bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells
Authors: Wallace Darren P. , Christensen Marcy , Reif Gail , Belibi Franck , Thrasher Brantley , Herrell Duke , Grantham Jared J.
Published: March 2015

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDiand hIMCDtcells. The effects of AVP and PGE2were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDiand hIMCDt. Basal short-circuit current ( ISC) of hIMCDimonolayers was 1.4 ± 0.5 μA/cm2and was inhibited by benzamil, a Na+channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased ISC; the current was reduced by blocking PKA, apical Cl−channels, basolateral NKCC1 (a Na+-K+-2Cl−cotransporter), and basolateral Cl−/HCO[Formula: see text]exchangers. In fluid transport studies, hIMCDimonolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDtmonolayers, basal current was not different from zero and cAMP had no effect on ISC. We conclude that AVP and PGE2stimulate cAMP-dependent Cl−secretion by hIMCDicells, but not hIMCDtcells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.
Chronic renal hypoxia after acute ischemic injury: effects ofl-arginine on hypoxia and secondary damage
Authors: Basile David P. , Donohoe Deborah L. , Roethe Kelly , Mattson David L.
Published: March 2015

Ischemic acute renal failure (ARF) results in the permanent loss of peritubular capillaries and predisposes the progression of chronic renal failure. The present study was undertaken to determine whether renal hypoxia, which may represent an important mediator in disease progression, is persistently exacerbated after recovery from ARF. Rats were subjected to ischemia-reperfusion injury and allowed to recover for 5 or 20 wk. Immunohistochemistry of the hypoxia-sensitive marker 2-pimonidizole at 5 wk revealed an overall increase in incorporation in the outer medullary region after recovery from ARF compared with sham-operated controls. Unilateral nephrectomy, in combination with ischemia-reperfusion injury resulted in greater 2-pimonidizole staining than that observed in the bilateral injury model. In addition, in the unilateral ischemia-nephrectomy model, proteinuria, interstitial fibrosis, and renal functional loss developed significantly faster than in the bilateral model of ARF when animals were allowed to recover for 20 wk. l-Arginine in the drinking water (∼0.5 g/day) increased total renal blood flow ∼30%, decreased pimonidizole staining, and attenuated manifestations of chronic renal disease. These data suggest that a reduction in the peritubular capillary density after ARF results in a persistent reduction in renal Po2and that hypoxia may play an important role in progression of chronic renal disease after ARF.
Altered expression of renal acid-base transporters in rats with lithium-induced NDI
Authors: Kim Young-Hee , Kwon Tae-Hwan , Christensen Birgitte M. , Nielsen Jakob , Wall Susan M. , Madsen Kirsten M. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

Prolonged lithium treatment of humans and rodents often results in hyperchloremic metabolic acidosis. This is thought to be caused by diminished net H+secretion and/or excessive back-diffusion of acid equivalents. To explore whether lithium treatment is associated with changes in the expression of key renal acid-base transporters, semiquantitative immunoblotting and immunocytochemistry were performed using kidneys from lithium-treated ( n = 6) and control ( n = 6) rats. Rats treated with lithium for 28 days showed decreased urine pH, whereas no significant differences in blood pH and plasma [Formula: see text] levels were observed. Immunoblot analysis revealed that lithium treatment induced a significant increase in the expression of the H+-ATPase (B1-subunit) in cortex (190 ± 18%) and inner stripe of the outer medulla (190 ± 9%), and a dramatic increase in inner medulla (900 ± 104%) in parallel to an increase in the expression of type 1 anion exchanger (400 ± 40%). This was confirmed by immunocytochemistry and immunoelectron microscopy, which also revealed increased density of intercalated cells. Moreover, immunoblotting and immunocytochemistry revealed a significant increase in the expression of the type 1 electrogenic [Formula: see text] cotransporter (NBC) in cortex (200 ± 23%) and of the electroneutral NBCn1 in inner stripe of the outer medulla (250 ± 54%). In contrast, there were no changes in the expression of Na+/H+exchanger-3 or of the [Formula: see text] exchanger pendrin. These results demonstrate that the expression of specific renal acid-base transporters is markedly altered in response to long-term lithium treatment. This is likely to represent direct or compensatory effects to increase the capacity for [Formula: see text] reabsorption, [Formula: see text] reabsorption, and proton secretion to prevent the development of systemic metabolic acidosis.
Angiotensin II clamp prevents the second step in renal apical NHE3 internalization during acute hypertension
Authors: Leong Patrick K. K. , Yang Li E. , Holstein-Rathlou Niels-Henrik , McDonough Alicia A.
Published: March 2015

Acute hypertension inhibits proximal tubule (PT) sodium reabsorption. The resultant increase in NaCl delivery to the macula densa suppresses renin release. We tested whether the sustained pressure-induced inhibition of PT sodium reabsorption requires a renin-mediated decrease in ANG II levels. Plasma ANG II concentration of anesthesized Sprague-Dawley rats was clamped by simultaneous infusion of the ANG I-converting enzyme inhibitor captopril (12 μg/min) and ANG II (20 ng · kg−1· min−1). Blood pressure was increased 50 mmHg for 20 min by arterial constriction ± ANG II clamp or by sham operation. This acute hypertension increased urine output and endogenous Li+clearance, and these responses were blunted 40–50% in ANG II clamped rats. Acute hypertension provoked a rapid redistribution of Na+/H+exchanger isoform 3 (NHE3) out of apical brush-border membranes (21 ± 4% decrease of total NHE3 abundance) to endosomal/lysosomal membranes (16 ± 6% increase of total). In ANG II-clamped rats, acute hypertension also provoked disappearance of NHE3 from the apical membranes (27 ± 2% decrease of total), but NHE3 was shifted to membranes enriched in intermicrovillar cleft and dense apical tubules ( step 1) rather than endosomal/lysosomal membranes ( step 2). This difference was independently confirmed by confocal analysis. In contrast, the pressure-induced redistribution of Na+-Picotransporter type 2 was not altered by ANG II clamp. We conclude that the responses to acute hypertension, including diuresis and redistribution of PT NHE3 into intracellular membranes, require a responsive renin-angiotensin system and that the responses may be induced by the sustained increase in NaCl delivery to the macula densa during acute hypertension.
Altered expression of renal NHE3, TSC, BSC-1, and ENaC subunits in potassium-depleted rats
Authors: Elkjær Marie-Louise , Kwon Tae-Hwan , Wang Weidong , Nielsen Jakob , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

The purpose of this study was to examine whether hypokalemia is associated with altered abundance of major renal Na+transporters that may contribute to the development of urinary concentrating defects. We examined the changes in the abundance of the type 3 Na+/H+exchanger (NHE3), Na+-K+-ATPase, the bumetanide-sensitive Na+-K+-2Cl−cotransporter (BSC-1), the thiazide-sensitive Na+-Cl−cotransporter (TSC), and epithelial sodium channel (ENaC) subunits in kidneys of hypokalemic rats. Semiquantitative immunoblotting revealed that the abundance of BSC-1 (57%) and TSC (46%) were profoundly decreased in the inner stripe of the outer medulla (ISOM) and cortex/outer stripe of the outer medulla (OSOM), respectively. These findings were confirmed by immunohistochemistry. Moreover, total kidney abundance of all ENaC subunits was significantly reduced in response to the hypokalemia: α-subunit (61%), β-subunit (41%), and γ-subunit (60%), and this was confirmed by immunohistochemistry. In contrast, the renal abundance of NHE3 in hypokalemic rats was dramatically increased in cortex/OSOM (736%) and ISOM (210%). Downregulation of BSC-1, TSC, and ENaC may contribute to the urinary concentrating defect, whereas upregulation of NHE3 may be compensatory to prevent urinary Na+loss and/or to maintain intracellular pH levels.
Substance P via NK1receptor facilitates hyperactive bladder afferent signaling via action of ROS
Authors: Chien Chiang-Ting , Yu Hong-Jeng , Lin Tser-Bin , Lai Ming-Kuen , Hsu Su-Ming
Published: March 2015

We explored whether substance P (SP) via neurokinin (NK) receptor facilitates bladder afferent signaling and reactive oxygen species (ROS) formation in bladder in association with neurogenic inflammation. We evaluated ROS activity and cystometrograms as well as pelvic nervous activity in anesthetized rat bladder with SP stimulation. Our results showed that endogenous SP via NK1, not NK2, receptor mediated a micturition reflex. An increase in SP by electrical stimulation of the pelvic nerve or an increase in exogenous SP by intra-arterial or intrathecal administration can facilitate myogenic and neurogenic bladder contractions. Furthermore, exaggerated SP release increased ROS in the bladder and whole blood via increased mast cell degranulation, intercellular adhesion molecule expression, and leukocyte adhesion, a primary source of ROS in the inflamed bladder. Treatment with NK1-receptor antagonists or ROS scavengers reduced bladder intercellular adhesion molecule expression and ROS and ameliorated the hyperactive bladder response. Our study indicates that the mechanism by which SP participates in the neurogenic bladder may be complicated by its proinflammatory activity and its ability to stimulate ROS generation.
Overload proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of immune cells
Authors: Alvarez Violeta , Quiroz Yasmir , Nava Mayerly , Pons Héctor , Rodríguez-Iturbe Bernardo
Published: March 2015

Recent evidence suggests that salt-sensitive hypertension develops as a consequence of renal infiltration with immunocompetent cells. We investigated whether proteinuria, which is known to induce interstitial nephritis, causes salt-sensitive hypertension. Female Lewis rats received 2 g of BSA intraperitoneally daily for 2 wk. After protein overload (PO), 6 wk of a high-salt diet induced hypertension [systolic blood pressure (SBP) = 156 ± 11.8 mmHg], whereas rats that remained on a normal-salt diet and control rats (without PO) on a high-salt diet were normotensive. Administration of mycophenolate mofetil (20 mg · kg−1· day−1) during PO resulted in prevention of proteinuria-related interstitial nephritis, reduction of renal angiotensin II-positive cells and oxidative stress (superoxide-positive cells and renal malondialdehyde content), and resistance to the hypertensive effect of the high-salt diet (SBP = 129 ± 12.2 mmHg). The present studies support the participation of renal inflammatory infiltrate in the pathogenesis of salt-sensitive hypertension and provide a direct link between two risk factors of progressive renal damage: proteinuria and hypertension.
Targeted disruption of the bradykinin B2receptor gene in mice alters the ontogeny of the renin-angiotensin system
Authors: Yosipiv Igor V. , Dipp Susana , El-Dahr Samir S.
Published: March 2015

First published July 12, 2001; 10.1152/ajprenal.0020.2001.—Angiotensin II type 1 (AT1) receptor knockout (KO) mice exhibit an activated kallikrein-kinin system (KKS) that serves to attenuate the severity of the renal vascular phenotype in these mice (Tsuchida S, Miyazaki Y, Matsusaka T, Hunley TE, Inagami T, Fogo A, and Ichikawa I, Kidney Int 56: 509–516, 1999). Conversely, gestational high salt suppresses the fetal renin-angiotensin system (RAS) and provokes aberrant renal development in bradykinin B2-KO mice (El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S, Physiol Genomics 3: 121–131, 2000). Thus the cross talk between the RAS and KKS may be critical for normal renal maturation. To further define the developmental interactions between the KKS and RAS, we examined the consequences of B2receptor gene ablation on the expression of RAS components. Renal renin mRNA levels are 50% lower in newborn B2-KO than wild-type (WT) mice. Also, the age-related decline in renin mRNA is greater in B2-KO than WT mice (3.5- vs. 2-fold, P < 0.05). Although renal angiotensinogen (Ao) protein levels are higher in newborn B2-KO than WT mice, Ao mRNA levels are not, suggesting accumulation of Ao as a result of decreased renin-mediated cleavage. Similar age-related increases (8-fold) in angiotensin I-converting enzyme (ACE) activity are observed in B2-KO and WT mice. Renal AT1protein levels are not different in B2-KO and WT mice. Furthermore, the developmental increases in renal kallikrein mRNA and enzymatic activity are more pronounced in B2-KO compared with WT mice (mRNA: 8- vs. 3-fold; activity: 13- vs. 6-fold, P < 0.05). We conclude that 1) bradykinin stimulates renin gene expression, 2) renal kallikrein is regulated via a negative feedback loop involving the B2receptor, and 3) Ao, ACE, and AT1are not bradykinin-target genes.
Regulation of the apical Cl−/HCO 3 − exchanger pendrin in rat cortical collecting duct in metabolic acidosis
Authors: Petrovic Snezana , Wang Zhaohui , Ma Liyun , Soleimani Manoocher
Published: March 2015

Pendrin is an apical Cl−/OH−/HCO[Formula: see text] exchanger in β-intercalated cells (β-ICs) of rat and mouse cortical collecting duct (CCD). However, little is known about its regulation in acid-base disorders. Here, we examined the regulation of pendrin in metabolic acidosis, a condition known to decrease HCO[Formula: see text]secretion in CCD. Rats were subjected to NH4Cl loading for 4 days, which resulted in metabolic acidosis. Apical Cl−/HCO[Formula: see text] exchanger activity in β-ICs was determined as amplitude and rate of intracellular pH change when Cl was removed in isolated, microperfused CCDs. Intracellular pH was measured by single-cell digital ratiometric imaging using fluorescent pH-sensitive dye 2′,7′-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein-AM. Pendrin mRNA expression in kidney cortex was examined by Northern blot hybridizations. Expression of pendrin protein was assessed by indirect immunofluorescence. Microperfused CCDs isolated from acidotic rats demonstrated ∼60% reduction in apical Cl−/HCO[Formula: see text] exchanger activity in β-ICs ( P < 0.001 vs. control). Northern blot hybridizations indicated that the mRNA expression of pendrin in kidney cortex decreased by 68% in acidotic animals ( P < 0.02 vs. control). Immunofluorescence labeling demonstrated significant reduction in pendrin expression in CCDs of acidotic rats. We conclude that metabolic acidosis decreases the activity of the apical Cl−/HCO[Formula: see text] exchanger in β-ICs of the rat CCD by reducing the expression of pendrin. Adaptive downregulation of pendrin in metabolic acidosis indicates the important role of this exchanger in acid-base regulation in the CCD.
NHE3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis
Authors: Gekle Michael , Serrano Oscar K. , Drumm Karina , Mildenberger Sigrid , Freudinger Ruth , Gassner Birgit , Jansen Hans Willi , Christensen Erik I.
Published: March 2015

Receptor-mediated, clathrin-dependent endocytosis (RME) is important for macromolecular transport and regulation of cell-surface protein expression. Pharmacological studies have shown that the plasma membrane transport protein Na+/H+exchanger 3 (NHE3), which shuttles between the plasma membrane and the early endosomal compartment by means of clathrin-mediated endocytosis, contributes to endosomal pH homeostasis and endocytic fusion events. Furthermore, it is known that NHE3 is phosphorylated and inhibited by cAMP-dependent kinase (protein kinase A). Here, we show, in a cellular knockout/retransfection approach, that NHE3 supports RME and confers cAMP sensitivity to RME, using megalin/cubilin-mediated albumin uptake in opossum kidney cells. RME, but not fluid-phase endocytosis, was dependent on NHE3 activity and expression. Furthermore, NHE3 deficiency or inhibition reduced the relative surface expression of megalin without altering total expression. In wild-type cells, cAMP inhibits NHE3 activity, leads to endosomal alkalinization, and reduces RME. In NHE3-deficient cells, endosomal pH is not sensitive to NHE3 inhibition, and cAMP does not affect endosomal pH or RME. NHE3 transfection into deficient cells restores RME and the effects of cAMP. Thus our data show that NHE3 is important for cAMP sensitivity of clathrin-dependent RME.
A rat kidney tubule suspension for the study of vasopressin-induced shuttling of AQP2 water channels
Authors: Shaw Stephen , Marples David
Published: March 2015

AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.
Experimental and clinical rationale for use of MMF in nontransplant progressive nephropathies
Authors: Zatz Roberto , Noronha Irene Lourdes , Fujihara Clarice Kazue
Published: March 2015

The incidence of progressive nephropathies and, consequently, the population suffering from end-stage renal disease have increased steadily in recent years, posing an ever-growing cost, in both human and financial terms, to society. There is mounting evidence that, in both immune-mediated and nonimmune-mediated chronic nephropathies, renal inflammatory events are key to the propagation and perpetuation of renal injury. Mycophenolate mofetil (MMF) is an antilymphocyte agent recently introduced in clinical practice for the prevention of allograft rejection. The present review discusses clinical and experimental evidence that the anti-inflammatory action of MMF can be advantageously used to arrest immune- and nonimmune-mediated progressive injury of native kidneys as well.
Estrogen-induced proliferation of urothelial cells is modulated by nerve growth factor
Authors: Teng Jian , Wang Zun-Yi , Bjorling Dale E.
Published: March 2015

Both nerve growth factor (NGF) and estrogen have been shown to stimulate proliferation of various cell types. Human urothelial cells (HUC) express the α- and β-subtypes of the estrogen receptor (ERαand ERβ) as well as tyrosine kinase A (trkA), the high-affinity receptor for NGF. We investigated interactions between estrogen and NGF relative to cell proliferation using primary cultures of HUC. 17β-estradiol (E2) stimulated NGF synthesis by HUC, and E2 (50 nM), the ERαagonist 16α-iodo-17β-estradiol (10 nM), or the ERβagonist genistein (50 nM) each stimulated HUC proliferation, an effect that was abolished by the estrogen antagonist ICI-182,780 (100 nM). NGF (1–100 ng/ml) stimulated HUC proliferation, and this was abolished by NGF antiserum (0.1 μl/ml) or the trkA antagonist K252a (100 nM). HUC proliferation stimulated by E2 was also abolished by NGF antiserum or K252a. Finally, we observed that treatment of HUC with NGF (50 ng/ml) or E2 (50 nM) stimulated trkA phosphorylation, and this was abolished by K252a (100 nM) or NGF antiserum (0.1 μl/ml). These data indicate that the effects of ER activation on HUC proliferation at least partly involve activation of trkA by NGF.
NHERF-1 is required for renal adaptation to a low-phosphate diet
Authors: Weinman Edward J. , Boddeti Anuradha , Cunningham Rochelle , Akom Michael , Wang Fengying , Wang Yu , Liu Jie , Steplock Deborah , Shenolikar Shirish , Wade James B.
Published: March 2015

The sodium-dependent renal phosphate transporter (Npt2, Na-Pi IIa) is the major regulated phosphate transporter in the renal proximal convoluted tubule. Npt2 associates with a number of PDZ-containing proteins including Na+H+exchanger regulatory factor-1 (NHERF-1). To determine whether NHERF-1 is involved in the acute regulation of phosphate transport, wild-type and NHERF-1 (–/–) mice were stabilized on a high-phosphate diet and then acutely changed to a low-phosphate diet. At 24 h after the change to a low-phosphate diet, there was a significant decrease in the urinary excretion of phosphate in both groups but the urinary excretion of phosphate in NHERF-1 (–/–) mice was significantly higher than in wild-type animals (1,097 ± 356 vs. 255 ± 54 ng/min, P < 0.05). Renal mRNA levels and total cellular Npt2 protein did not differ between the animal groups or in response to the changes in diet. Renal brush-border membrane (BBM) expression of Npt2 protein, however, was lower in NHERF-1 (–/–) mice compared with wild-type. In addition, with both the high- and low-phosphate diets, there was increased detection of Npt2 in submicrovillar domains that were particularly prominent in NHERF-1 (–/–) mice compared with wild-type animals. On the other hand, a change from a low-phosphate diet to a high-phosphate diet was associated with a similar increase in the urinary excretion of phosphate in wild-type and NHERF-1 (–/–) animals. These experiments demonstrate that full renal adaptation to a low-phosphate diet requires NHERF-1, which serves to increase BBM expression of Npt2.
Basolateral membrane Cl−-, Na+-, and K+-coupled base transport mechanisms in rat MTALH
Authors: Bourgeois Soline , Massé Sandrine , Paillard Michel , Houillier Pascal
Published: March 2015

Mechanisms involved in basolateral HCO[Formula: see text] transport were examined in the in vitro microperfused rat medullary thick ascending limb of Henle (MTALH) by microfluorometric monitoring of cell pH. Removing peritubular Cl−induced a cellular alkalinization that was inhibited in the presence of peritubular 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and blunted in the absence of external CO2/HCO[Formula: see text]. The alkalinization elicited by removing peritubular Cl−persisted in the bilateral absence of Na+, together with a voltage clamp. When studied in Cl−-free solutions, lowering peritubular pH induced a base efflux that was inhibited by peritubular DIDS or by the absence of external CO2/HCO[Formula: see text]. Removing peritubular Na+elicited a cellular acidification that was accounted for by stimulation of a DIDS- and ethylisopropylamiloride (EIPA)-insensitive Na+-HCO[Formula: see text] cotransport and inhibition of a basolateral Na+/H+exchange. Increasing bath K+induced an intracellular alkalinization that was inhibited in the absence of external CO2/HCO[Formula: see text]. At 2 mM, peritubular Ba2+, which inhibits the K+-Cl−cotransport, did not induce any change in transepithelial voltage but elicited a cellular alkalinization and inhibited K+-induced cellular alkalinization, consistent with the presence of a basolateral, electroneutral Ba2+-sensitive K+-Cl−cotransport that may operate as a K+-HCO[Formula: see text] cotransport. This cotransport was inhibited in the peritubular presence of furosemide, [(dihydroindenyl)oxy]alkanoic acid, 5-nitro-2-(3-phenylpropylamino)benzoate, or DIDS. At least three distinct basolateral HCO[Formula: see text] transport mechanisms are functional under physiological conditions: electroneutral Cl−/HCO[Formula: see text] exchange, DIDS- and EIPA-insensitive Na+-HCO[Formula: see text] cotransport, and Ba2+-sensitive electroneutral K+-Cl−(HCO[Formula: see text]) cotransport.
Manganese superoxide dismutase gene therapy protects against irradiation-induced cystitis
Authors: Kanai Anthony J. , Zeidel Mark L. , Lavelle John P. , Greenberger Joel S. , Birder Lori A. , De Groat William C. , Apodaca Gerard L. , Meyers Susan A. , Ramage Richard , Epperly Michael W.
Published: March 2015

Urinary bladder cystitis occurs in patients receiving radiation therapy for pelvic tumors. Radiation-induced formation of superoxide radicals is believed to damage the urothelium, exposing the underlying bladder smooth muscle to urine, culminating in nerve irritation and muscle dysfunction. We tested whether overexpression of MnSOD could decrease superoxide levels and protect the bladder from radiation damage. Pelvic irradiation led to sloughing of urothelial umbrella cells, with decreased transepithelial resistance, increased water and urea permeabilities, and increased expression of inducible nitric oxide synthase. Six months after irradiation, cystometrograms showed elevated intravesical pressures and prolonged voiding patterns. However, urothelia transfected with the MnSOD transgene recovered from radiation injury more rapidly, and detrusor function was much closer to that of control bladders than irradiated bladders without the transgene. We conclude that MnSOD gene therapy is protective, which could lead to its use in mitigating radiation cystitis and preventing dysfunction of the urinary bladder.
CFTR-dependent and -independent swelling-activated K+ currents in primary cultures of mouse nephron
Authors: Belfodil Radia , Barrière Hervé , Rubera Isabelle , Tauc Michel , Poujeol Chantal , Bidet Michel , Poujeol Philippe
Published: March 2015

The role of CFTR in the control of K+ currents was studied in mouse kidney. Whole cell clamp was used to identify K+ currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K+ current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K+ currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K+ currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca2+-sensitive swelling-activated K+ conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl− and K+channels.
Phospholemman expression in extraglomerular mesangium and afferent arteriole of the juxtaglomerular apparatus
Authors: Wetzel Randall K. , Sweadner Kathleen J.
Published: March 2015

The molecular mechanisms with which the juxtaglomerular apparatus accomplishes its twin functions, acute regulation of glomerular blood flow and secretion of renin, are still not clearly understood. Least understood is the role of the extraglomerular mesangial (EM) cells, also known as lacis or Goormaghtigh cells, which lie sandwiched between the macula densa and the afferent and efferent arterioles. Here, we report that immunoreactivity for phospholemman (FXYD1), a single-span membrane protein homologous to the gamma (γ) sub-unit of the Na,K-ATPase, is found in the kidney in EM cells with the Na,K-ATPase β2-subunit and in cortical blood vessels and the afferent arteriole with Na,K-ATPase α2and β2. Phospholemman's distribution in EM cells is distinct from that of the Na,K-ATPase γ-subunit, which is found on the basolateral surface of macula densa cells with Na,K-ATPase α1and β1. Phospholemman is a major kinase target, and its location in the juxtaglomerular apparatus suggests that it is involved in tubuloglomerular feedback.
Novel regulation of cell [Na+] in macula densa cells: apical Na+ recycling by H-K-ATPase
Authors: Peti-Peterdi János , Bebok Zsuzsa , Lapointe Jean-Yves , Bell P. Darwin
Published: March 2015

Na-K-ATPase is the nearly ubiquitous enzyme that maintains low-Na+, high-K+concentrations in cells by actively extruding Na+ in exchange for K+. The prevailing paradigm in polarized absorbing epithelial cells, including renal nephron segments and intestine, has been that Na-K-ATPase is restricted to the basolateral membrane domain, where it plays a prominent role in Na+absorption. We have found, however, that macula densa (MD) cells lack functionally and immunologically detectable amounts of Na-K-ATPase protein. In fact, these cells appear to regulate their cytosolic [Na+] via another member of the P-type ATPase family, the colonic form of H-K-ATPase, which is located at the apical membrane in these cells. We now report that this constitutively expressed apical MD colonic H-K-ATPase can function as a Na(H)-K-ATPase and regulate cytosolic [Na+] in a novel manner. This apical Na+-recycling mechanism may be important as part of the sensor function of MD cells and represents a new paradigm in cell [Na+] regulation.
Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+exchanger expression in distal convoluted tubule cells
Authors: Magyar Clara E. , White Kenneth E. , Rojas Raul , Apodaca Gerard , Friedman Peter A.
Published: March 2015

The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+exchanger regulate intracellular Ca2+concentrations and mediate Ca2+efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1–4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+exchanger, mediate basal or hormone-stimulated Ca2+efflux by distal tubules.
HCaRGis a novel regulator of renal epithelial cell growth and differentiation causing G2M arrest
Authors: Devlin Alison M. , Solban Nicolas , Tremblay Sandra , Gutkowska Jolanta , Schürch Walter , Orlov Sergei N. , Lewanczuk Richard , Hamet Pavel , Tremblay Johanne
Published: March 2015

We recently identified a novel calcium-regulated gene, HCaRG, that is highly expressed in the kidney and maps to a chromosomal locus determining kidney weight in rats. The mRNA levels of HCaRG negatively correlate with the proliferative status of the kidney cells. To investigate its role in renal epithelial cellular growth directly, we studied the human embryonic kidney cell line (HEK-293) stably transfected with either plasmid alone or plasmid containing rat HCaRG. [3H]thymidine incorporation was significantly lower in HCaRG clones. Although HCaRG clones exhibited some enhanced susceptibility to cell death, this was not the primary mechanism of reduced proliferation. Cell cycle analysis revealed a G2M phase accumulation in HCaRG clones that was associated with upregulation of p21Cip1/WAF1and downregulation of p27Kip1. HCaRG clones had a greater protein content, larger cell size, and released 4.5- to 8-fold more of an atrial natriuretic peptide-like immunoreactivity compared with controls. In addition, HCaRG clones demonstrated the presence of differentiated junctions and a lower incidence of mitotic figures. Genistein treatment of wild-type HEK-293 cells mimicked several phenotypic characteristics associated with HCaRGoverexpresssion, including increased cell size and increased release of atrial natriuretic peptide. Taken together, our results suggest that HCaRG is a regulator of renal epithelial cell growth and differentiation causing G2M cell cycle arrest.
Differential effects of Npt2a gene ablation and X-linked Hyp mutation on renal expression of Npt2c
Authors: Tenenhouse Harriet S. , Martel Josée , Gauthier Claude , Segawa Hiroko , Miyamoto Ken-ichi
Published: March 2015

The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (Pi) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-Pi cotransport. In mice homozygous for the disrupted Npt2a gene ( Npt2– /–), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2+/+ littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to β-actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. Pi deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2+/+ mice but failed to elicit a further increase in Npt2c protein in Npt2– /– mice. Pi restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2– /– mice and Hyp mice and that the Npt2c response to low-Pi challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2– /– mice and suggest that Npt2c likely accounts for residual BBM Na-Pi cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression.
Thromboxane synthase and TP receptor mRNA in rat kidney and brain: effects of salt intake and ANG II
Authors: Wilcox Christopher S. , Welch William J.
Published: March 2015

A TP receptor (TP-R) mimetic causes salt-sensitive hypertension and renal afferent arteriolar vasoconstriction. TP-Rs mediate effects of ANG II on renal vascular resistance and drinking. Therefore, we investigated the hypothesis that thromboxane A2synthase (TxA2-S) and/or TP-R expression is regulated by salt and/or ANG II. Rats ( n= 6) received high-salt (HS) or low-salt (LS) diets. Additional HS-diet rats received ANG II while other HS- and LS-diet rats received the AT1receptor (AT1-R) antagonist losartan. Excretion of thromboxane B2by conscious rats was increased with the HS diet compared with the LS diet (126 ± 10 vs. 48 ± 5 pmol/24 h, respectively; P < 0.01). The mRNA abundance for TP-Rs (relative to β-actin) in the kidney cortex was enhanced 30% by the HS diet ( P< 0.001) and was reduced 50% by the addition of ANG II ( P < 0.001). However, during losartan administration, the effects of salt were reversed; mRNA more than doubled during the LS diet ( P < 0.001). Similarly, the mRNA abundance for TP-Rs in the brain stem was reduced by 50% with the addition of ANG II ( P < 0.001) and during losartan administration was almost doubled by the LS diet ( P < 0.001). The mRNA abundance for TxA2-S in the kidney cortex also was increased many times with the HS diet ( P < 0.001). In contrast, the mRNA for TxA2-S in the brain was unaffected by salt. ANG II did not affect TxA2-S at either site. During losartan administration, TxA2-S increased modestly in the brain stem with the LS diet. mRNA abundance for TP-Rs in the kidney cortex and brain stem is suppressed by ANG II acting on AT1-Rs. In the absence of AT1-Rs, expression of TP-Rs at both sites is enhanced by LS intake. In contrast, ANG II does not affect the mRNA abundance for TxA2-S. Expression of TxA2-S is enhanced by HS intake in the kidney cortex but by LS intake in the brain stem only during losartan administration. Thus TP-Rs are strongly dependent on ANG II acting on AT1-Rs, whereas TxA2-S is regulated differentially in the kidney cortex and brain stem by salt intake.
Paradoxes of nitric oxide in the diabetic kidney
Authors: Komers Radko , Anderson Sharon
Published: March 2015

As an important modulator of renal function and morphology, the nitric oxide (NO) system has been extensively studied in the diabetic kidney. However, a number of studies in different experimental and clinical settings have produced often confusing data and contradictory findings. We have reviewed a wide spectrum of findings and issues that have amassed concerning the pathophysiology of the renal NO system in diabetes, pointed out the controversies, and attempted to find some explanation for these discrepancies. Severe diabetes with profound insulinopenia can be viewed as a state of generalized NO deficiency, including in the kidney. However, we have focused our hypotheses and conclusions on the events occurring during moderate glycemic control with some degree of treatment with exogenous insulin, representing more the clinically applicable state of diabetic nephropathy. Available evidence suggests that diabetes triggers mechanisms that in parallel enhance and suppress NO bioavailability in the kidney. We hypothesize that during the early phases of nephropathy, the balance between these two opposing forces is shifted toward NO. This plays a role in the development of characteristic hemodynamic changes and may contribute to consequent structural alterations in glomeruli. Both endothelial (eNOS) and neuronal NO synthase can contribute to altered NO production. These enzymes, particularly eNOS, can be activated by Ca2+-independent and alternative routes of activation that may be elusive in traditional methods of investigation. As the duration of exposure to the diabetic milieu increases, factors that suppress NO bioavailability gradually prevail. Increasing accumulations of advanced glycation end products may be one of the culprits in this process. In addition, this balance is continuously modified by actual metabolic control and the degree of insulinopenia.
Specificity of human and rat orthologs of the concentrative nucleoside transporter, SPNT
Authors: Gerstin Karin M. , Dresser Mark J. , Giacomini Kathleen M.
Published: March 2015

To understand the roles that nucleoside transporters play in the in vivo distribution of clinically important nucleoside analogs, the substrate specificity of each transporter isoform should be determined. In the present work, we studied the substrate specificities of the human and rat orthologs of the Na+-dependent purine-selective nucleoside transporter (SPNT; concentrative nucleoside transporter 2), for nucleosides, nucleobases, and base- and ribose-modified nucleoside analogs. The two-electrode voltage-clamp technique in Xenopus laevisoocytes expressing these transporters was used. Purine nucleosides and uridine induced currents in oocytes expressing rat SPNT (rSPNT) or human SPNT1 (hSPNT1). The rank order of magnitude of nucleoside-induced currents was guanosine > uridine > adenosine > inosine and guanosine > uridine > inosine > adenosine for rSPNT- and hSPNT1-expressing oocytes, respectively. Uridine analogs (modified at the 5-position of the base) induced little or no current, suggesting that these compounds are only poorly transported by either transporter. Cladribine induced currents in oocytes expressing rSPNT ( K0.5= 57 ± 12 μM) but not hSPNT1. The ribose-modified nucleoside analogs, adenine arabinoside, and 2′,3′-dideoxyadenosine induced currents in rSPNT-expressing, but not in hSPNT1-expressing, oocytes. These data suggest that there are notable species differences in the specificity of SPNT for synthetic nucleoside analogs.
Effect of partial outlet obstruction on rabbit urinary bladder smooth muscle function
Authors: Su Xiaoling , Stein Raymund , Stanton Michaela C. , Zderic Stephen , Moreland Robert S.
Published: March 2015

Bladder outlet obstruction secondary to benign prostate hyperplasia is associated with many cellular changes. This study was designed to determine whether these changes involve the contractile apparatus. Bladder smooth muscles from rabbits subjected to partial outlet obstruction for 2 wk were mounted for isometric force, isotonic shortening velocity, and myosin light chain (MLC) phosphorylation levels. Muscle strips from obstructed bladders exhibited spontaneous phasic activity; muscle strips from control bladders did not. Muscle strips from obstructed bladders exhibited increased sensitivity and higher levels of stress in response to the cumulative addition of KCl or carbachol compared with control. During noncumulative addition of KCl or carbachol, no differences in sensitivity were noted. Muscle strips from obstructed bladders had elevated basal MLC phosphorylation levels and stimulation produced small increases in MLC phosphorylation compared with control. Vmaxduring KCl stimulation of muscle strips from obstructed bladders was 10-fold lower than control. Our results suggest that bladder outlet obstruction produces a muscle cell that develops higher levels of force but with greatly reduced cross bridge cycling rates.
Renal nerve stimulation augments effect of intraluminal angiotensin II on proximal tubule transport
Authors: Quan Albert , Baum Michel
Published: March 2015

The proximal tubule synthesizes and secretes angiotensin II into the lumen, where it regulates transport. Renal denervation abolishes the effect of angiotensin II on proximal tubule transport. Using in vivo microperfusion, we examined whether renal nerve stimulation modulates the effect of angiotensin II on transport. The effect of angiotensin II was assessed by measuring the decrease in volume reabsorption with the addition of 10−4M luminal enalaprilat. Luminal enalaprilat did not alter volume reabsorption (2.80 ± 0.18 vs. 2.34 ± 0.14 nl · mm−1· min−1). However, with renal nerve stimulation, enalaprilat decreased volume reabsorption (3.45 ± 0.22 vs. 1.67 ± 0.20 nl · mm−1· min−1, P < 0.0005). The absolute and percent decrements in volume reabsorption with luminal enalaprilat were higher with renal nerve stimulation than with native innervation (1.78 ± 0.19 vs. 0.46 ± 0.23 nl · mm−1· min−1, P < 0.02, and 51.8 ± 5.0 vs. 14.6 ± 7.4%, P < 0.05, respectively). Renal nerve stimulation did not alter the glomerular filtration rate or renal blood flow. Renal nerve stimulation augments the stimulatory effect of intraluminal angiotensin II. The sympathetic renal nerves modulate the proximal tubule renin-angiotensin system and thereby regulate proximal tubule transport.
Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells
Authors: Kwon Osun , Phillips Carrie L. , Molitoris Bruce A.
Published: March 2015

Although altered renal vascular reactivity is known to occur after ischemia, the structural basis explaining the phenomenon has not been clarified. To evaluate for structural damage to the renal vasculature in ischemic acute renal failure (ARF), F-actin in the renal vasculature of rat kidneys and cultured vascular smooth muscle cells was examined using confocal fluorescence microscopy. The left renal artery was clamped for 15 or 45 min in Sprague-Dawley rats. In other experimental groups, 45 min of renal arterial clamping was followed by 1 or 3 h of reperfusion. Control kidneys were procured without any preceding interventional procedure. F-actin was labeled with either fluorescein or Texas red-conjugated phalloidin. Serial optical sections were collected by confocal microscopy, and image volumes were rendered three dimensionally. The degree of cytoskeletal damage in the vasculature was assessed by semiquantitative scoring of the staining for F-actin. Disorganization/disarray of F-actin, reflected by disruption and clumping of the actin filaments, was observed in arteries, arterioles, and the vasa rectae of the kidney after ischemia or ischemia-reperfusion. Smooth muscle cells from arteries and arterioles showed significant damage to F-actin after either 15 or 45 min of ischemia in a duration-dependent manner. The actin cytoskeleton tended to recover from damage from 45 min of ischemia 1 and 3 h after reperfusion. The vasa rectae did not demonstrate significant damage to F-actin after 15- or 45-min ischemia. However, significant damage to the vasa rectae was manifest 3 h after the reperfusion following 45 min of ischemia. In summary, disorganization/disarray of F-actin in vascular smooth muscle cells of the kidney was observed after ischemia or ischemia-reperfusion. A similar finding was observed in cultured vascular smooth muscle cells. We suggest that this disorganization of the actin cytoskeleton may play a contributory role in the loss of autoregulation of renal blood flow and the aberrant vascular reactivity in postischemic ARF.
Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms
Authors: Kanakiriya Sharan K. R. , Croatt Anthony J. , Haggard Jill J. , Ingelfinger Julie R. , Tang Shiow-Shih , Alam Jawed , Nath Karl A.
Published: March 2015

This study examined the effect of hemin on the expression of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalized rat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose- and time-dependent induction of HO-1 and MCP-1 mRNA. HO activity contributed to MCP-1 mRNA expression at early time points (4–6 h) because inhibition of HO activity by zinc protoporphyrin (ZnPP) prevented hemin-induced expression of MCP-1 mRNA. Catalytically active intracellular iron was markedly increased in hemin-treated IRPTCs and contributed to the induction of HO-1 and MCP-1 mRNA because an iron chelator blocked hemin-induced upregulation of both genes, whereas a cell-permeant form of iron directly induced these genes. N-acetylcysteine completely blocked hemin-induced expression of HO-1 and MCP-1 mRNA, thereby providing added evidence for redox regulation of expression of these genes. The redox-sensitive transcription factor NF-κB was recruited in hemin-induced upregulation of MCP-1 because two different compounds that abrogate the activation of NF-κB (TPCK and BAY 11-7082) completely blocked hemin-induced upregulation of MCP-1 mRNA. In contrast to this HO-mediated induction of MCP-1 through redox-sensitive, iron-dependent, and NF-κB-involved pathways observed after 4–6 h, hemin also elicited a delayed induction of MCP-1 at 18 h through HO-independent pathways. We conclude that hemin is a potent inducer of MCP-1 in IRPTCs: HO-dependent, heme-degrading pathways lead to an early, robust, and self-remitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1.