Mechanism Of Kidney Fibrosis In A Kim-1Transgenic Model

Background Kim-1(kidney injury molecule-1) is a type I transmembrane glycoprotein, cloned as the receptor for hepatitis A virus (HAVcr-1). Kim-1contains a six-cysteine Ig-like domain and a mucin domain in its extracellular region. Kim-1is not expressed in normal kidney but is markedly upregulated in the injured proximal tubular epithelial cells of the human and rodent kidney several hours after acute kidney injury(AKI). Kim-1was firstly discovered by Dr.Bonventre that significantly increased in rats with AKI. Since then, Kim-1has been reported as a novel bio-marker of AKI by a large amount of studies.In situ hybridization and immunohistochemistry revealed that KIM-1is expressed in proliferative and dedifferentiated epithelial cells in regenerating proximal tubules after injury, especially in the outer medulla where the S3segments of the proximal tubule, which are highly susceptible to insult, are localized. Shedding of Kim-1ectodomain from injury proximal tubular cells into the urine or culture medium is regulated by active MAP kinases. In preclinical and clinical studies performed in the recent10years (refer to review1, please), urinary Kim-1serves as an earlier diagnostic indicator of kidney injury when compared with any of the conventional biomarkers, e.g., plasma creatinine; BUN; glycosuria; increased proteinuria; or increased urinary NAG, y-GT, or AP levels. Studies in man indicate that tissue expression and urinary excretion of the ectodomain of KIM-1are sensitive and specific markers of injury as well as predictors of outcome. In the meeting of the Acute Kidney Injury Network (AKIN) in2008, seven markers, including urinary KIM-1, were listed as being on the leading edge of predictive biomarkers of AKI.Recently, studies on Kim-1molecular functions revealed that Kim-1could serve as a "participant" as well as a "consequence" in various kidney injuries. Ichimura et al. has reported that Kim-1can transform epithelial cells into semiprofessional phagocytes. The extracellular Kim-1Ig variable domain binds and internalizes oxidized lipid as well as phosphatidylserine exposed on the outer leaflet of luminal apoptotic cells, thereby aiding in nephron repair and tissue remodeling through clearance of cells and debris. Therefore, Kim-1plays an important role in regulation of inflammation and tissue repair following epithelial injury. Kim-1is expressed in chronic kidney diseases (CKD) where it colocalizes with areas of fibrosis and inflammation and its expression correlates directly with the outcome of disease. The expression of Kim-1in chronic and progressive kidney disease and its association with inflammation and fibrosis suggests that Kim-1might play a pathogenic role in CKD and antagonizing Kim-1signaling might ameliorate renal fibrosis. The molecular mechanisms of progressive CKD remain obscure, which significantly limits the therapeutic achievement in clinics. Understanding the role of Kim-1in the development of CKD might lead to the discovery of novel therapeutic targets in fibrotic kidney disease.Our study will focus on the molecular mechanism of Kim-1in injured kidney. Kim-1expression is tightly regulated, so over expression in an organ where it is not normally expressed, or at a time when it is not normally expressed may be very informative about its normal function. Our experiment includes3parts:1. Establishment of mouse model with epithelial Kim-1overexpression and its phenotype identification;2. Mechanisms of Kim-1induced kidney injury;3. Role of mTOR pathway in kidney injury induced by chronic Kim-1overexpression. Chapter I Establishment of Kim-1transgenic model and its phenotype identificationObjective To create a transgene mouse with conditional epithelial expression of Kim-1and study the effect of Kim-1expression on kidney.Methods A Kim-1cDNA was inserted into the Notl site of the Z/AP plasmid and linearized Z/Kim-AP transgene was introduced into FVB zygotes by pronuclear injection. Three independent founder lines were obtained, all exhibited Six2-GC-dependent transgene expression and the line with highest outer medulla and cortex transgene expression was selected for further analysis. Z/Kim-AP transgenic mice were crossed with the Six2-GFPCre mouse (Six2-GC) to generate bigenic Kim-1REC-tg(Kim-1Renal Epithelial Cell transgenic) mice. In all cases, littermate control phenotypes were compared. Kim-1REC-tg were genotyped using Kim-1and Cre primers. Physiologic measurements were performed at different time point, including serum creatinine, serum electrolytes, hematocrit, urinary Kim-1, urinary NAG, proteinuria, blood pressure, body weight and survival rate. Electron microscope examination and immunohistochemistry were used to evaluate the kidney injuries.Results Kim-1REC-tg were born at expected mendelian ratios and expressed Kim-1mRNA at birth. At birth, kidneys from Kim-1REC-tg mice were23%smaller by weight than littermate controls. This was associated with46%fewer nephrons in Kim-1REC-tg kidneys without significant difference in glomerular diameter. Kidney histology at P1was normal. A detailed histologic analysis at P14revealed normal glomeruli,interstitium and vasculature with focal coarse vacuolization and focal epithelial degeneration noted only in Kim-1REC-tg kidneys. By4weeks, however, a pathy mononuclear interstitial infiltrate was evident with occasional hyaline casts, focal tubular damage and few focal segmental glomerulosclerosis. By12weeks, interstitial inflammation predominated with tubular dedifferentiation and necrosis, microcystic tubular dilation, hyaline casts and severe fibrosis. Serum creatinine was equal between transgenic and control mice at P14, but rose progressively thereafter from6weeks. Kim-1REC-tg mice died spontaneously of progressive renal failure at a median age of11weeks. Urinary analysis revealed progressive proteinuria occurred in Kim-1REC-tg by4weeks.Kim-1REC-tg also developed profound and progressive anemia (by6weeks), hypertension with cardiac hypertrophy (by14-16weeks), and severe electrolyte disturbance (hyperkalemia and hyperphosphatemia, by11weeks).Conclusion1.Kim-1REC-tg is the first transgene model for studying a functional role of Kim-1in epithelial cells;2. Chronic epithelial Kim-1expression leads to progressive tubular-interstitial injury and spontaneous renal failure;3. Kim-1REC-tg is a novel model for chronic kidney disease that reflects the progressive nature, vascular and endocrine aspects of the human disease. Chapter Ⅱ Epithelial over-expression of Kim-1leads to progressive inflammation and kidney fibrosisObjective To investigate the effects of Kim-1expression on kidney parenchymal and interstitial cells, and the molecular mechanisms of progressive inflammation and fibrosis induced by Kim-1.Methods Ischemia reperfusion injury (IRI) was induced in C57BL/6J mice. After72hours, kidney tissue was analyzed by immunohistochemical costaining of Ki67and Kim-1. Ki67+tubular or glomerular cells and CD3+interstitial cells in Kim-1REC-tg cortex were analyzed respectively. Immunofluorescent staining was performed to evaluate infiltration of F4/80+cells in Kim-1REC-tg. mRNA levels of a panel of growth factors and inflammatory factors at different ages were detected by realtime-PCR. To distinguish between synthesis of TNF-by epithelial cells or infiltrating leukocytes, primary epithelial cultures were established from4week-old Kim-1REC-tg kidneys or control kidneys. Supernatant of epithelial cultures were collected after4-5days. TNF-α protein in the supernatant was detected by ELISA assays.Results Immunohistochemistry in IRI kidneys revealed that most Kim-1positive epithelia co-expressed Ki67at72hours post injury, indicating significant epithelial proliferation. Co-localization of Ki67and Kim-1was also observed in cyst or epithelial cells of Kim-1REC-tg by2weeks or4weeks. Ki67+epithelial cells significantly increased in all ages of Kim-1REC-tg, compared with controls. Ki67+glomerular cells in4-week Kim-1REC-tg were significantly more than controls. Herein, Kim-1expression significantly induces proliferation of kidney parenchymal cells. Realtime PCR data indicated Kim-1stimulated the increment of a panel of growth factors in an early stage of2weeks and more robustly at4weeks. Together, Kim-1might induce the proliferation of kidney parenchymal cells by stimulating the secretion of growth factors. Immunofluorent analysis revealed progressive infiltration of F4/80+cells in Kim-1REC-tg, especially in the area with Kim-1expression. CD3+T cells appeared abundantly in the interstitial of4-week Kim-1REC-tg mice and more severe after12weeks. Migration of CD3+T cells can also be observed into the tubular lumens. Realtime PCR revealed a pro-inflammation in2-week Kim-1REC-tg, which turned out to be an inflammatory cascade by4weeks and10weeks. Immunohistochemistry and realtime PCR indicate progressive inflammation occurs in Kim-1REC-tg, where Kim-1may play a chemotactic role in leukocytes infiltration. There were substantially increased levels of TNF-a protein in the supernatant of epithelial cultures from Kim-1REC-tg kidneys compared to controls, indicating that epithelial cells were producing this cytokine and supporting a model in which chronic expression of Kim-1drives a pro-inflammatory response through epithelial secretion of cytokines and chemokines.Conclusion Kim-1overexpression induces the epithelial synthesis of multiple growth factors and proinflammatory factors and leads to progressive inflammation and fibrosis in Kim-1REC-tg. Chapter III Role of mTOR signaling in kidney injury induced by chronic Kim-1over-expression.Objective To investigate the role of mTOR pathway in kidney injury of Kim-1REC-tg induced by epithelial Kim-1overexpressionMethods Kidney activation of mTOR/S6K pathway in2to5-weeks Kim-11REC-tg was analyzed by realtime PCR and immunohistochemistrey.4-week Kim-1REC-tg mice were randomly assigned to rapamycin group (n=6) or vehicle group (n=6). Littermates were selected as Kim-1negative control groups, respectively (n=5). Administration of rapamycin (2mg/kg/day) or vehicle was performed by intraperitoneal injection for a6-week trial. After6weeks, serum creatinine level was examined and pathological analysis was performed to evaluate kidney injuries. Immunohistochemistry was applied to analyzed PS6K expression, cell proliferation (Ki67), leukocytes infiltration (F4/80and CD3) and fibrosis (αSMA).Results mTOR/S6K pathway activated in Kim-1REC-tg as early as2weeks, compared with the littermates (Kim-1negative), and more robustly up-regulated by4weeks. Immunohistochemistry (IHC) also confirmed high expression of PS6K protein in epithelial and interstitial cells of Kim-1REC-tg. Rapamycin treatment significantly lowered down the serum creatinine and proteinuria of Kim-1REC-tg, compared with vehicle group. IHC and western blot revealed rapamycin inhibits the expression of PS6K and ameliorates tubular injuries, inflammation (F4/80and CD3) and fibrosis (αSMA) in Kim-1REC-tg.Conclusion1. mTOR/S6K signaling is significantly up-regulated in Kim-1REC-tg, pS6K activation is strongly associated with interstitial fiborsis of Kim-1REC-tg;2. Rapamycin ameliorates inflammation and fibrosis in Kim-1REC-tg by inhibiting activation of mTOR/S6K signaling;3. mTOR signaling mediates the kidney injury induced by chronic epithelial Kim-1overexpression.