Glucocorticoids Reverse Diluted Hyponatremia Through Inhibiting Arginine Vasopressin Pathway in Heart Failure Rats.

Background Arginine vasopressin dependent antidiuresis plays a key role in water-sodium retention in heart failure. In recent years, the role of glucocorticoids in the control of body fluid homeostasis has been extensively investigated. Glucocorticoid deficiency can activate V2R (vasopressin receptor 2), increase aquaporins expression, and result in hyponatremia, all of which can be reversed by glucocorticoid supplement. Methods and Results Heart failure was induced by coronary artery ligation for 8 weeks. A total of 32 rats were randomly assigned to 4 groups (n=8/group): sham surgery group, congestive heart failure group, dexamethasone group, and dexamethasone in combination with glucocorticoid receptor antagonist RU486 group. An acute water loading test was administered 6 hours after drug administration. Left ventricular function was measured by a pressure-volume catheter. Protein expressions were determined by immunohistochemistry and immunoblotting. The pressure-volume loop analysis showed that dexamethasone improves cardiac function in rats with heart failure. Western blotting confirmed that dexamethasone remarkably reduces the expressions of V2R, aquaporin 2, and aquaporin 3 in the renal-collecting ducts. As a result of V2R downregulation, the expressions of glucocorticoid regulated kinase 1, apical epithelial sodium channels, and the furosemide-sensitive Na-K-2Cl cotransporter were also downregulated. These favorable effects induced by dexamethasone were mostly abolished by the glucocorticoid receptor inhibitor RU486, indicating that the aforementioned effects are glucocorticoid receptor mediated. Conclusions Glucocorticoids can reverse diluted hyponatremia via inhibiting the vasopressin receptor pathway in rats with heart failure.

Aldosterone Decreases Vasopressin-Stimulated Water Reabsorption in Rat Inner Medullary Collecting Ducts.

Aldosterone indirectly regulates water reabsorption in the distal tubule by regulating sodium reabsorption. However, the direct effect of aldosterone on vasopressin-regulated water and urea permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether aldosterone regulates osmotic water permeability in isolated perfused rat IMCDs. Adding aldosterone (500 nM) to the bath significantly decreased osmotic water permeability in the presence of vasopressin (50 pM) in both male and female rat IMCDs. Aldosterone significantly decreased aquaporin-2 (AQP2) phosphorylation at S256 but did not change it at S261. Previous studies show that aldosterone can act both genomically and non-genomically. We tested the mechanism by which aldosterone attenuates osmotic water permeability. Blockade of gene transcription with actinomycin D did not reverse aldosterone-attenuated osmotic water permeability. In addition to AQP2, the urea transporter UT-A1 contributes to vasopressin-regulated urine concentrating ability. We tested aldosterone-regulated urea permeability in vasopressin-treated IMCDs. Blockade of gene transcription did not reverse aldosterone-attenuated urea permeability. In conclusion, aldosterone directly regulates water reabsorption through a non-genomic mechanism. Aldosterone-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. There may be a sex difference apparent in the inhibitory effect of aldosterone on water reabsorption in the inner medullary collecting duct. This study is the first to show a direct effect of aldosterone to inhibit vasopressin-stimulated osmotic water permeability and urea permeability in perfused rat IMCDs.

Albumin evokes Ca2+-induced cell oxidative stress and apoptosis through TRPM2 channel in renal collecting duct cells reduced by curcumin.

In proteinuric nephropathies of chronic kidney disease, the epithelial cells of the nephron including the collecting duct are exposed to high concentrations of luminal albumin. Albumin is taken up from collecting duct cells by endocytosis causing excessive reactive oxygen species (ROS) production and a proinflammatory response. Curcumin used in the traditional medicine possesses anti-inflammatory and antioxidant effects. ROS and ADP-ribose (ADPR) activate the cation channel TRPM2. We hypothesize, that albumin-induced cell stress and proinflammatory response are mediated by Ca2+ and can be reduced by curcumin. The cortical collecting duct (CCD) cells mpkCCDc14 exhibit spontaneous and inducible Ca2+ oscillations, which can be blocked by pre-treatment with curcumin. Curcumin accumulates in plasma membrane and intracellular vesicles, where it interferes with TRPM2 and decreases the influx of Ca2+. Albumin reduces cell viability and increases apoptosis, NF-κB activation, and mitochondrial membrane depolarization via Ca2+-dependent signaling, which results in increased ROS production. Albumin-induced cell stress is diminished by the inhibition of TRPM2 after administration of curcumin and ADPR (PARP1) inhibitors. Curcumin did not reduce the Ca2+ elevation induced by thapsigargin in Ca2+-free medium, but it reduced the function of store-operated Ca2+ channels and ATP-evoked Ca2+ response. In conclusion, albumin-induced oxidative stress is mediated by Ca2+-dependent signaling via TRPM2 and leads to cell damage and a proinflammatory response, strengthening the role of CCD cells in the progression of chronic kidney disease.

Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

Aliskiren increases aquaporin-2 expression and attenuates lithium-induced nephrogenic diabetes insipidus.

The direct renin inhibitor aliskiren has been shown to be retained and persist in medullary collecting ducts even after treatment is discontinued, suggesting a new mechanism of action for this drug. The purpose of the present study was to investigate whether aliskiren regulates renal aquaporin expression in the collecting ducts and improves urinary concentrating defect induced by lithium in mice. The mice were fed with either normal chow or LiCl diet (40 mmol·kg dry food-1·day-1 for 4 days and 20 mmol·kg dry food-1·day-1 for the last 3 days) for 7 days. Some mice were intraperitoneally injected with aliskiren (50 mg·kg body wt-1·day-1 in saline). Aliskiren significantly increased protein abundance of aquaporin-2 (AQP2) in the kidney inner medulla in mice. In inner medulla collecting duct cell suspension, aliskiren markedly increased AQP2 and phosphorylated AQP2 at serine 256 (pS256-AQP2) protein abundance, which was significantly inhibited both by adenylyl cyclase inhibitor MDL-12330A and by PKA inhibitor H89, indicating an involvement of the cAMP-PKA signaling pathway in aliskiren-induced increased AQP2 expression. Aliskiren treatment improved urinary concentrating defect in lithium-treated mice and partially prevented the decrease of AQP2 and pS256-AQP2 protein abundance in the inner medulla of the kidney. In conclusion, the direct renin inhibitor aliskiren upregulates AQP2 protein expression in inner medullary collecting duct principal cells and prevents lithium-induced nephrogenic diabetes insipidus likely via cAMP-PKA pathways.

Quantitative evaluation of Oryeongsan and its action on water regulation in renal inner medullary collecting duct cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Oryeongsan (ORS, Wulingsan) has been reported to possess renal protective effects from renal diseases such as diabetes-induced renal damage, and nephrocalcinosis. AIM OF THE STUDY: This study was conducted to evaluate the quantitative analysis and the inhibitory effect of ORS on hypertonic stress-induced water channel and apoptosis in murine inner medullary collecting duct cell line (mIMCD-3). MATERIALS AND METHODS: Chromatographic and NMR spectroscopic analysis were performed and water balance regulation was determined by Western blot, RT-PCR, and immunofluorescnece. RESULTS: Seven active principles (5-hydroxymethylfurfural, alismoxide, methyl(-)trans-cinnamate, adenine, guanosine, adenosine, and ferulic acid) in ORS were isolated and the structures were identified mainly by NMR spectroscopic analysis. In addition, contents of these metabolites in ORS were evaluated by HPLC analysis. Pretreatment with ORS significantly attenuated the hypertonic stress (175mM NaCl)-induced increase in protein levels of AQP2 and apical membrane insertion. ORS also attenuated osmolyte sodium-myo-inositol transporter (SMIT) expression and tonicity-responsive enhancer binding protein (TonEBP) mRNA under hypertonic stress. Those actions of ORS presented the similar effect of PKA inhibitor which AQP2 expression throughout the inhibition of vasopressin-mediated cAMP/PKA signal pathway. On the other hand, pretreatment with ORS attenuated hypertonic stress-induced cell death. Hypertonic stress-induced Bax or caspase-3 expression was decreased by ORS, resulting in anti-apoptotic effect. CONCLUSIONS: The present data suggest that the beneficial effect of ORS in water balance and apoptosis against hypertonic stress of renal collecting ducts.

Rapamycin inhibition of mTORC1 reverses lithium-induced proliferation of renal collecting duct cells.

Nephrogenic diabetes insipidus (NDI) is the most common renal side effect in patients undergoing lithium therapy for bipolar affective disorders. Approximately 2 million US patients take lithium of whom ∼50% will have altered renal function and develop NDI (2, 37). Lithium-induced NDI is a defect in the urinary concentrating mechanism. Lithium therapy also leads to proliferation and abundant renal cysts (microcysts), commonly in the collecting ducts of the cortico-medullary region. The mTOR pathway integrates nutrient and mitogen signals to control cell proliferation and cell growth (size) via the mTOR Complex 1 (mTORC1). To address our hypothesis that mTOR activation may be responsible for lithium-induced proliferation of collecting ducts, we fed mice lithium chronically and assessed mTORC1 signaling in the renal medulla. We demonstrate that mTOR signaling is activated in the renal collecting ducts of lithium-treated mice; lithium increased the phosphorylation of rS6 (Ser240/Ser244), p-TSC2 (Thr1462), and p-mTOR (Ser2448). Consistent with our hypothesis, treatment with rapamycin, an allosteric inhibitor of mTOR, reversed lithium-induced proliferation of medullary collecting duct cells and reduced levels of p-rS6 and p-mTOR. Medullary levels of p-GSK3ß were increased in the renal medullas of lithium-treated mice and remained elevated following rapamycin treatment. However, mTOR inhibition did not improve lithium-induced NDI and did not restore the expression of collecting duct proteins aquaporin-2 or UT-A1.

Effect of Poria cocos on hypertonic stress-induced water channel expression and apoptosis in renal collecting duct cells.

ETHNOPHARMACOLOGICAL RELEVANCE: A major physiological role of the kidney is to regulate body water and urine concentration. Aquaporin-2 (AQP2), a family of water channels, plays an important role in the urinary concentrating process and regulation of water balance in the kidney. The dried sclerotia of Poria cocos Wolf has been known to have a diuretic effect and used for the treatment of chronic edema and nephrosis. AIM OF THE STUDY: This study was conducted to evaluate the inhibitory effect of the sclerotia of Poria cocos (WPC) on hypertonic stress-induced AQP2 expression and apoptosis in inner medullary collecting duct cell lines (IMCD-3). MATERIALS AND METHODS: Hypertonic stress was induced by 175mM NaCl. Inhibitory effect of WPC on hypertonic stress-induced AQP2 expression and apoptosis were determined by western blot, RT-PCR, and immunofluorescence. RESULTS: Hypertonic stress (175mM NaCl) increased in the levels of AQP2 expression by hypertonicity in IMCD-3 cells. WPC attenuated the hypertonicity-induced increase in protein and mRNA levels of AQP2 in a concentration-dependent manner. Pretreatment with WPC attenuated hypertonicity-induced cell death. Hypertonicity increased serum- and glucocorticoid-inducible protein kinase (Sgk1) phosphorylation, however, WPC attenuated the hypertonicity-induced Sgk1 activation. Tonicity-responsive enhancer binding protein (TonEBP) mRNA was also recovered by WPC under hypertonic stress. Pretreatment with WPC presented the similar effect of PKA inhibitor which decreased hypertonic stress-induced AQP2 expression. Hypertonicity increased cAMP levels and the changes were blocked by WPC. On the other hand, hypertonic stress-induced Bax or caspase-3 expression was decreased by WPC, resulting in anti-apoptotic effect. CONCLUSIONS: These results provided evidence that the beneficial effect of WPC in water balance against in vitro hypertonic stress of renal collecting ducts. In addition, WPC exhibits anti-apoptotic property response to hypertonic stress. Thus, these data suggests that WPC has benefit for the therapeutic approach to the inhibition of renal disorder.

Long-term aldosterone treatment induces decreased apical but increased basolateral expression of AQP2 in CCD of rat kidney.

The purpose of the present studies was to determine the effects of high-dose aldosterone and dDAVP treatment on renal aquaporin-2 (AQP2) regulation and urinary concentration. Rats were treated for 6 days with either vehicle (CON; n = 8), dDAVP (0.5 ng/h, dDAVP, n = 10), aldosterone (Aldo, 150 microg/day, n = 10) or combined dDAVP and aldosterone treatment (dDAVP+Aldo, n = 10) and had free access to water with a fixed food intake. Aldosterone treatment induced hypokalemia, decreased urine osmolality, and increased the urine volume and water intake in ALDO compared with CON and dDAVP+Aldo compared with dDAVP. Immunohistochemistry and semiquantitative laser confocal microscopy revealed a distinct increase in basolateral domain AQP2 labeling in cortical collecting duct (CCD) principal cells and a reduction in apical domain labeling in Aldo compared with CON rats. Given the presence of hypokalemia in aldosterone-treated rats, we studied dietary-induced hypokalemia in rats, which also reduced apical AQP2 expression in the CCD but did not induce any increase in basolateral AQP2 expression in the CCD as observed with aldosterone treatment. The aldosterone-induced basolateral AQP2 expression in the CCD was thus independent of hypokalemia but was dependent on the presence of sodium and aldosterone. This redistribution was clearly blocked by mineralocorticoid receptor blockade. The increased basolateral expression of AQP2 induced by aldosterone may play a significant role in water metabolism in conditions with increased sodium reabsorption in the CCD.

Chronic dDAVP infusion in rats decreases the expression of P2Y2 receptor in inner medulla and P2Y2 receptor-mediated PGE2 release by IMCD.

Activation of P2Y2 receptor (P2Y2-R) in inner medullary collecting duct (IMCD) of rat decreases AVP-induced water flow and releases PGE(2). We observed that dehydration of rats decreases the expression of P2Y2 receptor in inner medulla (IM) and P2Y2-R-mediated PGE(2) release by IMCD. Because circulating vasopressin (AVP) levels are increased in dehydrated condition, we examined whether chronic infusion of desmopressin (dDAVP) has a similar effect on the expression and activity of P2Y2-R. Groups of rats were infused with saline or dDAVP (5 or 20 ng/h sc, 5 or 6 days) via osmotic minipumps and euthanized. Urine volume, osmolality, and PGE(2) metabolite content were determined. AQP2- and P2Y2- and V2-R mRNA and/or protein in IM were quantified by real-time RT-PCR and immunoblotting, respectively. P2Y2-R-mediated PGE(2) release by freshly prepared IMCD was assayed using ATPgammaS as a ligand. Chronic dDAVP infusion resulted in low-output of concentrated urine and significantly increased the AQP2 protein abundance in IM. On the contrary, dDAVP infusion at 5 or 20 ng/h significantly decreased P2Y2-R protein abundance (approximately 40% of saline-treated group). In parallel, the relative expression of P2Y2-R vs. AQP2- or V2-R mRNA was significantly decreased. Furthermore, the P2Y2-R-mediated PGE(2) release by IMCD was significantly decreased in rats infused 20 ng/h but not 5 ng/h of dDAVP. Urinary PGE(2) metabolite excretion, however, did not change with dDAVP infusion. In conclusion, chronic dDAVP infusion decreases the expression and activity of P2Y2-R in IM. This may be due to a direct effect of dDAVP or dDAVP-induced increase in medullary tonicity.

Rosiglitazone activates renal sodium- and water-reabsorptive pathways and lowers blood pressure in normal rats.

Synthetic agonists of the peroxisomal proliferator-activated receptor subtype gamma (PPAR-gamma) are highly beneficial in the treatment of type II diabetes. However, they are also associated with fluid retention and edema, potentially serious side effects of unknown origin. These studies were designed to test the hypothesis that rosiglitazone (RGZ, PPAR-gamma agonist) may activate sodium- and water-reabsorptive processes in the kidney, possibly in response to a drop in mean arterial blood pressure (MAP), as well as directly through PPAR-gamma. Targeted proteomics of the major renal sodium and water transporters and channel proteins was used to identify potentially regulated sites of renal sodium and water reabsorption. RGZ (47 or 94 mg/kg diet) was fed to male, Sprague-Dawley rats (approximately 270g) for 3 days. MAP, measured by radiotelemetry, was decreased significantly in rats fed either level of RGZ, relative to control rats. Delta MAP from baseline was -3.2 +/- 1.2 mm Hg in rats fed high-dose RGZ versus + 3.4 +/- 0.8 for rats fed control diet. RGZ did not affect feed or water intake, but rats treated with high-dose RGZ had decreased urine volume (by 22%), sodium excretion (44%), kidney weight (9%), and creatinine clearance (35%). RGZ increased whole kidney protein abundance of the alpha-1 subunit of Na-K-ATPase, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the sodium hydrogen exchanger (NHE3), the aquaporins 2 and 3, and endothelial nitric-oxide synthase. We conclude that both increases in renal tubule transporter abundance and a decrease in glomerular filtration rate likely contribute to the RGZ-induced sodium retention.

BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways.

Bone morphogenetic protein-7 (BMP7) controls ureteric bud and collecting duct morphogenesis in a dose-dependent manner (Piscione TD, Yager TD, Gupta IR, Grinfeld B, Pei Y, Attisono L, Wrana JL, and Rosenblum ND. Am J Physiol Renal Physiol 273: F961-F975, 1997). We defined cellular and molecular mechanisms underlying these effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting tubule morphogenesis. Low-dose (0.25 nM) BMP7 significantly increased tubule number and cell proliferation. Similar to BMP2, high-dose (10 nM) BMP7 inhibited cell proliferation and stimulated apoptosis. To define molecular mechanisms, we identified signaling events downstream of BMP7. High-dose BMP7, but not low-dose BMP7, activated Smad1 in mIMCD-3 cells. Moreover, the inhibitory effects of high-dose BMP7 and BMP2, but not the stimulatory effects of low-dose BMP7, on tubulogenesis and cell proliferation were significantly reduced in mIMCD-3 cells stably expressing Smad1(Delta458), a dominant negative mutant form of Smad1, but not in cells stably expressing wild-type Smad1. We conclude that BMP7 exerts dose-dependent effects on ureteric bud or collecting duct cell proliferation and apoptosis by signaling via Smad1-dependent and Smad1-independent pathways.

Influence of parathyroidectomy and calcium on rat renal function.

Parathyroid hormone (PTH) has multiple effects on water and electrolyte transport along the nephron. However, the influences of PTH and calcium on the urinary concentration ability are not fully understood. In this study, clearance and microperfusion studies were performed in thyroparathyroidectomized (TPTX) rats either supplemented (TPTX+Ca(2+)) or not with calcium added to the ingested food as CaCl(2) (1.6 g/100 g). Acid-base data and renal functional parameters were measured in TPTX and TPTX+Ca(2+) rats. Additional studies were performed in the isolated inner medullary collecting tubules of intact and TPTX rats to evaluate the osmotic permeability of this segment in the presence of 10(-6) M PTH added to the bath. In these experiments the possible influence of PTH on antidiuretic hormone induced changes of the osmotic permeability in TPTX and TPTX+Ca(2+) rats was also investigated. In the TPTX+Ca(+) group, the glomerular filtration rate increased significantly when compared to the TPTX group (6.04 +/- 0.42 vs. 4.88 +/- 0.20 ml.min(-1).kg(-1); p < 0.05), but the U/P inulin ratio remained lower than control values (30.8 +/- 1.48 vs. 54.0 +/- 3.5; p < 0.05), which suggests that normal levels of PTH are necessary to maintain the concentrating ability. In a group of TPTX rats, an acute infusion of PTH (0.5 microg.min(-1).kg(-1)) significantly decreased the urinary flow and increased the renal plasma flow, results that agree with the vasomodulator action of this hormone on the renal vasculature. A significant increase in the fractional K(+) excretion observed in the TPTX+Ca(2+) group as compared with both control and TPTX, groups suggests that the excreted load of Ca(2+) may interfere with tubular K(+) handling in the absence of PTH. PTH (10(-6) M) added to the bath of the isolated inner medullary collecting tubules did not change the osmotic permeability, of intact, TPTX, and TPTX+Ca(2+) rats. Furthermore, it did not modify the antidiuretic hormone induced changes in the osmotic permeability. These results suggest that this segment of the nephron is PTH insensitive as far as water and ion transport are concerned.

Protective effect of L-arginine on hypercholesterolemia-enhanced renal ischemic injury.

The effects of hypercholesterolemia on ischemic renal failure were evaluated in rats subjected to 60 min of left renal artery clamping and contralateral nephrectomy. One group of rats (HC) was kept on a cholesterol-supplemented diet for 3 weeks before renal injury and compared to a group fed a regular diet (ND). Two days after renal ischemia, inulin clearance (C(in), ml/min per 100 g BW) was lower in HC-rats (0.033 +/- 0.011) than in ND-rats (0.227 +/- 0.037; P < 0.01). indicating that hypercholesterolemia potentiated renal ischemic injury. Twenty-one days after renal ischemia the C(in) of HC-rats did not differ from ND-rats, suggesting that hypercholesterolemia did not limit late recovery. Since nitric oxide production is impaired in HC, L-arginine (50 mg/kg BW i.v.) was administered immediately after ischemia. Two days after ischemia, L-arg did not protect ND-rats from ischemia, while the C(in) and renal blood flow were higher in L-arg-treated HC rats than in untreated HC rats (C(in) = 0.125 +/- 0.013 rats vs. 0.033 +/- 0.011; P < 0.001) (RBF = 3.96 +/- 0.64 vs. 2.40 +/- 0.20 ml/min per 100 g BW; P < 0.05), indicating that L-arg protects HC rats from renal ischemia. The administration of D-arginine to ND rats induced a significant decrease of the C(in) and a significant increase of FE H2O, FE Na and FE K compared to the L-arginine and not treated groups. Cultures of inner medullary collecting duct cells from ND rats were resistant to 24-h hypoxia. In contrast, IMCD cell cultures from HC rats showed higher LDH release after 24-h hypoxia than normoxic cells (69.2 +/- 3.4 vs. 30.9 +/- 3.6%, P < 0.001); 1 mM L-arg added to the medium attenuated LDH release (44.3 +/- 2.4%, P < 0.01). These data demonstrate that HC predisposes renal tubular cells to hypoxic injury and L-arg protects cells of HC.

Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells.

Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha 1- and beta 1-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH2O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha 1-subunit mRNA accumulation and five- to eightfold increases in the beta 1-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH2O, the maximal effects on both alpha 1- and beta 1-subunit mRNA accumulation occurred at 500 mosmol/kgH2O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha 1- and beta 1-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha 1- and beta 1-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH2O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha 1- and beta 1-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha 1- and beta 1-subunit mRNA leads to an increase in Na- K -ATPase activity; and 3) the above upregulation of alpha1- and beta 1-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.

Expression cloning of the aldosterone target cell-specific 11 beta-hydroxysteroid dehydrogenase from rabbit collecting duct cells.

11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor from occupancy by endogenous glucocorticoids. We have recently described a novel isoform of 11-OHSD in the renal aldosterone target cells (11 beta-OHSD/CD) that differs from the previously characterized isoform (11 beta-OHSD-1). Unlike 11-OHSD-1, the collecting duct enzyme catalyzes irreversible dehydrogenation, has a very high affinity for its substrate, and is tissue-specific. We report here the isolation, sequence, and characterization of a complementary DNA (cDNA) encoding the rabbit collecting duct 11 beta-OHSD/CD or 11 beta-OHSD type 2. The cDNA, isolated using expression screening in Xenopus oocytes, is 1.9 kilobases in length and encodes a protein of 406 amino acids with a predicted molecular mass of 44,130 daltons. The cloned enzyme has a Michaelis constant (Km) for corticosterone of 6.6 +/- 3 nM, catalyzes exclusively dehydrogenation, and uses only NAD as cofactor. The cloned enzyme shows 85% and 75% amino acid identity to the recently cloned human type 2 11 beta-OHSD and sheep kidney 11 beta-OHSD, respectively, whereas the overall homology to rat liver 11 beta-OHSD-1 is less than 20% The messenger RNA for this 11 beta-OHSD is expressed at very high levels in the renal collecting duct and at much lower levels in the colon. The intrarenal distribution was determined by reverse-transcription polymerase chain reaction in isolated nephron segments or cell types. The messenger RNA is present only in aldosterone target cells within the kidney, at highest levels in principal cells, at lower levels in intercalated cells, and in inner medullary cells. These data suggest that the 11 beta-OHSD cDNA from rabbit collecting duct cells encodes the enzyme that confers aldosterone selectivity to mineralocorticoid target cells.

Changes in pH affect Cl- removal and AVP action in collecting tubules.

We examined what mechanisms are involved in the alteration by chloride (Cl-) removal of arginine vasopressin (AVP)-induced cellular cAMP production, and cellular free calcium ([Ca2+]i) mobilization in rat renal papillary collecting tubule cells in culture, using two buffer systems: bicarbonate and non-bicarbonate buffers. The first study was performed in the bicarbonate-supplemented buffer. Removal of Cl-, which was replaced by methylsulfonate or gluconate, increased cellular pH (pHi) from 7.19 to 7.26. AVP increased cellular cAMP production in a dose-dependent manner; 1 x 10(-9) and 1 x 10(-7) M AVP-induced increases in cellular cAMP production were significantly enhanced by the Cl- removal. Also, 1 x 10(-7) M AVP-mobilized [Ca2+]i was augmented by the Cl- removal (181.3 vs. 224.5 nM, P < 0.05). The second study was carried out with the Krebs-Ringer buffered saline (KRB). Removal of Cl- lowered pHi from 7.20 to 7.09. AVP-induced increases in cellular cAMP production were significantly reduced in the Cl(-)-free KRB compared to those in the KRB. The reduction was obtained with KRB containing less than 25 mM Cl-. Similar results were obtained with 2 x 10(-8) M forskolin, a diterpene activator of adenylate cyclase. 1 x 10(-7) M AVP-mobilized [Ca2+]i was also diminished by the Cl(-)-free KRB. These results indicate that Cl- depletion affects the cellular response to AVP mediated via the changes in pHi in renal papillary collecting tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Compensatory hypertrophy and adaptation in the cortical collecting duct.

The cortical collecting duct (CCD) undergoes hypertrophy and functional adaptation following reduction of renal mass. The nature and mechanisms of these changes have been investigated using microperfusion of isolated CCD from rabbit remnant kidneys. By 1 week after reduction of renal mass, tubule hypertrophy and increased sodium transport are fully developed. The transport adaptations are specific or selective, since bicarbonate transport in these CCD is unchanged. Mineralocorticoids may play an important role in the hypertrophy and increased sodium transport, since plasma aldosterone increases early after reduction of renal mass. Also, adrenalectomy abolishes the changes in size and sodium transport, even with supplementation of aldosterone to unstressed physiologic levels. Epidermal growth factor also has immediate effects on CCD sodium transport; however, the direction of the effect is opposite--an inhibition of transport.

Optimal concentration of cellular free calcium for AVP-induced cAMP in collecting tubules.

The role of Ca2+ in the cellular action of arginine vasopressin (AVP) has been demonstrated in renal papillary collecting tubule. We further examined whether an optimal concentration of cellular free Ca2+ [( Ca2+]i) exists for AVP-induced cAMP production in rat renal papillary collecting tubule cells in culture. [Ca2+]i was measured using fura-2. When cells were exposed for one hour to the media supplemented with 0 mM Ca2+ (containing 1 mM EGTA), 1, 2, 3, 4, 6.4, or 8 mM Ca2+, basal [Ca2+]i ranged as below: 24.9 +/- 5.6, 90.7 +/- 7.4, 107.4 +/- 9.8, 146.1 +/- 13.7, 162.0 +/- 14.6, 241.5 +/- 32.8, and 234.9 +/- 29.6 nM, respectively. When medium Ca2+ was 1 mM, 1 x 10(-7) M AVP increased [Ca2+]i to 181.5 +/- 13.2 nM from 90.7 +/- 7.4 nM (P less than 0.01). AVP-induced increases in [Ca2+]i were obtained with the varying Ca2+ media described above, though the increases in [Ca2+]i were quantitatively variable. AVP-induced cellular cAMP production was examined during three minute incubation period in the presence of 5 x 10(-4) M 3-isobutyl-1-methylxanthine. Basal level of cellular cAMP was 87.6 +/- 7.9 fmol/micrograms protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of element content in isolated papillary collecting duct cells by electron probe microanalysis.

A method was developed to measure the element content of freshly isolated papillary collecting duct (PCD) cells by electron probe microanalysis in a scanning electron microscope. After isolation, the cells were transferred onto a Thermanox support by centrifugation and the extracellular medium was removed by brief exposure to buffered ammonium acetate; cryofixation, freeze-drying, and coating with carbon followed. Under visual control in the scanning electron microscope the Na, Cl, K and P content of cell clusters (about 30 cells/cluster) was then measured by X-ray microanalysis. Cells incubated in control medium showed potassium:sodium ratios identical to those determined previously in cryosections of the same cells. In ouabain-treated cells sodium influx and potassium efflux was demonstrated. Potassium left the cells with a t1/2 of 21.7 min. The t1/2 of Na influx was 12.6 min for the first 15 min of incubation, whereafter further influx was markedly slower. Ouabain-induced sodium influx was inhibited 40% by amiloride. These results indicate that X-ray microanalysis can be applied to analyze the ion content of isolated cell clusters derived from the papillary collecting duct. Using ouabain and amiloride as inhibitors the suitability of the method to identify transport systems is demonstrated.