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Cystinosis is an autosomal recessive disease resulting from mutations in ctns, which encodes for cystinosin, a proton-coupled cystine transporter that exports cystine from lysosomes. The major clinical form, infantile cystinosis, is associated with renal failure due to the malfunctioning of the renal proximal tubule (RPT). To examine the hypothesis that the malfunctioning of the cystinotic RPT arises from defective differentiation, human-induced pluripotent stem cells (hiPSCs) were generated from human dermal fibroblasts from an individual with infantile cystinosis, as well as a normal individual. The results indicate that both the cystinotic and normal hiPSCs are pluripotent and can form embryoid bodies (EBs) with the three primordial germ layers. When the normal hiPSCs were subjected to a differentiation regime that induces RPT formation, organoids containing tubules with lumens emerged that expressed distinctive RPT proteins, including villin, the Na+/H+ Exchanger (NHE) isoform 3 (NHE3), and the NHE Regulatory Factor 1 (NHERF1). The formation of tubules with lumens was less pronounced in organoids derived from cystinotic hiPSCs, although the organoids expressed villin, NHE3, and NHERF1. These observations can be attributed to an impairment in differentiation and/or by other defects which cause cystinotic RPTs to have an increased propensity to undergo apoptosis or other types of programmed cell death. [ABSTRACT FROM AUTHOR]

Prostaglandins (PGs) play a number of roles in the kidney, including regulation of salt and water reabsorption. In this report, evidence was obtained for stimulatory effects of PGs on Na-K-ATPase in primary cultures of rabbit renal proximal tubule (RPT) cells. The results of our real-time PCR studies indicate that in primary RPTs the effects of [PGE.sub.2], the major renal PG, are mediated by four classes of PGE (EP) receptors. The role of these EP receptors in the regulation of Na-K-ATPase was examined at the transcriptional level. Na-K-ATPase consists of a catalytic [alpha]-subunit encoded by the ATP1A1 gene, as well as a [beta]-subunit encoded by the ATPIB1 gene. Transient transfection studies conducted with pH[beta]1-1141 Luc, a human ATPIB1 promoter/luciferase construct, indicate that both [PGE.sub.1] and [PGE.sub.2] are stimulatory. The evidence for the involvement of both the cAMP and [Ca.sup.2+] signaling pathways includes the inhibitory effects of the myristolylated PKA inhibitor PKI, the adenylate cyclase (AC) inhibitor SQ22536, and the PKC inhibitors Go 6976 and Ro-32-0432 on the [PGE.sub.1] stimulation. Other effectors that similarly act through cAMP and PKC were also stimulatory to transcription, including norepinephrine and dopamine. In addition to its effects on transcription, a chronic incubation with [PGE.sub.1] was observed to result in an increase in Na-K-ATPase mRNA levels as well as an increase in Na-K-ATPase activity. An acute stimulatory effect of [PGE.sub.1] on Na-K-ATPase was observed and was associated with an increase in the level of Na-K-ATPase in the basolateral membrane. doi: 10.1152/ajprenal.00467.2009.

The accumulation of uric acid, an end-product of purine metabolism, is responsible for the many deleterious effects observed in gouty arthritis, including renal injury. Here, we present evidence that under conditions of hyperuricemia (> [10.sup.-4] M uric acid) [[sup.3]H]thymidine incorporation into primary renal proximal tubule cells (PTCs) is inhibited, and we delineate the signaling pathways involved. Elevated uric acid was observed to stimulate MAPK phosphorylation. The uric acid induced p38 MAPK phosphorylation was also blocked by H-7 (a PKC inhibitor), indicating that p38 MAPK was a downstream target of PKC. Evidence that cytoplasmic phospholipase [A.sub.2] (cPL[A.sub.2]) was involved further downstream included 1) the stimulatory effect of uric acid on [[sup.3]H]-labeled arachidonic acid (AA) release; 2) the stimulation of AA release in response to uric acid was blocked by the PKC inhibitor H-7 as well as by the p38 MAPK inhibitor SB 203580; and 3) the uric acid-induced inhibition of [[sup.3]H]thymidine incorporation was prevented by SB 203580, as well as by the cPL[A.sub.2] inhibitor arachidonyl trifluoromethyl ketone, and mepacrine (another PL[A.sub.2] inhibitor). Evidence of a uric acid-induced activation of NF-[kappa]B as well as PL[A.sub.2] was obtained. Moreover the uric acid-induced inhibition of [[sup.3]H]thymidine incorporation was also blocked by two NF-[kappa]B inhibitors, pyrrolidine dithiocarbamate and SN 50. However, SN 50 did not block the uric acid induced [[sup.3]H]AA release. Thus the inhibition of [[sup.3]H]thymidine incorporation caused by uric acid can be explained by two distinct mechanisms, the activation of NF-[kappa]B as well as the activation of PL[A.sub.2]. kidney; hyperuricemia

Renal prostaglandins modulate the activity of a number of the transport systems in the kidney, including the Na-K-ATPase. Not only do prostaglandins have acute affects on renal Na-K-ATPase, but in addition prostaglandins have chronic affects, which include regulation at the transcriptional level. Previously, we have presented evidence that one such prostaglandin, PG[E.sub.1] stimulates the transcription of the human Na-K-ATPase [[beta].sub.1]-subunit gene in Madin-Darby canine kidney cells via cAMP- and [Ca.sup.2+]-mediated pathways (Taub M, Borsick M, Geisel J, Matlhagela K, Rajkhowa T, and Allen C. Exp Cell Res 299: 1-14, 2004; Matlhagela K, Borsick M, Rajkhowa T, and Taub M. J Biol Chem 280: 334-346, 2005). Evidence was presented indicating that PG[E.sub.1] stimulation was mediated through the binding of cAMP-regulatory element binding protein (CREB) to a prostaglandin-responsive element (PGRE) as well as Sp1 binding to an adjacent Sp1 site. In this report, we present evidence from EMSAs and DNA affinity precipitation studies that another PGRE present in the Na-K-ATPase [[beta].sub.1]-subunit promoter similarly binds CREB and Sp1. The evidence that indicates a requirement for CREB as well as Sp1 for gene activation through both PGREs (PGRE1 and PGRE3) includes studies with a dominant negative CREB (KCREB), Drosophila SL2 cells, and PGRE mutants. The results of these studies are indicative of a synergism between Sp1 and CREB in mediating regulation by PGRE3; while regulation occurring through PGRE1 also involves Sp1 and CREB, the mechanism appears to be distinct. Madin-Darby canine kidney cells; transport; gene regulation; kidney; eicosanoids do:10.1152/ajprenal.00452.2005

Oxidative stress plays an important role in the pathogenesis of renal diseases such as diabetic nephropathy. The metabolism of excessive intracellular glucose may involve a number of processes. One consequence of excessive intracellular glucose levels is an increased rate of oxidative phosphorylation under hyperglycemic conditions, whereas another consequence is an increase in the metabolism of glucose to sorbitol by aldose reductase. In addition, hyperglycemia may result in the activation of NADPH oxidase, the production of superoxide anion, and hydrogen peroxide ([H.sub.2][O.sub.2]). In this report, we investigate the mechanisms responsible for the [H.sub.2][O.sub.2] production that occurs as the consequence of hyperglycemia and the effect of [H.sub.2][O.sub.2] on the activity of the [Na.sup.+]/glucose cotransport system (SGLT) in primary cultures of renal proximal tubule cells (PTCs). When primary PTCs were cultured in the presence of high glucose, one consequence was that the [Na.sup.+]/glucose cotransport system was inhibited, as indicated by uptake studies utilizing [alpha]-methyl-D-glucoside ([alpha]-MG), a nonmetabolizable analog of D-glucose. Pretreatment of the cultures with either 1) aminoguanidine or pyridoxamine [inhibitors of the accumulation of advanced glycation end products (AGEs)], 2) rotenone (an inhibitor of the mitochondrial electron transport chain), or 3) apocynin or diphenylene iodonium (DPI; inhibitors of NADPH oxidase) blocked the observed changes that occurred as a consequence of the incubation of the PTCs with high glucose. Included among these changes were the observed increase in [H.sub.2][O.sub.2] levels, as well as an increase in lipid peroxide production, and a decrease both in the activity of catalase and in the level of glutathione (GSH), endogenous antioxidants. The high glucose-induced decrease in the level of the [Na.sup.+]/glucose cotransporter was similarly prevented by either aminoguanidine, rotenone, or apocynin. Thus the inhibitory effect of high glucose on both the level of the [Na.sup.+]/glucose cotransport system and the activity of the [Na.sup.+]/ glucose cotransport system can be explained, at least in part, as being due to the effects of the [H.sub.2][O.sub.2], the consequent formation of AGEs, the increase in mitochondrial metabolism, and in NADPH oxidase activity in the PTCs. Other related changes observed in the PTCs that could be reversed by treatment with either aminoguanidine, pyridoxamine, rotenone, apocynin, or DPI included an increase in transforming growth factor-[[beta].sub.1] secretion and the activation of the NF-[kappa]B signal transduction pathway. reactive oxygen species; kidney; hydrogen peroxide; diabetic nephropathy; NF-[kappa]B; TGF-[[beta].sub.1]