Cystine loading induces Fanconi's syndrome in rats: in vivo and vesicle studies

Abstract

To better understand the link between lysosomal cystine accumulation and the renal impairment seen in cystinosis, we have studied the effect of cystine loading in vivo, on renal function of rats, and in brush-border membrane vesicles (BBMV) prepared from the kidney cortex of the treated rats. Intraperitoneal injection of cystine dimethyl ester (CDME) (400 mumol, twice a day, for 5 days) led to an increased urine volume and excretion of glucose, phosphate, and protein. Kinetic analysis of alpha-methylglucoside initial flux in BBMV showed reduction in maximal transport capacity (Vmax, from 10.1 +/- 1.3 to 8.5 +/- 0.7 nmol.min-1.mg protein-1; P < 0.01) with no change in Michaelis constant (Km, 4.80 +/- 0.08 and 4.90 +/- 0.05 mM). The number of phlorizin binding sites declined (from 6.5 +/- 0.7 to 4.1 +/- 0.4 pmol/mg protein; P < 0.01) with no significant change in the affinity for phlorizin (0.64 +/- 0.08 and 0.59 +/- 0.06 microM). In the cortex homogenate, cystine concentration, which was undetectable in controls, increased to 0.97 +/- 0.09 nmol 1/2 cystine/mg protein. Two hours after CDME administration, ATP content declined to approximately 50% of control values. This decline was transient, and ATP content was recovered to control values 5 h after CDME administration. The treatment did not affect ouabain-sensitive adenosinetriphosphatase activity (40.0 +/- 3.9 and 38.6 +/- 4.7 nmol Pi.mg protein-1.min-1) or the number and affinity of ouabain binding sites (Bmax = 1.48 +/- 0.25 and 1.44 +/- 0.18 pmol/mg, and Kd = 0.68 +/- 0.09 and 0.72 +/- 0.12 microM, respectively). (ABSTRACT TRUNCATED AT 250 WORDS)

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

“…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). Most of proximal tubular transport is Na ϩ -coupled; depending therefore on the Na ϩ -gradient from the extrato the intracellular space, the role of energy metabolism, Na ϩ / K ϩ -ATPase activity, intracellular phosphate, and ATP levels in this experimental model have been investigated (14,18,31,33,34). 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).…”

“…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).…”

“…Most of proximal tubular transport is Na ϩ -coupled; depending therefore on the Na ϩ -gradient from the extrato the intracellular space, the role of energy metabolism, Na ϩ / K ϩ -ATPase activity, intracellular phosphate, and ATP levels in this experimental model have been investigated (14,18,31,33,34). 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).…”

“…These findings indicate that at least 30 min of CDME accumulation are necessary in these human proximal tubule cells to interfere with the Na ϩ -alanine cotransporter. 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.…”

“…Inhibition of glucose transport was also shown in LLC-PK 1 cells (15). Corresponding to the decreased glucose uptake, cystine loading resulted in a reduced number of glucose transporters on the apical membrane of LLC-PK 1 cells (15) and in brush border membrane vesicles from rats after intraperitoneal CDME injections (16). Salmon et al (13) used the CDME model to show that reduction in proximal tubular transport was due to an inhibition of active transport but not to an increase in proximal tubular permeability, as it was previously supposed for the Fanconi syndrome caused by maleic acid (17).…”