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)

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)

A 35-year-old man had severe exercise intolerance and cramps. Venous blood lactate did not rise after ischemic exercise, and electromyographically silent contracture of hand muscles appeared. Histochemistry and electronmicroscopy of a muscle biopsy revealed subsarcolemmal and intermyofibrillar accumulation of glycogen. Biochemical studies showed moderately increased amount of glycogen. Total phosphorylase activity was normal, but the active form "a" was 27% of normal. Phosphorylase kinase activity was 12% of the normal value and was normal in leukocytes and erythrocytes.

We studied the effect of lysosomal loading with cystine (by incubation with cystine dimethyl ester, CDME) on the functions of renal epithelial cells in culture (LLC-PK₁). The concentrating capacity (the ratio of the intra- and extracellular concentrations) of the nonmetabolized glucose analog, α-methylglucoside (AMG), was reduced after incubation with CDME in a dose- and time-dependent manner. Consistent with these findings was a 30% reduction in the number of the sodium-coupled D-glucose transporters assessed by phlorizin binding. Since the impairment in glucose transport was less effective with other amino acid methyl ester analogues, LLC-PK₁ cells treated with CDME can be used to study the cellular mechanisms responsible for the impairment of kidney function in cystine accumulation diseases, e.g., cystinosis.

Pediat. Two sisters showed a similar pattern, whereas one brother had normal PK activity. The patient's liver protein kinase activity was normal. Addition of exogenous protein kinase did not affect PK activity, whereas exogenous PK restored phosphorylase activity to normal.These findings indicate that these patients are affected by a rare variant of PK deficiency, which involves both muscle and liver and which apparently is not sex linked. It is possible that this defect represents an unusual mutation of a subunit of the phosphorylase kinase enzyme. Speculation