The Leptospira Outer Membrane Protein LipL32 Induces Tubulointerstitial Nephritis-Mediated Gene Expression in Mouse Proximal Tubule Cells

Abstract

Abstract. Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-␣ (TNF-␣) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 g/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-␣ mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-B (NF-B) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

Attenuated Nephritis in Inducible Nitric Oxide Synthase Knockout C57BL/6 Mice and Pulmonary Hemorrhage in CB17 SCID and Recombination Activating Gene 1 Knockout C57BL/6 Mice Infected with Leptospira interrogans

“…The only significant difference observed between the Inos KO and the WT C57BL/6 mice was that the transgenic animals did not develop interstitial nephritis to the same degree or severity as the WT control. This result is in accordance with the hypothesis that the expression of proinflammatory markers by renal tubular cells in vitro after exposure to leptospiral products may be related to the development of interstitial nephritis in vivo (20)(21)(22). The present study confirms previous reports, using mice of other backgrounds (C3H and C3H/HeJ), that immunodeficiency results in high susceptibility to acute infection, rapidly progressing to death (14,19).…”

“…In vitro studies have previously reported the activation of a broad range of inflammatory genes, such as those for transcription factor NF-B, inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1, and tumor necrosis factor alpha, by renal tubular cells in response to exposure to leptospire-derived products. These findings have been interpreted as a molecular trigger for interstitial nephritis (20)(21)(22). The genetic deficiency of iNOS has not been investigated in vivo.…”

Insuficiência renal aguda associada à leptospirose

“…15 Arterial hypotension is common. 16,17 The hemodynamic status and alterations in most patients with severe leptospirosis are similar to those observed in patients with sepsis. Because of systemic vasodilation, the plasma levels of aldosterone and antidiuretic hormone are high.…”

Detection of pathogenic leptospires by real-time quantitative PCR

“…More recently, a realtime PCR was developed using TaqMan chemistry (Smythe et al, 2002) which targeted an 87 bp section of the 16S rRNA gene of Leptospira spp. In this study, we report the development of a real-time assay using SYBR green chemistry, in which the target is the gene for the major outer-membrane lipoprotein LipL32 (Haake et al, 2000), which appears to be an important virulence factor (Yang et al, 2002), confined to pathogenic strains of all Leptospira spp. †Present address: Saskatchewan Health, Provincial Laboratory, 3211 Albert Street, Regina, Saskatchewan, Canada S4S 5W6.…”