Anti-heparan sulfate antibody and functional loss of glomerular heparan sulfate proteoglycans in lupus nephritis.

Background The purpose of this study was to evaluate the features of heparan sulfate proteoglycans (HSPGs) as agrins of the glomerular basement membrane (GBM) and circulating anti-heparan sulfate (HS) antibodies in lupus nephritis, comparing titers among the following groups: lupus nephritis (LN), non-renal lupus, non-lupus nephritis, and healthy controls. Methods The stage of nephritis was determined based on the kidney biopsy. Alcian blue staining and immunohistochemical (IHC) staining for agrin were performed for histological evaluation of GBM HSPGs in normal glomeruli, non-lupus membranous glomerulonephritis (MGN), and lupus MGN. The results were used for measurement of the serum anti-HS antibody titers using an enzyme-linked immunosorbent assay (ELISA) in the following groups: 38 healthy controls, 38 non-lupus nephritis, 37 non-renal lupus, and 38 LN. Results Glomerulus HSPGs were stained bluish-green along the GBM with Alcian blue. However, IHC staining against agrin was almost completely negative in the lupus MGN group compared with the normal and non-lupus MGN groups, which showed brown staining of GBM. A higher level of anti-HS IgG was detected in LN compared with other groups, respectively. Higher titers were associated with the presence of SLE and nephritis. A higher degree of proteinuria normalized to glomerular filtration rate (eGFR) was observed in association with higher anti-HS antibody titers in LN. Conclusion This study demonstrated a functional loss of GBM HSPGs and higher levels of circulating anti-HS antibodies as a characteristic feature of lupus nephritis, suggesting their involvement in the pathogenesis of lupus nephritis and proteinuria.

C-reactive protein, immunoglobulin G and complement co-localize in renal immune deposits of proliferative lupus nephritis.

The pattern recognition molecules C-reactive protein (CRP) and C1q are of big interest in relation to the pathogenesis of systemic lupus erythematosus (SLE). Circulating autoantibodies against CRP and C1q are frequently found in SLE patients with active disease, particularly in lupus nephritis (LN), and rising levels reportedly relate to disease activity and outcome. If CRP-, or dsDNA- and/or C1q-containing immune complexes (ICs) are pathogenic in LN, glomerular IgG-deposits would be expected to co-localize with these antigens. In search for proof of this concept, renal biospsies from patients with active LN (n = 5) were examined with high-resolution immunogold electron microscopy. Renal biopsies from patients with Henoch-Schönlein purpura, pauci-immune nephritis and renal cancer served as controls. IgG antibodies against CRP, C1q and nucleosomes were analyzed in pre-post flare sera. We could demonstrate that CRP, C1q, C3c and dsDNA were co-localized with IgG in electron dense deposits in the glomerular basement membrane/subendothelial space in all of the 5 LN patients. Deposits of IgG, CRP, complement and dsDNA were 10-fold higher in LN compared to controls. All SLE patients had circulating anti-nucleosome antibodies; 4/5 had serum antibodies against CRP, dsDNA, and C1q at biopsy/flare. Despite a limited number of cases, the results support the notion of a pathogenic role not only for anti-dsDNA antibodies, but also for anti-CRP and anti-C1q in LN. The glomerular ICs may have been generated by deposition of circulating ICs or by in situ IC formation.

[Expression of neonatal Fc receptor on human nephritis and rat nephritis models].

OBJECTIVE: To study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models: anti-Thy1.1 nephritis and Heymann nephritis. METHODS: Thirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010, including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy, 12 cases of IgA nephropathy and 6 cases of lupus nephritis. Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls. Laser capture microdissection and real-time RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides, and immunohistochemistry (IHC) of FcRn by SuperVision method was performed. In addition, rat models of mesangial proliferative nephritis (anti-Thy1.1 nephritis) and passive membranous nephropathy (Heymann nephritis) were established and FcRn was examined in renal tissues by IHC. RESULTS: The FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls (P < 0.05). FcRn protein expression by IHC was seen in lupus nephritis (6/6), membranous nephropathy (6/9) and IgA nephropathy (7/12), significantly higher than that of normal controls (0/5), P < 0.05. Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8, 0/4 respectively) and not statistically difference from that of normal controls. Furthermore, FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls. CONCLUSIONS: Expression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thy1.1 nephritis and Heymann nephritis. FcRn may play a role in the development of immune-induced glomerulonephritis.

Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice.

OBJECTIVE: The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways. METHODS: To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis. RESULTS: We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo. CONCLUSION: We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.

Analysis and classification of B-cell infiltrates in lupus and ANCA-associated nephritis.

Intrarenal B cell infiltrates resembling secondary lymphoid tissue have been found in several forms of inflammatory kidney disease. Their role in renal inflammation is not well defined, perhaps because B cell clusters have been regarded as a single entity while being quite heterogeneous. Therefore we characterized intrarenal lymphoid clusters of 32 patients diagnosed with lupus nephritis and 16 with ANCA associated nephritis. We identified four increasingly organized levels of intrarenal aggregates from scattered B cells to highly compartmentalized B cell clusters with central follicular dendritic cell networks. Most B cells displayed a mature non-antibody producing phenotype with antigen presenting ability. In regions of B cell infiltration, expression of the lymphoid chemokine BCA-1 was found in cells of a dendritic-like morphology and most B cells expressed the corresponding receptor CXCR5. Biopsies containing B cells had significantly higher levels of BCA-1 mRNA expression compared to those without, suggesting a role of BCA-1 and CXCR5 for B cell infiltration into the kidney. Our study proposes a new classification of B cell clusters in lupus and ANCA associated nephritis which might help to study the function of intrarenal B cell clusters in a more differentiated manner.

Lupus eritematoso sistémico / Lupus erythematosus

El lupus eritematoso sistémico (LES) es una enfermedad autoinmune compleja, caracterizada por la múltiple presencia de autoanticuerpos, algunos de ellos claramente relacionados con manifestaciones típicas de la enfermedad. El LES puede aparecer a cualquier edad, pero afecta fundamentalmente a mujeres jóvenes en edad fértil. La patogénesis del lupus continúa sin conocerse, no obstante se han relacionado factores genéticos, ambientales, hormonales, así como también diversas alteraciones celulares y una pérdida en el equilibrio de las citoquinas. El cuadro clínico es muy heterogéneo, pudiendo afectar a casi cualquier órgano. Las principales manifestaciones clínicas son la afectación articular, la cutánea, la glomerulonefritis, la serositis (pleuritis y/o pericarditis), la afectación del sistema nervioso central y en ocasiones la trombosis. El compromiso renal marca claramente el pronóstico de los pacientes con LES. Estudios recientes muestran una mejoría en las tasas de sobrevida de los pacientes con LES, así como también una reducción en el número de brotes. Durante los últimos años se vienen desarrollando nuevas terapias para el LES que parecen tener unas tasas de respuestas esperanzadoras.

Toll-like receptors: emerging concepts in kidney disease.

PURPOSE OF REVIEW: To summarize the recent advances in the role of Toll-like receptors (TLRs) in innate immunity, with a special focus on recent studies addressing the expression and function of TLRs in kidney disease. RECENT FINDINGS: Pathogen-recognition receptors including TLRs mediate immune activation upon pathogen recognition in different extracellular and intracellular compartments. In contrast to professional antigen-presenting cells, renal cells express a limited pattern of TLR (i.e. express TLR1-TLR6 but lack expression of the endosomal TLR7-TLR9). TLRs on renal cells contribute to the innate immune response in renal infection. Furthermore, recent studies provide experimental evidence for the functional role of TLRs in immune complex disease and autoimmunity. Furthermore, the recognition of endogenous molecules released from injured cells such as biglycan or heat-shock proteins may contribute to acute tubular injury and seem to provide adjuvant activity for renal inflammation. Furthermore, TLR7 and TLR9 are involved in the pathogenesis of lupus nephritis. SUMMARY: The field of TLR research elucidates the molecular mechanisms of infection-associated kidney diseases but may also further support the concept that innate immunity significantly contributes to the so-called types of nonimmune kidney diseases.

Nrf2 deficiency improves autoimmune nephritis caused by the fas mutation lpr.

BACKGROUND: Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinate transcriptional induction of antioxidant and phase II drug metabolizing enzymes. We previously reported that Nrf2-deficient female mice develop lupus-like autoimmune nephritis (Kidney Int 60:1343-1353, 2001). The result suggested that nrf2 is a possible candidate gene in determining susceptibility to autoimmune diseases. MRL/lpr mice, defective in Fas-mediated apoptosis, develop glomerulonephritis due to the production of autoantibodies. METHODS: To investigate the mechanism whereby Nrf2 contributes to the susceptibility to autoimmune diseases, we generated nrf2-/-lpr/lpr mice. RESULTS: Unexpectedly, the lifespan of nrf2-/-lpr/lpr female mice was markedly prolonged and these mice showed an improvement in nephritis compared to nrf2+/+lpr/lpr female mice. Immunologic abnormalities and hypergammaglobulinemia were also alleviated in nrf2-/-lpr/lpr female mice. Furthermore, lymphadenopathy was suppressed as a result of increased apoptosis. To elucidate the molecular mechanism causing a stimulation of apoptosis, we analyzed the response made by nrf2-/-lpr/lpr mice to death signals. We show that nrf2-/-lpr/lpr mice are sensitive to tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis. Since intracellular glutathione levels are decreased in Nrf2-deficient cells, it is probable that a prolonged depletion in glutathione levels leads to the enhancement in TNF-alpha-mediated apoptosis. CONCLUSION: These results indicate that a deficiency in Nrf2 enhances TNF-alpha-mediated apoptosis which in-turn ameliorates the abnormal apoptotic response that arises from a mutation in the lpr gene. Therefore, Nrf2 deficiency acts as a suppressor of the autoimmune accelerating gene lpr.

Phenotyping renal leukocyte subsets by four-color flow cytometry: characterization of chemokine receptor expression.

To investigate mechanisms of cell-mediated injury in renal inflammatory disease it is critical to determine the surface phenotype of infiltrating renal leukocyte subsets. However, the cell-specific expression of many leukocyte receptors is difficult to characterize in vivo. Here, we report a protocol based on flow cytometry that allows simultaneous characterization of surface receptor expression on different subsets of infiltrating renal leukocytes. The described technique combines an adapted method to prepare single cell suspensions from whole kidneys with subsequent four-color flow cytometry. We recently applied this technique to determine the differential expression of murine chemokine receptors CCR2 and CCR5 on infiltrating renal leukocyte subsets. In this article, we summarize our current findings on the validity of the method as compared with immunohistology and in situ hybridization in two murine models of nonimmune (obstructive nephropathy) and immune-mediated (lupus nephritis) inflammatory renal disease. Flow cytometry analysis revealed an accumulation of CCR5-, but not CCR2-positive lymphocytes in inflamed kidneys, compared to the peripheral blood. Particularly renal CD8+ cells expressed CCR5 (79% in obstructed kidneys, 90% in lupus nephritis). In both models, infiltrating renal macrophages were positive for CCR2 and CCR5. These data corresponded to immunohistological and in situ hybridization results. They demonstrate that flow cytometric analysis of single cell suspensions prepared from inflamed kidneys is a rapid and reliable technique to characterize and quantify surface receptor expression on infiltrating renal leukocyte subsets.