CELL GROWTH, NA/H EXCHANGE, AND CYCLINS IN IDDM PATIENTS

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The development of nephropathy is the strongest predictor of premature death and cardiovascular disease in insulin-dependent diabetic patients (IDDM). The risk of nephropathy, which occurs in about 25% of IDDM patients, appear, at least in part, to be genetically determined. An increase in the activity of the Na+/H+ antiporter and intrinsic growth nephropathy. The overall theme of this proposal is the examination of the growth phenotype expressed in skin fibroblasts from IDDM patients and its relationship to nephropathy. Since overactivity of the Na+/H+ antiporter is a feature of cultured skin fibroblasts from IDDM patients with nephropathy and the NHE-1 isoform of this transporter is involved in cell growth, Na+/H+ antiporter activity, the relative abundance of NHE-1 protein, and its phosphorylation levels will be evaluated. We hypothesize that over-expression of NHE-1 and cyclin D1 protein in skin fibroblasts grown in early passage in culture from IDDM patients with nephropathy may be key features of a growth phenotype which may serve as a surrogate predictor for nephropathy. The proposed studies will also help unravel the mechanism(s) whereby DNA synthesis is enhanced in fibroblasts from IDDM patients with nephropathy. FACS analysis will be used to determine which phase of the cell-cycle is abnormal in fibroblasts from IDDM patients with nephropathy. The retinoblastoma protein (pRB), acting in its un- phosphorylated form, inhibits cell-cycle progression at the GS/1 boundary. To examine G1 transit in the cell cycle, we will assess pRB phosphorylation, the abundance of cyclins D1 and E as well as cyclin D1 kinase (CD1K) activity using pRB as the substrate in the basal stage and after stimulation with angiotensin II or serum. As cyclin D1 encodes the regulatory subunit of a holoenzyme required for pRB phosphorylation in the G1 phase of the cell cycle, the relative abundance of this cyclin, if increased in cells from IDDM patients with nephropathy, could be a determinant of shortened G1 transit and thus enhanced DNA synthesis. We will therefore characterize cyclin D1 expression and CD1K activity in the fibroblasts of IDDM patients with and without nephropathy as compared to those from age-matched controls [and individuals with essential hypertension] to determine where abnormalities of this and/or other G1 cyclins and cyclin dependent kinase inhibitor proteins (KIP's contribute to the altered growth phenotype observed in IDDM patients with nephropathy. If cyclin D1 levels and activity are increased in the fibroblasts from IDDM patients with nephropathy, we will test the significance of the increased cyclin D1 explicitly by altering cyclin D1 expression through the microinjection of inducible antisense and sensory vectors we have recently characterized.