The repair process for renal functional recovery following toxic and ischemic acute renal failure is dependent upon regeneration of the proximal tubule epithelium and its differentiation into a mature functional phenotype. The major focus of this research proposal is directed towards understanding the molecular determinants of proximal tubule cell differentiation and tubulogenesis. Since renal proximal tubule cells have the ability to regenerate after severe nephrotoxic or ischemic injury to form a fully functional differentiated epithelium, renal proximal tubule stem cells must exist in the adult kidney. In this regard, Specific Aim 1 is to develop techniques to isolate and grow renal proximal tubule stem cells in tissue culture. Cell selection techniques are based upon retinoic acid induced laminin production or cell surface expression of integrins and utilization of magnetic fractionation of cell adhesion characteristics.
Specific Aim 2 is based upon the observation of the importance of the basement membrane protein, laminin, in renal tubulogenesis. Planned experiments will attempt to identify the microdomains of laminin responsible for the development of the differentiated renal tubule cell phenotype and lumen formation. Cell culture methodology, immunofluorescent techniques, inhibitory short polypeptide sequences and monoclonal blocking antibodies to binding sites of laminin will be utilized to determine microdomains in the laminin molecule which determines kidney tubulogenesis.
Specific Aim 3 will focus on the integrin family of plasma membrane spanning proteins, which are critical cell surface laminin receptors to identify the integrins responsible for the laminin interaction which results in the differentiated epithelial cell type of the renal proximal tubule. Experiments will utilize cell culture methodology, immunofluorescent microscopy and monoclonal blocking antibodies to specific integrin subunits to identify the intergin(s) responsible for laminin induced tubulogenesis and pattern formation in proximal tubule cells.
Specific Aim 4 is based on the thesis that laminin gene expression may be dependent upon transcriptional factors induced by retinoic acid as a differentiating factor. Since homeobox genes have been demonstrated to be important in the pattern formation in the developing embryo and have been shown to be activated by retinoic acid, the identification of specific homeobox genes expressed in mouse proximal tubule cells in tissue culture in response to various growth factors will be detailed before and after the ability of proximal tubule cells to promote tubulogenesis. These planned experiments have the potential to define the molecular determinants of kidney tubulogenesis as a final differentiation step in renal repair after ATN. The linkages between stem cells, laminin, integrins, retinoic acid and homeobox genes may begin to define the sophisticated spatial and temporal features that are required to direct and regulate a correct architectural sequence for renal tubule pattern formation, morphogenesis and, eventually, organogenesis.
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