Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells

Abstract

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…Incubation with CDME for Ͼ30 min led, however, to an inhibition of Na ϩ /H ϩ -exchanger activity by 78%. An inhibition of the Na ϩ / H ϩ -exchanger was previously shown in cystine-loaded LLC-PK 1 cells (19). The Na ϩ /H ϩ -exchanger NHE-3 is responsible for a large fraction of the Na ϩ reabsorbed by the proximal tubule.…”

“…The activity of the Na ϩ /K ϩ -ATPase was not inhibited under V max conditions (14,18), but a reduction in intracellular phosphate (32,35) and ATP content (14,18,34) was supposed to lead consecutively to an inhibition of Na ϩ / K ϩ -ATPase activity (14,18,34). According to these findings, an increase in the intracellular Na ϩ -concentration parallel with a decrease in the intracellular K ϩ -concentration in LLC-PK 1 cells (19) and for rat proximal tubules was reported (18). An increase in backflux due to an increase in proximal tubular permeability, as it was supposed previously for the maleic acid-induced Fanconi syndrome (17), could be excluded.…”

“…This model therefore mimics the biochemical hallmark of cystinosis (7). So far with this model uptake measurements with radiolabeled solutes were performed in rat (10,18,31,34) and rabbit (11,13,14,32,33) proximal tubules and with cultured porcine LLC-PK 1 cells (15,19,30). These studies showed an inhibition of volume absorption (13,14), reduction of the transepithelial potential difference (13), and inhibition of transport of different solutes (7,10,(13)(14)(15).…”

“…Previously a concentration-and time-dependent reduction of the uptake capacity of ␣-methylglucoside, a nonmetabolized glucose analog, by LLC-PK 1 cells was shown for CDME (16). In earlier studies, different incubation times from 10 min (10) to 2 (19) or 5 d (15) and CDME-concentrations from 0.1 (15) to 2 mM (10,18,19,31) were used. In our experiments, incubation of IHKE-1 cells with 1 mM CDME for 3 h led to intracellular cystine contents of 20 nmol of cystine/mg protein similar to those found in cystinosis patients (36).…”

“…Na ϩ /K ϩ -ATPase was not directly inhibited by CDME loading (14,18), but a decrease in intracellular phosphate and ATP-levels of cystine-loaded tubules with subsequent inhibition of Na ϩ /K ϩ -ATPase activity could be shown (14,15,18). According to these findings, there was an increase in intracellular Na ϩ -concentration parallel to a decrease in intracellular K ϩ -concentration in LLC-PK 1 cells (19) and in rat proximal tubules (18). This study's aim was to investigate potential effects of CDME on basal membrane voltages (V m ) of immortalized proximal tubular cells of human origin (IHKE-1 cells) (20) and to learn whether CDME differently affects acutely or after incubation some of the main Na ϩ -dependent transporters that are responsible for solute uptake across the apical membrane of these human proximal tubule cells.…”