both. This is the first report of LCH associated with severe nephrotic syndrome caused by membranous nephropathy.     

Complement activation products in the urine from proteinuric patients

The presence of plasma proteins in the tubular lumen has variety of adverse effects on the tubular cells. Among various plasma proteins filtered through glomerular barrier, complement has been proven as the possible candidate inducing tubulointerstitial injury. To study the role of intratubular complement activation in proteinuric patients, complement activation products (CAP) at C3 level (iC3b and Bb) and C9 level (membrane attack complex) were measured in both plasma and urine of patients with minimal change nephrotic syndrome (MCNS), focal glomerular sclerosis, IgA nephropathy, membranous nephropathy, and diabetic nephropathy. For evaluation of the effect of metabolic acidosis on the intratubular complement activation, urinary CAP were measured before and after sodium bicarbonate administration in patients with renal insufficiency. The following results were obtained: (1) Patients with focal glomerular sclerosis and diabetic nephropathy showed the highest level of urinary CAP excretion rate (unit/creatinine), while MCNS revealed no increase. (2) Patients with membranous nephropathy showed a unique finding, i.e., isolated increase of membrane attack complex excretion. (3) There was no significant correlation between urine and plasma levels of CAP. (4) Except for MCNS patients, the urinary excretion rate of CAP significantly increased when the level of proteinuria exceeded the nephrotic range, and it was significantly correlated with the serum creatinine level. (5) Urinary CAP excretion rate significantly decreased 2 wk after sodium bicarbonate administration without affecting the level of proteinuria or plasma CAP. These results suggest that the degree of intratubular complement activation correlates with the level of proteinuria, type of glomerular disease, impairment of renal function, and metabolic acidosis.     

The effect of craniotomy on lymphocyte subpopulations

The effect of craniotomy on cell mediated immunity was studied by using monoclonal antibodies and flow cytometry. Twelve patients undergoing craniotomy were investigated. Six patients had malignant brain tumor, four benign tumor, one AVM and one aneurysm. Examined lymphocyte monoclonal antibodies were Leu4, Leu2a and Leu3a. In addition, two color analysis using two monoclonal antibodies, Leu3a and Leu8, or Leu2a and Leu15, was performed. This technique can classify Leu3a positive cell into helper T cell and inducer T cell and Leu2a positive cell into suppressor T cell and cytotoxic T cell. The number of Leu4 positive cell (T cell) decreased during operation. The number of Leu3a positive cell (OKT4) also decreased at 30 minutes after incision and moderately recovered during operation. Leu2a positive cell (OKT8) did not show significant change. As the result, the decline of Leu3a/Leu2a ratio (OKT4/OKT8 ratio) was found after incision and this ratio gradually returned to preoperative level during operation. By two color analysis, decrease of inducer and helper T cell in Leu3a positive cell, and increase of suppressor T cell, and decrease of cytotoxic T cell in Leu2a positive cell were observed. The results of this study show that stress of craniotomy affected the immunological state.     

Predominance of type-2 immune response in idiopathic membranous nephropathy. Cytoplasmic cytokine analysis

BACKGROUND: The Th1/Th2 paradigm is proving increasingly useful in the understanding of infectious diseases and many autoimmune diseases. Th1 cells predominantly produce interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and are instrumental in initiating delayed-type hypersensitivity and activating macrophages. Th2 cells secrete other cytokines, such as IL-4, IL-5, IL-10 and IL-13 that trigger B-cell activation and immunoglobulin synthesis. It has been shown that in patients with membranous nephropathy, there may be a predominance of Th2, because of the presence of IgG, particularly IgG4, which belongs to a subclass of the type-2 immune response, and complement deposits in glomeruli. In this study, we investigated the immunoresponse of helper T cells, i.e. Th predominance in patients with idiopathic membranous nephropathy. METHODS: We used flow cytometry to assess the levels of circulating Th cells in patients with idiopathic membranous nephropathy (n = 8) and in normal individuals (n = 23) based on the expression of intracellular type-1 and type-2 cytokines. Because the production of each of these cytokines has a specific time course, we observed the cytokine synthesis at 3, 6, 9 and 12 h after stimulation. RESULTS: The percentages of IL-2+/CD4+ cells from patients with idiopathic membranous nephropathy were significantly lower than those from normal individuals at 6, 9 and 12 h, with the difference becoming more significant over time. IFN-gamma+/CD4+ cells and IL-4+/CD4+ cells were not significantly different between the two groups. In patients with idiopathic membranous nephropathy, the percentages of IL-10+/CD4+ cells were significantly higher than those in normal individuals at each point in time. CONCLUSION: Increased IL-10-producing Th cells may lead to suppression of delayed-type hypersensitivity and activate suppressor cells and IgG4 synthesis, resulting in idiopathic membranous nephropathy.     

Treatment-related changes in urinary excretion of high and low molecular weight proteins in patients with idiopathic membranous nephropathy and renal insufficiency.

BACKGROUND: In patients with idiopathic membranous nephropathy, an increased urinary excretion of high (IgG) and low [beta(2)-microglobulin (beta(2)M), alpha(1)-microglobulin (alpha(1)M)] molecular weight proteins predicts prognosis and precedes renal insufficiency. We have studied the changes in the urinary excretion of these proteins in patients with idiopathic membranous nephropathy and renal insufficiency during and after treatment with cyclophosphamide and steroids, and investigated their value in predicting long-term outcome. METHODS: Standardized measurements of urinary IgG, albumin, beta(2)M and alpha(1)M were performed at 0, 2, 6 and 12 months in 11 patients, at 12 months in 25 patients and in 17 of these last patients after 2-5 years. RESULTS: We observed a rapid improvement of glomerular permselectivity and tubular protein reabsorption within 2 months after the start of therapy. Despite a partial remission of proteinuria within 12 months in most patients, evidence of tubulo-interstitial injury remained apparent. Neither absolute levels of urinary IgG, beta(2)M or alpha(1)M at baseline or at 12 months nor the percentage reduction between baseline and 12 months clearly predicted the occurrence of a remission or a relapse to nephrotic range proteinuria. In the case of a persistent stable remission, we observed a gradual decrease of urinary beta(2)M towards normal values. CONCLUSIONS: In patients with idiopathic membranous nephropathy and renal insufficiency, treatment with cyclophosphamide and steroids resulted in an improvement of glomerular permeability and tubular proteinuria. Tubular proteinuria remained present for many years, even in patients with stable remission of proteinuria. Measurements of urinary proteins at 12 months after treatment start lacked predictive accuracy.     

Nephrotic syndrome due to membranous nephropathy associated with metastatic prostate cancer: rapid remission after initial endocrine therapy

A case of severe nephrotic syndrome (urinary protein excretion 12.9 g/day) due to membranous nephropathy associated with untreated prostate cancer and multiple bone metastases is described. A combination of initial endocrine treatment and steroid therapy resulted in normalization of prostate-specific antigen levels followed by a rapid decrease of urinary protein excretion within 4 months. No proteinuria was subsequently detected. Seven months after the initiation of therapy, the patient remained well with complete clinical remission from the nephrotic syndrome. This rapid achievement of remission may have been due to tumor shrinkage by androgen ablation in addition to steroid therapy of the membranous nephropathy. The nephrotic syndrome is a rare complication of prostate cancer, and, to the best of our knowledge, no previous cases have been reported of membranous nephropathy as one of the first disease manifestations.     

In vitro B-lymphocyte switch disturbance from IgM into IgG in IgM mesangial nephropathy

In vitro immunoglobulin (Ig) synthesis using a co-culture technique after activation of lymphocytes with pokeweed mitogen, T-cell subsets and interleukin-2 (IL-2) production was studied in 10 children who suffered from IgM mesangial nephropathy (IgMN), 10 children who suffered from minimal change nephrotic syndrome (MCNS) with hypercellularity and 6 children who suffered from MCNS with normal cellularity during the acute nephrotic phase. Reduced in vitro IgG production was found in the presence of OKT8 cells from all groups of patients. However, in vitro IgM production was increased only in OKT8 cells from IgMN and MCNS patients with hypercellularity. In vitro Tac expression on the OKT8 cells, IL-2 production, T-cell subsets including Leu2a⁺15⁺ (suppressor T-cells), Leu2a⁺DR⁺ (activated suppressor T-cells) and Leu3a⁺8⁺ (suppressor T-cell inducer) were all increased in IgMN and MCNS patients with hypercellularity. There was a significant correlation between in vitro IgM production by co-culture technique and IL-2 production. These results strongly suggest the hyperfunction of isotype-specific suppressor T-cells which may affect the switch of IgM B-cells to IgG B-cells in IgMN and MCNS patients with hypercellularity and may be used to explain in part the clinical findings of lower serum IgG and increased IgM in those patients.     

儿童肾病综合征T细胞亚群检测的临床意义

目的：探讨儿童原发性肾病综合征（NS）T细胞亚群检测的临床意义。方法：对25例NS活动期和缓解期患儿应用流式细胞仪检测T2细胞亚群的变化。结果：NS活动期组CD3^ ,CD4^ ,CD4^ /CD8^ 比值，NK[CD(16 56)]^ 细胞均明显低于缓解期组（P＜0．01）和对照组（P＜0．01）。结论：说明NS细胞免疫功能减低，T细胞亚群检测可作为肾病综合征活动指标之一。     

Depletion of clusterin in renal diseases causing nephrotic syndrome

BACKGROUND: Clusterin is a lipoprotein that has anti-complement effects in membranous nephropathy (MN). In focal segmental glomerulosclerosis (FSGS), it inhibits permeability plasma factor activity and could influence proteinuria. Moreover, with aging, knockout mice for clusterin develop a progressive glomerulopathy with sclerosis. METHODS: Since little is known about clusterin metabolism in humans, we determined clusterin levels and composition in the sera and urine of 23 patients with MN, 25 with FSGS and 23 with steroid-responsive nephrotic syndrome (NS). Renal localization was evaluated by immunofluorescence and morphometry. RESULTS: Serum clusterin was markedly reduced in active MN, in FSGS and in children with NS compared to controls; after stable remission of proteinuria, nearly normal levels were restored. Among various biochemical variables, serum clusterin was inversely correlated with hypercholesterolemia. Urinary clusterin, representing a 0.01 fraction of serum, was higher in the urine from normal subjects and FSGS patients in remission with proteinuric MN, FSGS and idiopathic NS; clusterin was inversely correlated with proteinuria. In all cases, urinary and serum clusterin was composed of the same 80 kD isoforms. Finally, a decrease in focal segmental or global clusterin staining was found in FSGS glomeruli, especially in areas of sclerosis. Instead, in MN an overall increment of staining was observed that ranged from mild/focal to very intense/diffuse. CONCLUSIONS: The overall pool of clusterin is reduced in glomerular diseases causing nephrotic syndrome, with hypercholesterolemia appearing as the unifying feature. Depletion of clusterin should negatively affect the clinical outcome in nephrotic patients and efforts should be aimed at normalizing clusterin overall pool.     

Nonimmunosuppressive therapy of membranous nephropathy

Idiopathic membranous nephropathy (MN) has a variable rate of progression to end-stage renal failure, with a significant number of patients going into spontaneous remission without therapy. For those who have persistent nephrotic proteinuria or manifest deterioration of renal function, steroids and immunosuppressive drugs are used. However, their long-term efficacy is challenged by a meta-analysis presented here. A different approach to reduction of proteinuria, a recognized progression promoter, is based on the notion that angiotensin II inhibition controls proteinuria and slows progression. Further, a more complex approach is required than simple administration of an angiotensin-converting enzyme (ACE) inhibitor: a multidrug approach to remission of nephrotic syndrome therefore is described here.     

Interleukin-15 and interleukin-12 have an additive effect on the release of vascular permeability factor by peripheral blood mononuclear cells in normals and in patients with nephrotic syndrome.

BACKGROUND: The vascular permeability factor (VPF) is a lymphokine that has been shown to play a role in minimal-change nephrotic syndrome (MCNS). A better understanding of the mechanisms that upregulate VPF release is of basic importance to control the immune system in nephrotic syndrome (NS). Interleukin (IL)-15 is a key inducer of differentiation of uncommitted T helper cells, which regulates cellular immunity. The cytokine IL-15 appears to mimic the stimulatory activity of IL-2 on T cells. PATIENTS AND METHODS: In the present report, we studied the ability of IL-15, alone or in combination with IL-12, to influence the release of VPF by peripheral blood mononuclear cells (PBMC) from nephrotic patients. We have analyzed the release of VPF by concanavalin-A- (Con A) stimulated PBMC in normals, 16 patients with MCNS and 16 patients with IgA nephropathy (IgAN). RESULTS: In both patient groups 50% had a proteinuria below 0.8 g/day. We demonstrate that nephrotic, but not non-nephrotic patients (both MCNS and IgAN), exhibit a high VPF release, which can be stimulated further by IL-15 + IL-12. To determine the specificity of the stimulatory effect, neutralizing anti-IL-15 and anti-IL-12 antibodies were preincubated with IL- 15 and IL-12 prior to the addition of responder cells, respectively. The antibodies completely inhibited the effects of IL-15 and IL-12. CONCLUSION: These results indicate that IL-15 plus IL-12 acted additively to augment VPF release. These biological interactions between IL-15 and IL-12 may be important in the pathophysiology of VPF in vitro.