Inhibition of Na+-Dependent Transporters in Cystine-Loaded Human Renal Cells

Abstract

Abstract. Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V m ) of IHKE-1 cells were Ϫ30.7 Ϯ 0.4 mV (n ϭ 151). CDME concentration-dependently altered V m with an initial depolarization (2.7 Ϯ 0.2 mV; n ϭ 76; 1 mM CDME) followed by a more pronounced hyperpolarization (Ϫ9.9 Ϯ 1.0 mV; n ϭ 49). Three Na ϩ -dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 Ϯ 0.7 mV (n ϭ 59), and phosphate (1.8 mM) depolarized by 9.7 Ϯ 1.1 mV (n ϭ 18).Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V m by 7.1 Ϯ 0.3 mV (n ϭ 21) followed by a repolarization by 2.9 Ϯ 0.3 mV/min (n ϭ 17), reflecting Na ϩ /H ϩ -exchanger activity. Acute addition of 1 mM CDME did not alter the alanine-and propionate-induced changes in V m , but it reduced the phosphate-induced depolarization by 37 Ϯ 9% (n ϭ 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 Ϯ 4% (n ϭ 30), 45 Ϯ 9% (n ϭ 9), and 78 Ϯ 15% (n ϭ 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V m of IHKE-1 cells and that at least three Na ϩ -dependent transporters are inhibited, the Na ϩ -phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na ϩ -gradient are involved in the development of the Fanconi syndrome of cystinosis.

Cystine Dimethylester Model of Cystinosis: Still Reliable?

“…In concordance with this idea, a recent study using the IHKE-1 cell line demonstrated that CDME had an acute effect on the basal membrane potential, possibly due to activated K ϩ conductance (16). In the present study, we investigated the effect of CDME loading on mitochondrial ATP-generating capacity, superoxide production, and viability of control fibroblasts compared with cystinotic fibroblasts accumulating cystine due to a known genetic defect of the CTNS gene.…”

Decreased Intracellular ATP Content and Intact Mitochondrial Energy Generating Capacity in Human Cystinotic Fibroblasts

“…This effect was distinct from those on Na ϩ -dependent alanine transporter, which was inhibited after at least 30 min of incubation with CDME. As the hyperpolarization induced by CDME could be blocked by Ba 2ϩ , the authors concluded that CDME activated a K ϩ conductance (34).…”

Lysosomal Cystine Storage Augments Apoptosis in Cultured Human Fibroblasts and Renal Tubular Epithelial Cells

“…These two tissues are the first to be affected in nephropathic cystinosis (2), and it is feasible that the order of tissues involved in the disease reflects the intrinsic sensitivity of each to apoptosis. CDME is known to disrupt renal tubule cell function in animal models and cultured cells (31)(32)(33), and it could be that exposure to CDME alone alters the redox potential sufficiently to trigger the apoptotic response (Figures 5 and 6). CDME also causes inhibition of Na ϩ -dependent transporters (33).…”

“…CDME is known to disrupt renal tubule cell function in animal models and cultured cells (31)(32)(33), and it could be that exposure to CDME alone alters the redox potential sufficiently to trigger the apoptotic response (Figures 5 and 6). CDME also causes inhibition of Na ϩ -dependent transporters (33). Free cystine has been known to be nephrotoxic in experimental animals since 1925 (34); however, the mechanism remains to be determined.…”