Systemic Approach in Leptospirosis Renal Disease by Toll-Like Receptor Knock-out Mice and Proteomics

Leptospirosis is a widespread zoonosis which frequently occurs in tropical countries. Kidneys are the preferential site where leptospira accumulates, in the interstitium and also in renal tubule epithelial cells. Renal invasion may lead to mild or severe renal tubulointerstitial nephritis responsible for acute or chronic renal failure. In the past years, our research groups had devoted ourselves on the roles/mechanisms of leptospiral components in the induction of renal tubulointerstitial inflammation and possibly renal fibrosis. Two major breakthroughs were achieved in recently two years. First, we had shown that purified leptospiral outer membrane proteins (LOMP) from pathogenic Leptospira shermani were specifically recognized by Toll-like receptors 2 (TLR2), induced the preferential activation of the MAP kinase p38 pathway, and thereby the up-regulation of proinflammatory cytokines/chemokines involved in the chemoattraction of leukocytes in cultured murine proximal renal tubule cells. Second, we clearly demonstrated the stimulatory effect of LOMP on extracellular matrix (ECM) production by enhancing ECM synthesis through a TGF-beta1/Smad-dependent pathway in human proximal renal tubule cells and further implied the role of leptospira infection on the process of tubulointerstitial fibrosis. Dr. Werts had demonstrated that LPS purified from L. interrogans was specifically recognized by TLR2 in human macrophages and lipid A isolated from L. interrogans, however, was able to activate murine peritoneal macrophages through TLR4. These findings suggest that the roles of TLR2, but also TLR4, in the recognition of leptospira to initiate innate and adaptive immune responses in the host. To delineate the reciprocal roles of TLRs, we would like to develop a collaboration 3-years project: Year 1- To establish novel murine models reproducing the main features of acute human leptospirosis in wild-type and genetic knock-out mice (tlr2-/- , tlr4-/- genes, and double tlr2-/- and tlr4-/- knock-out mice). Year 2- To obtain the primary renal tubule cultured cells from kidneys of tlr2-/- or tlr4-/- genes knock-out mice to study their roles/mechanisms in tubulointerstitial inflammation and fibrosis in vitro. Year 3- To obtain animals chronically infected with sub-lethal leptospira in order to systematically analyze the kinetics of inflammatory responses that may lead to renal tubulointerstitial nephritis/fibrosis. Our group from the Kidney Research Center of the Chang Gung Memorial Hospital working on leptospirosis has decided to engage a collaboration with the INSERM (Paris, France) research group directed by Dr. A. Vandewalle who also established a collaborative study with Dr. C. Werts (Pasteur Institute, Paris, France) to analyze the consequences of the administration of L. interrogans to wild type C57BL/6 mice and knock-out mice for the tlr2-/- or tlr4-/- genes and double tlr2-/- and tlr4-/- knock-out (DKO mice) exhibiting the similar C57BL/6 genetic background. The complementary knowledge of our group in clinical human and experimental leptospirosis in vitro, of the team group of A. Vandewalle in renal pathophysiology and renal cell biology, and the experience of Dr. C. Werts in leptospiral LPS and innate immunity should permit at best to realise the project proposed. The establishment of a suitable murine model (wild and TLRs knock-out mice) of acute or chronic leptospirosis, and systems biologic approach by transcriptome and proteomic techniques on the nature course of leptospirosis will extend our knowledge on the roles and multiple pathways of innate immunity (esp. TLRs) and related molecular mechanisms involved in infection-related acute renal inflammation and chronic renal fibrosis.

Project ID:PC9709-0545
External Project ID:NSC97-2314-B182-043-MY3