Role Of Surfactant Proteins A And D In Urinary Tract Infection And Innate Immunity In Kidney

Background and Objectives:Surfactant proteins A and D (SP-A, SP-D) are members of the C-type lectin (collectin) protein family. Except for surfactant-related physiological function (lowering surface tension at the air-liquid interface of the lung and surfactant homeostatesis), SP-A and SP-D play an important role in innate immunity and modulate of inflammation in the lung, as well as other organs. Both SP-A and SP-D can opsonize many kinds of pathogens and enhance pathogen uptakes by macrophages. SP-A and SP-D directly bind to rough lipopolysaccharide (LPS) and other componets present on the surface of bacteria and other micro-organisms. SP-A and SP-D also inhibit the growth of gram-negative bacteria by increasing membrane permeability and have ability to kill bateria. In addition, SP-A and SP-D bind to different receptors on the surface of several types of immune cells and modulate inflammatory processes and innate immune function through influening NF-κB and other signaling pathways.The expression of SP-A and SP-D was originally identified and characterized in the lung. Recent studies demonstrate that SP-A and SP-D express in several extrapulmonary tissues, such as gastrointestinal tract, female reproductive tract and kidney. However, little is known about their biological function of SP-A and SP-D in the kidney, as well as possible role in the pathogenesis of kidney diseases, such as UTI. Recently, our study showed that urinary SP-A and SP-D levels were decreased in female patients with recurrent urinary tract infection compared to healthy controls, and genetic polymorphisms (19A1a allele and223Gln allele) in SP-A gene are associated with the susceptibility to recurrent urinary tract infection in chinese women, suggesting that SP-A and SP-D may be implicated in the pathophysiology in kidney disease.In this work, in first objective is to investigate the role of SP-A and SP-D in urinary tract infection mouse model, in which SP-A and SP-D double knoukout (SP-A/D KO) mice are used, and then we examine the effects of SP-A and SP-D on the bacterial growth inhibition in vitro. Second objective is to analyze SP-D expression in cultured human renal proximal tubular epithelial cells (HK-2), and to examine the effect of SP-D on proinflammatory cytokine production after lipopolysaccharide (LPS) stimulus, as well as to study the effect of SP-D in the pathogenesis of tubulointerstitial fibrosis. Methods For Part1:SP-A and SP-D double knockout (SP-A/D KO) and age-matched wild-type (WT) C57BL/6female mice (7-10weeks) were used for this study. The expression of SP-A and SP-D in kidney was detected by Western blot and Immunohistochemistry (IHC) with mouse SP-A and SP-D antibodies. The level of p-p38and p38protein were measured and compared by Western blot in kidney of SP-A/D KO and WT mice. Fifty μl of Uropathogenic Escherichia coli (UPEC,5x106CFU/mouse) or buffer was delivered into the bladder of female mice. At24and48hrs after inoculation, CFU of E. coli in the kidney and urine of the treated and control mice were measured. Histological, cellular and molecular analyses were performed by several methods of H/E staining; IHC, Western blot and Enzyme-linked immunosorbent assay (ELISA). The effects of SP-A and SP-D on bacterial growth were studied in vitro. For Part2:We examined the expression of both SP-D mRNA and protein in human kidney and in vitro HK-2cells by immunohistochemistry, Western blotting analysis, reverse transcription PCR, and real-time PCR. To explore the potential role of SP-D in the pathogenesis of tubulointerstitial fibrosis in kidney infection, we examined the production of monocyte chemoattractant protein-1(MCP-1) in HK-2cells after LPS treatment, and studied interaction of SP-D and MCP-1expression in vitro.Results Part1:In the UTI model, SP-A/D KO and WT sham mice showed normally histologic renal structure. Infected SP-A/D KO mice with UPEC demonstrate increased susceptibility to UTI as evidenced by1) higher CFU in kidney and urine at24and48hrs,2) increased inflammatory cell infiltration in kidney;3) Increased keratinocyte-derived chemokine (IL-8) in the kidney at24hrs; but not for SP-A/D KO mice;4) Higher p38MAPK phosphorylation level in kidney of SP-A/D KO mice;5) In addition, in vitro E. coli growth was inhibited3-fold by40μg/ml SP-A and2-fold by20μg/ml SP-D. Part2:1) SP-D expression in human kidney tissue and HK-2cells;2) LPS treatment inhibited SP-D expression in HK-2cells;3) Overexpression of SP-D significantly reduced the LPS-induced expression of MCP-1in transfected cells.Conclusions SP-A and SP-D can attenuate renal inflammation in this murine UTI model and inhibit UPEC growth in vivo and in vitro. And SP-D in the kidney may function as an anti-inflammatory factor in renal tubular epithelial cells and may modulate tubulointerstitial fibrosis in kidney disease.