bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic metabolic acidosis upregulates rat kidney Na-HCO 3 − cotransporters NBCn1 and NBC3 but not NBC1
Authors: Kwon Tae-Hwan , Fulton Christiaan , Wang Weidong , Kurtz Ira , Frøkiær Jørgen , Aalkjær Christian , Nielsen Søren
Published: March 2015

Several members of the Na-HCO[Formula: see text] cotransporter (NBC) family have recently been identified functionally and partly characterized, including rkNBC1, NBCn1, and NBC3. Regulation of these NBCs may play a role in the maintenance of intracellular pH and in the regulation of renal acid-base balance. However, it is unknown whether the expressions of these NBCs are regulated in response to changes in acid-base status. We therefore tested whether chronic metabolic acidosis (CMA) affects the abundance of these NBCs in kidneys using two conventional protocols. In protocol 1, rats were treated with NH4Cl in their drinking water (12 ± 1 mmol · rat−1· day−1) for 2 wk with free access to water ( n = 8). Semiquantitative immunoblotting demonstrated that whole kidney abundance of NBCn1 and NBC3 in rats with CMA was dramatically increased to 995 ± 87 and 224 ± 35%, respectively, of control levels ( P < 0.05), whereas whole kidney rkNBC1 was unchanged (88 ± 14%). In protocol 2, rats were given NH4Cl in their food (10 ± 1 mmol · rat−1· day−1) for 7 days, with a fixed daily water intake ( n = 6). Consistent with protocol 1, whole kidney abundances of NBCn1 (262 ± 42%) and NBC3 (160 ± 31%) were significantly increased compared with controls ( n = 6), whereas whole kidney rkNBC1 was unchanged (84 ± 17%). In both protocols, immunocytochemistry confirmed upregulation of NBCn1 and NBC3 with no change in the segmental distribution along the nephron. Consistent with the increase in NBCn1, measurements of pH transients in medullary thick ascending limb (mTAL) cells in kidney slices revealed two- to threefold increases in DIDS- sensitive, Na+-dependent HCO[Formula: see text] uptake in rats with CMA. In conclusion, CMA is associated with a marked increase in the abundance of NBCn1 in the mTAL and NBC3 in intercalated cells, whereas the abundance of NBC1 in the proximal tubule was not altered. The increased abundance of NBCn1 may play a role in the reabsorption of NH[Formula: see text] in the mTAL and increased NBC3 in reabsorbing HCO[Formula: see text].
Mouse TonEBP-NFAT5: expression in early development and alternative splicing
Authors: Maouyo Djikolngar , Kim Jee Y. , Lee Sang D. , Wu Yanhong , Woo Seung K. , Kwon Hyug M.
Published: March 2015

Tonicity-responsive enhancer binding protein (TonEBP)- nuclear factor of activated T cell family 5 is a DNA binding protein that plays a key role in the response of cells to hypertonicity. However, TonEBP is expressed and active in tissues that are in an isotonic milieu. To explore the biological role of TonEBP, we cloned mouse TonEBP that shares 92% of amino acids with the human counterpart. TonEBP is expressed in embryonic stem cells and throughout the stages of fetal development. Immunohistochemical analysis shows expression of TonEBP in most, if not all, developing tissues, including the brain, colon, heart, muscle, and eyes. Widespread alternative splicing in exons 2–4 was detected throughout development and in different adult tissues. As a result, four different polypeptides are produced with different lengths at the NH2terminus. Two of the isoforms differ in their ability to stimulate transcription. In conclusion, the presence of TonEBP mRNA during mouse embryogenesis suggests that TonEBP functions at all stages of mouse development, as well as in isotonic adult tissues.
Antigen retrieval reveals widespread basolateral expression of syntaxin 3 in renal epithelia
Authors: Breton Sylvie , Inoue Takeaki , Knepper Mark A. , Brown Dennis
Published: March 2015

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a key role in docking and fusion of intracellular transport vesicles and may regulate apical and basolateral membrane protein delivery in epithelial cells. In a previous study, syntaxin 3 (a target SNARE) protein was detectable in the kidney only in intercalated cells. We now report a more widespread distribution of syntaxin 3 in a variety of renal epithelial cells after antigen retrieval. Sections of rat kidney were treated with SDS and incubated with antisyntaxin 3 antibodies. Strong basolateral membrane staining was seen in descending and ascending thin limbs of Henle, thick ascending limbs of Henle, the macula densa, distal and connecting tubules, and all cells of the collecting duct including A- and B-intercalated cells. The papillary surface epithelium and the transitional epithelium of the ureter were also stained, but proximal tubules were negative. Western blotting revealed a strong signal at 37 kDa in all regions, and the antigen was restricted to membrane fractions. SDS treatment was not necessary to reveal syntaxin 3 in intercalated cells. These data show that syntaxin 3 might be involved in basolateral trafficking pathways in most renal epithelial cell types. The exclusive basolateral location of syntaxin 3 in situ, however, contrasts with the apical location of this SNARE protein in some kidney epithelial cells in culture.
ERK mediates inhibition of Na+/H+exchange and HCO 3 − absorption by nerve growth factor in MTAL
Authors: Watts Bruns A. , Good David W.
Published: March 2015

Mitogen-activated protein (MAP) kinases mediate a variety of critical cellular events, but their role in the regulation of epithelial transport is largely undefined. Recently, we demonstrated that nerve growth factor (NGF) inhibits HCO[Formula: see text] absorption in the rat medullary thick ascending limb (MTAL) through an unusual mechanism: 1) NGF inhibits basolateral membrane Na+/H+exchange activity, an effect opposite to the stimulation of Na+/H+exchange by growth factors in other cells; and 2) inhibition of basolateral Na+/H+exchange results secondarily in inhibition of apical Na+/H+exchange, thereby inhibiting HCO[Formula: see text] absorption. In this study, we examined the role of MAP kinases in mediating inhibition by NGF. In tissue strips from the inner stripe of the outer medulla and in microdissected MTALs, NGF increased extracellular signal-regulated kinase (ERK) activity twofold but had no effect on c-Jun NH2-terminal kinase (JNK) or p38 MAP kinase activity. The selective MAP kinase kinase (MEK1/2) inhibitors U0126 and PD-98059 abolished the NGF-induced ERK activation and largely eliminated (≥60%) the effects of NGF to inhibit basolateral Na+/H+exchange activity and transepithelial HCO[Formula: see text] absorption in perfused MTALs. The MEK1/2 inhibitors did not affect inhibition of HCO[Formula: see text]absorption by bath ethylisopropyl amiloride, indicating that ERK activation is not involved in mediating interaction between the basolateral and apical Na+/H+exchangers. These results demonstrate that NGF inhibits basolateral Na+/H+exchange activity and HCO[Formula: see text] absorption in the MTAL through activation of the ERK signaling pathway. These findings identify a novel action of ERK to inhibit Na+/H+exchange activity and establish a role for MAP kinase pathways in the acute regulation of Na+/H+exchange activity and transepithelial acid secretion in renal tubules.
Age dependency of renal function in CD-1 mice
Authors: Luippold Gerd , Pech Bärbel , Schneider Swetlana , Osswald Hartmut , Mühlbauer Bernd
Published: March 2015

Renal function was studied in mice of different ages. In metabolic cage experiments, the renal electrolyte excretion was similar in young ( n = 8; 5- to 7-wk-old) and adult ( n = 6; 20- to 22-wk-old) CD-1 (ICR) BR mice, whereas spontaneous drinking volume and urinary flow rate were significantly higher in the adult compared with the young mice. Subsequently, the renal functional reserve was investigated by amino acid (AA) infusion (10%) in anesthetized young ( n = 8) and adult ( n= 6) mice. Because the body weight of adult mice was significantly higher than that of young animals, one group of adult mice ( n = 8) received 12.5% AA to ensure that the dose of AA related to body weight was similar in both groups. Young animals constantly infused with Ringer solution served as time controls ( n = 8). Glomerular filtration rate (GFR) at baseline was similar in each group. Because of AA, GFR significantly increased in young mice but not in both groups of adult animals, whereas in time controls GFR remained constant. Urinary flow rate and sodium excretion were elevated by AA in young and adult mice. We conclude that in CD-1 mice the first signs of age-related changes in kidney function concern alterations in renal hemodynamics, whereas renal tubular function appears to be preserved.
Responses of proximal tubule sodium transporters to acute injury-induced hypertension
Authors: Yang Li E. , Leong Patrick K. K. , Ye Shaohua , Campese Vito M. , McDonough Alicia A.
Published: March 2015

Renal injury-induced by phenol injection activates renal sympathetic afferent pathways, increases norepinephrine release from the posterior hypothalamus, activates renal efferent pathways, and provokes a rapid and persistent hypertension. This study aimed to determine whether phenol injury provoked a redistribution of proximal Na+transporters from internal stores to the apical cell surface mediated by sympathetic activation, a response that could contribute to generation or maintenance of hypertension. Anesthetized rats were cannulated for arterial blood pressure tracing and saline infusion and then 50 μl 10% phenol or saline was injected into one renal cortex ( n = 7 each). Fifty minutes after injection, kidneys were removed and renal cortex membranes from injected kidneys were fractionated on sorbitol gradients and pooled into three windows (WI–WIII) that contained enriched apical brush border (WI); mixed apical, intermicrovillar cleft and dense apical tubules (WII); and intracellular membranes (WIII). Na+transporter distributions were determined by immunoblot and expressed as percentage of total in gradient. Acute phenol injury increased blood pressure 20–30 mmHg and led to redistribution of Na+/H+exchanger type 3 (NHE3) out of WIII (from 22.79 ± 4.75 to 10.79 ± 2.01% of total) to WI (13.07 ± 1.97 to 27.15 ± 4.08%), Na+-Picotransporter 2 out of WII (68.72 ± 1.95 to 59.76 ± 2.21%) into WI (9.5 ± 1.62 to 18.7 ± 1.45%), and a similar realignment of dipeptidyl-peptidase IV immunoreactivity and alkaline phosphatase activity to WI. Renal denervation before phenol injection prevented the NHE3 redistribution. By confocal microscopy, NHE3 localized to the brush border after phenol injection. The results indicate that phenol injury provokes redistribution of Na+transporters from intermicrovillar cleft/intracellular membrane pools to apical membranes associated with sympathetic nervous system activation, which may contribute to phenol injury-induced hypertension.
Advective transport of nitric oxide in a mathematical model of the afferent arteriole
Authors: Smith Kayne M. , Moore Leon C. , Layton Harold E.
Published: March 2015

Endothelium-derived nitric oxide (NO) is thought to be short-lived in blood because of rapid removal from plasma, mainly by binding to Hb. The extent to which removal limits NO advection is unclear, especially for blood flow in the renal afferent arteriole (AA), which has a transit time of 3–30 ms. A mathematical model of AA fluid dynamics and myogenic response that includes NO diffusion, advection, degradation, and vasorelaxant action was used to estimate NO advective transport. Model simulations indicate that advective transport of locally produced NO is sufficient to yield physiologically significant NO concentrations along much of the AA. Advective transport is insensitive to NO scavenging by Hb because the NO-Hb binding rate is slow relative to AA transit time. Hence, plasma NO concentration near the vessel wall is influenced by both diffusion from endothelial cells and advection from upstream sites. Simulations also suggest that NO advection may constitute a mechanism to stabilize arteriolar flow in response to a localized vasoconstriction accompanied by enhanced NO release.
Concerted action of ENaC, Nedd4–2, and Sgk1 in transepithelial Na+transport
Authors: Kamynina Elena , Staub Olivier
Published: March 2015

The epithelial Na+channel (ENaC), located in the apical membrane of renal aldosterone-responsive epithelia, plays an essential role in controlling the Na+balance of extracellular fluids and hence blood pressure. As of now, ENaC is the only Na+transport protein for which genetic evidence exists for its involvement in the genesis of both hypertension (Liddle's syndrome) and hypotension (pseudohypoaldosteronism type 1). The regulation of ENaC involves a variety of hormonal signals (aldosterone, vasopressin, insulin), but the molecular mechanisms behind this regulation are mostly unknown. Two regulatory proteins have gained interest in recent years: the ubiquitin-protein ligase neural precursor cell-expressed, developmentally downregulated gene 4 isoform Nedd4–2, which negatively controls ENaC cell surface expression, and serum glucocorticoid-inducible kinase 1 (Sgk1), which is an aldosterone- and insulin-dependent, positive regulator of ENaC density at the plasma membrane. Here, we summarize present ideas about Sgk1 and Nedd4–2 and the lines of experimental evidence, suggesting that they act sequentially in the regulatory pathways governed by aldosterone and insulin and regulate ENaC number at the plasma membrane.
Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose inXenopus laevisoocytes
Authors: Blasco Tatiana , Aramayona José J. , Alcalde Ana I. , Catalán Julia , Sarasa Manuel , Sorribas Victor
Published: March 2015

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and α-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.
Regulation of renal cortical cyclooxygenase-2 in young rats
Authors: Zhang Ming-Zhi , Wang Su-wan , Cheng Huifang , Zhang Yahua , McKanna James A. , Harris Raymond C.
Published: March 2015

Cyclooxygenase-2 (COX-2) is involved in kidney morphogenesis and is transiently elevated in the immature kidney. In adult rats, renal cortical COX-2 expression is tonically suppressed by mineralocorticoids (MC) and glucocorticoids (GC) and induced by chronic salt restriction. Young rats have low levels of GC and are in a state of relative volume depletion. The present study was designed to investigate the mechanisms underlying elevated cortical COX-2 expression in the immature kidney. Supplementation of GC or MC suppressed cortical COX-2 expression in suckling rats. GC suppression was significantly, but not completely, prevented by either an MC receptor antagonist or a GC receptor antagonist. MC suppression was completely prevented by a mineralocorticoid receptor antagonist. Salt supplementation suppressed cortical COX-2 expression in a dose- and time-dependent pattern in the suckling rats. Cortical COX-2 expression in the weanling rats was upregulated by a low-salt diet and downregulated by a high-salt diet. These results suggest that relative volume depletion and reduced GC levels are involved in elevated cortical COX-2 expression in the immature rodent kidney.
Injury in renal ischemia-reperfusion is independent from immunoglobulins and T lymphocytes
Authors: Park Pierce , Haas Mark , Cunningham Patrick N. , Bao Lihua , Alexander Jessy J. , Quigg Richard J.
Published: March 2015

Ischemia-reperfusion injury (IRI) is a complex and incompletely understood process involving a cascade of events that culminates in apoptotic and/or necrotic cell death. Natural IgM antibodies and complement have been implicated in the pathogenesis of IRI in a variety of organ systems as have T lymphocytes in renal IRI. To investigate the role of Ig and T lymphocytes in renal IRI, recombination-activating gene (RAG)-1-deficient mice were studied. RAG-1(−/−) mice were not protected from acute renal failure induced by 27.5 min of bilateral renal ischemia and subsequent reperfusion [serum urea nitrogen levels 30 h after reperfusion, 155.2 ± 5.6 and 152.8 ± 11.4 mg/dl in RAG-1(−/−) and wild-type mice, respectively; n = 13 each]. Histological examination showed acute tubular necrosis and neutrophilic infiltration with no significant differences between groups. In contrast with other organ systems, Igs were not found in kidneys at time points ranging from 1 min to 30 h after ischemia. Thus Igs and mature T lymphocytes do not appear to play a significant role in the pathogenesis of IRI in the kidney.
Partial bladder outlet obstruction alters Ca2+sensitivity of force, but not of MLC phosphorylation, in bladder smooth muscle
Authors: Stanton Michaela C. , Clement Michele , Macarak Edward J. , Zderic Stephen A. , Moreland Robert S.
Published: March 2015

Partial bladder outlet obstruction in the rabbit produces changes in bladder function similar to those seen clinically in patients with obstructive uropathies. Whole organ function is significantly altered, as are the smooth muscle cells inside the bladder wall. This study was designed to determine whether outlet obstruction alters smooth muscle function at the level of contractile filaments. Rabbit bladders were partially obstructed for 2 wk. Triton X-100 was used to provide a detergent-skinned bladder smooth muscle preparation that would allow control of the intracellular environment while the ability to shorten and develop force is maintained. Ca2+-force and Ca2+-myosin light chain (MLC) phosphorylation relations and maximal velocity of shortening were determined. The Ca2+sensitivity of force was significantly lower in tissues from animals subjected to outlet obstruction compared with tissues from control animals. In contrast, no difference was noted in the Ca2+sensitivity of MLC phosphorylation. Maximal levels of stress and MLC phosphorylation were similar in both animal groups. Maximal velocity of shortening was significantly slower in tissues from outlet-obstructed animals at all Ca2+concentrations compared with tissues from control animals. Ultrastructurally, detergent skinning had little effect on structural integrity. Moreover, tissues from obstructed animals showed an increase in the number of sarcolemmal attachment plaque structures. We suggest that partial bladder outlet obstruction produces deleterious (e.g., decrease in Ca2+sensitivity of force) and compensatory (e.g., increase in membrane attachment plaques) changes in bladder smooth muscle cells.
Correction of age-related polyuria by dDAVP: molecular analysis of aquaporins and urea transporters
Authors: Combet Sophie , Geffroy Nancy , Berthonaud Véronique , Dick Bernhard , Teillet Laurent , Verbavatz Jean-Marc , Corman Bruno , Trinh-Trang-Tan Marie-Marcelle
Published: March 2015

Senescent female WAG/Rij rats exhibit polyuria without obvious renal disease or defects in vasopressin plasma level or V2receptor mRNA expression. Normalization of urine flow rate by 1-desamino-8-d-arginine vasopressin (dDAVP) was investigated in these animals. Long-term dDAVP infusion into 30-mo-old rats reduced urine flow rate and increased urine osmolality to levels comparable to those in control 10-mo-old rats. The maximal urine osmolality in aging rat kidney was, however, lower than that in adult kidney, despite supramaximal administration of dDAVP. This improvement involved increased inner medullary osmolality and urea sequestration. This may result from upregulation of UT-A1, the vasopressin-regulated urea transporter, in initial inner medullary collecting duct (IMCD), but not in terminal IMCD, where UT-A1 remained low. Expression of UT-A2, which contributes to medullary urea recycling, was greatly increased. Regulation of IMCD aquaporin (AQP)-2 (AQP2) expression by dDAVP differed between adult and senescent rats: the low AQP2 abundance in senescent rats was normalized by dDAVP infusion, which also improved targeting of the channel; in adult rats, AQP2 expression was unaltered, suggesting that IMCD AQP2 expression is not regulated by dDAVP directly. Increased AQP3 expression in senescent rats may also be involved in improved urine-concentrating capacity owing to higher basolateral water and urea reabsorption capacity.
Mild hyperuricemia induces glomerular hypertension in normal rats
Authors: Sánchez-Lozada Laura G. , Tapia Edilia , Avila-Casado Carmen , Soto Virgilia , Franco Martha , Santamaría José , Nakagawa Takahiko , Rodríguez-Iturbe Bernardo , Johnson Richard J. , Herrera-Acosta Jaime
Published: March 2015

Mildly hyperuricemic rats develop renin-dependent hypertension and interstitial renal disease. Hyperuricemia might also induce changes in glomerular hemodynamics. Micropuncture experiments under deep anesthesia were performed in Sprague-Dawley rats fed a low-salt diet (LS group), fed a low-salt diet and treated with oxonic acid (OA/LS group), and fed a low-salt diet and treated with oxonic acid + allopurinol (OA/LS/AP group) for 5 wk. The OA/LS group developed hyperuricemia and hypertension compared with the LS group: 3.1 ± 0.2 vs. 1.1 ± 0.2 mg/dl ( P < 0.01) and 143 ± 4 vs. 126 ± 2 mmHg ( P < 0.01). Hyperuricemic rats developed increased glomerular capillary pressure compared with the LS rats: 56.7 ± 1.2 vs. 51.9 ± 1.4 mmHg ( P < 0.05). Pre- and postglomerular resistances were not increased. Histology showed afferent arteriolar thickening with increased α-smooth muscle actin staining of the media. Allopurinol prevented hyperuricemia (1.14 ± 0.2 mg/dl), systemic (121.8 ± 2.8 mmHg) and glomerular hypertension (50.1 ± 0.8 mmHg), and arteriolopathy in oxonic acid-treated rats. Linear regression analysis showed that glomerular capillary pressure and arteriolar thickening correlated positively with serum uric acid and systolic blood pressure. Glomerular hypertension may be partially mediated by an abnormal vascular response to systemic hypertension due to arteriolopathy of the afferent arteriole.
The isolated polycystin-1 cytoplasmic COOH terminus prolongs ATP-stimulated Cl–conductance through increased Ca2+entry
Authors: Wildman Scott S. , Hooper Kimberly M. , Turner Clare M. , Sham James S. K. , Lakatta Edward G. , King Brian F. , Unwin Robert J. , Sutters Michael
Published: March 2015

The precise steps leading from mutation of the polycystic kidney disease ( PKD1) gene to the autosomal dominant polycystic kidney disease (ADPKD) phenotype remain to be established. Fluid accumulation is a requirement for cyst expansion in ADPKD, suggesting that abnormal fluid secretion into the cyst lumen might play a role in disease. In this study, we sought to establish a link between polycystin-1 (the PKD1 gene product) and ATP-stimulated Cl–secretion in renal tubule cells. To do this, we performed a whole cell patch-clamp analysis of the effects of expression of the isolated cytoplasmic COOH-terminus of polycystin-1 in stably transfected mouse cortical collecting duct cells. The truncated polycystin-1 fusion protein prolonged the duration of ATP-stimulated Cl–conductance and intracellular Ca2+responses. Both effects were dependent on extracellular Ca2+. It was determined that expression of the truncated polycystin-1 fusion protein introduced, or activated, an ATP-induced Ca2+entry pathway that was undetectable in transfection control cell lines. Our findings are concordant with increasing evidence for a role of polycystin-1 in cell Ca2+homeostasis and indicate that dysregulated Ca2+entry might promote Cl–secretion and cyst expansion in ADPKD.
Central role for Rho in TGF-β1-induced α-smooth muscle actin expression during epithelial-mesenchymal transition
Authors: Masszi András , Di Ciano Caterina , Sirokmány Gábor , Arthur William T. , Rotstein Ori D. , Wang Jiaxu , McCulloch Christopher A. G. , Rosivall László , Mucsi István , Kapus András
Published: March 2015

New research suggests that, during tubulointerstitial fibrosis, α-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-β1(TGF-β1) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-β1-induced EMT in LLC-PK1cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-β1treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, β-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-β1induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-β1-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG (CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-β1-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ROK pathway, whereas the transcriptional effects are partially ROK independent.
NO inhibits Na+-K+-2Cl−cotransport via a cytochromeP-450-dependent pathway in renal epithelial cells (MMDD1)
Authors: He Hao , Podymow Tiina , Zimpelmann Joseph , Burns Kevin D.
Published: March 2015

Nitric oxide (NO) exerts direct effects on nephron transport. We determined the effect of NO on Na+-K+-2Cl−cotransport in a cell line (MMDD1) with properties of macula densa. Na+-K+-2Cl−cotransport was measured as bumetanide-sensitive86Rb+uptake in the presence of ouabain. MMDD1 cells expressed mRNA for the neuronal isoform of nitric oxide synthase, as well as NKCC1 and NKCC2(B) isoforms of the Na+-K+-2Cl−cotransporter. Preincubation of cells with the NO donors sodium nitroprusside (SNP) or S-nitroso- N-acetylpenicillamine (SNAP) caused concentration-dependent inhibition of Na+-K+-2Cl−cotransport. Both apical and basolateral Na+-K+-2Cl−cotransport was inhibited by NO donors. SNP or SNAP had no significant effect on cellular levels of cGMP, cAMP, cytosolic calcium, or phosphorylation of ERK1 and ERK2. In contrast, the inhibitors of cytochrome P-450, 1-aminobenzotriazole (ABT; 10−3M) or ketoconazole (1.5 × 10−5M), completely reversed the inhibitory effect of SNAP on apical or basolateral Na+-K+-2Cl−cotransport [apical: control 1.18 ± 0.15 vs. SNAP (10−4M) 0.41 ± 0.05 pmol · mg−1· 5 min−1; P < 0.001; SNAP (10−4M) + ABT 1.32 ± 0.10 pmol · mg−1· 5 min−1; P = not significant vs. control; n = 5]. The cytochrome P-450 epoxyeicosatrienoic acid (EET) metabolite 14,15-EET (5 × 10−7M) inhibited both apical and basolateral cotransport, whereas 8,9-EET and 11,12-EET had no significant effect. Although 20-hydroxyeicosatetraenoic acid inhibited apical cotransport, the inhibitor of ω-hydroxylase activity HET0016 did not reverse SNAP-mediated inhibition of apical cotransport. These data indicate that NO inhibits apical and basolateral Na+-K+-2Cl−cotransport in MMDD1 cells. The results suggest that the inhibitory pathway is independent of cGMP and might involve stimulation of a cytochrome P-450-dependent pathway.
Glomerular heteroporous membrane modeling in third trimester and postpartum before and during amino acid infusion
Authors: Milne J. E. C. , Lindheimer M. D. , Davison J. M.
Published: March 2015

Human pregnancy is associated with substantial increments in glomerular filtration rate (GFR) and renal plasma flow (RPF). We have previously demonstrated that permselectivity to neutral dextrans is altered in pregnancy, theoretical analysis of the dextran sieving curves suggesting that elevated GFR is due to increased RPF and decreased glomerular oncotic pressure (πGC) with no evidence of increased transglomerular hydrostatic pressure difference (ΔP). These conclusions have been challenged, with claims that the rise in GFR is primarily a result of a decrement in πGC. With refined laboratory and infusion protocols, we have reexplored the determinants of ultrafiltration in a serial study of 11 healthy women in late pregnancy (LP) and 4 mo postpartum (PP), both in the baseline state and after increasing GFR and RPF by infusion of amino acids. Results were analyzed using two computer modeling programs. Increased GFR in LP (38%, P < 0.05) was due to a combination of elevated RPF (22%) and a decrement in πGCand associated with an increased ultrafiltration coefficient, without evidence of increased ΔP, and additional amino acid-provoked GFR increments ( P < 0.05) produced similar findings. In addition, refined methodology permitted collection of sufficient data on excreted large-radii dextrans (>60 Å) to better define the nondiscriminatory “shunt” pathway (ω0) and the standard deviation of pore size ( S) about the mean radius of the distribution. Thus it was possible to demonstrate that the physiological increase in total protein excretion in LP is associated with a prominent shunt and an upward shift in breadth of distribution of pore sizes. This ability to quantify ω0and S will now permit better evaluation of the pathophysiological changes in the glomerulus associated with pregnancy in women with renal disease and in gravidas developing preeclampsia.
Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B12
Authors: Birn Henrik , Willnow Thomas E. , Nielsen Rikke , Norden Anthony G. W. , Bönsch Christian , Moestrup Søren K. , Nexø Ebba , Christensen Erik I.
Published: March 2015

Megalin has previously been shown to bind and mediate endocytosis of transcobalamin (TC)-B12. However, the physiological significance of this has not been established, and other TC-B12binding proteins have been suggested to mediate renal uptake of this vitamin complex. The present study demonstrates by the use of megalin-deficient mice that megalin is, in fact, essential for the normal renal reabsorption of TC-vitamin B12and for renal accumulation of this highly conserved vitamin. Megalin-deficient mice excrete increased amounts of TC and B12in the urine, revealing a defective renal tubular uptake of TC-B12. The urinary B12excretion is increased ∼4-fold, resulting in an ∼28-fold higher renal B12clearance. This is associated with an ∼4-fold decrease in B12content in megalin-deficient kidney cortex. Thus megalin is important to prevent urinary loss of vitamin B12. In addition, light- and electron-microscopic immunocytochemistry demonstrate lysosomal accumulation of B12in rat and mouse proximal tubules. In rats this accumulation is correlated with vitamin intake. Thus renal lysosomal B12accumulation is dependent on vitamin status, indicating a possible reserve function of this organelle in the rat kidney.
Expression of adenosine receptors in the preglomerular microcirculation
Authors: Jackson Edwin K. , Zhu Chongxue , Tofovic Stevan P.
Published: March 2015

The purpose of this study was to systematically investigate the abundance of each of the adenosine receptor subtypes in the preglomerular microcirculation vs. other vascular segments and vs. the renal cortex and medulla. Rat preglomerular microvessels (PGMVs) were isolated by iron oxide loading followed by magnetic separation. For comparison, mesenteric microvessels, segments of the aorta (thoracic, middle abdominal, and lower abdominal), renal cortex, and renal medulla were obtained by dissection. Adenosine receptor protein and mRNA expression were examined by Western blotting, Northern blotting, and RT-PCR. Our results indicate that compared with other vascular segments and renal tissues, A1 and A2B receptor protein and mRNA are abundantly expressed in the preglomerular microcirculation, whereas A2A and A3 receptor protein and mRNA are barely detectable or undetectable in PGMVs. We conclude that, relative to other vascular and renal tissues, A1 and A2Breceptors are well expressed in PGMVs, whereas A2A and A3 receptors are notably deficient. Thus A1 and A2B receptors, but not A2A or A3receptors, may importantly regulate the preglomerular microcirculation.
PAR-2 elicits afferent arteriolar vasodilation by NO-dependent and NO-independent actions
Authors: Trottier Greg , Hollenberg Morley , Wang Xuemei , Gui Yu , Loutzenhiser Kathy , Loutzenhiser Rodger
Published: March 2015

Proteinase-activated receptors (PARs) are a novel class of G protein-coupled receptors that respond to signals through endogenous proteinases. PAR activation involves enzymatic cleavage of the extracellular NH2-terminal domain and unmasking of a new NH2terminus, which serves as an anchored ligand to activate the receptor. At least four PAR subtypes have been identified. In the present study, we used the in vitro perfused hydronephrotic rat kidney to examine the effects of activating PAR-2 on the afferent arteriole. The synthetic peptide SLIGRL-NH2, which corresponds to the exposed ligand sequence and selectively activates PAR-2, did not alter basal afferent arteriolar diameter but caused a concentration-dependent vasodilation (3–30 μM) of arterioles preconstricted by angiotensin II (0.1 nM). A modified peptide sequence (LSIGRL-NH2, inactive at PAR-2) had no effect. This vasodilation was characterized by an initial transient component followed by a smaller sustained response. A similar pattern of vasodilation was seen when SLIGRL-NH2was administered to isolated perfused normal rat kidney. The sustained component of the PAR-2-induced afferent arteriolar vasodilation was eliminated by nitric oxide (NO) synthase inhibition (100 μM nitro-l-arginine methyl ester). In contrast, the transient vasodilation persisted under these conditions. This transient response was not observed when afferent arterioles were preconstricted with elevated KCl, suggesting involvement of an endothelium-derived hyperpolarizing factor. Finally, RT-PCR revealed the presence of PAR-2 mRNA in isolated afferent arterioles. These findings indicate that PAR-2 is expressed in the afferent arteriole and that its activation elicits afferent arteriolar vasodilation by NO-dependent and NO-independent mechanisms.
Differential, inducible gene targeting in renal epithelia, vascular endothelium, and viscera of Mx1Cre mice
Authors: Schneider André , Zhang Yahua , Guan Youfei , Davis Linda S. , Breyer Matthew D.
Published: March 2015

The Cre/loxP transgenic system may be used to achieve temporally and/or spatially regulated gene deletion. The Mx1Cremouse expresses Cre recombinase under control of the IFN-inducible Mx1 promoter. Mx1Cre mice were crossed with a reporter strain (ROSA26tm1Sor) in which β-galactosidase activity is expressed only after Cre-mediated recombination to determine the cellular pattern of Cre-mediated genetic recombination in the kidney and other tissues. Widespread recombination was observed in vascular endothelium as well as in the liver and spleen. Recombination was restricted to subsets of stromal cells in uterus, duodenum, colon, aorta, and kidney. In the cortex, χ-galactosidase activity was detected in a subset of tubules and all glomerular cells, including endothelium, mesangium, and podocytes. No χ-galactosidase activity was detected in proximal tubules. Costaining of kidneys with segment-specific markers demonstrated induction of χ-galactosidase activity in collecting duct, with sporadic labeling of the thick ascending limb but no significant labeling of distal convoluted tubules. We conclude that Mx1-driven gene recombination is spatially as well as temporally restricted. The Mx1Cretransgene should prove a useful reagent to achieve temporally regulated recombination in endothelial, glomerular, and distal renal epithelia in mice.
Properties of an inwardly rectifying K+ channel in the basolateral membrane of mouse TAL
Authors: Paulais Marc , Lourdel Stéphane , Teulon Jacques
Published: March 2015

We investigated the properties of K+ channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K+ channel (K+-to-Na+permeability ratio ∼22), having an inward conductance ( G in) of 44 pS and an outward conductance ( G out) of ∼10 pS ( G in/ G out ∼ 4). Channel activity ( NP o) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg2+-free medium, the channel had a G in of 50 pS and was weakly inwardly rectifying ( G in/ G out ∼ 1). Cytosolic Mg2+ reduced G out, yielding a G in/ G out of 3.8 at 1.3 mM Mg2+. Internal Na+ also yielded a G in/ G out of 1.6 at 20 mM Na+. Spermine reduced NP o on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration ( K i) = 0.2 μM] was much greater than at hyperpolarizing voltages ( K i = 26 μM). Half-inactivation by 0.5 μM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NP o of inside-out membrane patches was observed, with a p K of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NP o of cell-attached patches. This channel is probably a major component of K+ conductance in the CTAL basolateral membrane.
Local electric stimulation causes conducted calcium response in rat interlobular arteries
Authors: Salomonsson Max , Gustafsson Finn , Andreasen Ditte , Jensen Boye L. , Holstein-Rathlou Niels-Henrik
Published: March 2015

The purpose of the present study was to investigate the conducted Ca2+response to local electrical stimulation in isolated rat interlobular arteries. Interlobular arteries were isolated from young Sprague-Dawley rats, loaded with fura 2, and attached to pipettes in a chamber on an inverted microscope. Local electrical pulse stimulation (200 ms, 100 V) was administered by means of an NaCl-filled microelectrode (0.7–1 MΩ) juxtaposed to one end of the vessel. Intracellular Ca2+concentration ([Ca2+]i) was measured with an image system at a site ∼500 μm from the location of the electrode. The expression of mRNA for pore-forming units CaV3.1 and CaV3.2 of voltage-sensitive T-type channels was investigated by using RT-PCR. Current stimulation elicited a conducted [Ca2+]iresponse. A positive electrode (relative to ground) increased [Ca2+]ito 145 ± 7% of baseline, whereas the response was absent when the electrode was negative. This response was not dependent on perivascular nerves, because the conducted response was unaffected by TTX (1 μM). The conducted [Ca2+]iresponse was abolished by an ambient Ca2+free solution and blunted by nifedipine (1 μM). Rat interlobular arteries exhibited conducted [Ca2+]iresponse to current stimulation. This response was dependent on Ca2+entry. L-type Ca2+channels may play a role in this process.
Regulation of the voltage-gated K+ channel KCNA10 by KCNA4B, a novel β-subunit
Authors: Tian Shulan , Liu Weimin , Wu Yanling , Rafi Hamid , Segal Alan S. , Desir Gary V.
Published: March 2015

Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming α-subunits and accessory β-subunits. Several β-subunits have been identified and shown to interact with specific α-subunits to modify their levels of expression or some of their kinetic properties. The aim of the present study was to isolate accessory proteins for KCNA10, a novel Kv channel α-subunit functionally related to Kv and cyclic nucleotide-gated cation channels. Because one distinguishing feature of KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by coinjection with KCNA10 in Xenopus oocytes. One of these clones (KCNA4B), when injected alone in oocytes, produced no detectable current. However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold. In addition, the current became more sensitive to activation by cAMP. KCNA4B can be coimmunoprecipitated with the COOH terminus of KCNA10 and full-length KCNA10. It encodes a soluble protein (141 aa) with no amino acid homology to known β-subunits but with limited structural similarity to the NAD(P)H-dependent oxidoreductase superfamily. KCNA4Bis located on chromosome 13 and spans ∼16 kb, and its coding region is made up of five exons. In conclusion, KCNA4B represents the first member of a new class of accessory proteins that modify the properties of Kv channels.
Natriuretic peptides and acute renal failure
Authors: Vesely David L.
Published: March 2015

Atrial natriuretic peptides (ANPs) are a family of peptide hormones, e.g., ANP, long-acting natriuretic peptide, vessel dilator, and kaliuretic peptide synthesized by the ANP gene. Brain natriuretic peptide (BNP) and C-type natriuretic peptide are also members of this family but are synthesized by separate genes. Within the kidney, the ANP prohormone's posttranslational processing is different from that of other tissues, resulting in an additional four amino acids added to the NH2terminus of ANP (e.g., urodilatin). Each of these natriuretic and diuretic peptides increases within the circulation with acute renal failure (ARF). Renal transplantation but not hemodialysis returns their circulating concentrations to those of healthy individuals. BNP and adrenomedullin, a 52-amino acid natriuretic peptide, have beneficial effects on glomerular hypertrophy and glomerular injury but do not improve tubular injury (i.e., acute tubular necrosis). Vessel dilator ameliorates acute tubular necrosis with regeneration of the brush borders of proximal tubules. Vessel dilator decreases mortality in ARF from 88 to 14% at day 6 of ARF, even when given 2 days after renal failure has been established.
Gene structure of urea transporters
Authors: Bagnasco Serena M.
Published: March 2015

Urea plays various roles in the biology of diverse organisms. The past decade has produced new information on the molecular structure of several urea transporters in various species. Availability of DNA probes has revealed that the presence of urea transporters is not confined to the mammalian kidney but is also evident in testis and brain, raising new questions about the possible physiological role of urea in these organs. Cloning of the genes encoding the two closely related mammalian urea transporters UT-A and UT-B has helped in identifying molecular mechanisms affecting expression of urea transporters in the kidney, such as transcriptional control for UT-A abundance. On the basis of analysis of genomic sequences of individuals lacking the UT-B transporter, mutations have been found that explain deficits in their capacity to concentrate urine. More urea transporters are being characterized in marine organisms and lower vertebrates, and studying the role and regulation of urea transport from an evolutionary perspective can certainly enrich our understanding of renal physiology.
Role of PKC and calcium in modulation of effects of angiotensin II on sodium transport in proximal tubule
Authors: Du Zhaopeng , Ferguson William , Wang Tong
Published: March 2015

It has been well documented that low concentrations of ANG II (10−11 to 10−10 M) stimulate, whereas high concentrations of ANG II (10−8 to 10−5 M) inhibit Na+transport in proximal tubules of rat and rabbit kidneys. Measured ANG II concentration in proximal tubular fluid is in the nanomolar range. In the present study, we investigated the role of PKC, intracellular Ca2+, and cAMP in modulating the effects of luminal ANG II on Na+ absorption by microperfusion techniques in rabbit superficial segment of proximal tubules in vitro. We confirmed that ANG II (10−9 M) had no change on fluid absorption ( J v); however, fluid absorption increased significantly when 10−9 M ANG II and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular calcium mobilization, were added together. In contrast, ANG II significantly decreased J v when PKC was inhibited. When 10−9 M ANG II was present together with 1-(5-isoquinolinesulfonyl)-2-mehtylpiperazine and TMB-8, no significant change of J v occurred. Inhibition of endogenous cAMP activity by a PKA inhibitor did not change either basal or ANG II-stimulated fluid absorption. Our results indicate that ANG II regulates Na+ absorption by a cAMP-independent mechanism and that PKC and intracellular calcium both play a critical role in modulating the effects of physiological concentration of ANG II on proximal tubule transport. Balance between these two cytosolic messengers modulates the effects of ANG II on fluid absorption in the proximal tubule.
Structure and characterization of the mouse UT-A gene (Slc14a2)
Authors: Fenton R. A. , Cottingham C. A. , Stewart G. S. , Howorth A. , Hewitt J. A. , Smith C. P.
Published: March 2015

The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A ( Slc14a2) and UT-B ( Slc14a1). By genomic library screening and P1 artificial chromosome “shotgun” sequencing, we have determined the structure of the mouse UT-A gene. The gene is >300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5′-region of the gene is the UT-Aα promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-Aβ, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 μM dibutryl cAMP, 25 μM forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-Aα promoter construct 6.2-fold [from 0.026 ± 0.003 to 0.160 ± 0.004, relative light units (RLU)/μg protein] and a 2.4-kb UT-Aβ promoter construct 9.5-fold (from 0.020 ± 0.002 to 0.190 ± 0.043 RLU/μg protein) above that in untreated controls. Interestingly, only the UT-Aβ promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kgH2O with NaCl caused a significant increase (from 0.060 ± 0.004 to 0.095 ± 0.010 RLU/μg protein) in UT-Aα promoter activity but had no effect on the UT-Aβ promoter. A tonicity-responsive enhancer was identified in UT-Aα and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.