bluuebunny/crossref_metadata_2025 huggingface dataset:
Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats
Authors: Marples David , Christensen Birgitte Mønster , Frøkiaer Jørgen , Knepper Mark A. , Nielsen Søren
Published: December 2017

To examine the involvement of vasopressin and dehydration in the regulation of aquaporin-2 (AQP2) expression in rat kidney, we investigated the effects of treatment for 60 h with the specific V2-receptor antagonist OPC-31260 (OPC), alone and in conjunction with dehydration for the last 12 h. Changes in AQP2 protein and mRNA expression in kidney inner medulla were determined by Western and Northern blotting, and AQP2 distribution was analyzed by immunocytochemistry and immunoelectron microscopy. Treatment with OPC increased urine output fourfold, with a reciprocal decrease in urine osmolality. AQP2 expression decreased to 52 ± 11% of control levels ( n = 12, P < 0.05), and AQP2 was found predominantly in intracellular vesicles in collecting duct principal cells. This is consistent with efficient blockade of the vasopressin-induced AQP2 delivery to the plasma membrane and with the observed increased diuresis. Consistent with this, AQP2 mRNA levels were also reduced in response to prolonged OPC treatment (30 ± 10% of control levels, n = 9). Five days of treatment with furosemide, despite producing even greater polyuria than OPC, was not associated with downregulation of AQP2 levels, demonstrating that AQP2 downregulation is not secondary to increased urine flow rate or loss of medullary hypertonicity. During 12-h thirsting in the continued presence of OPC, urine output dropped dramatically, to levels not significantly different from that seen in (nonthirsted) control animals. In parallel with this, AQP2 levels rose to control levels. Control experiments confirmed continued effective receptor blockade. These results indicate that the V2-receptor antagonist causes a modest decrease in AQP2 expression that is not a consequence of increased urine flow rate or washout of medullary hypertonicity. However, this decrease is much less marked than that seen in some forms of acquired nephrogenic diabetes insipidus. In conjunction with the effects of thirsting, this suggests that modulation of AQP2 expression is mediated partly, but not exclusively, via V2receptors.
Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2)
Authors: MacGregor Gordon G. , Xu Jason Z. , McNicholas Carmel M. , Giebisch Gerhard , Hebert Steven C.
Published: December 2017

The activity of the cloned renal K+ channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313–9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability ( P o), whereas the NH2-terminal mutation at site S25A showed no change in P o compared with wild-type ROMK2. The decrease in P o for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was ∼66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.
Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-α, -δ, and -ε
Authors: Dlugosz John A. , Munk Snezana , Zhou Xiaopeng , Whiteside Catharine I.
Published: December 2017

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 ± 3%, 56 ± 3%, and 78 ± 2% of basal (means ± SE, n = 40–50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 ± 7 to 220 ± 30 nM or 97 ± 10 to 192 ± 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 ± 3% and 48 ± 6% of time 0, respectively). Cells pretreated with 10 μM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-α, -δ, and -ε to 173 ± 30%, 162 ± 26%, and 166 ± 11% of basal ( P< 0.05 vs. basal), respectively, and decreased PKC-δ and PKC-ε in the cytosol to 56 ± 11% and 37 ± 6% of basal, respectively ( P < 0.05). ET-1 increased particulate PKC-δ and PKC-ε to 172 ± 15% and 187 ± 33% of basal ( P < 0.05), respectively. PKC-α in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-ζ was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-α, -δ, and -ε, but not PKC-ζ.
Reflex effects on components of synchronized renal sympathetic nerve activity
Authors: DiBona Gerald F. , Jones Susan Y.
Published: December 2017

The effects of peripheral thermal receptor stimulation (tail in hot water, n = 8, anesthetized) and cardiac baroreceptor stimulation (volume loading, n = 8, conscious) on components of synchronized renal sympathetic nerve activity (RSNA) were examined in rats. The peak height and peak frequency of synchronized RSNA were determined. The renal sympathoexcitatory response to peripheral thermal receptor stimulation was associated with an increase in the peak height. The renal sympathoinhibitory response to cardiac baroreceptor stimulation was associated with a decrease in the peak height. Although heart rate was significantly increased with peripheral thermal receptor stimulation and significantly decreased with cardiac baroreceptor stimulation, peak frequency was unchanged. As peak height reflects the number of active fibers, reflex increases and decreases in synchronized RSNA are mediated by parallel increases and decreases in the number of active renal nerve fibers rather than changes in the centrally based rhythm or peak frequency. The increase in the number of active renal nerve fibers produced by peripheral thermal receptor stimulation reflects the engagement of a unique group of silent renal sympathetic nerve fibers with a characteristic response pattern to stimulation of arterial baroreceptors, peripheral and central chemoreceptors, and peripheral thermal receptors.
Urea and NaCl differentially regulate FAK and RAFTK/PYK2 in mIMCD3 renal medullary cells
Authors: Zhang Zheng , Avraham Hava , Cohen David M.
Published: December 2017

Two cytosolic tyrosine kinases, focal adhesion kinase (FAK) and the newly described FAK homolog, related adhesion focal tyrosine kinase (RAFTK, also called PYK2 and CAKβ), have been implicated in signaling to multiple mitogen-activated protein kinase (MAPK) pathways. Therefore, the ability of NaCl and urea to activate these kinases was investigated by in vitro kinase assay and anti-phosphotyrosine immunoblotting. RAFTK was promptly but only transiently activated by urea (within 1 min; 45%), whereas NaCl activated this kinase at 1, 5, 15, and 30 min of treatment (35–60%). In contrast, FAK exhibited only subtle regulation by the two solutes; however, the time course of induction was distinct for each solute. NaCl activated FAK at 1, 5, and 15 min (25–40%), whereas urea-inducible FAK activation (30%) was not evident until fully 15 min of treatment. At 5 min of treatment with increasing concentrations of solute, both urea and NaCl activated RAFTK in a dose-dependent and comparable fashion, culminating in an approximately twofold activation at 800 mosmol/kgH2O solute. Consistent with these data, solute treatment also enhanced tyrosine phosphorylation of RAFTK.
Peptide YY inhibits vasopressin-stimulated chloride secretion in inner medullary collecting duct cells
Authors: Breen Christopher M. , Mannon Peter J. , Benjamin Bruce A.
Published: December 2017

mIMCD-k2 cells are derived from the inner medullary collecting duct of a mouse and exhibit electrogenic sodium absorption and cAMP- and vasopressin (AVP)-stimulated electrogenic chloride secretion [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 ( Renal Fluid Electrolyte Physiol. 37): F347–F355, 1995; and N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 ( Renal Fluid Electrolyte Physiol. 37): F854–F861, 1995]. The purpose of the present study was to determine how peptide YY (PYY) affects electrogenic Na+and Cl−current in mIMCD-k2 cells. Short-circuit currents ( Isc) were measured across monolayers of mIMCD-k2 cells mounted in Ussing-type chambers. PYY did not alter baseline Isc, nor did it alter Iscin chloride-free conditions, indicating no effect on electrogenic sodium transport. Baseline chloride current in these cells is low; therefore, chloride short-circuit current[Formula: see text] was stimulated with AVP (10 nM) added to the basolateral surface and 10 μM amiloride added to the apical surface. Although apical applications of PYY had no effect, basolateral application of PYY caused attenuation of[Formula: see text], with the maximal inhibitory dose (100 nM) causing 52 ± 1.3% inhibition (IC50= 0.11 nM). Inhibition by PYY of [Formula: see text] is mediated through the Y2receptor subtype, as PYY-(3–36) was the only PYY analog tested that caused inhibition and was equipotent to PYY. Inhibition by PYY of[Formula: see text] was abolished following incubation with pertussis toxin. We also show that PYY inhibits AVP-stimulated cAMP accumulation, with a maximal inhibitory dose (100 nM) causing a 38% ± 6% inhibition (IC50= 0.16 nM), comparable to inhibition by PYY of [Formula: see text]. We conclude that PYY acts through either Gior Goto inhibit adenylate cyclase activity, leading to a decrease in AVP-stimulated chloride current.
Hypertonicity activates MAP kinases and inhibits HCO 3 − absorption via distinct pathways in thick ascending limb
Authors: Watts Bruns A. , Di Mari John F. , Davis Roger J. , Good David W.
Published: December 2017

Mitogen-activated protein (MAP) kinases are activated by osmotic stress in a variety of cells, but their function and regulation in renal tubules is poorly understood. The present study was designed to examine the osmotic regulation of MAP kinases in the medullary thick ascending limb (MTAL) of the rat and to determine their possible role in the hyperosmotic inhibition of[Formula: see text] absorption in this segment. Tissues from the inner stripe of the outer medulla and microdissected MTALs were incubated at 37°C in control (290 mosmol/kgH2O) or hyperosmotic (300 mM added mannitol) solution for 15 min. Activities of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase were then measured using immune complex assays. Hyperosmolality increased p38 MAP kinase activity (2.3-fold) and ERK activity (2.0-fold) but had no effect on JNK activity (1.1-fold). Exposure to hyperosmolality for various times showed that the activation of p38 MAP kinase was rapid (≤5 min) and was sustained for up to 60 min, whereas the activation of ERK was transient (ERK activity peaked at 15 min, then declined to basal levels at 30 min). Pretreatment with the MAP kinase kinase inhibitor PD98059 (15 μM) blocked the hyperosmotic activation of p38 MAP kinase and ERK but did not prevent hyperosmotic inhibition of[Formula: see text] absorption. These results show that hyperosmolality differentially activates p38 MAP kinase and ERK in the MTAL. In contrast, we found no evidence for involvement of JNK in the early response to hyperosmotic stress. Eliminating the activation of p38 MAP kinase and ERK does not prevent hyperosmotic inhibition of [Formula: see text]absorption, suggesting that hyperosmolality inhibits apical membrane Na+/H+exchange (NHE3) activity via a signaling pathway distinct from these MAP kinase pathways.
Dietary K+ restriction upregulates total and Sch-28080-sensitive bicarbonate absorption in rat tIMCD
Authors: Wall Susan M. , Mehta Pramod , DuBose Thomas D.
Published: December 2017

In tubules from the terminal segment of the inner medullary collecting duct (tIMCD) from rats with chronic metabolic acidosis, our laboratory has shown that bicarbonate absorption ( J tCO2) is inhibited by removal of K+ from the luminal fluid or by the addition of Sch-28080 to the perfusate. The present study asked whether total and/or Sch-28080-sensitive J tCO2 is regulated by changes in systemic K+ homeostasis. Rat tIMCD tubules were perfused in vitro in symmetrical,[Formula: see text]/CO2-buffered solutions containing 10 mM KCl + 6 mM NH4Cl. Total and Sch-28080-sensitive J tCO2 were measured in rats with varying K+intake. In K+-replete rats, baseline J tCO2was 2.1 ± 0.3 pmol ⋅ mm−1 ⋅ min−1( n = 6). In rats fed a K+-deficient diet for 3 days, J tCO2 was 5.4 ± 0.7 pmol ⋅ mm−1 ⋅ min−1( n = 16, P < 0.05). To determine the mechanism for the increase in [Formula: see text]absorption observed with K+restriction, the Sch-28080-sensitive component of J tCO2 was measured in each treatment group. Following the addition of Sch-28080 (10 μM) to the perfusate, a 40% reduction in J tCO2 was observed in K+-restricted rats. J tCO2 was not reduced following the addition of Sch-28080 in rats with normal K+ intake. Because Sch-28080-sensitive J tCO2 was increased in K+-restricted rats, Sch-28080-sensitive J tCO2 was studied further in tIMCD tubules from rats in this treatment group. In K+-restricted rats, J tCO2 decreased by 20% following the addition of 5 mM ouabain to the perfusate. This ouabain-induced decline in J tCO2 was observed both in the presence and in the absence of Sch-28080. We conclude that total and Sch-28080-sensitive net acid secretion is increased with dietary K+restriction. However, since ∼50% of J tCO2 is insensitive to both Sch-28080 and ouabain, future studies will be necessary to define other mechanisms of luminal acidification in the rat tIMCD.
Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney andC. elegans
Authors: Holtzman Eli J. , Kumar Sumit , Faaland Carol A. , Warner Fern , Logue Paul J. , Erickson Sara J. , Ricken Gesa , Waldman Jeremy , Kumar Shiv , Dunham Philip B.
Published: December 2017

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.
Effects of antihypertensive drugs on autoregulation of RBF and glomerular capillary pressure in SHR
Authors: Kvam Fred Ivan , Ofstad Jarle , Iversen Bjarne M.
Published: December 2017

The relationship between systemic blood pressure and glomerular capillary pressure (Pgc) in spontaneously hypertensive rats (SHR) during treatment with antihypertensive drugs is still unclear. The effects of an angiotensin-converting enzyme inhibitor (enalapril), two calcium channel antagonists (nifedipine and verapamil), and an α1-receptor blocker (doxazosin) on renal blood flow (RBF) autoregulation, Pgc, and renal segmental resistances were therefore studied in SHR. Recordings of RBF autoregulation were done before and 30 min after intravenous infusion of the different drugs, and Pgcwas thereafter measured with the stop-flow technique. When the mean arterial pressure (MAP) was reduced to ∼120 mmHg by infusions of doxazosin or enalapril, the lower pressure limit of RBF autoregulation was reduced significantly. Nifedipine or verapamil abolished RBF autoregulation. Doxazosin did not change Pgc (43.6 ± 1.4 vs. 46.7 ± 1.5 mmHg in controls, P > 0.5), enalapril lowered (41.3 ± 0.8 mmHg, P < 0.01), and the calcium channel antagonists increased Pgc[53.7 ± 1.4 mmHg (nifedipine) and 54.8 ± 1.2 mmHg (verapamil), P < 0.01]. When MAP was reduced to ∼85 mmHg by drugs, Pgc was reduced to 43.3 ± 1.7 mmHg after nifedipine ( P > 0.2 vs. control), whereas Pgc after enalapril was 38.5 ± 0.5 mmHg ( P < 0.05 vs. control). Enalapril reduced Pgc mainly by reducing efferent resistance. During treatment with calcium channel antagonists, Pgc became strictly dependent on MAP. Monotherapy with nifedipine may increase Pgc and by this mechanism accelerate glomerulosclerosis if a strict blood pressure control is not obtained.
Selective increase of cyclooxygenase-2 expression in a model of renal ablation
Authors: Wang Jun-Ling , Cheng Hui-Fang , Zhang Ming-Zhi , McKanna James. A. , Harris Raymond C.
Published: December 2017

Previous studies have suggested a possible role for prostaglandins (PGs) in mediating alterations in nephron structure and function ensuing after renal ablation. Two isoforms of cyclooxygenase (COX) have been described: constitutive (COX-1) and inducible (COX-2). We examined expression of these isoforms following subtotal renal ablation (5/6 ablation, RA) in rats. In renal cortex, COX-2 mRNA and immunoreactive protein (IP) increased progressively compared with sham-operated littermates. In contrast, there were no significant changes in COX-1 mRNA expression. In normal kidney, cortical COX-1 IP was immunolocalized predominantly to mesangial cells and collecting tubules, whereas COX-2 IP was found in a subset of cortical thick ascending limb of Henle’s loop (CTAL) cells in the region of the macula densa (MD). Following RA, significantly increased COX-2 IP was detected in the MD and surrounding CTAL cells. In addition, fainter immunoreactive COX-2 was detected in scattered visceral epithelial cells and mesangial cells of the glomerulus. Immunoblotting of isolated glomeruli demonstrated a selective increase of glomerular immunoreactive COX-2 expression following RA. No change of COX-1 expression was seen. To determine COX activity, isolated glomeruli were incubated with arachidonic acid and PGE2measured by enzyme immunoassay (EIA). Compared with sham, glomeruli from 2 wk RA produced significantly more PGs. SC-58560, a selective COX-1 inhibitor, did not inhibit PG production in the remnant glomeruli at concentrations up to 10−4M, whereas SC-58236, a relatively selective COX-2 inhibitor, significantly inhibited PG production by RA glomeruli. In preliminary studies, to define mechanisms of altered expression of glomerular COX-2, rat mesangial cells were incubated with serum from sham or 2 wk RA. There were significant increases in COX-2 expression in response to 2 wk RA serum. In summary, these results indicate selective increases in renal cortical COX-2 expression following renal ablation.
Role of superoxide in apoptosis induced by growth factor withdrawal
Authors: Lieberthal Wilfred , Triaca Veronica , Koh Jason S. , Pagano Patrick J. , Levine Jerrold S.
Published: December 2017

We have examined the role of reactive oxygen species (ROS) in apoptosis induced by growth factor deprivation in primary cultures of mouse proximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose insulin directly inhibit apoptosis of MPT cells deprived of growth factors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by growth factor withdrawal is inhibited by a number of antioxidants and scavengers of ROS. Growth factor deprivation also results in activation of caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis. The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apoptosis induced by growth factor deprivation. However, zVAD-fmk had no effect on the increased levels of superoxide associated with growth factor deprivation. Thus we provide novel evidence that ROS play an important role in mediating apoptosis associated with growth factor deprivation. ROS appear to act upstream of caspases in the apoptotic pathway. We hypothesize that oxidant stress, induced by growth factor withdrawal, represents a signaling mechanism for the default pathway of apoptosis.
Reduced AQP1, -2, and -3 levels in kidneys of rats with CRF induced by surgical reduction in renal mass
Authors: Kwon Tae-Hwan , Frøkiaer Jørgen , Knepper Mark A. , Nielsen Søren
Published: December 2017

Urinary concentration characteristically decreases in response to a reduction in renal mass in chronic renal failure (CRF). In the present study, we examined whether there are changes in the expression of aquaporins in rats where CRF was induced by 5/6 nephrectomy. Plasma creatinine levels were significantly elevated consistent with significant CRF: 135.7 ± 15.1 ( n = 17, CRF) vs. 33.9 ± 1.1 μmol/l ( n = 11, sham), P < 0.05. Two weeks after 5/6 nephrectomy, the remnant kidneys were hypertrophied, and total renal mass increased to 65 ± 3% of sham levels ( P < 0.05). Urine production increased markedly from 40 ± 2 to 111 ± 3 μl ⋅ min−1⋅ kg−1in CRF rats ( P < 0.05), whereas urine osmolality and solute-free water reabsorption decreased significantly. Quantitative immunoblotting of total kidney membrane fractions revealed a significant decrease in total kidney AQP2 expression in CRF rats to 43 ± 12% of sham levels ( P < 0.05). A similar reduction was observed for AQP1 and AQP3. Furthermore, the increased urine output and decreased urine osmolality persisted in CRF rats despite 7 days treatment with 1-desamino-[8-d-arginine]vasopressin (DDAVP, 0.1 μg/h sc) compared with untreated sham-operated controls. Also, there was no change in AQP2 expression (which remained at 38 ± 3% of sham levels, P < 0.05), urine output, or urine osmolality between CRF rats with or without DDAVP treatment. Immunocytochemistry confirmed the decreased AQP2 expression in collecting duct principal cells in CRF rats, with a predominant apical labeling. In conclusion, the results demonstrated that there was a significant vasopressin-resistant downregulation of AQP2 and AQP3 as well as downregulation of AQP1 associated with the polyuria in CRF rats.
Dopamine D4receptor isoform mRNA and protein are expressed in the rat cortical collecting duct
Authors: Sun Duo , Wilborn Teresa W. , Schafer James A. ,   (With the Technical Assistance of Mary L. Watkins)
Published: December 2017

We reported previously [ Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F391–F400, 1996] that dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+transport in the rat cortical collecting duct (CCD) apparently through a D4dopamine receptor. The present experiments used RT-PCR of total RNA extracted from microdissected rat CCD to determine whether the D4and D1Adopamine receptor isoforms are expressed. Specific primers were used to amplify three regions of the D4cDNA. All three gave products with 98–100% nucleotide identity to the known rat D4sequence; however, there was an extra 6-bp insert at the 3′ end of the second transmembrane region that was identical to the human and mouse sequences but which had not been documented in the rat sequence. D4receptor protein was also localized exclusively to the CCD and medullary collecting ducts by immunohistochemistry. Two regions of the D1Adopamine receptor message were also amplified by RT-PCR of RNA from rat CCD and were verified by sequencing and immunohistochemistry. We conclude that both D4and D1Adopamine receptors are expressed in the rat CCD, but the physiological effects are attributable to a D4receptor.
SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles
Authors: Inoue Takeaki , Nielsen Søren , Mandon Beatrice , Terris James , Kishore Bellamkonda K. , Knepper Mark A.
Published: December 2017

Vesicle targeting proteins (“SNAREs”) have been proposed to direct vasopressin-induced trafficking of aquaporin-2 water channels in kidney collecting ducts. A newly identified SNARE protein, SNAP-23, is proposed to mediate vesicle targeting to the plasma membrane in diverse tissues. The current studies were done to determine whether SNAP-23 is expressed in collecting ducts with an intracellular distribution compatible with a role in aquaporin-2 trafficking. RT-PCR demonstrated SNAP-23 mRNA in microdissected collecting ducts and other tubular segments including the proximal tubule and thick ascending limb. Immunoblotting using a polyclonal antibody raised against a COOH-terminal peptide revealed a solitary band at an apparent molecular mass of 30 kDa in renal medullary membrane fractions and inner medullary collecting duct suspensions. Differential centrifugation revealed that SNAP-23 is present in membrane fractions including the low-density fraction enriched in intracellular vesicles. Immunocytochemistry revealed SNAP-23 labeling at both the apex and the cytoplasm of collecting duct principal cells. Immunoblotting of intracellular vesicles immunoisolated using an aquaporin-2 antibody revealed the presence of both SNAP-23 and synaptobrevin-2 (VAMP-2) in aquaporin-2-bearing vesicles. We conclude that SNAP-23 is strongly expressed in collecting duct principal cells, consistent with a role in vasopressin-regulated trafficking of aquaporin-2. However, localization of SNAP-23 in both intracytoplasmic vesicles and plasma membranes suggests a function different from that originally proposed for SNAP-25 in synaptic vesicle targeting.
Expression of Grb7 growth factor receptor signaling protein in kidney development and in adult kidney
Authors: Leavey Sean F. , Arend Lois J. , Dare Heidi , Dressler Gregory R. , Briggs Josie P. , Margolis Benjamin L.
Published: December 2017

Grb7, a signaling protein whose physiological function is unknown, binds receptor tyrosine kinases important for normal kidney development. By investigating and correlating Grb7 gene expression with that reported for Grb7-binding receptors, we provide clues to Grb7 function(s). RT-PCR and immunoblot were used to demonstrate Grb7 gene and protein expression in the mature kidney. Additional RT-PCR studies detected gene expression in all microdissected adult nephron segments examined, except glomeruli, and in the mouse metanephric kidney from embryonic day 11( E11) through to day 17( E17). In situ hybridization at E14 demonstrated the following cellular pattern of localization: Grb7 mRNA in metanephric epithelia of mesenchymal and ureteric bud origin; no expression in the undifferentiated mesenchyme; and little expression in podocyte-destined cells or primitive glomeruli. Grb7 mRNA was also present in the epithelia of the lung and gut at E14. Thus Grb7 may have a basic function in growth factor signaling in terminally differentiated epithelia along the nephron and in developing epithelia in the kidney, lung, and gut. It is localized in a pattern permissive for a role in Her2 and Ret receptor signaling.
Renal cortical Na+-K+-ATPase activity and abundance is decreased in normal pregnant rats
Authors: Mahaney J. , Felton C. , Taylor D. , Fleming W. , Kong J. Q. , Baylis C.
Published: December 2017

During late pregnancy, the rat undergoes massive plasma volume expansion due to cumulative renal sodium retention. In the present study, conducted in virgin, mid- ( days 11–13), and late-pregnant ( days 18–20) rats, we measured both Na+-K+-ATPase activity (by coupled enzyme assay) and abundance of the α-subunits of the Na+-K+-ATPase (by Western and slot blot analyses) in renal cortex, medulla, and brain stem. Unexpectedly, Na+-K+-ATPase in renal cortex, in both stages of pregnancy, is reduced versus the virgin, consistent with our finding of a reduced quantity of the α1-subunit. In renal medulla, there is a small rise in activity at midterm, but there is no difference in either activity or abundance of the α1-subunit in late pregnancy, when renal Na retention is maximal. In brain stem, where only α2- and α3-subunits are evident, pregnancy has no impact on enzyme activity or abundance of either isoform. In conclusion, the outcome of these experiments was unexpected in that we did not observe increased renal Na+-K+-ATPase activity in late pregnancy in the rat. In fact, in renal cortex, Na+-K+-ATPase activity and abundance are reduced. Whatever promotes net sodium retention in pregnancy must be capable of overwhelming this and several other strong natriuretic signals.
A tyrosine-based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney
Authors: Wang Tong , Courtois-Coutry Nathalie , Giebisch Gerhard , Caplan Michael J.
Published: December 2017

Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the β-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase β-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the β-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase β-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 ± 0.13 meq/l in transgenic and 4.12 ± 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 ± 3.62% in transgenic and 50.1 ± 4.78% in control; n = 9, P < 0.01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase β-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.
Role of nitric oxide in the control of renin secretion
Authors: Kurtz Armin , Wagner Charlotte
Published: December 2017

Because of the significant constitutive expression of NO synthases in the juxtaglomerular apparatus, nitric oxide (NO) is considered as a likely modulator of renin secretion. In most instances, NO appears as a tonic enhancer of renin secretion, acting via inhibition of cAMP degradation through the action of cGMP. Depending on as yet unknown factors, the stimulatory effect of NO on renin secretion may also switch to an inhibitory one that is compatible with the inhibition of renin secretion by cGMP-dependent protein kinase activity. Whether NO plays a direct regulatory role or a more permissive role in the control of renin secretion remains to be answered.
Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X7receptors
Authors: Schulze-Lohoff Eckhard , Hugo Christian , Rost Sylvia , Arnold Susanne , Gruber Angela , Brüne Bernhard , Sterzel Ralf Bernd
Published: December 2017

Mesangial cells undergo cell death both by apoptosis and necrosis during glomerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its receptors may regulate cell death of cultured mesangial cells. Addition of extracellular ATP (300 μM to 5 mM) to cultured rat mesangial cells for 90 min caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidyl transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA fragmentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 expression were reproduced by the purinergic P2Z/P2X7receptor agonist, 3′- O-(4-benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X7receptor blocker, oxidized ATP. Transcripts encoding the P2Z/P2X7receptor were expressed by cultured mesangial cells as determined by Northern blot analysis. P2Z/P2X7receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X7receptors by extracellular ATP causes apoptosis and necrosis of cultured mesangial cells. Activation of purinergic P2Z/P2X7receptors may play a role in causing death of mesangial cells during glomerular disease.
Isolated superfused juxtaglomerular cells from rat kidney: a model for study of renin secretion
Authors: Albinus Margitta , Finkbeiner Erhard , Sosath Birgit , Osswald Hartmut
Published: December 2017

Freshly isolated rat juxtaglomerular cells (JGC) were superfused to study renin secretion rate (RSR) at the cellular level. Effluates from the superfusion chamber collected in 20-min intervals showed a time-dependent decline in RSR from 85.5 ± 32 to 4.0 ± 2.4 ng ANG I ⋅ ml−1⋅ h−1⋅ mg protein−1⋅ min−1within 100 min of collection (mean ± SE, n = no. of JGC preparations/superfusion chambers = 9/18). Addition of adenosine deaminase type II (ADA II, 3 U/1.4 mg protein) to the superfusion medium increased RSR more than fourfold to 402 ± 100 ng in the first collection period, which dropped to 237.5 ± 67 ng ANG I ⋅ ml−1⋅ h−1⋅ mg protein−1⋅ min−1( n = 9/18) within 100 min. This ADA II effect was rapid in onset and fully reversible. When the purified ADA type VII, with a 40-fold higher specific activity, was added to the superfusate, RSR was increased only by 96 ± 17.8% compared with controls. This ADA VII (5 U/30 μg) effect could be mimicked by the selective adenosine A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10−6mol/l). Since albumin stimulated RSR in a concentration-dependent fashion, to an extent similar to that of ADA II, we assume that the ADA II effect was largely unspecific in nature. We conclude that 1) superfusion of isolated JGC from rats is suitable for investigations of renin secretion at the cellular level, 2) the increase in RSR by ADA II appears to be only in part due to deamination of endogenously generated adenosine, and 3) albumin in the superfusate induces a similar stimulatory effect as ADA II.
Endogenous nitric oxide inhibits chloride transport in the thick ascending limb
Authors: Plato Craig F. , Stoos Barbara A. , Wang Ding , Garvin Jeffrey L.
Published: December 2017

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. We first determined the effect of exogenously added NO on net chloride flux ( JCl). The NO donor spermine NONOate (SPM; 10 μM) decreased JClfrom 101.2 ± 9.6 to 65.0 ± 7.7 pmol ⋅ mm−1⋅ min−1, a reduction of 35.5 ± 6.4%, whereas controls did not decrease over time. To determine whether endogenous NO affects cortical TALH transport, we measured the effect of adding the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME), the substratel-arginine (l-Arg), or its enantiomerd-arginine (d-Arg) on JCl.l-NAME andd-Arg did not alter JCl; in contrast, addition of 0.5 mM l-Arg decreased JClby 40.2 ± 10.4% from control. The inhibition of chloride flux by 0.5 mM l-Arg was abolished by pretreatment with l-NAME, indicating that cortical TALH NOS is active, but production of NO is substrate-limited in our preparation. Furthermore, cortical TALH chloride flux increased following removal of 0.5 mMl-Arg from the bath, indicating that the reductions in chloride flux observed in response tol-Arg are not the result of NO-mediated cytotoxicity. We conclude that 1) exogenous NO decreases cortical TALH JCl; 2) cortical TALHs produce NO in the presence of l-Arg, which decreases JCl; and 3) the response of cortical TALHs to l-Arg is reversible in vitro. These data suggest an important role for locally produced NO, which may act via an autocrine mechanism to directly affect TALH sodium chloride transport. Thus TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO observed in vivo.
Immunolocalization of the electrogenic Na+- HCO 3 − cotransporter in mammalian and amphibian kidney
Authors: Schmitt Bernhard M. , Biemesderfer Daniel , Romero Michael F. , Boulpaep Emile L. , Boron Walter F.
Published: December 2017

Electrogenic cotransport of Na+and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928–1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a ∼130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl−/[Formula: see text]exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at ∼130 kDa in both rat and rabbit, as well as a single ∼160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-μm cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.
Renal type I inositol 1,4,5-trisphosphate receptor is reduced in streptozotocin-induced diabetic rats and mice
Authors: Sharma Kumar , Wang Lewei , Zhu Yanqing , DeGuzman Aurora , Cao Gao-Yuan , Lynn Richard B. , Joseph Suresh K.
Published: December 2017

The mechanisms underlying glomerular hypertrophy and hyperfiltration in diabetes remain unclear. We have previously demonstrated that the cytokine transforming growth factor-β1 (TGF-β1) is increased in early diabetic kidney disease and TGF-β1 inhibits the expression of the inositol 1,4,5-trisphosphate (IP3)-gated calcium channel, the type I IP3receptor (IP3R), in mesangial cells. To test the hypothesis that reduced type I IP3R may be important in diabetic kidney disease, we evaluated type I IP3R expression in the kidney of streptozotocin-induced diabetic rats and mice. Two-week-old diabetic rats have decreased renal type I IP3R protein and mRNA levels. Immunostaining of normal rat kidney demonstrated presence of type I IP3R in glomerular and vascular smooth muscle cells, whereas diabetic rats had reduced staining in both compartments. Reduction of type I IP3R also occurred in parallel with renal hypertrophy, increased creatinine clearance, and increased renal TGF-β1 expression in the diabetic rats. Two-week-old diabetic mice also had reduced renal type I IP3R protein and mRNA expression in association with renal hypertrophy and increased TGF-β1 mRNA expression. These findings demonstrate that there is reduced type I IP3R in glomerular and vascular smooth muscle cells in the diabetic kidney, which may contribute to the altered renal vasoregulation and renal hypertrophy of diabetes.
Epidermal growth factor inhibits Na-Picotransport in weaned and suckling rats
Authors: Arar Mazen , Zajicek Hubert K. , Elshihabi Ihsan , Levi Moshe ,   (With the Technical Assistance of Myrna Gonzales and Paul Wilson)
Published: December 2017

In the present study, we determined the effect of epidermal growth factor (EGF; 10 μg/100 g body wt) on sodium gradient-dependent phosphate transport (Na-Picotransport) regulation in suckling (12-day-old) and weaned (24-day-old) rats. Weaned rats had higher proximal tubular brush border membrane vesicle (BBMV) Na-Picotransport activity (232 ± 16 in weaned vs. 130 ± 9 pmol ⋅ 10 s−1⋅ mg protein−1in suckling rats, P < 0.05). Chronic treatment with EGF induced inhibition of BBMV Na-Picotransport in both suckling (130 ± 9 vs. 104 ± 7 pmol ⋅ 10 s−1⋅ mg protein−1, P < 0.05) and weaned rats (232 ± 16 vs. 145 ± 9 pmol ⋅ 10 s−1⋅ mg protein−1, P < 0.005). The inhibitory effect was selective for Na-Picotransport as there was no inhibition of Na-glucose cotransport. Weaned rats had a higher abundance of BBMV NaPi-2 protein than suckling rats (increase of 54%, P < 0.001) and a twofold increase in NaPi-2 mRNA. The EGF-induced inhibition of Na-Pitransport was paralleled by decreases in NaPi-2 protein abundance in both weaned (decrease of 26%, P < 0.01) and suckling (decrease of 27%, P < 0.01) animals. In contrast, there were no changes in NaPi-2 mRNA abundance. We conclude that proximal tubule BBMV Na-Picotransport activity, NaPi-2 protein abundance, and NaPi-2 mRNA abundance are higher in weaned than in suckling rats. EGF inhibits Na-Picotransport activity in BBMV isolated from suckling and weaned rats, and this inhibition is mediated via a decrease in NaPi-2 protein abundance, in the absence of a change in NaPi-2 mRNA.
Suppressed impact of nitric oxide on renal arteriolar function in rats with chronic heart failure
Authors: Ikenaga Hideki , Ishii Naohito , Didion Sean P. , Zhang Kun , Cornish Kurtis G. , Patel Kaushik P. , Mayhan William G. , Carmines Pamela K.
Published: December 2017

We performed experiments to test the hypothesis that experimental heart failure (HF) is associated with altered nitric oxide (NO)-dependent influences on the renal microvasculature, including diminished modulation of constrictor responses to ANG II. Eight to ten weeks after inducing HF in rats by coronary artery ligation, we administered enalaprilat to suppress ANG II synthesis and studied renal arteriolar function using the in vitro blood-perfused juxtamedullary nephron technique. In kidneys from sham-operated rats, NO synthase inhibition [100 μM Nω-nitro-l-arginine (l-NNA)] reduced afferent arteriolar diameter by 4.1 ± 0.6 μm and enhanced ANG II responsiveness (10 nM ANG II decreased afferent diameter by 10.1 ± 1.4 μm before and 12.8 ± 1.6 μm duringl-NNA treatment; P < 0.05). In kidneys from HF rats,l-NNA did not alter afferent arteriolar baseline diameter or ANG II responsiveness (10 nM ANG II decreased diameter by 12.5 ± 1.5 μm before and 12.5 ± 2.3 μm during l-NNA). The effects of l-NNA on efferent arteriolar function were also abated in HF rats. In renal cortex of HF rats, NO synthase activity was decreased by 63% and superoxide dismutase activity was diminished by 39% relative to tissue from sham-operated rats. Urinary nitrate/nitrite excretion was also reduced in HF rats. Thus both diminished synthesis and augmented degradation are likely to contribute to a decreased renal microvascular impact of endogenous NO during chronic HF, the consequences of which include loss of NO-dependent modulation of ANG II-induced vasoconstriction.
Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity
Authors: Frøkiaer Jørgen , Marples David , Valtin Heinz , Morris John F. , Knepper Mark A. , Nielsen Søren
Published: December 2017

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 ± 7% (±SE) of those in control mice ( n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels ( P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.