Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.

Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-ß1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-ß1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.

The effect of oral contraceptives on the nitric oxide system and renal function.

We have demonstrated that oral contraceptive (OC) users exhibit elevated angiotensin II levels and angiotensin II type 1 receptor expression, indicative of renin-angiotensin system (RAS) activation, yet the renal and systemic consequences are minimal, suggesting that there is increased vasodilatory activity, counteracting the effect of RAS activation. We hypothesized that the nitric oxide (NO) system would be upregulated in OC users and that this would be reflected by a blunted hemodynamic response to l-arginine infusion. All subjects were studied after a 7-day controlled sodium and protein diet. Inulin and para-aminohippurate clearance techniques were used to assess renal function. l-Arginine was infused at 100, 250, and 500 mg/kg, each over 30 min. Skin endothelial NO synthase mRNA expression was assessed by real-time PCR. While OC nonusers exhibited significant increases in effective renal plasma flow (670.8 +/- 35.6 to 816.2 +/- 59.7 ml.min(-1).1.73 m(-2)) and glomerular filtration rate (133.4 +/- 4.3 to 151.0 +/- 5.7 ml.min(-1).1.73 m(-2), P = 0.04) and declines in renal vascular resistance (81.1 +/- 6.1 to 63.5 +/- 6.2 mmHg.ml(-1).min, P = 0.001) at the lower l-arginine infusion rates, the responses in OC users were blunted. While l-arginine reduced mean arterial pressure at the 250 and 500 mg/kg doses in OC nonusers, OC users only exhibited a decrease in mean arterial pressure at the highest infusion rate. In contrast, tissue endothelial NO synthase mRNA levels were higher in the OC users (P = 0.04). In summary, these findings suggest that the NO system is upregulated by OC use in young, healthy women. Increased activity of the NO pathway may modulate the hemodynamic effects of RAS activation in OC users.

Angiotensin-(1-7) inhibits angiotensin II-stimulated phosphorylation of MAP kinases in proximal tubular cells.

Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE, which is not blocked by ACE inhibitors. High amounts of ACE2 are present in the proximal tubule, and ACE2 catalyzes generation of angiotensin 1-7 (Ang-(1-7)) by this segment. Ang-(1-7) binds to a receptor distinct from the AT1 or AT2 Ang II receptor, identified as the mas receptor. We studied the effects of Ang-(1-7) on Ang II-mediated cell signaling pathways in proximal tubule. In primary cultures of rat proximal tubular cells, activation of mitogen-activated protein kinases (MAPK) was detected by immunoblotting, in the presence or absence of agonists/antagonists. Transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay. Ang II (5 min, 10(-7) M) stimulated phosphorylation of the three MAPK (p38, extracellular signal-related kinase (ERK 1/2), and c-Jun N-terminal kinase (JNK)). While incubation of proximal tubular cells with Ang-(1-7) alone did not significantly affect MAPK phosphorylation, Ang-(1-7) (10(-7) M) completely inhibited Ang II-stimulated phosphorylation of p38, ERK 1/2, and JNK. This inhibitory effect was reversed by the Ang-(1-7) receptor antagonist, D-Ala7-Ang-(1-7). Ang II significantly increased production of TGF-beta1 in proximal tubular cells, an effect that was partly inhibited by Ang-(1-7). Ang-(1-7) had no significant effect on cyclic 3',5'-adenosine monophosphate production in these cells. In summary, Ang-(1-7) inhibits Ang II-stimulated MAPK phosphorylation in proximal tubular cells. Generation of Ang-(1-7) by proximal tubular ACE2 could thereby serve a protective role by counteracting the effects of locally generated Ang II.

Effect of potassium depletion on proximal tubule AT1 receptor localization in normal and remnant rat kidney.

BACKGROUND: Since both potassium depletion and renal ablation result in proximal tubule hypertrophy and the angiotensin II type 1 (AT1) receptor has been localized in rat proximal tubules, we explored the possibility that the AT1 receptor intracellular distribution is modulated by potassium depletion in proximal tubular cells of 5/6 nephrectomized (Nx) rats. METHODS: Four groups of rats were studied: sham operated, potassium-depleted sham-operated rats, 5/6 Nx rats two weeks postsurgery, and potassium-depleted 5/6 Nx rats two weeks postsurgery. After the morphometry of proximal tubular cells was defined, by using immmunogold electron microscopy techniques the subcellular distribution of AT1 receptors were visualized and quantitated. RESULTS: Hypertrophy of proximal tubule cells due to both 5/6 Nx and potassium depletion was documented. Furthermore, to our knowledge for the first time, the results showed that in potassium depletion, with and without superimposed 5/6 Nx, the AT1 receptor density in proximal tubular cells was dramatically enhanced in the apical membrane, the basal membrane, and in nuclei. CONCLUSION: In normal rats and those subjected to renal ablation, these immunocytochemical data provide intracellular proximal tubule AT1 receptor localization and demonstrate loci of increased receptor density after potassium depletion.

Hyperbaric oxygen in the treatment of calciphylaxis: a case series.

BACKGROUND: Calciphylaxis, also referred to as calcific uraemic arteriolopathy, is a syndrome associated with end-stage renal disease (ESRD), and causes necrotic skin ulcers, often leading to a fatal outcome. Hyperbaric oxygen (HBO(2)) therapy has been used to enhance wound healing, but its role in the treatment of calciphylaxis is unclear. METHODS: We undertook a retrospective study of patients on renal replacement therapy with biopsy-proven calciphylaxis who were treated with HBO(2) between March 1997 and February 2000. RESULTS: Five patients were treated with HBO(2): three patients were on continuous ambulatory peritoneal dialysis (CAPD) and two were on chronic haemodialysis therapy. None of the patients had uncontrolled hyperparathyroidism and none underwent parathyroidectomy. The patients each received 25-35 treatments of HBO(2) at 2.5 atmospheres for 90 min per treatment. Two of these patients had complete resolution of extensive necrotic skin ulcers, with no adverse effects of HBO(2) therapy. Both had improvement in wound area transcutaneous oxygen pressure (P(tc)O(2)) with administration of 100% oxygen when measurements were taken at normobaric and hyperbaric pressures. In the other three patients receiving HBO(2), the skin lesions did not resolve. P(tc)O(2) was measured in two of these patients, neither of whom showed improvement with 100% oxygen administered at normobaric pressure. CONCLUSIONS: The data support a role for HBO(2) in the treatment of some patients with calciphylaxis, particularly as in the absence of uncontrolled secondary hyperparathyroidism there are few therapeutic options.

Angiotensin II AT(2) receptors inhibit growth responses in proximal tubule cells.

Angiotensin II (ANG II) subtype 2 (AT(2)) receptors are expressed in the adult kidney, but the effects of AT(2) receptor activation are unclear. The proximal tubule cell line LLC-PK(1) was transfected with a plasmid containing cDNA for the rat AT(2) receptor. In transfected cells, specific binding of (125)I-labeled ANG II was detected (dissociation constant = 0.81 nM), with inhibition by the AT(2) antagonist PD-123319, and no effect of the AT(1) antagonist losartan. ANG II (10(-7) M) significantly inhibited mitogen-activated protein kinase (MAPK) activity in transfected cells, associated with decreased phosphorylation of the extracellular signal-related kinases ERK1 and ERK2. ANG II stimulated phosphotyrosine phosphatase activity within 5 min, an effect blocked by PD-123319 and the phosphatase inhibitor vanadate. In transfected cells, ANG II inhibited epidermal growth factor-stimulated [(3)H]thymidine incorporation, an effect reversed by vanadate. In contrast, vanadate did not block ANG II-stimulated apoptosis of transfected cells. In summary, AT(2) receptors in proximal tubule cells inhibit MAPK activity and stimulate phosphotyrosine phosphatase. AT(2) receptor-induced inhibition of mitogenesis is mediated by phosphatase activation, whereas effects on apoptosis are insensitive to phosphatase inhibition. The data suggest that AT(2) receptors inhibit cell growth via distinct signaling pathways in the proximal tubule.

Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo.

To directly determine intratubular nitric oxide concentrations ([NO]) in vivo, we modified amperometric integrated electrodes (WPI P/N ISO-NOP007), which are highly sensitive to NO and not affected by ascorbic acid, nitrite, L-arginine, or dopamine. Although reactive lengths were as short as 5 microm long, the electrode still responded rapidly. With the use of kidney surface fluid as the "zero point," the electrode tip was inserted into tubular segments along the track of a perforation made by a beveled glass pipette. The surface fluid zero point was usually stable as distal, late proximal, and early proximal tubule [NO] levels were measured sequentially in the same nephron. In eight normal rats, distal, late proximal, and early proximal [NO] concentrations were each approximately 110 nM. In contrast, in nine 5/6 nephrectomized rats 2 wk postsurgery, although [NO] also did not differ among distal, late proximal, and early proximal segments, levels were approximately fourfold higher than those in normal rats and were significantly reduced after N(G)-monomethyl-L-arginine administration. These are the first quantitative in vivo tubular fluid [NO] measurements and show a significant increase in tubular fluid [NO] after renal ablation.

Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells.

Angiotensin IV (ANG IV), the COOH-terminal hexapeptide fragment of angiotensin II (ANG II), binds to specific sites in the kidney, distinct from type 1 (AT(1)) and type 2 (AT(2)) receptors and designated type 4 (AT(4)) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK(1)/Cl(4). In these cells, we found no specific binding of [(125)I]-ANG II. In contrast, ANG IV dose dependently competed for [(125)I]-labeled ANG IV binding, with no displacement by either ANG II, the AT(1) receptor antagonist losartan, or the AT(2) antagonist PD-123319. Saturation binding indicated the presence of AT(4) receptors of high affinity [dissociation constant (K(d)) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did not increase cytosolic calcium concentration in these cells. In contrast, immunoprecipitation and immunoblotting studies revealed that ANG IV caused dose-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-FAK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not affected by pretreatment with either losartan or PD-123319, and ANG II (10(-7) M) did not induce protein tyrosine phosphorylation. Our results indicate that LLC-PK(1)/Cl(4) cells express ANG IV receptors, which we demonstrate for the first time are linked to tyrosine phosphorylation of focal adhesion-associated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cells.

Early streptozotocin-diabetes mellitus downregulates rat kidney AT2 receptors.

The interaction of ANG II with intrarenal AT1 receptors has been implicated in the progression of diabetic nephropathy, but the role of intrarenal AT2 receptors is unknown. The present studies determined the effect of early diabetes on components of the glomerular renin-angiotensin system and on expression of kidney AT2 receptors. Three groups of rats were studied after 2 wk: 1) control (C), 2) streptozotocin (STZ)-induced diabetic (D), and 3) STZ-induced diabetic with insulin implant (D+I), to maintain normoglycemia. By competitive RT-PCR, early diabetes had no significant effect on glomerular mRNA expression for renin, angiotensinogen, or angiotensin-converting enzyme (ACE). In isolated glomeruli, nonglycosylated (41-kDa) AT1 receptor protein expression (AT1A and AT1B) was increased in D rats, with no change in glycosylated (53-kDa) AT1 receptor protein or in AT1 receptor mRNA. By contrast, STZ diabetes caused a significant decrease in glomerular AT2 receptor protein expression (47.0 +/- 6.5% of C; P < 0.001; n = 6), with partial reversal in D+I rats. In normal rat kidney, AT2 receptor immunostaining was localized to glomerular endothelial cells and tubular epithelial cells in the cortex, interstitial, and tubular cells in the outer medulla, and inner medullary collecting duct cells. STZ diabetes caused a significant decrease in AT2 receptor immunostaining in all kidney regions, an effect partially reversed in D+I rats. In summary, early diabetes has no effect on glomerular mRNA expression for renin, angiotensinogen, or ACE. AT2 receptors are present in glomeruli and are downregulated in early diabetes, as are all kidney AT2 receptors. Our data suggest that alterations in the balance of kidney AT1 and AT2 receptor expression may contribute to ANG II-mediated glomerular injury in progressive diabetic nephropathy.

Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat.

BACKGROUND: Enhanced intrarenal angiotensin II (Ang II) activity may contribute to diabetic nephropathy. The proximal tubule is a proposed site of significant intrarenal Ang II production. We determined the effect of early diabetes on mRNA expression of components of the proximal tubule renin-angiotensin system. METHODS: Three groups of male Sprague-Dawley rats were studied after two weeks: (1) control (C), (2) streptozotocin-induced diabetes (STZ), and (3) STZ-induced diabetes, with normoglycemia maintained by insulin implants (STZ-I). Competitive reverse transcription-polymerase chain reaction was used to assay mRNA for renin, angiotensinogen, and angiotensin-converting enzyme in suspensions of proximal tubules; plasma and kidney levels of Ang II were measured by radioimmunoassay, and Western analysis of Ang II subtype 1 (AT1) receptors was performed. RESULTS: STZ rats tended to have increased plasma and intrarenal levels of Ang II compared with C and STZ-I rats. In proximal tubules, mRNA for renin was significantly increased in STZ rats, with reversal to control values in STZ-I rats (C, 2432 +/- 437 vs. STZ, 5688 +/- 890 fg/0.25 microg RNA, P < 0.05 vs. C, N = 9, vs. STZ-I, 1676 +/- 376 fg/0.25 microg RNA, P = NS vs. C). In STZ rats, the AT1 receptor antagonist losartan caused a further fivefold increase in proximal tubule renin mRNA, associated with proximal tubular renin immunostaining. STZ had no significant effect on mRNA expression for angiotensinogen or angiotensin-converting enzyme in proximal tubules. By Western blot analysis, cortical and proximal tubule AT1 receptor protein expression was significantly decreased in STZ rats. CONCLUSIONS: These data suggest activation of the proximal tubule renin-angiotensin system in early STZ diabetes, mediated at least partly by enhanced expression of renin mRNA. Increased local production of Ang II could contribute to tubulointerstitial injury in this model.

Angiotensin II and its receptors in the diabetic kidney.

Studies using either angiotensin-converting enzyme inhibitors or type 1 (AT(1)) angiotensin II (ANG II)-receptor blockers indicate that ANG II is a mediator of progressive injury in diabetic nephropathy. However, suppression of the systemic renin-angiotensin system (RAS) generally has been shown in diabetes mellitus. Evidence suggests that intrarenal RASs within glomeruli and proximal tubules may be activated with hyperglycemia, leading to stimulation of local ANG II production, which may exert feedback inhibition of systemic renin release. Once formed, intrarenal ANG II exerts most of its well-described effects through binding to AT(1) receptors that are abundantly present in cells of the glomeruli, tubules, vasculature, and interstitium. Thus, AT(1)-receptor activation increases vascular resistance, reduces renal blood flow, and stimulates production of extracellular matrix in the mesangium and tubulointerstitium. Recent studies suggest that the adult kidney also expresses type 2 (AT(2)) ANG II receptors in glomeruli, tubular segments, and vasculature. AT(2)-receptor activation is associated with increased intrarenal nitric oxide production, stimulation of natriuresis, and inhibition of cell growth and matrix synthesis, effects that oppose those of kidney AT(1) receptors. A number of studies have shown a reduction in kidney AT(1)-receptor expression in diabetic nephropathy, suggesting that the balance between AT(1)- and AT(2)-receptor-mediated cell-signaling events may be a determinant of progression rate in diabetic nephropathy and that unopposed stimulation of AT(2) receptors by ANG II with use of AT(1)-receptor blockers may contribute to the beneficial properties of these agents. Determination of the expression pattern of AT(2) receptors in diabetes and further definition of the role of AT(2) receptors in opposing the detrimental effects of AT(1) receptors may lead to more selective targeting of the RAS in diabetic nephropathy.

Localization of protein inhibitor of neuronal nitric oxide synthase in rat kidney.

We have recently demonstrated that in rats with 5/6 nephrectomy (5/6 Nx), renal cortical and inner medullary neuronal NOS (nNOS) expression is downregulated, associated with decreased urinary excretion of nitric oxide (NO) products. Recently, a novel 89-amino acid protein [protein inhibitor of nNOS (PIN)] was isolated from rat brain and shown to inhibit nNOS activity. The present studies localized PIN in the rat kidney and determined the effect of 5/6 Nx on PIN expression. By RT-PCR, PIN mRNA was detected in the kidney cortex and inner medulla. Immunohistochemistry revealed staining for PIN in glomerular and vasa rectae endothelial cells. PIN was also localized to the apical membranes of inner medullary collecting duct (IMCD) cells. Two weeks after 5/6 Nx, inner medullary PIN expression was significantly upregulated (sham, 0.18+/-0.07 vs. 5/6 Nx, 0.58+/-0.13 arbitrary units; n = 6, P<0.02), as determined by Western blotting. In summary, our data show that PIN, a specific inhibitor of nNOS activity, is expressed in the IMCD, a site of high nNOS expression in the kidney. PIN expression is upregulated in the inner medulla of 5/6 Nx rats. Inhibition of nNOS activity by PIN may have important implications for the regulation of NO synthesis in the IMCD of normal and remnant kidneys.

Surviving rat distal tubule bicarbonate reabsorption: effects of chronic AT(1) blockade.

To determine the in vivo effects of chronic ANG II type 1 (AT(1))-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption (J(HCO(3))) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. J(HCO(3)) was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive H(+)-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of H(+)-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that H(+)-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining J(HCO(3)), we perfused three inhibitors simultaneously [5-(N, N-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: J(HCO(3)) was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule J(HCO(3)) of surviving nephrons is largely mediated by AT(1) receptor-dependent synthesis and insertion of apical H(+)-ATPase pumps in A-type intercalated cells.

Distal tubule bicarbonate reabsorption in intact and remnant diabetic kidneys.

BACKGROUND: In the diabetic patient, hyperkalemia and hyperchloremic metabolic acidosis has been attributed to one or more of the following factors associated with diabetic nephropathy: hypoaldosteronism, altered potassium homeostasis, or a distal tubular (DT) defect in hydrogen ion secretion. To evaluate maximal in vivo DT acidification in streptozotocin (STZ) diabetes, unidirectional bicarbonate reabsorption (JHCO3) was measured in DTs after acid loading and in surviving DT after 2/3 nephrectomy (Nx). METHODS: Acid gavage induced hyperchloremic metabolic acidosis in four groups of rats: diabetic rats with hyperglycemia two (a) and (b) eight weeks after STZ injection, (c) diabetic rats with tight glucose control two weeks after STZ injection and insulin pump implantation; and (d) control nondiabetic rats. Another group of diabetic rats underwent (e) Nx one week after STZ injection; these rats were neither acid loaded nor pump implanted. RESULTS: In the acidotic rats, the plasma potassium concentration, the plasma and urine acid-base parameters in the three STZ diabetic groups was not different from control rats, whereas JHCO3 fluxes were brisk without important differences between groups. In Nx rats, although the plasma potassium concentration and acid-base status were normal, surviving JHCO3 fluxes were still brisk and not different from the acid-loaded rats. CONCLUSIONS: These in vivo measurements indicate there is no impairment in DT unidirectional bicarbonate reabsorption in the intact or remnant STZ diabetic kidney.

Downregulation of neuronal nitric oxide synthase in the rat remnant kidney.

Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42+/-15.60 versus 5/6 Nx: 47.55+/-12.78 arbitrary units, P<0.05, n = 4) and inner medulla (sham: 147.70+/-26.96 versus 5/6 Nx: 36.95+/-17.24 arbitrary units, P<0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66+/-0.04-fold of sham values, P<0.05, n = 6) and inner medulla (0.74+/-0.12-fold of sham values, P<0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.

Prostanoid signaling, localization, and expression of IP receptors in rat thick ascending limb cells.

It is widely held that only one prostacyclin (IP) receptor exists that can couple to guanine stimulatory nucleotide binding proteins (Gs) leading to activation of adenyl cyclase. Although IP receptor mRNA is expressed in vascular arterial smooth muscle cells and platelets, with lower level expression in mature thymocytes, splenic lymphocytes, and megakaryocytes, there is no molecular evidence for IP receptor expression in renal epithelial cells. The purpose of the present study was to obtain molecular evidence for the expression and localization of the IP receptor and to study the signaling pathways of IP receptor in rat medullary thick ascending limb (MTAL). Biochemical studies showed that IP prostanoids do not increase cAMP in rat MTAL. However, in the presence of vasopressin, inhibition of cAMP formation by prostacyclin (PGI2) analogs is pertussis toxin sensitive and does not activate protein kinase C. In situ hybridization studies localized IP receptor mRNA expression to MTAL in the rat kidney outer medulla. The results of RT-PCR of freshly isolated RNA from MTAL, with primers specific for the mouse IP receptor cDNA, produced an amplification product of the correct predicted size that contained an expected Nco I endonuclease restriction site. We conclude that rat renal epithelial cells express the IP receptor, coupled to inhibition of cAMP production.

Effect of dietary salt on neuronal nitric oxide synthase in the inner medullary collecting duct.

Nitric oxide (NO) derived from neuronal NO synthase (nNOS) in the kidney inner medulla has been implicated in the regulation of arterial blood pressure. The purpose of the present study was to determine the effect of high dietary NaCl on the expression of nNOS in the rat inner medullary collecting duct (IMCD). After 3 days or 3 wk of high (4.0%)-NaCl diet in rats, urinary NO-2/NO-3 excretion significantly increased. In freshly microdissected IMCD, nNOS was readily detected by immunofluorescence with polyclonal antibody, an effect that was completely blocked by neutralization of antibody with immunizing antigen. In rats fed a 4.0% NaCl diet for 3 days, IMCD nNOS mRNA, detected by RT-PCR, did not change from control values (0.3% NaCl, 19.84 +/- 1.57 x 10(3), vs. 4.0% NaCl, 20.44 +/- 3.14 x 10(3) cpm; P = not significant, n = 3). By Western blotting however, nNOS protein expression significantly increased (0.3% NaCl, 0.51 +/- 0.12, vs. 4.0% NaCl, 0.92 +/- 0.14 arbitrary units; P < 0. 05, n = 5). After 3 wk of 4.0% dietary NaCl, expression of nNOS mRNA and protein in IMCD did not differ significantly from control values. In contrast to these data, renal cortical expression of nNOS mRNA and protein was significantly decreased after 4.0% NaCl diet for 3 days. High dietary NaCl had no significant effect on expression of mRNA for inducible NO synthase (iNOS) in IMCD after either 3 days or 3 wk. In summary, our data indicate that nNOS mRNA and protein are expressed in IMCD and that high dietary NaCl differentially regulates nNOS expression in IMCD and cortex. The early increase in nNOS protein in IMCD may contribute to enhanced local production of NO and thereby represent an adaptive response to salt intake.

K depletion stimulates in vivo HCO3 reabsorption in surviving rat distal tubules.

To evaluate whether K depletion enhances in vivo bicarbonate reabsorption (JtCO2) in surviving distal tubules (DT), we compared DT JtCO2 in five-sixths nephrectomized rats (Nx) with and without dietary K depletion (Nx-K). Furthermore, to identify possible mechanisms of increased JtCO2, we perfused inhibitors of proton secretion in both Nx and Nx-K rats. JtCO2 (102 +/- 8 pmol.min-1.mm-1) was significantly increased in Nx-K vs. Nx rats (65 +/- 7 pmol.min-1.mm-1, P < 0.05) but unaffected by 10(-6) M losartan perfusion (94 +/- 6 pmol.min-1.mm-1, P = not significant). Although 10(-5) M Sch-28080 also had no significant effect, 5 x 10(-9) M concanamycin A perfusion significantly decreased JtCO2 in Nx-K rats to 65 +/- 8 pmol.min-1. mm-1 (P < 0.05). Morphometric evaluation and H(+)-ATPase immunogold labeling of Nx-K A-type intercalated cells revealed cellular hypertrophy, elaborated apical microplicae, and enhanced H(+)-ATPase apical polarization. Accordingly, these combined studies confirm that K depletion enhances JtCO2 in surviving DT by stimulating H(+)-ATPase activity, independent of the AT1 receptor.

Potassium depletion stimulates mRNA expression of proximal tubule AT1 angiotensin II receptors.

Dietary potassium (K+) deficiency is associated with blood pressure elevation and impaired urinary sodium excretion. Since angiotensin II is a potent stimulator of tubular sodium transport, we studied the effect of low [K+] on expression of kidney AT1 angiotensin receptors. In rabbits fed a K+-deficient diet for 14 days, plasma [K+] was significantly reduced compared to rabbits fed a standard diet (control: 4.06 +/- 0.12 vs. K+-deficient: 2.66 +/- 0.19 mmol/l; p < 0.001; n = 6-9). By Northern hybridization or RNase protection assays, dietary K+ deficiency caused an increase in mRNA expression for AT1 receptors in kidney cortex (43.5 +/- 12.9% increase vs. control; p < 0.04; n = 8), and in proximal tubule segments in suspension (76.4 +/- 28.8% increase vs. control; p < 0.005; n = 6). K+ deficiency had no effect on AT1 receptor mRNA expression in liver, or on mRNA expression of beta-actin in kidney cortex, proximal tubule suspensions, or liver. To determine if low extracellular [K+] might directly modulate AT1 receptor mRNA expression, primary cultures of rabbit proximal tubule cells were incubated for 1, 3, 6 or 24 h in media with or without 5 mmol/l K+. Incubation of cells in 0 mmol/l K+ caused a 99.2 +/- 32.9% increase in AT1 receptor mRNA expression at 3 h (p < 0.001; n = 14), returning to control levels by 24 h. Incubation of proximal tubule cells in 0 mmol/l K+ also caused a significant increase in basolateral membrane specific binding of [125I]-angiotensin II (p < 0.05; n = 4). These results indicate that dietary K+ deficiency and low extracellular [K+] stimulate expression of kidney AT1 angiotensin II receptors. Increased AT1 receptor mRNA and protein expression in proximal tubule may promote enhanced sodium reabsorption in K+ deficiency.