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Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-[kappa]B (NFKB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0-36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NF[kappa]B, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NF[kappa]B p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 rain. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NF[kappa]B, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation. cytokine mix; cycloheximide

Reid, Alicia C., Christina J. Mackins, Nahid Seyedi, Roberto Levi, and Randi B. Silver. Coupling of angiotensin II [AT.sub.1] receptors to neuronal NHE activity and carrier-mediated norepinephrine release in myocardial ischemia. Am J Physiol Heart Circ Physiol 286: H1448-H1454, 2004. First published December 18, 2003; 10.1152/ ajpheart.01062.2003.--In ischemia, cardiac sympathetic nerve endings (cSNE) release excessive amounts of norepinephrine (NE) via the nonexocytotic [Na.sup.+]-dependent NE transporter (NET). NET, normally responsible for NE reuptake into cSNE, reverses in myocardial ischemia, releasing pathological amounts of NE. This carrier-mediated NE release can be triggered by elevated intracellular [Na.sup.+] levels in the axoplasm. The fact that ischemia activates the intracellular pH regulatory [Na.sup.+]/[H.sup.+] exchanger (NHE) in cSNE is pivotal in increasing intraneuronal [Na.sup.+] and thus activating carrier-mediated NE release. Angiotensin (ANG) II levels are also significantly elevated in the ischemic heart. However, the effects of ANG II on cSNE, which express the ANG II receptor, [AT.sub.1]R, are poorly understood. We hypothesized that ANG II-induced [AT.sub.1]R activation in cSNE may be positively coupled to NHE activity and thereby facilitate the pathological release of NE associated with myocardial ischemia. We tested this hypothesis in a cSNE model, human neuroblastoma cells stably transfected with rat recombinant [AT.sub.1A] receptor (SH-SY5Y-[AT.sub.1A]). SH-SY5Y-[AT.sub.1A] constitutively expresses amiloride-sensitive NHE and the NET. NHE activity was assayed in BCECF-loaded SH-SY5Y-[AT.sub.1A] as the rate of the [Na.sup.+]-dependent alkalinization in response to an acute acidosis. ANG II activation of [AT.sub.1]R markedly increased NHE activity in SH-SY5Y-[AT.sub.1A] via a [Ca.sup.2+]-dependent pathway and promoted carrier-mediated NE release. In addition, in guinea pig cSNE expressing native [AT.sub.1]R, ANG II elicited carrier-mediated NE release. In SH-SY5Y-[AT.sub.1A] and cSNE, amiloride inhibited the ANG II-mediated release of NE. Our results provide a link between [AT.sub.1]R and NHE in cSNE, which can exacerbate carrier-mediated NE release during protracted myocardial ischemia. acidosis; [Na.sup.+] -dependent norepinephrine transporter; SH-SY5Y cells; cardiac sympathetic nerves

Interactions between transforming growth factor-[beta] (TGF-[beta]) and nitric oxide (NO) are important in the pathophysiology of unilateral ureteral obstruction (UUO). Dopamine (DA) is a vasoactive renal mediator active at the [D.sub.1A] receptor ([D.sub.1A]R), which has not been studied in UUO; therefore, we examined the interactions among DA, TGF-[beta], and NO in UUO. In vivo, UUO was carried out in rats with or without concurrent treatment with 1D11, a monoclonal antibody to TGF-[beta], for 14 days. In vitro, NRK-52E cells (normal rat kidney tubules) were treated with DA, and NO and TGF-[beta] release were examined. UUO resulted in a 70% decrease in the expression of renal [D.sub.1A]R, confirmed by both Western blot analysis and immunohistochemistry. 1D11 treatment restored expression to 60% of control values. DA treatment decreased NRK-52E release of TGF-[beta] by 80%; conversely, DA significantly increased NO release from NRK-52E cells. These results suggest that DA modulates the release of cytokines, which are involved in the fibrotic and apoptotic sequelae of UUO, and that these effects are independent of DA's known vasoactive properties. kidney; obstructive uropathy; apoptosis; unilateral ureteral obstruction; [D.sub.1A] receptor

Ratiometric fluorescence imaging of the intracellular pH was used to monitor the activity of the hydrogen-potassium exchanger in the intercalated cells of the cortical collecting duct of rats under conditions of sodium chloride deprivation. Results reveal that salt deprivation increases the activity of the hydrogen-potassium exchanger in the presence of an acid load. This increase in hydrogen-potassium exchanger expression is not related to plasma levels of aldosterone.

The role of the H-K-adenosinetriphosphatase (H-K-ATPase) in the intercalated cells of neonatal rabbit cortical collecting duct (CCD) was analyzed with fluorescent hydrogen-ion concentration-sensitive dyes. Analysis of neonatal rabbit CCDs and fully differentiated rhodamine peanut lectin (PNA)-differentiated intercalated cells indicated the presence of H-K-ATPases in the early stages of development. Furthermore, H-K-ATPase activity in neonatal rabbit CCDs were inhibited by renal cytostolic proteins.

The function of renal H-K-adenosine triphosphatase (ATPase) in relation to acidosis-induced net hydrogen secretion was investigated. Measurements of the rate of K-dependent intracellular pH recovery in split open rabbit cortical collecting duct (CCD) intercalated cells (ICs) with chronic metabolic acidosis (CMA) using the single-cell fluorescence technique showed a higher K-dependent pH recovery rate in CMA ICs than in control ICs. It was concluded that CMA enhanced the functional activity of H-K-ATPase.

The influence exerted by exogenous adenosine 3.'5'-cyclic monophosphate (cAMP) on apical Na channels in rat cortical tubule is investigated. The investigation is performed using fura 23 fluorescence measurements and patch/clamp techniques associated with intracellular calcium ions. The investigation reveals that a reduction in the number of open channels is mediated by the cellular metabolism stimulated by enhanced Na transport.

Alterations in aldosterone levels in cortical collecting tubule (CCTs) of rats were used to study the activity of Na channels in apical membranes. A low Na-diet or injection of diuretic furosemide altered the aldosterone levels. The patch-clamp technique was employed to study the mean open channels per patch. An increase in the induction of channels (NPo) occurred after a week of low-salt diet or after three hours of injection of furosemide. Plasma aldoserone and NPo levels were reduced with the resumption of salt depletion for two weeks. A partial revival in aldosterone levels after 14 hours implied dissociation of channel activity and aldosterone concentration with a revived salt-diet.

The patch-clamp method was used to study Na channels in the apical membrane of the rat renal cortical collecting tubule and channel activity was regulated in cell-attached patches on tubules that were exposed to reveal the luminal surface. Alterations in Na influx cause various cellular alterations. The regulation of channel open probability by the speed of Na entry into the cell acts as a feedback function to sustain cellular ion homeostasis. This could help to diffuse Na reabsorption uniformly in the nephron.

Channel metabolism, cell Ca, and pHi under identical states in the rat cortical collecting tubule were calculated by patch-clamp and fluorescence methods. Intracellular calcium (Cai) increases due to alterations in the driving forces for Na/Ca exchange during the restriction of Na discharge from the cell by the ouabain-sensitive Na pump. The apical Na channels are restricted by Cai enhancement, possibly through a circuitous function. Na channel restriction under these states could also be caused by membrane depolarization.

Cortical collecting tubules (CCT) isolated from rabbit kidneys were examined for the demonstration of a proton-potassium antiporter inintercalated cells using fluoresent techniques. Removal of sodium and addition of potassium in the an experimental ultrafiltrate used with acid loaded CCT resulted in a partial and Sch 28080-sensitive recovery of the intracellular hydronium ion concentration in intercalated cells. This suggest the presence ofa proton-potassium antiporter in these cells. The activity of this antiporter is blocked by ouabain indicating that the transporter is an adenosine triphosphatase.

Potassium depletion increases proton pump ([H.sup.+]-ATPase) activity in intercalated cells of cortical collecting duct. Am J Physiol Renal Physiol 279: F195-F202, 2000.--Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases ([H.sup.+]-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically [K.sup.+]-depleted rats and that upregulation of tubular [H.sup.+]-ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, [H.sup.+]-ATPase activity was determined in individually identified ICs from control and chronically [K.sup.+]-depleted rats (9-14 days on a low-[K.sup.+] diet) by monitoring [K.sup.+]- and [Na.sup.+]-independent [H.sup.+] extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH ([pH.sub.i]) indicator 2',7'-bis (2-carboxyethyl)-5(6)-arboxyfluorescein, and [pH.sub.i] was determined by using ratiometric fluorescence imaging. The rate of [pH.sub.i] recovery in ICs in response to an acute acid load, a measure of plasma membrane [H.sup.+]-ATPase activity, was increased after [K.sup.+] depletion to almost three times that of controls. Furthermore, the lag time before the start of [pH.sub.i] recovery after the cells were maximally acidified fell from 93.5 [+ or -] 13.7 s in controls to 24.5 [+ or -] 2.1 s in [K.sup.+]-depleted rats. In all ICs tested, [Na.sup.+]- and [K.sup.+]-independent [pH.sub.i] recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the [H.sup.+]-ATPase. Analysis of the cell-to-cell variability in the rate of [pH.sub.i] recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from [K.sup.+]-depleted rats. Immunocytochemical analysis of collecting ducts from control and [K.sup.+]-depleted rats showed that [K.sup.+]-depletion increased the number of ICs with tight apical [H.sup.+]ATPase staining and decreased the number of cells with diffuse or basolateral [H.sup.+]-ATPase staining. Taken together, these data indicate that chronic [K.sup.+] depletion induces a marked increase in plasma membrane [H.sup.+]ATPase activity in individual ICs. proton-adenosinetriphosphatase; bafilomycin; intracellular pH; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; immunocytochemistry

Mennitt, Patricia A., Gustavo Frindt, Randi B. Silver, and Lawrence G. Palmer. Potassium restriction downregulates ROMK expression in rat kidney. Am J Physiol Renal Physiol 278: F916-F924, 2000.--The ROMK family of proteins has biophysical properties and distribution within the kidney similar to those of secretory potassium channels of the distal nephron. To study the regulation of ROMK during variations in dietary potassium, we measured the abundance of ROMK protein in rat kidney by immunoblotting. Neither 2 nor 5 days of a high-potassium diet had an effect on protein abundance in the cortex or medulla. Potassium deprivation (2 or 5 days) decreased ROMK protein content in both cortical and medullary fractions, to 51 and 40% of controls, respectively. To see whether the Na-K-2Cl cotransporter is similarly affected by potassium restriction, we analyzed immunoblots by using an antibody for the rat type 1 bumetanide-sensitive cotransporter (BSC-1). Like ROMK, BSC-1 protein content was found to decrease significantly in the renal medulla of potassium-deprived rats. In the thick ascending limb of Henle's loop, a decrease in ROMK and BSC-1 could result in decreased reabsorption of NaCl, a finding associated with hypokalemia. ascending limb of henle's loop; cortical collecting tubule; secretory potassium channel; potassium excretion; sodium-potassium-chlorine cotransporter; type 1 bumetanide-sensitive cotransporter