Protein load impairs factor H binding promoting complement-dependent dysfunction of proximal tubular cells.

Intrarenal complement activation plays an important role in the progression of chronic kidney disease. A key target of the activated complement cascade is the proximal tubule, a site where abnormally filtered plasma proteins and complement factors combine to promote injury. This study determined whether protein overloading of human proximal tubular cells (HK-2) in culture enhances complement activation by impairing complement regulation. Addition of albumin or transferrin to the cells incubated with diluted human serum as a source of complement caused increased apical C3 deposition. Soluble complement receptor-1 (an inhibitor of all 3 activation pathways) blocked complement deposition while the classical and lectin pathway inhibitor, magnesium chloride-EGTA, was, ineffective. Media containing albumin as well as complement had additive proinflammatory effects as shown by increased fractalkine and transforming growth factor-beta mRNA expression. This paralleled active C3 and C5b-9 generations, effects not shared by transferrin. Factor H, one of the main natural inhibitors of the alternative pathway, binds to heparan sulfate proteoglycans. Both the density of heparan sulfate and factor H binding were reduced with protein loading, thereby enhancing the albumin- and serum-dependent complement activation potential. Thus, protein overload reduces the ability of the tubule cell to bind factor H and counteract complement activation, effects instrumental to renal disease progression.

Involvement of renal tubular Toll-like receptor 9 in the development of tubulointerstitial injury in systemic lupus.

OBJECTIVE: Toll-like receptor 9 (TLR-9), a receptor for CpG DNA, has been implicated in the activation of immune cells in lupus. We undertook this study to determine whether the expression of TLR-9 in resident renal cells in lupus nephritis is related to the development of tubulointerstitial injury. METHODS: TLR-9 was analyzed in selectively retrieved renal tissue from (NZB x NZW)F1 mice at different stages of disease by laser capture microdissection combined with real-time quantitative reverse transcriptase-polymerase chain reaction, and in renal biopsy specimens from lupus nephritis patients by immunohistochemistry. We investigated for the molecular component responsible for TLR-9 activation by cultured proximal tubular cells in serum from patients with lupus. RESULTS: Renal tissue from NZB x NZW mice displayed robust TLR-9 expression localized to proximal tubular cells. TLR-9 levels correlated with proteinuria and tubulointerstitial injury to the extent that a cyclin-dependent kinase inhibitor, while reducing proteinuria and renal structural damage, prevented tubular TLR-9 generation in lupus mice. Consistently, exaggerated TLR-9 staining was found in proximal tubular cells of lupus patients, which correlated with tubulointerstitial damage. DNA-containing immune complexes purified from sera of patients with lupus induced TLR-9 in cultured proximal tubular cells. This was prevented by CCGG-rich short oligonucleotides, specific antagonists of CpG DNA, indicating that the DNA component of immune complexes was required for TLR-9 stimulation. CONCLUSION: These findings suggest that tubular TLR-9 activation has a pathogenetic role in tubulointerstitial inflammation and damage in experimental and human lupus nephritis, and they indicate a novel target for future therapies.

Shigatoxin-induced endothelin-1 expression in cultured podocytes autocrinally mediates actin remodeling.

Shigatoxin (Stx) is the offending agent of post-diarrheal hemolytic uremic syndrome, characterized by glomerular ischemic changes preceding microvascular thrombosis. Because podocytes are highly sensitive to Stx cytotoxicity and represent a source of vasoactive molecules, we studied whether Stx-2 modulated the production of endothelin-1 (ET-1), taken as candidate mediator of podocyte dysfunction. Stx-2 enhanced ET-1 mRNA and protein expression via activation of nuclear factor kappaB (NF-kappaB) and activator protein-1 (Ap-1) to the extent that transfection with the dominant-negative mutant of IkappaB-kinase 2 or with Ap-1 decoy oligodeoxynucleotides reduced ET-1 mRNA levels. We propose a role for p38 and p42/44 mitogen-activated protein kinases (MAPKs) in mediating NF-kappaB-dependent gene transcription induced by Stx-2, based on data that Stx-2 phosphorylated p38 and p42/44 MAPKs and that MAPK inhibitors reduced transcription of NF-kappaB promoter/luciferase reporter gene construct induced by Stx-2. Stx-2 caused F-actin redistribution and intercellular gaps via production of ET-1 acting on ETA receptor, because cytoskeleton changes were prevented by ETA receptor blockade. Exogenous ET-1 induced cytoskeleton rearrangement and intercellular gaps via phosphatidylinositol-3 kinase and Rho-kinase pathway and increased protein permeability across the podocyte monolayer. These data suggest that the podocyte is a target of Stx, a novel stimulus for the synthesis of ET-1, which may control cytoskeleton remodeling and glomerular permeability in an autocrine fashion.