ABSTRACT
Nephropathic cystinosis is an autosomal recessive disorder caused by mutations in the CTNS gene [1], which encodes for a transporter (cystinosin) responsible for cystine efflux from lysosomes. In cystinotic renal proximal tubules (RPTs), the defect in cystinosin function results in reduced reabsorption of solutes by apical Na(+)/solute cotransport systems, including the Na(+)/phosphate (Pi) cotransport system [2]. However the underlying molecular mechanisms are unknown, given the lack of an appropriate cellular model. To obtain such a model system, we have knocked down cystinosin with siRNA in primary RPT cell cultures. An 80% reduction in cystinosin strongly inhibited Na(+) dependent Pi uptake (70%). Although this finding could be explained by a direct effect on transporters as well as by altered energetics (the ATP level dropped by 52%), our results demonstrate a lack of involvement of Na, K-ATPase, and a reduction in the number of NaPi2a transporters.
Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Kidney Tubules, Proximal/metabolism , Phosphates/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Transport Systems, Neutral/genetics , Animals , Cells, Cultured , Cystinosis , Energy Metabolism , Fanconi Syndrome , Gene Knockdown Techniques , Ion Transport , Nephrotic Syndrome/genetics , Nephrotic Syndrome/metabolism , RNA, Small Interfering/genetics , Rabbits , Sodium/metabolism , Sodium-Phosphate Cotransporter Proteins, Type IIa/geneticsABSTRACT
Both oxidative stress and epidermal growth factor (EGF) contribute to the initiation and progression of renal proximal tubular dysfunction under pathophysiologic conditions. Thus, this study was performed (1) to examine both the individual, and the combined effects of H2O2 and EGF on alpha-methyl-D-glucopyranoside uptake (alpha-MG uptake) in the primary cultured renal proximal tubule cells (PTCs), and (2) to elucidate the involvement of p44/42 mitogen activated protein kinase (MAPK) and phospholipase A2 in mediating these actions. Both H2O2 and EGF inhibited alpha-MG uptake individually, while the combination of H2O2 and EGF further potentiated the inhibitory effect on alpha-MG uptake, which was elicited by each agent. H2O2 not only caused a rapid increase in the phosphorylation of p44/42 MAPK, but also promoted the translocation of cytosolic phospholipase A2 (cPLA2) from the cytosolic to particulate fraction, and stimulated cellular [3H]-arachidonic acid (AA) release. EGF similarly activates phosphorylation of p44/42 MAPK and stimulates [3H]-AA release. When PTCs were exposed to 100 microM H2O2 and 50 ng/ml EGF simultaneously, a further increase in the phosphorylation of p44/42 MAPK, of [3H]-AA release, and of prostaglandin E2 (PGE2) production was elicited as compared with the effects of each individual agonist alone. Moreover, the additive phosphorylation of p44/42 MAPK, [3H]-AA release, and PGE2 production by H2O2 and EGF was almost completely inhibited by the p44/42 MAPK inhibitor, PD 98059. In conclusion, these results are consistent with the hypothesis that under conditions of oxidative stress, the H2O2-induced inhibition of alpha-MG uptake in the renal proximal tubule is mediated through a modulation of the EGF signaling pathway, promoting further phosphorylation of p44/42 MAPK, activation of PLA2.
