bluuebunny/crossref_metadata_2025 huggingface dataset:
EP1and EP4receptors mediate prostaglandin E2actions in the microcirculation of rat kidney
Authors: Purdy Kit E. , Arendshorst William J.
Published: December 2017

Vasodilator prostaglandin PGE2protects the kidney from excessive vasoconstriction during contraction of extracellular fluid volume and pathophysiological states. However, it is not yet clear which of the four known E-prostanoid (EP) receptors is localized to resistance vessels and mediates net vasodilation. In the present study, we assessed the presence, signal transduction, and actions of EP receptor subtypes in preglomerular arterioles of Sprague-Dawley rat kidneys. RNA encoding EP1, an EP1-variant, and EP4receptors was identified by RT-PCR in freshly isolated preglomerular microvessels; cultured preglomerular vascular smooth muscle cells (VSMC) had EP1-variant and EP4RNA but lacked EP1. EP2and EP3receptors were undetectable in both vascular preparations. In studies of cell signaling, stimulation of cAMP by various receptor agonists is consistent with primary actions of PGE2on the EP4receptor, with no inhibition of cAMP by EP1receptors. Studies of cytosolic calcium concentration in cultured renal VSMC support an inhibitory role of EP4during ANG II stimulation. In vivo renal blood flow (RBF) studies indicate that the EP4receptor is the primary receptor mediating sustained renal vasodilation produced by PGE2, whereas the EP1receptor elicits transient vasoconstriction. The EP1-variant receptor does not appear to possess any cAMP or cytosolic calcium signaling capable of affecting RBF. Collectively, these studies demonstrate that the EP4receptor is the major receptor in preglomerular VSMC. EP4mediates PGE2-induced vasodilation in the rat kidney and signals through Gsproteins to stimulate cAMP and inhibit cytosolic calcium concentration.
Molecular and functional properties of two-pore-domain potassium channels
Authors: Lesage Florian , Lazdunski Michel
Published: December 2017

The two-pore-domain K+ channels, or K2P channels, constitute a novel class of K+channel subunits. They have four transmembrane segments and are active as dimers. The tissue distribution of these channels is widespread, and they are found in both excitable and nonexcitable cells. K2P channels produce currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K+ channel blockers. These properties designate them as background K+ channels. They are expected to play a major role in setting the resting membrane potential in many cell types. Another salient feature of K2P channels is the diversity of their regulatory mechanisms. The weak inward rectifiers TWIK-1 and TWIK-2 are stimulated by activators of protein kinase C and decreased by internal acidification, the baseline TWIK-related acid-sensitive K+ (TASK)-1 and TASK-2 channels are sensitive to external pH changes in a narrow range near physiological pH, and the TWIK-related (TREK)-1 and TWIK-related arachidonic acid-stimulated K+ (TRAAK) channels are the first cloned polyunsaturated fatty acids-activated and mechanogated K+ channels. The recent demonstration that TASK-1 and TREK-1 channels are activated by inhalational general anesthetics, and that TRAAK is activated by the neuroprotective agent riluzole, indicates that this novel class of K+ channels is an interesting target for new therapeutic developments.
In vivo role of CLC chloride channels in the kidney
Authors: Uchida Shinichi
Published: December 2017

Chloride channels in the kidney are involved in important physiological functions such as cell volume regulation, acidification of intracellular vesicles, and transepithelial chloride transport. Among eight mammalian CLC chloride channels expressed in the kidney, three (CLC-K1, CLC-K2, and CLC-5) were identified to be related to kidney diseases in humans or mice. CLC-K1 mediates a transepithelial chloride transport in the thin ascending limb of Henle's loop and is essential for urinary concentrating mechanisms. CLC-K2 is a basolateral chloride channel in distal nephron segments and is necessary for chloride reabsorption. CLC-5 is a chloride channel in intracellular vesicles of proximal tubules and is involved in endocytosis. This review will cover the recent advances in research on the CLC chloride channels of the kidney with a special focus on the issues most necessary to understand their physiological roles in vivo, i.e., their intrarenal and cellular localization and their phenotypes of humans and mice that have their loss-of-function mutations.
A2Aadenosine receptor-mediated inhibition of renal injury and neutrophil adhesion
Authors: Okusa Mark D. , Linden Joel , Huang Liping , Rieger Jayson M. , Macdonald Timothy L. , Huynh Long P.
Published: December 2017

We sought to determine the mechanisms responsible for the reduced renal tissue injury by agonists of A2Aadenosine receptors (A2A-ARs) in models of ischemia-reperfusion (I/R) injury. DWH-146e, a selective A2A-AR agonist, was administered subcutaneously to Sprague-Dawley rats and C57BL/6 mice via osmotic minipumps, and animals were subjected to I/R. I/R led to an increase in plasma creatinine and kidney neutrophil infiltration. Infusion of DWH-146e at 10 ng · kg−1· min−1produced a 70% reduction in plasma creatinine as well as a decrease in neutrophil density in outer medulla and cortex and myeloperoxidase activity in the reperfused kidney. Myeloperoxidase activity in kidney correlated with the degree of renal injury. P-selectin and intercellular adhesion molecule 1 (ICAM-1) immunoreactivity were most prominent in endothelial cells of peritubular capillaries and interlobular arteries of cortex and outer and inner medulla of vehicle-treated mice whose kidneys were subjected to I/R. DWH-146e treatment led to a pronounced decrease in P-selectin- and ICAM-1-like immunoreactivity. These data are consistent with our hypothesis that A2A-AR agonists limit I/R injury due to an inhibitory effect on neutrophil adhesion.
Renin expression in COX-2-knockout mice on normal or low-salt diets
Authors: Yang Tianxin , Endo Yoshimi , Huang Yuning G. , Smart Ann , Briggs Josie P. , Schnermann Jürgen
Published: December 2017

Experiments were performed in mice to investigate whether cyclooxygenase-2 (COX-2) in epithelial cells near the tubulovascular contact point (macula densa and TAL cells) may regulate renin gene expression in juxtaglomerular granular cells. Renin activity, afferent arteriolar granularity, and renin mRNA were determined in wild-type mice and in COX-2-knockout mice on control and low-NaCl diets. Renin activity in microdissected glomeruli assessed as angiotensin I formation in the presence of excess substrate and afferent arteriolar granularity determined by direct visualization and immunostaining were significantly reduced in COX-2 −/− compared with wild-type animals. Similarly, renal cortical mRNA levels were lower in COX-2 −/− than in wild-type mice. Maintaining mice on a low-salt diet for 14 days induced an increase in renin mRNA, afferent arteriolar granularity, and renin activity in wild-type mice. In contrast, renin mRNA and renin granularity did not significantly increase in low-salt-treated COX-2 −/− mice, whereas the increase in juxtaglomerular renin enzyme activity was markedly attenuated, but not fully blocked. In additional experiments we found that COX-2 mRNA was increased in angiotensin type 1A receptor-knockout mice compared with wild-type mice. We conclude that COX-2 in the tubulovascular contact region is a critical determinant of renin synthesis in granular cells under resting conditions and that it participates in the stimulation of renin expression caused by a low-NaCl intake.
Basolateral localization of organic cation transporter 2 in intact renal proximal tubules
Authors: Sweet Douglas H. , Miller David S. , Pritchard John B.
Published: December 2017

The localization of organic cation transporter 2 (OCT2) within renal cells is the subject of considerable controversy, resulting in marked uncertainty as to its function. To resolve this issue, we made an OCT2/green fluorescent protein (GFP) fusion construct (rOCT2-GFP) and determined its localization within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as well as greatly increased specific, potential-driven uptake of [14C]tetraethylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC-PK1 and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGFP-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distributed cytoplasmic signal with no plasma membrane fluorescence. In contrast, cells transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT2-GFP) showed predominantly plasma membrane fluorescence, which was most prominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MDCK/rOCT2-GFP) maintained in long-term culture showed a greatly increased basal and lateral membrane fluorescence. When grown on porous supports, MDCK/rOCT2-GFP monolayers showed specific, potential-driven TEA uptake from the basal side. Finally, expression and distribution of rOCT2-GFP were investigated in isolated killifish ( Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lateral membrane fluorescence, with no detectable signal at the apical membrane. In contrast, tubules expressing a luminal sodium-dicarboxylate cotransporter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubules expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. These results indicate that rOCT2 is basolateral in renal proximal tubule cells.
Pathways for angiotensin-(1—7) metabolism in pulmonary and renal tissues
Authors: Allred Alicia J. , Diz Debra I. , Ferrario Carlos M. , Chappell Mark C.
Published: December 2017

Two of the primary sites of actions for angiotensin (ANG)-(1—7) are the vasculature and the kidney. Because little information exists concerning the metabolism of ANG-(1—7) in these tissues, we investigated the hydrolysis of the peptide in rat lung and renal brush-border membrane (BBM) preparations. Radiolabeled ANG-(1—7) was hydrolyzed primarily to ANG-(1—5) by pulmonary membranes. The ANG-converting enzyme (ACE) inhibitor lisinopril abolished the generation of ANG-(1—5), as well as that of smaller metabolites. Kinetic studies of the hydrolysis of ANG-(1—7) to ANG-(1—5) by somatic (pulmonary) and germinal (testes) forms of rat ACE yielded similar values, suggesting that the COOH-domain is responsible for the hydrolysis of ANG-(1—7). Pulmonary metabolism of ANG-(1—5) yielded ANG-(3—5) and was independent of ACE but may involve peptidyl or dipeptidyl aminopeptidases. In renal cortex BBM, ANG-(1—7) was rapidly hydrolyzed to mono- and dipeptide fragments and ANG-(1—4). Aminopeptidase (AP) inhibition attenuated the hydrolysis of ANG-(1—7) and increased ANG-(1—4) formation. Combined treatment with AP and neprilysin (Nep) inhibitors abolished ANG-(1—4) formation and preserved ANG-(1—7). ACE inhibition had no effect on the rate of hydrolysis or the metabolites formed in the BBM. In conclusion, ACE was the major enzymatic activity responsible for the metabolism of ANG-(1—7) in the lung, which is consistent with the ability of ACE inhibitors to increase the half-life of circulating ANG-(1—7) and raise endogenous levels of the peptide. An alternate pathway of metabolism was revealed in the renal cortex, where increased AP and Nep activities, relative to ACE activity, promote conversion of ANG-(1—7) to ANG-(1—4) and smaller fragments.
Immunolocalization of electroneutral Na-HCO3−cotransporter in rat kidney
Authors: Vorum Henrik , Kwon Tae-Hwan , Fulton Christiaan , Simonsen Brian , Choi Inyeong , Boron Walter , Maunsbach Arvid B. , Nielsen Søren , Aalkjær Christian
Published: December 2017

An electroneutral Na-HCO3−cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ∼180 kDa, in rat kidney membranes. Peptide- N-glycosidase F deglycosylation reduced the band to ∼140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBCN1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3−-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3−transport in these cells.
Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury
Authors: Weinberg Joel M. , Venkatachalam Manjeri A. , Roeser Nancy F. , Saikumar Pothana , Dong Zheng , Senter Ruth A. , Nissim Itzhak
Published: December 2017

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with α-ketoglutarate (α-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of α-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with α-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of α-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.
Prostaglandins regulate acid-induced cell-mediated bone resorption
Authors: Krieger Nancy S. , Parker Walter R. , Alexander Kristen M. , Bushinsky David A.
Published: December 2017

Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 μM indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium PGE2levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and PGE2levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and PGE2. Net calcium flux was correlated directly with medium PGE2( r = 0.879, n = 29, P < 0.001). Exogenous PGE2, at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in PGE2levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic PGE2synthesis.
Extracellular Ca2+-sensing receptor is a promiscuous divalent cation sensor that responds to lead
Authors: Handlogten Mary E. , Shiraishi Naoki , Awata Hisataka , Huang Chunfa , Miller R. Tyler
Published: December 2017

The extracellular Ca2+-sensing receptor (CaR) responds to polycations, including Ca2+and neomycin. This receptor is a physiological regulator of systemic Ca2+metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb2+, Co2+, Cd2+, and Fe2+, are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A2(PLA2) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca2+, Ba2+, Cd2+, Co2+, Fe2+, Gd3+, Ni2+, Pb2+, and neomycin activated the CaR, but Hg2+and Fe3+did not. We analyzed the kinetics of activation of p42ERK and PLA2by the CaR in response to Ca2+, Co2+, and Pb2+. The EC50values ranged from ∼0.1 mM for Pb2+to ∼4.0 mM for Ca2+. The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca2+and Pb2+were additive for activation of the CaR. The EC50for Ca2+or Pb2+was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA2via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb2+, Cd2+, Co2+, and Fe2+for the kidney and other tissues where it is expressed.
Na+-K+-ATPase-mediated basolateral rubidium uptake in the maturing rabbit cortical collecting duct
Authors: Constantinescu Alexandru R. , Lane Jerome C. , Mak John , Zavilowitz Beth , Satlin Lisa M.
Published: December 2017

Within the renal cortical collecting duct (CCD), transepithelial Na+absorption and K+secretion are linked to basolateral Na+-K+-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na+-K+-ATPase-mediated86rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na+transport. Multiple CCDs (∼6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37°C for 10 min (±1–2.5 mM ouabain or 10 or 100 μM Schering-28080, an inhibitor of H+-K+-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of86Rb (±inhibitors). After 1 min at 37°C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na+-K+pump activity, increased ∼3-fold during the 1st 8 wk of postnatal life ( P< 0.03). The ∼2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 ± 0.45 to 5.02 ± 0.32 pmol · min−1· mm−1) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 ± 0.53 pmol · min−1· mm−1in adult animals ( P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H+-K+-ATPase is absent at the basolateral membrane. Na+-K+-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [γ-32P]ATP under maximum velocity ( Vmax) conditions, also increased in the differentiating CCD (from 316.2 ± 44.4 pmol · h−1· mm−1at 2 wk to 555.9 ± 105.1 at 4 wk to 789.7 ± 145.0 at 6 wk; r = 1.0 by linear regression analysis; P < 0.005). The parallel ∼2.5-fold increases in Na+-K+-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na+-K+-ATPase activity in the CCD remain to be defined.
Eicosanoid regulation of the renal vasculature
Authors: Imig John D.
Published: December 2017

Even though it has been recognized that arachidonic acid metabolites, eicosanoids, play an important role in the control of renal blood flow and glomerular filtration, several key observations have been made in the past decade. One major finding was that two distinct cyclooxygenase (COX-1 and COX-2) enzymes exist in the kidney. A renewed interest in the contribution of cyclooxygenase metabolites in tubuloglomerular feedback responses has been sparked by the observation that COX-2 is constitutively expressed in the macula densa area. Arachidonic acid metabolites of the lipoxygenase pathway appear to be significant factors in renal hemodynamic changes that occur during disease states. In particular, 12( S)- hydroxyeicosatetraenoic acid may be important for the full expression of the renal hemodynamic actions in response to angiotensin II. Cytochrome P-450 metabolites have been demonstrated to possess vasoactive properties, act as paracrine modulators, and be a critical component in renal blood flow autoregulatory responses. Last, peroxidation of arachidonic acid metabolites to isoprostanes appears to be involved in renal oxidative stress responses. The recent developments of specific enzymatic inhibitors, stable analogs, and gene-disrupted mice and in antisense technology are enabling investigators to understand the complex interplay by which eicosanoids control renal blood flow.
Activation of epithelial Na channels during short-term Na deprivation
Authors: Frindt Gustavo , Masilamani Shyama , Knepper Mark A. , Palmer Lawrence G.
Published: December 2017

The role of epithelial Na channels in the response of the kidney to short-term Na deprivation was studied in rats. Animals were fed either a control-Na (3.9 g/kg) or a low-Na ( 3.8 mg/kg) diet for 15 h. Urinary excretion of Na (μmol/min), measured in conscious animals in metabolic cages, was 0.45 ± 0.07 in controls and 0.04 ± 0.01 in Na-deprived animals. Glomerular filtration rate, measured as the clearance of creatinine, was unaffected by the change in diet, suggesting that the reduced Na excretion was the result of increased Na reabsorption. K excretion (μmol/min), increased after the 15-h period of Na deprivation from 0.70 ± 0.10 to 1.86 ± 0.19. Thus the decrease in urine Na was compensated for, in terms of electrical charge balance, by an increase in urine K. Plasma aldosterone increased from 0.50 ± 0.08 to 1.22 ± 0.22 nM. Principal cells from cortical collecting tubules isolated from the animals were studied by using the patch-clamp technique. Whole cell amiloride-sensitive currents were negligible in the control group (5 ± 4 pA/cell) but substantial in the Na-deprived group (140 ± 28 pA/cell). The abundance of the epithelial Na channel subunits, α, β, and γ in the kidney was estimated by using immunoblots. There was no change in the overall abundance of any of the subunits after the 15-h Na deprivation. However, the apparent molecular mass of a fraction of the γ-subunits decreased as was previously reported for long-term Na deprivation. Calculations of the rate of Na transport mediated by the Na channels indicated that activation of the channels during short-term Na deprivation could account in large part for the increased Na reabsorption under these conditions.
Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice
Authors: Ding Yueming , Sigmund Curt D.
Published: December 2017

We previously reported a novel transgenic model expressing human angiotensinogen from the kidney androgen-regulated protein promoter, and demonstrated sexually dimorphic expression. Herein, we investigated the hormonal regulation of this transgene. Testosterone increased transgene expression in female mice in a dose- and time-dependent manner and was not detectable 3-days after treatment was halted. High doses of estrogen were required to induce the transgene. Expression of transgene mRNA decreased after castration of male transgenic mice. As in females, however, transgene expression could be induced after administration of testosterone. Flutamide, an androgen receptor antagonist, dose dependently blocked transgene expression in males and blunted the induction caused by testosterone in females. Neither testosterone nor estrogen altered the proximal tubule cell-specific expression of the transgene. The data suggest that the level of transgene expression in this model can be controlled temporally and in magnitude by manipulating the levels of androgen. The fortuitous androgen regulation of this transgene can be used as a molecular “on-off” switch to control transgene expression and potentially manipulate blood pressure levels in this model.
Multiple aspects of mineralocorticoid selectivity
Authors: Farman Nicolette , Rafestin-Oblin Marie-Edith
Published: December 2017

Aldosterone regulates renal sodium reabsorption through binding to the mineralocorticoid receptor (MR). Because the glucocorticoid receptor (GR) is expressed together with the MR in aldosterone target cells, glucocorticoid hormones bound to GR may also intervene to modulate physiological functions in these cells. In addition, each steroid can bind both receptors, and the MR has equal affinity for aldosterone and glucocorticoid hormones. Several cellular and molecular mechanisms intervene to allow specific aldosterone regulatory effects, despite the large prevalence of glucocorticoid hormones in the plasma. They include the local metabolism of the glucocorticoid hormones into inactive derivatives by the enzyme 11β-hydroxysteroid dehydrogenase; the intrinsic properties of the MR that discriminate between ligands through differential contacts; the possibility of forming homo- or heterodimers between MR and GR, leading to differential transactivation properties; and the interactions of MR and GR with other regulatory transcription factors. The relative contribution of each of these successive mechanisms may vary among aldosterone target cells (epithelial vs. nonepithelial) and according to the hormonal context. All these phenomena allow fine tuning of cellular functions depending on the degree of cooperation between corticosteroid hormones and other factors (hormonal or tissue specific). Such interactions may be altered in pathophysiological situations.
Cellular regulation of endothelial nitric oxide synthase
Authors: Govers Roland , Rabelink Ton J.
Published: December 2017

Renal function is highly dependent on endothelium-derived nitric oxide (NO). Several renal disorders have been linked to impaired NO bioavailability. The enzyme that is responsible for the synthesis of NO within the renal endothelium is endothelial NO synthase (eNOS). eNOS-mediated NO generation is a highly regulated cellular event, which is induced by calcium-mobilizing agonists and fluid shear stress. eNOS activity is regulated at the transcriptional level but also by a variety of modifications, such as acylation and phosphorylation, by its cellular localization, and by protein-protein interactions. The present review focuses on the complex regulation of eNOS within the endothelial cell.
SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport
Authors: Wang Jian , Barbry Pascal , Maiyar Anita C. , Rozansky David J. , Bhargava Aditi , Leong Meredith , Firestone Gary L. , Pearce David
Published: December 2017

The epithelial Na+channel (ENaC) constitutes the rate-limiting step for Na+transport across tight epithelia and is the principal target of hormonal regulation, particularly by insulin and mineralocorticoids. Recently, the serine-threonine kinase (SGK) was identified as a rapidly mineralocorticoid-responsive gene, the product of which stimulates ENaC-mediated Na+transport. Like its close relative, protein kinase B (also called Akt), SGK's kinase activity is dependent on phosphatidylinositol 3-kinase (PI3K), a key mediator of insulin signaling. In our study we show that PI3K is required for SGK-dependent stimulation of ENaC-mediated Na+transport as well as for the production of the phosphorylated form of SGK. In A6 kidney cells, mineralocorticoid induction of the phosphorylated form of SGK preceded the increase in Na+transport, and specific inhibition of PI3K inhibited both phosphorylation of SGK and mineralocorticoid-induced Na+transport. Insulin both augmented SGK phosphorylation and synergized with mineralocorticoids in stimulating Na+transport. In a Xenopus laevisoocyte coexpression assay, SGK-stimulated ENaC activity was also markedly reduced by PI3K inhibition. Finally, in vitro-translated SGK specifically interacted with the ENaC subunits expressed in Escherichia coli as glutathione S-transferase fusion proteins. These data suggest that SGK is a PI3K-dependent integrator of insulin and mineralocorticoid actions that interacts with ENaC subunits to control Na+entry into kidney collecting duct cells.
Mechanisms underlying renoprotection during renin-angiotensin system blockade
Authors: Taal Maarten W. , Chertow Glenn M. , Rennke Helmut G. , Gurnani Anuradha , Jiang Tang , Shahsafaei Aliakbar , Troy Julia L. , Brenner Barry M. , Mackenzie Harald S.
Published: December 2017

Potential determinants of chronic renal disease (CRD) progression were studied in male Munich-Wistar rats subjected to 5/6 nephrectomy and treated with candesartan (Csn; n = 30) or enalapril (Ena; n = 27) from 5 wk postsurgery. Despite control of systolic blood pressure (SBP; 24 wk: Csn = 143 ± 9; Ena = 148 ± 8 mmHg), urinary protein excretion rates (UprV) increased over 24 wk (Csn = 92 ± 10; Ena = 99 ± 8mg/day). Glomerulosclerosis scores (GS) at 24 wk were similar for Csn (42 ± 7%) vs. Ena (42 ± 4%), values close to those of untreated controls at 12 wk (43 ± 4%). At 24 wk, SBP and UprV correlated strongly with GS, together accounting for 72% of the variance in GS. Renal cortex mRNA levels (determined by competitive RT-PCR) for transforming growth factor (TGF)-β1 and monocyte chemoattractant protein (MCP)-1 were elevated in Csn and Ena at 12 wk and remained higher at 24 wk vs. sham. Strong correlations were evident among TGF-β1, MCP-1, and interleukin-1β and renal injury at 24 wk. Cns and Ena are thus equally effective renoprotective agents in this model. During renin-angiotensin system inhibition, renoprotection is dependent on control of both SBP and UprV. Incomplete suppression of renal cytokine gene expression may also contribute to CRD progression.
Interactions of adenosine A1and A2areceptors on renal microvascular reactivity
Authors: Nishiyama Akira , Inscho Edward W. , Navar L. Gabriel
Published: December 2017

Adenosine vasoconstricts preglomerular arterioles via adenosine A1receptors. Because adenosine also activates adenosine A2receptors, its overall renal vascular actions are complex and not fully understood. The present study was performed to determine the relative contributions of adenosine A1and A2areceptors to the responsiveness of the renal microvasculature to adenosine. Afferent and efferent arteriolar diameters were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. Basal afferent and efferent arteriolar diameters averaged 17.1 ± 0.5 ( n = 35) and 17.8 ± 0.5 ( n = 20) μm, respectively. Superfusion with 0.1 and 1 μmol/l adenosine did not significantly alter afferent and efferent arteriolar diameters; however, 10 μmol/l adenosine significantly reduced afferent and efferent arteriolar diameters (−8.2 ± 0.8 and −5.7 ± 0.6%, respectively). The afferent and efferent arteriolar vasoconstrictor responses to adenosine waned at a dose of 100 μmol/l, such that diameters returned to values not significantly different from control within 2 min. During adenosine A1receptor blockade with 8-noradamantan-3-yl-1,3-dipropylxanthine (KW-3902: 10 μmol/l), 10 and 100 μmol/l adenosine significantly increased afferent diameter by, respectively, 8.1 ± 1.2 and 13.7 ± 1.3% ( n = 14) and efferent arteriolar diameter by 6.4 ± 1.3 and 9.3 ± 1.2% ( n = 8). The afferent and efferent arteriolar vasodilatory responses to adenosine in the presence of KW-3902 were significantly attenuated by addition of the adenosine A2areceptor antagonist 1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF-17837: 15 μmol/l, n = 7 and 6, respectively). The addition of KF-17837 alone significantly enhanced afferent ( n = 15) and efferent ( n = 6) arteriolar vasoconstrictor responses to 1, 10, and 100 μmol/l adenosine. These results indicate the presence of adenosine A1and A2areceptors on afferent and efferent arterioles of juxtamedullary nephrons, such that adenosine A2areceptor-mediated vasodilation partially buffers adenosine-induced vasoconstriction in both pre- and postglomerular segments of the renal microvasculature.
α1-Adrenergic receptors activate NHE1 and NHE3 through distinct signaling pathways in epithelial cells
Authors: Liu Fengming , Gesek Frank A.
Published: December 2017

The Na+/H+exchanger (NHE) regulates intracellular pH, cell volume, Na+absorption and H+secretion in epithelial cells of the renal proximal tubule (PT). α1-Adrenergic receptors (ARs) increase NHE activity in PT cells. The purpose of this study was to determine the mechanism of α1-AR activation of NHE isoforms expressed in PT cells. Northern and Western blotting demonstrate transcripts and protein expression of NHE1 and NHE3 in PT cells. An anti-NHE1 antibody predominately labels protein expressed at basal and lateral membranes. In contrast, NHE3 protein is expressed exclusively at the apical membrane. To determine NHE isoforms regulated by α1-ARs, antisense oligodeoxynucleotides (AS-ODNs) specific for NHE1 and NHE3 isoforms were introduced into cells with streptolysin O permeabilization. Cells incubated with AS-ODNs a total of three times exhibited a reduction in protein expression of ∼85%. Na uptake and changes in intracellular pH (pHi) were used as measures of NHE activity in PT cells. α1-AR stimulation increased Na uptake from 8.5 to 13.8 nmol · min−1· mg protein−1. AS-ODNs to NHE3 significantly reduced α1-AR stimulated Na uptake and increases in pHi; no effect was observed in sense-ODN-treated cells. Inhibition of NHE1 but not NHE3 expression abolishes amiloride-suppressible NHE activity. α1-AR stimulation of NHE1 is inhibited by the protein kinase C (PKC) inhibitor calphostin C whereas NHE3 activity is abolished by the mitogen-activated protein kinase (MAPK) inhibitor PD-98059. In PT cells transfected with MAPK kinase MEKK1COOH, a truncated version of MEKK1 that activates MAPK, NHE3 but not NHE1 activity is stimulated. We conclude that α1-ARs activate distinct signaling pathways to regulate specific NHE isoforms localized on opposite membranes in polarized renal epithelial cells. α1-AR activation of NHE1 is regulated by PKC whereas NHE3 is controlled by MAPK and serves to separately regulate pHi, Na absorption, and proton excretion in PT cells.
Segmental expression of somatostatin receptor subtypes sst1and sst2in tubules and glomeruli of human kidney
Authors: Balster Douglas A. , O'Dorisio M. Sue , Summers Monica A. , Turman Martin A.
Published: December 2017

Somatostatin is known to modulate mesangial and tubular cell function and growth, but the somatostatin receptor (sst) subtypes responsible for these effects have not been defined. There are at least five different sst receptor subtypes (sst1-sst5). We used RT-PCR to demonstrate that normal human kidney consistently expresses mRNA for sst1and sst2(9 of 9 donors). Some donors expressed sst4or sst5mRNA, but none expressed sst3mRNA. Expression of sst1and sst2was further assessed by staining serial sections of normal human kidney with sst1and sst2antisera, Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris lectin (proximal tubules), and Tamm-Horsfall protein (THP) antiserum (thick ascending limb of the loop of Henle). Specificity of antisera was demonstrated by transfection and absorption studies. Sst2, but not sst1, was expressed in glomeruli. Intense sst1and sst2staining localized exclusively to AH+and THP+tubules. Thus sst1and sst2subtype-selective analogs may be useful to beneficially modulate renal cell function in pathological conditions.
Posttranscriptional regulation of human iNOS by the NO/cGMP pathway
Authors: Pérez-Sala Dolores , Cernuda-Morollón Eva , Díaz-Cazorla Manuela , Rodríguez-Pascual Fernando , Lamas Santiago
Published: December 2017

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1β plus tumor necrosis factor (TNF)-α reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1β/TNF-α-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.
NO/cGMP signaling modulates regulation of Na+-K+-ATPase activity by angiotensin II in rat proximal tubules
Authors: Zhang Chaojie , Mayeux Philip R.
Published: December 2017

ANG II exerts a biphasic effect on Na+transport in the kidney through its effects on Na+-K+-ATPase activity. Beginning at 10−13M, ANG II increased Na+-K+-ATPase activity in freshly isolated rat proximal tubules to a maximum stimulation at 10−11M of 1.43 ± 0.08-fold above control. Stimulation decreased progressively at concentrations >10−10M to a value of 0.96 ± 0.1-fold at 10−7M. In the presence of additional l-arginine, the substrate for NO synthesis, the stimulatory effect of ANG II (10−11M) was lost. Conversely, N-monomethyl-l-arginine (l-NMMA), the nitric oxide (NO) synthase inhibitor, unmasked the stimulatory effect of ANG II at 10−7M (1.40 ± 0.1-fold). 1 H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one, the soluble guanylyl cyclase inhibitor, like l-NMMA, unmasked the stimulatory effect of ANG II at 10−7M (1.30 ± 0.1-fold). The intracellular cGMP concentration was increased 1.58 ± 0.28-fold at 10−7M ANG II. The ANG II AT1receptor antagonist SK&F 108566 blocked the stimulatory effect of ANG II at 10−11M. These data suggest that the NO/cGMP signaling pathway serves as a negative component in the regulation of Na+-K+-ATPase activity by ANG II.
Fasting downregulates renal water channel AQP2 and causes polyuria
Authors: Amlal Hassane , Chen Qian , Habo Khalid , Wang Zhaohui , Soleimani Manoocher
Published: December 2017

Starvation causes impairment in the urinary concentrating ability. The mechanism of this defect, however, remains unknown. We tested the possibility that food deprivation might affect the expression and activity of aquaporins (AQP1, 2), thereby impairing renal water reabsorption in the kidney. Rats fasted for 24 h exhibited severe polyuria (urine volume increased from 11 before fasting to 29 ml/24 h after fasting, P < 0.0001) along with failure to concentrate their urine (urine osmolality decreased from 1,485 before fasting to 495 mosmol/kgH2O after fasting, P < 0.0001). Refeeding for 24 h returned the urinary concentrating ability back to normal. Northern hybridization and immunoblot analysis demonstrated that fasting was associated with a decrease in AQP2 protein (−80%, P ≤ 0.002) and mRNA levels (−69%, P ≤ 0.003) in the outer medulla. In the cortex, fasting decreased AQP2 protein abundance by 60% ( P ≤ 0.004) but did not alter its mRNA expression. During the recovery phase, AQP2 expression returned to normal level in both tissues. In the inner medulla, the expression of AQP2 was not altered in fasting, but was increased significantly at both protein ( ± 92%) and mRNA ( ± 43%) levels during the recovery from fasting. The proximal nephron water channel (AQP1) was not affected in response to fasting or recovery from fasting. We conclude that 1) fasting impairs the urinary concentrating ability in rats, and 2) the renal water-handling defect in fasting results specifically from the downregulation of AQP2 in the cortical and outer medullary collecting duct.
A possible role for metalloproteinases in renal cyst development
Authors: Obermüller Nicholas , Morente Natividad , Kränzlin Bettina , Gretz Norbert , Witzgall Ralph
Published: December 2017

The expansion of cysts in polycystic kidneys bears several similarities to the invasion of the extracellular matrix by benign tumors. We therefore hypothesized that cyst-lining epithelial cells produce extracellular matrix-degrading metalloproteinases and that the inhibition of these enzymes may represent a potential target for therapeutic intervention. Using in situ hybridization, we first analyzed the expression of membrane-type metalloproteinase 1 (MMP-14), an essential matrix metalloproteinase, of its inhibitor TIMP-2, and of the cytokine transforming growth factor (TGF)-β2 in the ( cy/ +) rat model of autosomal-dominant polycystic kidney disease. Upregulated MMP-14 mRNA was predominantly located in cyst-lining epithelia and distal tubules, whereas TIMP-2 mRNA was confined almost exclusively to fibroblasts. TGF-β2, a cytokine known to regulate the expression of matrix metalloproteinases and their inhibitors, was also expressed by cyst wall epithelia. We then treated ( cy/ +) rats with the metalloproteinase inhibitor batimastat for a period of 8 wk. The treatment with the metalloproteinase inhibitor batimastat resulted in a significant reduction of cyst number and kidney weight. Our study suggests that metalloproteinase inhibitors represent a new therapeutic tool against polycystic kidney disease, which should be applicable independently of the background of the disease.
The concept of cellular “fight-or-flight” reaction to stress
Authors: Goligorsky Michael S.
Published: December 2017

As animals respond to environmental stress with a set of default reactions described as the “fight-or-flight” response, so do epithelial and endothelial cells when they are confronting stressors in their microenvironment. This review will summarize a growing body of data suggesting the existence of a set of stereotypical cellular reactions to stress, provide some examples of diseases that are characterized by excessive flight reactions, describe the cellular mechanisms whereby the fight-or-flight reaction is accomplished, as well as cellular mechanisms triggering either fight or flight. It is proposed that cell-matrix adhesion is a sensitive indicator of the severity of stress. This indicator is interfaced with several default programs for cellular survival or death, thus dictating the fate of the cell. Some diagnostic and therapeutic applications of the concept, presently used and potentially useful, are outlined. The essential feature of this concept is its ability to categorize cellular events in terms of either type of default reaction, predict the details of each, and potentially exploit them clinically.
Alternatively spliced isoform of apical Na+-K+-Cl−cotransporter gene encodes a furosemide-sensitive Na+-Cl−cotransporter
Authors: Plata Consuelo , Meade Patricia , Hall Amy , Welch Rick C. , Vázquez Norma , Hebert Steven C. , Gamba Gerardo
Published: December 2017

In the absence of vasopressin, medullary thick ascending limb cells express a K+-independent, furosemide-sensitive Na+-Cl−cotransporter that is inhibited by hypertonicity. The murine renal specific Na+-K+-2 Cl−cotransporter gene ( SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na+-K+-2 Cl−cotransporter (BSC1–9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (∼200 mosmol/kgH2O), mBSC1–4-injected oocytes do not express significant Na+uptake over H2O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1–4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1–4 oocytes are exposed to hypotonicity (∼100 mosmol/kgH2O), a significant increase in Na+uptake but not in86Rb+uptake is observed. The increased Na+uptake is Cl−dependent, furosemide sensitive, and cAMP sensitive but K+independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH2O ( r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na+-Cl−cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na+-Cl−-to-Na+-K+-2Cl−switching in thick ascending limb cells.
Altered membrane-cytoskeleton linkage and membrane blebbing in energy-depleted renal proximal tubular cells
Authors: Chen Jing , Wagner Mark C.
Published: December 2017

The effects of energy depletion on two membrane-cytoskeletal linker proteins (ezrin and myosin-1β) and membrane bleb formation were studied in isolated rabbit proximal tubule cells. Measurements of cytoskeletal-membrane interactions by using the laser optic trap method revealed a stronger association of control tubule membrane with the apical cytoskeleton compared with the basal cytoskeleton. Energy depletion weakened the apical membrane-cytoskeleton interactions to a greater degree. Biochemical studies demonstrated that energy depletion altered both ezrin and myosin-1β. The salt-insensitive ezrin fraction dissociated from the cytoskeleton; myosin-1β redistributed from the peripheral cytoskeleton to a perinuclear/nuclear complex. These changes in ezrin and myosin-1β and the weakening of the membrane-cytoskeleton interactions correlated with the release of brush-border membrane blebs observed by differential interference contrast microscopy. Permeability of membrane blebs was also evaluated during energy depletion and indicated an increased permeabilization of basal blebs to 3-kDa dextrans. These results support the hypothesis that alterations in membrane-cytoskeleton linkers facilitate the formation and detachment of blebs by weakening membrane-cytoskeleton interactions.