Diabetes mellitus is a major cause of mortality and morbidity as hyperglycemia adversely affects the biology of various organ systems during embryonic and adult life. Major complications in newborns include congenital anomalies, such as hypo/agenesis of the urogenital system, and diabetic nephropathy in adults. In diabetic nephropathy, both the glomerular and tubulo-interstitial compartments are involved, where an increase of extracellular matrix (ECM) is observed. ECM accumulation is due to increased synthesis of matrix proteins, e.g., collagen and fibronectin, and decreased activity of MMPs, and glycation of ECM proteins with formation of advanced glycation products (AGEs), leading to generation of reactive oxygen species (ROS), which via down-stream signals, such as TGF-beta, induce progressive glomerular and tubulo-interstitial injury. In addition, altered expression of other ECM proteins, e.g., osteopontin and fibronectin, has been reported. However, the role of a recently characterized ECM protein, tubulointerstitial nephritis antigen (TIN-ag), in diabetic nephropathy remains enigmatic. TIN-ag is a mosaic protein containing procathepsin B like domain, follistatin module, ATP binding motif, glycosylation sites and segmental homology with other ECM proteins. It is developmentally regulated, is expressed in progenitor of tubules, and modulates tubulogenesis in vitro. To decipher the role of TIN-ag in progressive renal injury during embryonic and adult life, experiments are proposed under the following specific aims: I. Role of TIN-ag in glucose-induced renal dysmorphogenesis will be investigated since treatment of fetal mice kidneys with high glucose or TIN-ag antisense yield similar phenotype, i.e., inhibited tubulogenesis, suggesting possible relevance of TIN-ag in the evolution of glucose-induced lesions. II. Role of TIN-ag in animal models overexpressing and masking TIN-ag gene will be investigated, using Tet-On/Off systems in transgenic mice and fetal Lewis rats. III. TIN-ag and its domain-specific interactions with other ECM proteins will be assessed since TIN-ag is masked in adult Lewis rats. Recombinant TIN-ag will be generated to study protein:protein interactions with isoforms of type-IV collagen and laminin, and novel interacting partners will be identified by yeast two-hybrid system. IV. Role of TIN-ag in various animal models of diabetic nephropathy, and mechanisms relevant to its increased expression will be investigated by morphologic and biochemical studies. In vitro culture system will be also employed to delineate the mechanisms of altered expression of TIN-ag in diabetic milieu. V. Role of TIN-ag in renal tubulo-interstitial pathobiology will be investigated by generating TIN-ag conditional and conventional knock out (KO) mice. For generation of the conditional KO mice, heterozygote gouty mice will be cross-bred with Ella Cre and then with Ksp Cre mice that would have lesions confined to kidney. Null alleles will be interbred to generate Null -/- mutant as a conventional KO mice with ablation of the TIN-ag gene.
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