Rituximab in immunologic glomerular diseases

Experimental data suggest that the B-cell antigen CD20 may play a significant role in the pathogenesis of many diseases including glomerular diseases. These and other findings underpin the central concept of B-cell-depleting therapies that target CD20 antigen as treatments for lupus nephritis, idiopathic membranous nephropathy, focal segmental glomerulosclerosis, cryglobulinemic glomerulonephritis, antibody mediated renal allograft rejection and recurrent glomerulonephritis in renal allograft. Use of rituximab as a B-cell depleting therapy has been associated with clinical improvement and has emerged as a possible adjunct or alternative treatment option in this field of nephrology.     

Rituximab in immunologic glomerular diseases

Experimental data suggest that the B-cell antigen CD20 may play a significant role in the pathogenesis of many diseases including glomerular diseases. These and other findings underpin the central concept of B-cell-depleting therapies that target CD20 antigen as treatments for lupus nephritis, idiopathic membranous nephropathy, focal segmental glomerulosclerosis, cryglobulinemic glomerulonephritis, antibody mediated renal allograft rejection and recurrent glomerulonephritis in renal allograft. Use of rituximab as a B-cell depleting therapy has been associated with clinical improvement and has emerged as a possible adjunct or alternative treatment option in this field of nephrology.     

"Topical nephropathy" and "tropical extramembranous glomerulonephritis" of unknown aetiology in Senegal.

A study of renal biopsy specimens obtained in Senegal from 24 children and six adults with nephrotic syndrome showed two unusual varieties of nephropathy--namely, an extramembranous glomerulonephritis associated with hypocomplementaemia (four cases), a combination previously described only in systemic lupus erythematosus, and a "tropical nephropathy" (16 cases). The latter, though lacking the diffuse glomerular deposits of immunoglobulin described in quartan malarial nephropathy (Q.M.N.), showed a curious progressive and segmental glomerulosclerosis, characterized by a "flaking" or fibrillary splitting of the glomerular capillary wall, seen in Q.M.N. Serological evidence of malaria was lacking in a third of the childhood cases.     

Schistosomal glomerulopathy and changes in the distribution of histological patterns of glomerular diseases in Bahia, Brazil

Distinct patterns of glomerular lesions, including membranoproliferative glomerulonephritis and focal segmental glomerulosclerosis, are associated with infection by Schistosoma mansoni or Schistosoma japonicum. Evidence suggests that immune complex deposition is the main mechanism underlying the different forms of schistosomal glomerulonephritis and that immune complex deposition may be intensified by portal hypertension. The relationship between focal segmental glomerulosclerosis and schistosomiasis remains poorly understood. A clinicopathologic classification of schistosomal glomerulopathies was proposed in 1992 by the African Association of Nephrology. In Brazil, mass treatment with oral medications has led to a decrease in the occurrence of schistosomal glomerulopathy. In a survey of renal biopsies performed in Salvador, Brazil, from 2003-2009, only 24 (4%) patients were identified as positive for S. mansoni infection. Among these patients, only one had the hepatosplenic form of the disease. Focal segmental glomerulosclerosis was found in seven patients and membranoproliferative glomerulonephritis was found in four patients. Although retrospective studies on the prevalence of renal diseases based on kidney biopsies may be influenced by many patient selection biases, a change in the distribution of glomerulopathies associated with nephrotic syndrome was observed along with a decline in the occurrence of severe forms of schistosomiasis.     

An Essential Role of the Universal Polarity Protein,aPKCλ, on the Maintenance of Podocyte Slit Diaphragms

Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin–podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3–atypical protein kinase C (aPKC)–PAR6β cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKCλ in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC–PAR3 complex associates with the nephrin–podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.     

−13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1

Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (−13907, −13910 and −13915) of the lactase gene in different ethnic populations. In contrast to the −13907*G and −13910*T SNPs, the −13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the −13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter–reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the −13907 to −13915 SNP region therefore remains a candidate interaction involved in lactase persistence.     

Changes in renal haemodynamics in the nephrotic syndrome

It has repeatedly been found that haemodynamic changes during hypoproteinaemia in the chronic phase of the nephrotic syndrome are different from those during hypoproteinaemia in the acute phase. In our series of patients, a decrease in the filtration fraction and relative hyperperfusion of the kidneys were associated with the presence of the nephrotic syndrome. No significant changes in renal haemodynamics were observed in patients with chronic glomerulonephritis without the nephrotic syndrome or in a group of healthy volunteers. The question of whether relative hyperperfusion of the kidneys in a repeatedly relapsing nephrotic syndrome can lead to the development of focal segmental glomerulosclerosis needs to be elucidated.     

Urinary Excretion of C3 Antigen in Glomerulonephritis

C3 and fibrin degradation products (F.D.P.) have been measured in early morning urine samples from 38 normal people and 123 patients with glomerulonephritis. Normal urine contained less than 0·3 μg of either antigen per ml. C3 and F.D.P. were both detected in the urine of many patients with glomerulonephritis. Levels above 1 μg/ml were exceptional in patients with “minimal change,” and the highest excretion of both antigens occurred in mesangiocapillary glomerulonephritis, membranous nephropathy, and focal glomerulosclerosis.Both C3 and F.D.P. excretion showed considerable variation with time, with parellel fluctuations in the two antigens. These fluctuations did not depend on the total protein leakage and suggest that the complement and clotting sequence are closely related in these glomerular disorders.     

C4d immunohistochemical staining is a sensitive method to confirm immunoreactant deposition in formalin-fixed paraffin-embedded tissue in membranous glomerulonephritis

Although the diagnosis of membranous glomerulonephritis (MGN) may be suspected on routine histology of formalin-fixed paraffin-embedded tissue, fresh-frozen tissue must be used to show the immunologic nature of the process by direct immunofluorescence (IF). The efficiency of IF or immunoperoxidase (IP) detection of IgG and C3 using paraffin sections is controversial. This study was designed to evaluate whether glomerular C4d deposition using an IP method in formalin-fixed paraffin-embedded tissue may be a useful marker for MGN. We showed characteristic glomerular, granular basement membrane deposition of C4d in 31 (100%) cases of idiopathic MGN and in 5 cases (100%) of pure class V membranous lupus nephritis, in which we had a positive diagnosis of the lesions for conventional IF study. Control cases were negative. Nineteen cases of different glomerulopathies, including IgA nephropathy, primary type I membranoproliferative glomerulonephritis, focal segmental glomerulosclerosis and minimal change disease showed diverse reproducible patterns of C4d deposition, without intrinsic background. Our results indicate that staining of formalin-fixed paraffin-embedded tissue for C4d can be used for confirmation of granular basement membrane immunoreactant deposition in cases of MGN. This proved to be a reliable method that could potentially obviate the need for rebiopsy in cases with absence of glomeruli in renal frozen sections or when other adjunct IF or IP methods on paraffin sections are negative. C4d immunostaining, using an IP method, deserves a place as an adjunct method in the biopsy diagnosis of MGN.     

Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy

Urinary microvesicles constitute a rich source of membrane‐bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein‐2 (LIMP‐2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP‐2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co‐localization of LIMP‐2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP‐2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP‐2 in glomeruli of patients with iMN.     

Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

Nephrotic syndrome, characterized by massive proteinuria, is caused by a large group of diseases including membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS). Although the underlying mechanisms are beginning to unravel, therapy is unspecific and far from efficient. It has been suggested that adrenocorticotropic hormone (ACTH) has beneficial effects in patients with MN and possibly in other nephrotic diseases. We have previously reported that ACTH may act directly on podocytes through the melanocortin 1 receptor (MC1R). In the present study, we evaluate the effect of highly specific MC1R agonists in two different nephrotic disease models. Experimental MN: Passive Heymann nephritis (PHN) was induced in rats that were treated for four weeks with MS05, a selective MC1R agonist, or saline. The degree of albuminuria was significantly reduced over time and the effect was sustained one week after treatment withdrawal (p<0.05). Experimental FSGS: Based on a dose-response study, two doses of adriamycin were used for induction of nephropathy in Balb/c mice. Mice were treated with either a synthetic MC1R agonist (BMS-470539), with α-melanocyte stimulating hormone (α-MSH) or with saline. There was no beneficial effect of treatment. In summary, MC1R agonists reduce albuminuria and improve morphology in experimentally induced MN whereas they have no effect in experimental FSGS. The results illustrate the differences in these podocytopathies in terms of signaling mechanisms underlying proteinuria, and progression of disease.     

Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.     

Gene polymorphism of vascular endothelial growth factor −1154 G>A is associated with hypertensive nephropathy in a Hispanic population

The aim of this study was to determine the association between hypertensive nephropathy and gene polymorphisms of vascular endothelial growth factor (VEGF) in a self-reported Hispanic patient group. A total of 155 Hispanic living kidney donors as controls and a total of 86 Hispanic kidney transplant patients, whose renal failure was attributed to hypertensive nephropathy after ruling out diabetes mellitus or other causes, were genotyped for four different single nucleotide polymorphisms of VEGF: −2578 C>A (rs699947), −1154 G>A (rs1570360), −460 C>T (rs833061), and +936 C>T (rs3025039). The homozygous mutant type (AA) of VEGF −1154 G>A (rs1570360) was found with significantly higher frequency in the hypertensive nephropathy patients than in controls. On the other hand, homozygous wild type (GG) was found less frequently in the hypertensive nephropathy patient group than in the control group. Linkage disequilibrium (LD) analyses revealed a high degree of LD among VEGF −2578 C>A (rs699947), VEGF −1154 G>A (rs1570360), and VEGF −460 C>T (rs833061). The haplotype analysis revealed that two haplotypes, CGTC and CATC (in the order of VEGF −2578 C>A (rs699947), −1154 G>A (1570360), −460 C>T (rs833061), and +936 C>T (3025039)), were significantly associated with hypertensive nephropathy in Hispanic patients. Hence, the −1154 G>A polymorphism (rs1570360) and two haplotypes (CGTC and CATC) of VEGF appear to be associated with hypertensive nephropathy in Hispanic patients who developed end-stage renal disease requiring kidney transplant.     

Intrarenal gene expression of proinflammatory chemokines and cytokines in chronic proteinuric glomerulopathies

Proteinuria has been recently shown to be an independent risk factor for the progression of chronic nephropathies, but the actual mechanisms by which urinary protein load damages renal tissue in humans remain unsolved. Using real-time RT-PCR method we evaluated intrarenal mRNA expression of various cytokines and chemokines in patients with biopsy-proven IgA nephropathy (IgAN, n=11), membranous nephropathy (MN, n=6) and focal and segmental glomerulosclerosis (FSGS, n=6) who exhibited proteinuria over 0.5 g/day. There was a significant positive correlation between the proteinuria extent and the intrarenal RANTES (regulated upon activation normal T cell expressed and secreted) mRNA expression in patients with IgAN, a similar trend was also observed in patients with MN and FSGS. There were no clear relationships between the proteinuria and intrarenal mRNA expression of tumor necrosis factor alpha, transforming growth factor beta1 and monocyte chemoattractant peptide-1. There were no differences in the pattern of cytokine mRNA expression between different glomerulopathies. In conclusion, our results support the hypothesis that lymphocytes, macrophages and their products provoke tissue injury in response to proteinuria independently of the nature of renal diseases in man.