bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of renal organic anion and cation transporters
Authors: Burckhardt Gerhard , Wolff Natascha A.
Published: December 2017

Here we review the structural and functional properties of organic anion transporters (OAT1, OAT2, OAT3) and organic cation transporters (OCTN1, OCTN2, OCT1, OCT2, OCT3), some of which are involved in renal proximal tubular organic anion and cation secretion. These transporters share a predicted 12-transmembrane domain (TMD) structure with a large extracellular loop between TMD1 and TMD2, carrying potential N-glycosylation sites. Conserved amino acid motifs revealed a relationship to the sugar transporter family within the major facilitator superfamily. Following heterologous expression, most OATs transported the model anion p-aminohippurate (PAH). OAT1, but not OAT2, exhibited PAH-α-ketoglutarate exchange. OCT1–3 transported the model cations tetraethylammonium (TEA), N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. OCTNs exhibited transport of TEA and/or preferably the zwitterionic carnitine. Substrate substitution as well as cis-inhibition experiments demonstrated polyspecificity of the OATs, OCTs, and OCTN1. On the basis of comparison of the structurally closely related OATs and OCTs, it may be possible to delineate the binding sites for organic anions and cations in future experiments.
Effect of intraperitoneal pressures on tissue water of the abdominal muscle
Authors: Zakaria El Rasheid , Lofthouse Joanne , Flessner Michael F.
Published: December 2017

A major factor that affects solute and water transport through tissue is the state of tissue hydration. The amount of interstitial water directly affects the transport coefficients for both diffusion and convection. To investigate the effect of simultaneous exposure of tissue to hydrostatic and osmotic pressures on the state of tissue hydration and the pattern of distribution of tissue water, we dialyzed rats with isotonic (290 mosmol/kg) or hypertonic (510 mosmol/kg) solution at intraperitoneal pressures (Pip) between 0 and 6 mmHg, and we infused isotopic markers intravenously and determined their equilibrium distribution volumes (VD) in the anterior abdominal muscle (AAM) by quantitative autoradiography. Total tissue water volume (θTW) was determined from dry-to-wet weight ratios. θurea, the VDof [14C]urea, equals the sum of the extracellular water volume (θEC, VDof [14C]mannitol) and intracellular water volume (θIC= θurea− θEC). If θif= interstitial water volume and θIV= vascular water volume (VDof131I-labeled IgG), then θEC= θif+ θIV. AAM hydrostatic pressure profiles were measured by a micropipette/servo-null system and demonstrated that elevation of Pipabove 3 mmHg significantly ( P < 0.05) increases mean tissue pressure (PT) to the same level regardless of intraperitoneal osmolality. The increase in PTresulted in a nonlinear tissue expansion primarily in the interstitium regardless of osmolality. From 0 to 6 mmHg, θif(in ml/g dry tissue) increased from 0.59 ± 0.02 to 1.7 ± 0.05 and to 1.5 ± 0.05 after isotonic and hypertonic dialysis, respectively, whereas θICincreased from 2.8 ± 0.08 to 3.0 ± 0.1 after isotonic dialysis and decreased to 2.6 ± 0.1 after hypertonic dialysis. After dialysis at 6 mmHg with isotonic or hypertonic solutions, θIVincreased from 0.034 ± 0.001 to 0.049 ± 0.001 and 0.042 ± 0.002, respectively. θureaduring hypertonic dialysis at Pipbetween 0 and 6 mmHg increased in a nonlinear fashion ( F = 26.3, P < 0.001), whereas θICinvariably decreased ( F = 11.1, P < 0.001) and θifdoubled from its control value at low Pip. In conclusion, elevation of intraperitoneal hydrostatic pressure causes tissue expansion, primarily in interstitium, irrespective of osmolality of the bathing solution. Tissue hydrostatic pressure is therefore the primary determinant of tissue properties with respect to hydration, which in turn affects diffusive and convective transport.
Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule
Authors: White Peter , Doctor R. Brian , Dahl Rolf H. , Chen Jing
Published: December 2017

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.
Decreased abundance of major Na+transporters in kidneys of rats with ischemia-induced acute renal failure
Authors: Kwon Tae-Hwan , Frøkiær Jørgen , Han Jin Suk , Knepper Mark A. , Nielsen Søren
Published: December 2017

Ischemia-induced acute renal failure (ARF) is known to be associated with significant impairment of tubular Na reabsorption. We examined whether temporary bilateral renal ischemia (30, 40, or 60 min) and reperfusion (1–5 days) affect the abundance of several renal Na transporters and urinary Na excretion (UNaV) in rats. In rats with mild ARF (30 min), immunoblotting revealed that proximal tubule type 3 Na+/H+exchanger (NHE-3) and type II Na-Picotransporter (NaPi-II) were significantly decreased to 28 ± 6 and 14 ± 6% of sham levels, respectively, at day 1. Moreover, Na+-K+-ATPase levels were also significantly decreased (51 ± 11%), whereas there was no significant decrease in type 1 bumetanide-sensitive cotransporter (BSC-1) and thiazide-sensitive cotransporter (TSC) levels. Consistent with reduced Na transporter abundance, fractional urinary Na excretion (FENa) was significantly increased in mild ARF (30 min) and UNaV was unchanged, despite a marked reduction in glomerular filtration rate. Na transporter levels and renal Na handling were normalized within 5 days. Severe ischemic injury (60 min) resulted in a marked decrease in the abundance of Na+-K+-ATPase, NHE-3, NaPi-II, BSC-1, and TSC at both days 1 and 5. Consistent with this, FENawas significantly increased at days 1 and 5. Intravenous K-melanocyte-stimulated hormone treatment partially prevented the ischemia-induced downregulation of renal Na transporters and reduced the high FENato control levels. We conclude that reduced levels of Na transporters along the nephron may play a critical role in the impairment of tubular Na reabsorption, and hence increased Na excretion, in ischemia-induced ARF.
Effects of 20-HETE and 19(S)-HETE on rabbit proximal straight tubule volume transport
Authors: Quigley Raymond , Baum Michel , Reddy Komandla Malla , Griener James C. , Falck J. R.
Published: December 2017

The kidney has the highest abundance of cytochrome P-450 of all extrahepatic organs. Within the kidney, the highest concentration of cytochrome P-450 is found in the proximal tubule. Whether 20- or 19( S)-hydroxyeicosatetraenoic acid (HETE), the major P-450 metabolites of arachidonic acid in the proximal tubule, affect transport in this segment has not been previously investigated. We examined the direct effects of 20- and 19( S)-HETE on volume absorption ( J v) in the rabbit proximal straight tubule (PST). Production of 20-HETE by rabbit PST was demonstrated by incubating microdissected tubules with [3H]arachidonic acid and separating the lipid extract by HPLC. There was significant conversion of [3H]arachidonic acid to 20-HETE in control tubules that was inhibited by 10− 5 M N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). Addition of exogenous 20-HETE had no effect on PST volume transport. However, inhibition of endogenous production of 20-HETE using DDMS stimulated transport. In the presence of DDMS, 20-HETE inhibited PST J v. 19( S)-HETE in the bathing solution stimulated PST J v alone and in the presence of DDMS. Thus ω- and ω-1-hydroxylase products of arachidonic acid have direct effects on PST transport. Endogenous production of 20-HETE may play a role in tonic suppression of transport and may therefore be an endogenous regulator of transport in the proximal tubule.
Adhesion of uric acid crystals to the surface of renal epithelial cells
Authors: Koka Rima M. , Huang Erick , Lieske John C.
Published: December 2017

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 12% of which contain uric acid (UA) either alone or admixed with calcium oxalates or calcium phosphates. UA crystals bind rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. The urinary glycoproteins osteopontin, nephrocalcin, and Tamm-Horsfall glycoprotein had no effect on binding of UA crystals to the cell surface, whereas other polyanions including specific glycosaminoglycans blocked UA crystal adhesion. Specific polycations also inhibited adhesion of UA crystals and appeared to exert their inhibitory effect by coating cells. However, removal of anionic cell surface molecules with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal binding, and sialic acid-binding lectins had no effect. These observations suggest that hydrogen bonding and hydrophobic interactions play a major role in adhesion of electrostatically neutral UA crystals to renal cells, unlike the interaction of calcium-containing crystals with negatively charged molecules on the apical cell surface via ionic forces. After adhesion to the plasma membrane, subsequent cellular events could contribute to UA crystal retention in the kidney and the development of UA or mixed calcium and UA calculi.
Characterization of the thiazide-sensitive Na+-Cl−cotransporter: a new model for ions and diuretics interaction
Authors: Monroy Adriana , Plata Consuelo , Hebert Steven C. , Gamba Gerardo
Published: December 2017

The thiazide-sensitive Na+-Cl−cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant ( Km) for Na+of 7.6 ± 1.6 mM and for Cl−of 6.3 ± 1.1 mM, and Hill coefficients for Na+and Cl−consistent with electroneutrality. The affinities of both Na+and Cl−were increased by increasing concentration of the counterion. The IC50values for thiazides were affected by both extracellular Na+and Cl−. The higher the Na+or Cl−concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both Na+and Cl−.
Store-operated calcium influx inhibits renin secretion
Authors: Schweda Frank , Riegger Günter A. J. , Kurtz Armin , Krämer Bernhard K.
Published: December 2017

On the basis of evidence that changes in the extracellular concentration of calcium effectively modulate renin secretion from renal juxtaglomerular cells, our study aimed to determine the effect of calcium influx activated by depletion of intracellular calcium stores on renin secretion. For this purpose we characterized the effects of the endoplasmatic Ca2+-ATPase inhibitors thapsigargin (300 nM) and cyclopiazonic acid (20 μM) on renin secretion from isolated perfused rat kidneys. We found that Ca2+-ATPase inhibition caused a potent inhibition of basal renin secretion as well as renin secretion activated by isoproterenol, bumetanide, and by a fall in the renal perfusion pressure. The inhibitory effect of Ca2+-ATPase inhibition on renin secretion was reversed within seconds by lowering of the extracellular calcium concentration into the submicromolar range but was not affected by lanthanum, gadolinium, flufenamic acid, or amlodipine. These data suggest that calcium influx triggered by release of calcium from internal stores is a powerful mechanism to inhibit renin secretion from juxtaglomerular cells. The store-triggered calcium influx pathway in juxtaglomerular cells is apparently not sensitive to classic blockers of the capacitative calcium entry pathway.
Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct
Authors: Silver Randi B. , Breton Sylvie , Brown Dennis
Published: December 2017

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+diet) by monitoring K+- and Na+-independent H+extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHirecovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHirecovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHirecovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.
A mathematical model of the outer medullary collecting duct of the rat
Authors: Weinstein Alan M.
Published: December 2017

A mathematical model of the outer medullary collecting duct (OMCD) has been developed, consisting of α-intercalated cells and a paracellular pathway, and which includes Na+, K+, Cl−, HCO3−, CO2, H2CO3, phosphate, ammonia, and urea. Proton secretion across the luminal cell membrane is mediated by both H+-ATPase and H-K-ATPase, with fluxes through the H-K-ATPase given by a previously developed kinetic model (Weinstein AM. Am J Physiol Renal Physiol 274: F856–F867, 1998). The flux across each ATPase is substantial, and variation in abundance of either pump can be used to control OMCD proton secretion. In comparison with the H+-ATPase, flux through the H-K-ATPase is relatively insensitive to changes in lumen pH, so as luminal acidification proceeds, proton secretion shifts toward this pathway. Peritubular HCO3−exit is via a conductive pathway and via the Cl−/HCO3−exchanger, AE1. To represent AE1, a kinetic model has been developed based on transport studies obtained at 38°C in red blood cells. (Gasbjerg PK, Knauf PA, and Brahm J. J Gen Physiol 108: 565–575, 1996; Knauf PA, Gasbjerg PK, and Brahm J. J Gen Physiol 108: 577–589, 1996). Model calculations indicate that if all of the chloride entry via AE1 recycles across a peritubular chloride channel and if this channel is anything other than highly selective for chloride, then it should conduct a substantial fraction of the bicarbonate exit. Since both luminal membrane proton pumps are sensitive to small changes in cytosolic pH, variation in density of either AE1 or peritubular anion conductance can modulate OMCD proton secretory rate. With respect to the OMCD in situ, available buffer is predicted to be abundant, including delivered HCO3−and HPO42−, as well as peritubular NH3. Thus, buffer availability is unlikely to exert a regulatory role in total proton secretion by this tubule segment.
Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney
Authors: Ecelbarger Carolyn A. , Kim Gheun-Ho , Terris James , Masilamani Shyama , Mitchell Carter , Reyes Ivan , Verbalis Joseph G. , Knepper Mark A.
Published: December 2017

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (α, β, and γ) in rat kidney. Seven-day infusion of 1-deamino-[8-d-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of β- and γ-ENaC (to 238% and 288% of vehicle, respectively), whereas α-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of β- and γ- but no significant change in α-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.
The transport properties of the human renal Na+- dicarboxylate cotransporter under voltage-clamp conditions
Authors: Yao Xiaozhou , Pajor Ana M.
Published: December 2017

The transport properties of the human Na+-dicarboxylate cotransporter, (hNaDC-1), expressed in Xenopus laevis oocytes were characterized using the two-electrode voltage clamp technique. Steady-state succinate-evoked inward currents in hNaDC-1 were dependent on the concentrations of succinate and sodium, and on the membrane potential. At −50 mV, the half-saturation constant for succinate ( K0.5succinate) was 1.1 mM and the half-saturation constant for sodium ( K0.5sodium) was 65 mM. The Hill coefficient was 2.3, which is consistent with a transport stoichiometry of 3 Na+:1 divalent anion substrate. The hNaDC-1 exhibits a high-cation selectivity. Sodium is the preferred cation and other cations, such as lithium, were not able to support transport of succinate. The preferred substrates of hNaDC-1 are fumarate ( K0.51.8 mM) and succinate, followed by methylsuccinate ( K0.52.8 mM), citrate ( K0.56.8 mM) and α-ketoglutarate ( K0.516 mM). The hNaDC-1 may also transport sodium ions through an uncoupled leak pathway, which is sensitive to phloretin inhibition. We propose a transport model for hNaDC-1 in which the binding of three sodium ions is followed by substrate binding.
Molecular pharmacology of renal organic anion transporters
Authors: Van Aubel Rémon A. M. H. , Masereeuw Rosalinde , Russel Frans G. M.
Published: December 2017

Renal organic anion transport systems play an important role in the elimination of drugs, toxic compounds, and their metabolites, many of which are potentially harmful to the body. The renal proximal tubule is the primary site of carrier-mediated transport from blood to urine of a wide variety of anionic substrates. Recent studies have shown that organic anion secretion in renal proximal tubule is mediated by distinct sodium-dependent and sodium-independent transport systems. Knowledge of the molecular identity of these transporters and their substrate specificity has increased considerably in the past few years by cloning of various carrier proteins. However, a number of fundamental questions still have to be answered to elucidate the participation of the cloned transporters in the overall tubular secretion of anionic xenobiotics. This review summarizes the latest knowledge on molecular and pharmacological properties of renal organic anion transporters and homologs, with special reference to their nephron and plasma membrane localization, transport characteristics, and substrate and inhibitor specificity. A number of the recently cloned transporters, such as the p-aminohippurate/dicarboxylate exchanger OAT1, the anion/sulfate exchanger SAT1, the peptide transporters PEPT1 and PEPT2, and the nucleoside transporters CNT1 and CNT2, are key proteins in organic anion handling that possess the same characteristics as has been predicted from previous physiological studies. The role of other cloned transporters, such as MRP1, MRP2, OATP1, OAT-K1, and OAT-K2, is still poorly characterized, whereas the only information that is available on the homologs OAT2, OAT3, OATP3, and MRP3–6 is that they are expressed in the kidney, but their localization, not to mention their function, remains to be elucidated.
Integrins in kidney development, function, and disease
Authors: Kreidberg Jordan A. , Symons Jordan M.
Published: December 2017

Integrins are heterodimeric cell surface receptors that mediate heterophilic cell-cell interactions and interactions between cells and the extracellular matrix (Hynes RO. Cell 69: 11–25, 1991). As such, they are involved in morphogenetic processes during development, as well as in the maintenance of normal tissue architecture in fully developed organs. Integrins are now recognized to be a large family of receptors, and several different integrins have been demonstrated as being expressed in the developing and adult kidney (Korhonen M, Ylkanne J, Laitinen L, and Virtanen I. Development 122: 3537–3547, 1996; Rahilly MA and Fleming S. J Pathol 167: 327–334, 1992). This review will summarize present knowledge about integrin expression in the developing, normal, and diseased kidney and attempt to provide a hypothetical framework for understanding integrin function in the urogenital system. Since the last time this area was reviewed (Hamerski DA and Santoro S. Curr Opin Nephrol Hypertens 8: 9–14, 1999), there have been significant publications on the roles of integrins in kidney development and disease. At present, there are many more questions than answers, and integrins present an area where many novel and exciting findings will emerge in the coming years.
Impairment of pressure-natriuresis and renal autoregulation in ANG II-infused hypertensive rats
Authors: Wang Chi-Tarng , Chin So Yeon , Navar L. Gabriel
Published: December 2017

Chronic infusions of initially subpressor doses of angiotensin II (ANG II) lead to progressive hypertension over a 2-wk period and to augmented intrarenal ANG II levels. The present study was performed to investigate total renal blood flow (RBF) and medullary blood flow (MBF) autoregulatory behavior and pressure-natriuresis in ANG II-infused hypertensive rats and how these are modified by concomitant treatment with an ANG II AT1receptor antagonist. ANG II-infused rats ( n = 27) were prepared by administration of ANG II at 60 ng/min via osmotic minipump for 13 days. Twelve of the ANG II-infused hypertensive rats were treated with losartan in the drinking water (30 mg · kg ·−1day−1). Rats were anesthetized with pentobarbital sodium (50 mg/kg, ip) and prepared for renal function measurements. An aortic clamp was placed above the junction of the left renal artery to reduce renal arterial pressure. Autoregulatory responses for renal plasma flow, overall RBF, and glomerular filtration rate were impaired in ANG II-infused hypertensive rats; however, MBF autoregulation was not disrupted. Most strikingly, pressure-natriuresis was markedly suppressed in ANG II-infused hypertensive rats. Chronic treatment with losartan prevented the impairment of the pressure-natriuresis relationship caused by chronic ANG II infusion. These findings demonstrate that chronic ANG II infusion leads to marked impairment of sodium excretion and suppression of the pressure-natriuresis relationship, which may contribute to the progressive hypertension that occurs in this model. These renal effects are prevented by simultaneous treatment with an AT1receptor blocker.
Endothelin inhibits thick ascending limb chloride flux via ETBreceptor-mediated NO release
Authors: Plato Craig F. , Pollock David M. , Garvin Jeffrey L.
Published: December 2017

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ETBreceptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux ( JCl). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ETBreceptors and release of NO. Exogenous ET-1 (1 nM) decreased JClfrom 118.3 ± 15.0 to 62.7 ± 13.6 pmol · mm−1· min−1(48.3 ± 8.2% reduction), whereas removal of ET-1 increased JClin a separate group of tubules from 87.6 ± 10.7 to 115.2 ± 10.3 pmol · mm−1· min−1(34.5 ± 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on JCl, we examined the effect of inhibiting of NO synthase (NOS) with NG-nitro-l-arginine methyl ester (l-NAME) on ET-1-induced changes in JCl. l-NAME (5 mM) completely prevented the ET-1-induced reduction in JCl, whereas d-NAME did not. l-NAME alone had no effect on JCl. These data suggest that the effects of ET-1 are mediated by NO. Blockade of ETBreceptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ETBreceptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on JCl(from 120.7 ± 12.6 to 75.4 ± 13.3 pmol · mm−1· min−1). In contrast, ETAreceptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH JCl(from 96.5 ± 10.4 to 69.5 ± 8.6 pmol · mm−1· min−1). Endothelin increased intracellular calcium from 96.9 ± 14.0 to 191.4 ± 11.9 nM, an increase of 110.8 ± 26.1%. We conclude that exogenous endothelin indirectly decreases TALH JClby activating ETBreceptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.
Renal interstitial hydrostatic pressure and pressure natriuresis in pregnant rats
Authors: Khraibi Ali A.
Published: December 2017

The objective of this study was to test the hypothesis that a decrease in renal interstitial hydrostatic pressure (RIHP) accounts for the blunted pressure natriuresis during pregnancy. RIHP was measured in nonpregnant (NP; n = 9), midterm pregnant (MP; 12–14 days after conception; n = 10), and late-term pregnant (LP; 18–21 days after conception; n = 12) female Sprague-Dawley rats at two renal perfusion pressure (RPP) levels (99 and 120 mmHg). At the lower RPP level, RIHP was 5.9 ± 0.3 mmHg for NP, 3.4 ± 0.4 mmHg for MP ( P < 0.05 vs. NP), and 2.9 ± 0.1 mmHg for LP ( P < 0.05 vs. NP) rats. The increase in RPP from 99 to 120 mmHg resulted in pressure natriuretic and diuretic responses in all groups; however, the increases in fractional excretion of sodium (ΔFENa), urine flow rate (ΔV), and ΔRIHP were significantly greater ( P < 0.05) in NP compared with both MP and LP rats. ΔFENa, ΔV, and ΔRIHP were 2.06 ± 0.28%, 81.44 ± 14.10 μl/min, and 3.0 ± 0.5 mmHg for NP; 0.67 ± 0.13%, 28.03 ± 5.28 μl/min, and 0.5 ± 0.2 mmHg for MP; and 0.48 ± 0.12%, 18.14 ± 4.70 μl/min, and 0.4 ± 0.1 mmHg for LP rats. In conclusion, RIHP is significantly lower in pregnant compared with nonpregnant rats at similar RPP levels. Also, the ability of pregnant rats to increase RIHP in response to an increase in RPP is blunted. These changes in RIHP may play an important role in the blunted pressure natriuresis and contribute to the conservation of sodium and water that is critical for fetal growth and development during normal pregnancy.
Vasopressin V2-receptor-dependent regulation of AQP2 expression in Brattleboro rats
Authors: Promeneur Dominique , Kwon Tae-Hwan , Frøkiær Jørgen , Knepper Mark A. , Nielsen Søren
Published: December 2017

The role of AVP-V2receptor (AVP-V2R)-dependent regulation of aquaporin-2 (AQP2) expression was evaluated in vasopressin-deficient Brattleboro (BB) rats. AQP2 levels were relatively high in BB rats (52 ± 8% of levels in Wistar rats), and treatment with the AVP-V2R antagonist SR-121463A (0.8 mg/day) for 48 h was associated with 1) increased urine output (170 ± 9%), 2), reduced AQP2 protein levels (42 ± 10% in whole kidney and 53 ± 8% in inner medulla), and 3) reduced AQP2 mRNA levels (36 ± 7%). In addition, the levels of AQP2 phosphorylated in the protein kinase A (PKA) consensus site (Ser256of AQP2) was reduced to 3 ± 1% of control levels. Lithium (Li) treatment of BB rats for 1 mo, known to reduce adenylyl cyclase (AC) activity, downregulated AQP2 protein levels (15 ± 6%) and increased urine output (220%). Downregulation of AQP2 expression in response to SR-121463A or Li treatment indicates that AQP2 expression in BB rats depends in part on activation of AVP-V2Rs and that the signaling cascade(s) involves AC and hence cAMP. Complete water restriction of BB rats produced only a small increase in AQP2 mRNA (235 ± 33%) and AQP2 protein (156 ± 22%) levels. Immunoelectron microscopy confirmed the increase in AQP2 abundance but revealed no change in AQP2 apical plasma membrane labeling in response to thirsting. In conclusion, the expression and phosphorylation of AQP2 in BB rats are in part dependent on AVP-V2R signaling, and AVP-V2-mediated regulation of AQP2 trafficking and expression is effectively decoupled in BB rats, indicating differences in AVP-V2R-mediated regulation of AQP2 trafficking and expression.
Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase
Authors: Defer Nicole , Best-Belpomme Martin , Hanoune Jacques
Published: December 2017

The present review focuses on the potential physiological regulations involving different isoforms of adenylyl cyclase (AC), the enzymatic activity responsible for the synthesis of cAMP from ATP. Depending on the properties and the relative level of the isoforms expressed in a tissue or a cell type at a specific time, extracellular signals received by the G protein-coupled receptors can be differently integrated. We report here on various aspects of such regulations, emphasizing the role of Ca2+/calmodulin in activating AC1 and AC8 in the central nervous system, the potential inhibitory effect of Ca2+on AC5 and AC6, and the changes in the expression pattern of the isoforms during development. A particular emphasis is given to the role of cAMP during drug dependence. Present experimental limitations are also underlined (pitfalls in the interpretation of cellular transfection, scarcity of the invalidation models, and so on).
A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation
Authors: Chen Ruihua , Mukhin Yurii V. , Garnovskaya Maria N. , Thielen Thomas E. , Iijima Yoshihiro , Huang Cancan , Raymond John R. , Ullian Michael E. , Paul Richard V.
Published: December 2017

We constructed an expression vector for a fusion protein [ANG II type 1a receptor-green fluorescent protein (AT1aR-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat AT1aR. Chinese hamster ovary (CHO) cells transfected with AT1aR-GFP demonstrated specific, high-affinity125I-labeled ANG II binding (IC5021 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with AT1aR or AT1aR-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT1R blocker losartan. ANG II-driven internalization of AT1aR-GFP in transfected CHO cells was demonstrated both by radioligand binding and by laser scanning confocal microscopy. Colocalization of GFP fluorescence with that of the nuclear stain TOTO-3 in confocal images was increased more than twofold after 1 h of ANG II exposure. We conclude that AT1aR-GFP exhibits similar pharmacological behavior to that of the native AT1aR. Our observations also support previous evidence for the presence of AT1aR in the nucleus and suggest that the density of AT1aR in the nucleus may be regulated by exposure to its ligand.
Vasopressin-dependent upregulation of aquaporin-2 gene expression in glucocorticoid-deficient rats
Authors: Saito Takako , Ishikawa San-E , Ando Fumiko , Higashiyama Minori , Nagasaka Shoichiro , Sasaki Sei , Saito Toshikazu
Published: December 2017

We determined alterations in renal aquaporin-2 (AQP2) gene expression in association with impaired water excretion in glucocorticoid-deficient rats. After adrenalectomy, Sprague-Dawley rats were administered aldosterone alone by osmotic pumps (glucocorticoid-deficient rats). As a control, both aldosterone and dexamethasone were administered. These animals were subjected to the studies on days 7–14. The expressions of AQP2 mRNA and protein in kidney of the glucocorticoid-deficient rats were increased by 1.6- and 1.4-fold compared with the control rats, respectively. An acute oral water load test verified the marked impairment in water excretion in the glucocorticoid-deficient rats. One hour after the water load, the expressions of AQP2 mRNA and protein were significantly reduced in the control rats, but they remained unchanged in the glucocorticoid-deficient rats. However, there was no alteration in [3H]arginine vasopressin (AVP) receptor binding and AVP V2receptor mRNA expression in the glucocorticoid-deficient rats. A V2-receptor antagonist abolished the increased expressions of AQP2 mRNA and protein in the glucocorticoid-deficient rats. These results indicate that augmented expression of AQP2 participates in impaired water excretion, dependent on AVP, in glucocorticoid deficiency.
Role of neuronal nitric oxide synthase (NOS1) in the pathogenesis of renal hemodynamic changes in diabetes
Authors: Komers Radko , Lindsley Jessie N. , Oyama Terry T. , Allison Kristen M. , Anderson Sharon
Published: December 2017

Nitric oxide (NO) has been implicated in the pathogenesis of renal hemodynamic changes in diabetes mellitus. However, the contribution of nitric oxide synthase (NOS) isoforms to intrarenal production of NO in diabetes remains unknown. To explore the role of NOS1 in the control of renal hemodynamics in diabetes, we assessed renal responses to inhibition of NOS1 with S-methyl-l-thiocitrulline (SMTC; administered into the abdominal aorta) in moderately hyperglycemic streptozotocin-diabetic rats (D) and their nondiabetic (C) and normoglycemic diabetic counterparts. The contribution of other NOS isoforms was also evaluated by assessing the responses to nonspecific NOS inhibition [ NG-nitro-l-arginine methyl ester (l-NAME)] in SMTC-treated diabetic rats. The number of NOS1-positive cells in macula densa of D and C kidneys was also evaluated by immunohistochemistry. D rats demonstrated elevated glomerular filtration rate (GFR) compared with C. SMTC (0.05 mg/kg) normalized GFR in D but had no effect in C. SMTC-induced reduction of renal plasma flow (RPF) was similar in C and D. Normoglycemic diabetic rats demonstrated blunted renal hemodynamic responses to NOS1 inhibition compared with hyperglycemic animals. Mean arterial pressure was stable in all groups. l-NAME induced a further decrease in RPF, but not in GFR, in D rats treated with SMTC. Immunohistochemistry revealed increased numbers of NOS1-positive cells in D. These observations suggest that NOS1-derived NO plays a major role in the pathogenesis of renal hemodynamic changes early in the course of diabetes. NOS1 appears to be the most important isoform in the generation of hemodynamically active NO in this condition.
Divalent cation transport by the distal nephron: insights from Bartter's and Gitelman's syndromes
Authors: Ellison David H.
Published: December 2017

Elucidation of the gene defects responsible for many disorders of renal fluid and electrolyte homeostasis has provided new insights into normal and abnormal physiology. Identifying the causes of Gitelman's and Bartter's syndromes has greatly enhanced our understanding of ion transport by thick ascending limb and distal convoluted tubule cells. Despite this information, several phenotypic features of these diseases remain confusing, even in the face of molecular insight. Paramount among these are disorders of divalent cation homeostasis. Bartter's syndrome is caused by dysfunction of thick ascending limb cells. It is associated with calcium wasting, but magnesium wasting is usually mild. Loop diuretics, which inhibit ion transport by thick ascending limb cells, markedly increase urinary excretion of both calcium and magnesium. In contrast, Gitelman's syndrome is caused by dysfunction of the distal convoluted tubule. Hypocalciuria and hypomagnesemia are universal parts of this disorder. Yet although thiazide diuretics, which inhibit ion transport by distal convoluted tubule cells, reduce urinary calcium excretion, they have minimal effects on urinary magnesium excretion, when given acutely. This review proposes mechanisms that may account for the differences between the effects of diuretic drugs and the phenotypic features of Gitelman's and Bartter's syndromes. These mechanisms are based on recent insights from another inherited disease of ion transport, inherited magnesium wasting, and from a review of the chronic effects of diuretic drugs in animals and people.
17β-Estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases
Authors: Noris Marina , Todeschini Marta , Zappella Sergio , Bonazzola Samantha , Zoja Carla , Corna Daniela , Gaspari Flavio , Marchetti Franco , Aiello Sistiana , Remuzzi Giuseppe
Published: December 2017

Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17β-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor l-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17β-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17β-estradiol staining was comparable to controls. Thus 17β-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.
Male gender increases sensitivity to proteinuria induced by mild NOS inhibition in rats: role of sex hormones
Authors: Verhagen A. Marjan G. , Attia Diana M. A. , Koomans Hein A. , Joles Jaap A.
Published: December 2017

Men are at greater risk for renal injury than women. We studied whether male rats are more sensitive to the hypertensive and proteinuric effects of chronic nitric oxide sythase (NOS) inhibition than female rats. In addition, we studied whether androgens or estrogens are responsible for differences in sensitivity to proteinuria induced by chronic NOS inhibition. Females and males were treated with 10, 20, 30, and 100 mg/l Nω-nitro-l-arginine (l-NNA) during 24 wk. Systolic blood pressure (SBP) and proteinuria were measured regularly and compared with time-control measurements in control females and males. In females and males treatment with 10 mg/l l-NNA had no effect on SBP or proteinuria. Treatment with 20, 30, and 100 mg/l l-NNA resulted in a dose-dependent increase in SBP that was similar in males and females. However, females treated with 20 and 30 mg/ll-NNA were resistant to the development of proteinuria: maximum values were 16 ± 7 and 46 ± 21, respectively, vs. 16 ± 3 mg/day in controls, whereas males treated with those doses showed an increase in proteinuria [139 ± 35 ( P< 0.05) and 318 ± 82 ( P < 0.01), respectively, vs. 55 ± 11 mg/day in controls]. Treatment with 100 mg/ll-NNA increased proteinuria similarly in both females and males. To study the role of sex hormones in differences in sensitivity to proteinuria induced by mild chronic NOS inhibition, treatment with 20 mg/l l-NNA was repeated in ovariectomized (Ovx) and orchidectomized rats. Ovariectomy did not affect the increase in SBP caused by 20 mg/l l-NNA, but, in contrast to intact females, this dose of l-NNA did cause Ovx rats to develop proteinuria (51 ± 16 vs. 16 ± 7 mg/day in control Ovx rats; P < 0.05). Orchidectomy completely prevented the increased SBP as well as proteinuria induced by 20 mg/ll-NNA in male rats. In conclusion, male rats are more sensitive than female rats to develop proteinuria induced by mild chronic NOS inhibition. Estrogens provide some protection in females, whereas androgens are responsible for the increased sensitivity of male rats to proteinuria induced by mild chronic NOS inhibition. Risk factors associated with a compromised nitric oxide system may be more detrimental to the kidney in men than in women.
Localization of organic cation transporters OCT1 and OCT2 in rat kidney
Authors: Karbach Ulrich , Kricke Jörn , Meyer-Wentrup Friederike , Gorboulev Valentin , Volk Christopher , Loffing-Cueni Dominique , Kaissling Brigitte , Bachmann Sebastian , Koepsell Hermann
Published: December 2017

Renal excretion and reabsorption of organic cations are mediated by electrogenic and electroneutral organic cation transporters, which belong to a recently discovered family of polyspecific transporters. These transporters are electrogenic and exhibit differences in substrate specificity. In rat, the renal expression of the polyspecific cation transporters rOCT1 and rOCT2 was investigated. By in situ hybridization, significant amounts of both rOCT1 and rOCT2 mRNA were detected in S1, S2, and S3 segments of proximal tubules. By immunohistochemistry, expression of the rOCT1 protein was mainly observed in S1 and S2 segments of proximal tubules, with lower expression levels in the S3 segments. At variance, rOCT2 protein was mainly expressed in the S2 and S3 segments. Both transporters were localized to the basolateral cell membrane. Neither rOCT1 nor rOCT2 was detected in the vasculature, the glomeruli, and nephron segments other than proximal tubules. The data suggest that rOCT1 and rOCT2 are responsible for basolateral cation uptake in the proximal tubule, which represents the first step in cation secretion.
Mechanisms underlying induction of heme oxygenase-1 by nitric oxide in renal tubular epithelial cells
Authors: Liang Mingyu , Croatt Anthony J. , Nath Karl A.
Published: December 2017

We examined whether nitric oxide-generating agents influence expression of heme oxygenase-1 (HO-1) in renal proximal tubular epithelial cells, LLC-PK1 cells, and the mechanisms underlying any such effects. In sublytic amounts, the nitric oxide donor sodium nitroprusside induced HO-1 mRNA and protein and HO activity in a dose-dependent and time-dependent fashion; this induction was specific for nitric oxide since the nitric oxide scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide significantly reduced such induction. The induction of HO activity by sodium nitroprusside, or by another nitric oxide donor, spermine NONOate, was markedly reduced by the iron chelator deferoxamine. Two different thiol-containing agents, N-acetylcysteine and dithiothreitol, blunted such induction of HO by nitric oxide. Downstream products of nitric oxide, such as peroxynitrite or cGMP, were not involved in inducing HO. In higher concentrations (millimolar amounts), sodium nitroprusside induced appreciable cytotoxicity as assessed by lactate dehydrogenase (LDH) release and lipid peroxidation, and both of these effects were markedly reduced by deferoxamine. Inhibition of HO did not affect the cytotoxic effects (measured by LDH release) of sodium nitroprusside. We thus provide the novel description of the induction of HO-1 in renal proximal tubular epithelial cells exposed to nitric oxide donors and provide the first demonstration in kidney-derived cells for the involvement of a redox-based mechanism in such expression. We also demonstrate that, in LLC-PK1 cells exposed to nitric oxide donors, chelatable iron is involved in eliciting the HO-1 response observed at lower concentrations of these donors, and in mediating the cytotoxic effects of these donors when present in higher concentrations.
Molecular modulation of inward and outward apical transporters ofl-dopa in LLC-PK1cells
Authors: Soares-Da-Silva P. , Serrão M. P.
Published: December 2017

The present study examined the nature of the apical inward and outwardl-3,4-dihydroxyphenylalanine (l-dopa) transporters in LLC-PK1cells and whether protein kinases differentially modulate the activities of these transporters. The apical inward transfer of l-dopa was promoted through an energy-dependent and sodium-insensitive transporter (Michaelis constant = 38 μM; maximum velocity = 2608 pmol · mg protein−1· 6 min−1). This transporter was insensitive to N-(methylamino)-isobutyric acid but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC; IC50= 251 μM). Modulators of protein kinase A (cAMP, forskolin, IBMX, and cholera toxin), protein kinase G (cGMP, zaprinast, LY-83583 and sodium nitroprusside), and protein kinase C (phorbol 12,13-dibutirate and chelerythrine) failed to affect the accumulation of l-dopa. The Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibitedl-dopa uptake (IC50of 72 and 55 μM, respectively). The inhibitory effect of calmidazolium on the accumulation of l-dopa was of the noncompetitive type. The organic anion inhibitor DIDS, but not p-aminohippurate, and the protein tyrosine kinase (PTK) inhibitor genistein significantly increased l-dopa accumulation, which was mainly due to inhibition of apical outward transfer of l-dopa. It is concluded that LLC-PK1cells take up l-dopa over the apical cell border through the L-type amino acid transporter, which appears to be under the control of Ca2+-calmodulin-mediated pathways. The apical outward transfer of l-dopa may be promoted through a DIDS-sensitive transport mechanism and appears to be under the tonic control of PTK.