Growth phenotype of cultured skin fibroblasts from IDDM patients with and without nephropathy and overactivity of the Na+/H+ antiporter

Abstract

An increased activity of the Na+/H+ antiporter in cells from patients with insulin-dependent diabetes mellitus (IDDM) has been proposed as a potential marker of nephropathy. We evaluated Na+/H+ antiporter activity and its relationship to DNA and protein synthesis in cultured skin fibroblasts from patients with IDDM classified as having either overt nephropathy or absence of nephropathy on the basis of urinary albumin excretion and kidney biopsy findings. In IDDM patients with overt nephropathy, Na+/H+ antiporter activity in serum stimulated cells was increased as compared to cells from control subjects (9.62 ± 0.89 vs. 5.67 ± 0.97 mmol H+/min, P < 0.005, respectively) and cells from IDDM patients without nephropathy (7.22 ± 0.67 mmol H+/min, P < 0.025). By contrast, in cells made quiescent by serum deprivation Na+/H+ antiporter activity was lower than in serum-stimulated cells and there were no significant differences between the three groups. DNA synthesis assessed by [3H] thymidine incorporation was increased in the IDDM group with nephropathy as compared to the group without nephropathy (138 ± 14 vs. 105 ± 13 cpm/1000 cells, respectively, P < 0.05) and as compared to control subjects (65 ± 11 cpm/1000 cells, P < 0.001). By contrast, protein synthesis assessed by [14C] L-leucine incorporation was not increased in fibroblasts from IDDM patients with nephropathy, suggesting that cellular hypertrophy is not a feature of their altered growth phenotype. After chronic inhibition of the Na+/D+ antiporter using EIPA (25 μM), [3H] thymidine incorporation was reduced bv about 20% both in cells from IDDM patients and controls. This parameter therefore remained higher in cells from IDDM patients with nephropathy than in those from controls (81 ± 16 vs. 40 ± 6 cpm/1000 cells, P < 0.05), while in cells from IDDM patients without nephropathy [3H] thymidine incorporation after EIPA (56 ± 7.0 cpm/1000 cells) was intermediate between cells from controls and IDDM patients with nephropathy. We argue that cultured skin fibroblasts from IDDM patients, with nephropathy display an abnormal growth phenotype characterized by cell hyperplasia. This growth phenotype is associated with overactivity of the Na+/H+ antiporter during serum stimulation but not when cells are made quiescent and persists after inhibition of the Na+/H+ antiporter. Our data, therefore, further shows that overactivity of the Na+/H+ antiporter is not required for the expression of the altered growth phenotype of cultured skin fibroblasts from IDDM patients with nephropathy.