Glycation and tubulointerstitial injury : in vitro and in vivo studies

Tubulointerstitial disease is a prominent phenomenon that underlies diabetic nephropathy and correlates closely with declining renal function. The formation of myofibroblasts represents a pivotal process in the development of tubulointerstitial fibrosis. Although the origin of these cells remains unclear, the cytokine transforming growth factor-p (TGF-P) has been shown to mediate the in vitro phenotypic transformation of tubular epithelial cells into myofibroblasts, a process known as transdifferentiation. The pathogenic link between chronic hyperglycaemia and the development of tubulointerstitial injury has not been fully elucidated, however diabetes mellitus is associated with the increased accumulation of a group of heterogeneous protein modifications termed advanced glycation endproducts (AGEs). Interactions between AGEs and specific receptors such as RAGE induce pleiotropic cellular effects including the increased expression of cytokines. This thesis explored the role of AGEs in inducing epithelial to myofibroblast transformation. Specific binding of I AGE-BSA to cell membranes prepared from a rat proximal tubule cell-line, was observed. Binding characteristics, immunoblotting and real time PCR experiments established that this binding site was RAGE. Whole cells exposed to AGEs including a physiological AGE-moiety, carboxymethy-lysine, demonstrated time and dose-dependent epithelial-myofibroblast transdifferentiation as determined by morphological changes and de-novo oc-smooth muscle actin expression. Reciprocal loss of expression of the epithelial antigens E-cadherin and zonula occludens was also seen. The addition of neutralising antibodies to RAGE or to TGF-6 abrogated AGE-mediated effects and prevented an observed AGE-RAGE augmentation of TGF-6 protein production. Furthermore, there was evidence of proximal tubular transdifferentiation in long-term diabetic rats and in a renal biopsy from a patient with type I diabetes. Administration of an AGE cross-link breaker, phenyl-4,5-dimethylthiazloium (ALT 711) reduced AGE accumulation, TGF-6 expression and transdifferentiation in diabetic rats. These studies provide a novel mechanism to explain the development of tubulointerstitial disease manifested in diabetic nephropathy and provide a new treatment target.