No abstract

Tubulointerstitial nephritis is a cardinal renal manifestation in leptospirosis and LipL32, the major lipoprotein component of leptospiral outer membrane proteins (OMPs), induces a robust inflammatory response in cultured renal proximal tubule cells through a nuclear factor-kappaB-related pathway. Here, we investigated whether Toll-like receptor (TLR), known to play a pivotal role in innate immunity, could mediate the inflammatory response induced by leptospiral OMPs in renal proximal tubule cells. TLR expression was analyzed by flow cytometry and indirect immunofluorescence in cultured mouse proximal tubule (pyruvate kinase simian virus 40-proximal straight (PKSV-PR)) cells. Reverse transcription-competitive polymerase chain reaction and enzyme-linked immunosorbent assay were undertaken to analyze the inducible effects of inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1 also termed CCL2) by pathogenic and non-pathogenic leptospiral OMPs and recombinant lipoproteins in either PKSV-PR cells or TLR-transfected human embryonic kidney (HEK) 293 cells. Anti-TLR antibodies were used for blocking experiments. Leptospira santarosai serovar Shermani OMPs and LipL32 induced a significant increase in TLR2 but not TLR4 expression in PKSV-PR cells. The increase in iNOS and CCL2/MCP-1 mRNA expressions could be prevented by an anti-TLR2 antibody, but not by an anti-TLR4 antibody. Furthermore, leptospiral OMPs stimulated both CCL2/MCP-1 mRNA and secreted protein in transfected HEK 293 cells with a TLR2-expressing plasmid, but had no effect in cells with a TLR4-expressing plasmid. In conclusion, these findings indicate that the stimulation of iNOS and CCL2/MCP-1 caused by pathogenic leptospiral OMPs, in particular LipL32, in proximal tubule cells requires TLR2 for the early inflammatory response.

The aim of this study was to characterize the mechanism of transforming growth factor (TGF)-β1-mediated alteration of renal proximal tubular cell phenotype. TGF-β1 altered cell phenotype, with cells appearing elongated and spindle shaped. This was associated with loss of cell-cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibers, and focal adhesions. Addition of the tyrosine phosphatase inhibitor sodium orthovanadate also led to rapid but transient loss of cell-cell contact, but it did not lead to a change of phenotype comparable to that seen following addition of TGF-β1. There was, however, no change in the formation of focal adhesions and no associated reorganization of the Factin cytoskeleton. Disruption of the actin cytoskeleton with cytochalasin D prevented phenotypic alterations following addition of TGF-β1. Transient transfection with Smad2/4 or Smad3/4 expression vectors did not alter cell phenotype. Previously, we demonstrated β-catenin translocation to proximal tubule cell nuclei and its association with Smad proteins following addition of TGF-β1, suggesting the possibility that TGF-β1 may modulate Wnt signaling. The Wnt-responsive Xtwn-reporter construct was, however, silent in response to TGF-β1. Similarly, a second Wnt/LEF-1-regulated element, Toplflash, which does not contain Smad binding sites, was insensitive to TGF-β1 signaling. In contrast, phenotypic changes in response to TGF-β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell-cell contact and adherens junction disassembly.

Both SOFA and RIFLE category showed high discriminative power in predicting hospital mortality in critically ill patients with cirrhosis. The RIFLE classification is a simple and easily applied evaluative tool with good prognostic abilities.