bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton remodeling in rat kidney MTAL
Authors: Bustamante Mauro , Roger Frank , Bochaton-Piallat Marie-Luce , Gabbiani Giulio , Martin Pierre-Yves , Féraille Eric
Published: March 2015

The kidney medulla is physiologically exposed to variations in extracellular osmolality. In response to hypertonic cell shrinkage, cells of the rat kidney medullary thick ascending limb of Henle's loop undergo p38 kinase-dependent regulatory volume increase (RVI). In the present study, we investigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases and reduces cellular volume, induced a sustained actin polymerization occurring after 10 min and concurrently with RVI. In contrast, hyperosmotic urea, which does not modify MAP kinase activity and cellular volume, did not induce sustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution of F-actin from a dense cortical ring to a diffuse network of actin bundles. Stabilization of actin filaments by jasplakinolide and inhibition of the generation of new actin filaments by swinholide A prevented RVI, whereas depolymerization of actin filaments by latrunculin B attenuated cell shrinkage and enhanced RVI. These actin-interfering drugs did not alter extracellular regulated kinase and p38 kinase activation under hypertonic conditions. Similar to swinholide A, inhibiting p38 kinase with SB-203580 abolished sustained actin polymerization, actin redistribution, and decreased RVI efficacy. We therefore propose that in rat kidney the medullary thick ascending limb of Henle's loop exposed to extracellular hypertonicity, p38 kinase activation induces depolymerization of the F-actin cortical ring and polymerization of a dense diffuse F-actin network that both contribute to increase RVI efficacy.
Renal fluid and electrolyte handling in BKCa-β1−/−mice
Authors: Pluznick Jennifer L. , Wei Peilin , Carmines Pamela K. , Sansom Steven C.
Published: March 2015

Large-conductance Ca2+-activated K+channels (BKCa) are composed of pore-forming α-subunits and one of four accessory β-subunits. The β1-subunit, found predominantly in smooth muscle, modulates the Ca2+sensitivity and pharmacological properties of BKCa. BKCa-β1 null mice (Mβ1−/−) are moderately hypertensive, consistent with the role of BKCain modulating intrinsic vascular tone. Because BKCaare present in various renal cells including the mesangium and cortical collecting ducts, we determined whether fluid or electrolyte excretion was impaired in Mβ1−/−under euvolemic, volume-expanded, or high-salt diet conditions. Under euvolemic conditions, no differences in renal function were found between Mβ1−/−and Mβ1+/+. However, glomerular filtration rate (GFR) and fractional K+excretion were significantly impaired in Mβ1−/−in response to acute volume expansion. In contrast, Mβ1−/−exhibited enhanced Na+excretion and fractional Na+excretion responses to acute volume expansion. Differences in renal function between Mβ1+/+and Mβ1−/−were not observed when chronically treated with a high-salt diet. These observations indicate that the β1-subunit of BKCacontributes to the increased GFR that accompanies an acute salt and volume load and raises the possibility that it is also involved in regulating K+excretion under these conditions.
“Step” vs. “dynamic” autoregulation: implications for susceptibility to hypertensive injury
Authors: Bidani Anil K. , Hacioglu Rifat , Abu-Amarah Isam , Williamson Geoffrey A. , Loutzenhiser Rodger , Griffin Karen A.
Published: March 2015

Renal autoregulatory (AR) mechanisms provide the primary protection against transmission of systemic pressures, and their impairment is believed to be responsible for the enhanced susceptibility to hypertensive renal damage in renal mass reduction (RMR) models. Assessment of AR capacity by the “step” change methodology under anesthesia was compared with that by “dynamic” methods in separate conscious control Sprague-Dawley rats and after uninephrectomy (UNX) and ¾ RMR (RK-NX) ( n = 7–10/group). Substantially less AR capacity was seen by the dynamic vs. the step methodology in control rats. Moreover, dynamic AR capacity did not differ among controls, UNX, and RK-NX rats (fractional gain in admittance ∼0.4–0.5 in all groups at frequencies in the range of 0.0025–0.025 Hz). By contrast, significant impairment of step AR was seen in RK-NX vs. control or UNX rats (AR indexes 0.7 ± 0.1 vs. 0.1 ± 0.02 and 0.2 ± 0.04, respectively, P < 0.01). We propose that the step and dynamic methods evaluate the renal AR responses to different components of blood pressure (BP) power with the step AR assessing the ability to buffer large changes in average BP (DC power), whereas the present “dynamic” methods assess the AR ability to buffer slow BP fluctuations (<0.25 Hz) superimposed on the average BP (AC power), a substantially smaller component of total BP power. We further suggest that step but not dynamic AR methods as presently performed provide a valid index of the underlying susceptibility to hypertensive glomerular damage after RMR.
Mesangial cell protein kinase C isozyme activation in the diabetic milieu
Authors: Whiteside Catharine I. , Dlugosz John A.
Published: March 2015

High-glucose-induced activation of mesangial cell protein kinase C (PKC) contributes significantly to the pathogenesis of diabetic nephropathy. Excess glucose metabolism through the polyol pathway leads to de novo synthesis of both diacylglyerol (DAG) and phosphatidic acid, which may account for increased mesangial cell PKC-α, -β, -δ, -ε, and -ζ activation/translocation observed within 48-h exposure to high glucose. Raised intracellular glucose causes generation of reactive oxygen species that may directly activate PKC isozymes and enhance their reactivity to vasoactive peptide signaling. In both diabetic rodent models of diabetes and cultured mesangial cells, PKC-β appears to be the key isozyme required for the enhanced expression of transforming growth factor-β1, initiation of early accumulation of mesangial matrix protein, and increased microalbuminuria. Enhanced collagen IV expression by mesangial cells in response to vasoactive peptide hormone stimulation, e.g., endothelin-1, requires PKC-β, -δ, -ε and -ζ. Loss of mesangial cell contractility to potent vasoactive peptides and coincident F-actin disassembly are due to high-glucose-activation of PKC-ζ. Inhibition of mesangial cell PKC isozyme activation in high glucose may prove to be the next important treatment for diabetic nephropathy.
Transepithelial transport and metabolism of glycine in S1, S2, and S3 cell types of the rabbit proximal tubule
Authors: Parks Lisa D. , Barfuss Delon W.
Published: March 2015

In the first of two sets of experiments, the lumen-to-cell and cell-to-bath transport rates for glycine were measured in the isolated-perfused medullary pars recta (S3 cells) of the rabbit proximal tubule at multiple luminal glycine concentrations (0–2.0 mM). The lumen-to-cell transport of glycine was saturated, which permitted the calculation of the transport maximum of disappearance rate of glycine from the lumen (pmol · min−1 · mm tubular length−1), K m (mM), and paracellular leak (pmol · min−1 · mm tubular length−1 · mM−1) values for this transport mechanism; these values were 4.3, 0.3, and 0.03, respectively. The cell-to-bath transport did not saturate but showed a linear relationship to cellular glycine concentration, 0.58 pmol · min−1 · mm tubular length−1 · mM−1. The second set of experiments characterized the transport rate, cellular accumulation, and metabolic rate of lumen-to-cell transported [3H]glycine in all segments (cell types) of the proximal tubule, pars convoluta (S1 cells), cortical pars recta (S2 cells), and medullary pars recta (S3 cells). These proximal tubular segments were isolated and perfused at a single glycine concentration of 11.2 μM. From the results of this study and previous work (Barfuss DW and Schafer JA. Am J Physiol 236: F149–F162, 1979), we conclude that the axial heterogeneity for glycine lumen-to-cell and cell-to-bath transport capacity extends to the medullary pars recta (S3 cells; S1 > S2 < S3 for lumen-to-cell transport and S1 > S2 > S3 for cell-to-bath transport). Also, we conclude that lumen-to-cell transported glycine can be metabolized and its metabolic rate displays axial heterogeneity (S1 > S2 > S3). The physiological significances of these transport and metabolic characteristics of the S3 cell type permits the medullary pars recta to effectively recover glycine from very low luminal glycine concentrations and makes glycine available for protective and maintenance metabolism of the medullary pars recta.
Role of an endoplasmic reticulum Ca2+-independent phospholipase A2in oxidant-induced renal cell death
Authors: Cummings Brian S. , McHowat Jane , Schnellmann Rick G.
Published: March 2015

Phospholipase A2(PLA2) hydrolyzes the sn-2 ester bond in phospholipids, releasing a fatty acid and a lysophospholipid. Recently, a novel 85-kDa membrane-bound-Ca2+-independent PLA2(iPLA2) was identified in insect and bacterial cells transfected with candidate PLA2sequences. However, few data exist demonstrating a membrane-bound-iPLA2in mammalian cells, its subcellular localization, or its physiological role. Herein, we demonstrate the expression of an 85-kDa endoplasmic reticulum (ER)-Ca2+-iPLA2(ER-iPLA2) in rabbit renal proximal tubule cells (RPTC) that is plasmalogen selective and is inhibited by the specific Ca2+-iPLA2inhibitor bromoenol lactone (BEL). RPTC exposed to tert-butylhydroperoxide for 24 h exhibited 20% oncosis compared with 2% in controls. Inhibition of ER-iPLA2with BEL before tert-butylhydroperoxide exposure resulted in 50% oncosis. To determine whether this effect was common to oxidants, we tested the ability of BEL to potentiate oncosis induced by cumene hydroperoxide, menadione, duraquinone, cisplatin, and the nonoxidant antimycin A. All oxidants tested produced oncosis after 24 h, and prior inhibition of ER-iPLA2potentiated oncosis at least twofold. In contrast, inhibition of ER-iPLA2did not alter antimycin A-induced oncosis. Lipid peroxidation increased from 1.4- to 5.2-fold in RPTC treated with BEL before oxidant exposure, whereas no change was seen in antimycin A-treated RPTC. These results are the first to demonstrate the expression and subcellular localization of an ER-iPLA2. These results also suggest that ER-iPLA2functions to protect against oxidant-induced lipid peroxidation and oncosis.
Urinary tumor necrosis factor contributes to sodium retention and renal hypertrophy during diabetes
Authors: DiPetrillo Keith , Coutermarsh Bonita , Gesek Frank A.
Published: March 2015

Nephropathy is a major contributor to overall morbidity and mortality in diabetic patients. Early renal changes during diabetes include Na retention and renal hypertrophy. Tumor necrosis factor (TNF) is elevated during diabetes and is implicated in the pathogenesis of diabetic nephropathy. We tested the hypothesis that TNF contributes to Na retention and renal hypertrophy during diabetes. Rats with streptozotocin-induced diabetes exhibit increased urinary TNF excretion, Na retention, and renal hypertrophy through the first 20 days of diabetes. Administration of a soluble TNF antagonist (TNFR:Fc) to diabetic rats reduces urinary TNF excretion and prevents Na retention and renal hypertrophy. TNF stimulates Na uptake in distal tubule cells isolated from diabetic rats, providing a possible mechanism for TNF-induced Na retention. We conclude that urinary TNF contributes to early diabetic nephropathy and may serve as a valuable diagnostic marker. Furthermore, inhibition of TNF during diabetes may attenuate early pathological changes in diabetic nephropathy.
Plasminogen activator inhibitor-1 and the kidney
Authors: Eddy Allison A.
Published: March 2015

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that was isolated 20 years ago. First recognized as an inhibitor of intravascular fibrinolysis, it is now evident that PAI-1 is a multifunctional protein with actions that may be dependent on or independent of its protease inhibitory effects. The latter often involve interactions between PAI-1 and vitronectin or the urokinase receptor. The protease-inhibitory actions of PAI-1 extend beyond fibrinolysis and include extracellular matrix turnover and activation of several proenzymes and latent growth factors. PAI-1 has been implicated in several renal pathogenetic processes, including thrombotic microangiopathies and proliferative and/or crescentic glomerulopathies. Most recently, it has become clear that PAI-1 also plays a pivotal role in progressive renal disease, both glomerulosclerosis and tubulointerstitial fibrosis. An active area of present research interest, untold stories are likely to be uncovered soon.
Characterization of Ca2+-activated Cl– currents in mouse kidney inner medullary collecting duct cells
Authors: Qu Zhiqiang , Wei Raymond W. , Hartzell H. Criss
Published: March 2015

Ca2+-activated Cl– (ClCa) channels were characterized biophysically and pharmacologically in a mouse kidney inner medullary collecting duct cell line, IMCD-K2. Whole cell recording was performed with symmetrical N-methyl-d-glucamine chloride (NMDG)-Cl in the intracellular and extracellular solutions, and the intracellular Ca2+ concentration ([Ca2+]i) was adjusted with Ca2+-EGTA buffers. The amplitude of the current was dependent on [Ca2+]i. [Ca2+]i <800 nM strongly activated outwardly rectifying Cl– currents, whereas high Ca2+ (21 μM) elicited time-independent currents that did not rectify. The currents activated at low [Ca2+] exhibited time-dependent activation and deactivation. The affinity of the channel for Ca2+ was voltage dependent. The EC50 for Ca2+ was ∼0.4 μM at +100 mV and ∼1.0 μM at –100 mV. The Cl– channel blocker niflumic acid in the bath equally inhibited both inward and outward currents reversibly, with a Ki = 7.6 μM. DIDS, diphenylamine-2-carboxylic acid, and anthracene-9-carboxylic acid reversibly inhibited outward currents in a voltage-dependent manner. DTT slowly inhibited the currents, but tamoxifen did not. Comparing the biophysical and pharmacological properties, we conclude that IMCD-K2 cells express the same type of ClCa channels as those we have described in detail in Xenopus laevis oocytes (Qu Z and Hartzell HC. J Biol Chem 276: 18423–18429, 2001).
Activation of D2-like receptors causes recruitment of tyrosine-phosphorylated NKA α1-subunits in kidney
Authors: Narkar Vihang A. , Hussain Tahir , Lokhandwala Mustafa F.
Published: March 2015

The present study investigates the cellular mechanisms responsible for dopamine D2-like receptor-mediated stimulation of Na+-K+-ATPase in the proximal tubules of the kidney. Previously, we showed that D2-like receptor-mediated increase in Na+-K+-ATPase involves an increase in the maximum rate of Na+-K+-ATPase activity ( Vmax). Therefore, we tested the hypothesis that D2-like receptor-mediated stimulation of Na+-K+-ATPase requires phosphorylation and recruitment of α1-subunits of the enzyme from cytosol to the membrane. This hypothesis was tested by Western blotting for Na+-K+-ATPase α1-subunits in proximal tubular membrane. Treatment of the proximal tubules with bromocriptine (D2-like receptor agonist) caused an increase in Na+-K+-ATPase α1-subunit abundance in the membrane preparations. This effect was blocked by genistein (tyrosine kinase inhibitor), suggesting a role for tyrosine phosphorylation. Moreover, bromocriptine caused an increase in tyrosine phosphorylation of membrane-bound Na+-K+-ATPase α1-subunits. This effect was blocked by bafilomycin A1 (vesicular trafficking inhibitor), which suggested that this increase was due to the recruitment of tyrosine-phosphorylated Na+-K+-ATPase α1-subunits. In conclusion, we have demonstrated that activation of D2-like receptors increases Na+-K+-ATPase activity by recruitment of the tyrosine-phosphorylated α1-subunits in the proximal tubules of the kidney.
AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors
Authors: Wang Weidong , Li Chunling , Kwon Tae-Hwan , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

The purpose of this study was to evaluate whether hypercalcemia is associated with downregulation of renal aquaporins (AQPs), including AQP1, AQP2, phosphorylated AQP2 (p-AQP2), AQP3, and AQP4, and if this is the case, to test whether cAMP-phosphodiesterase (PDE) inhibitor treatment can prevent AQP downregulation and prevent the development of polyuria. Vitamin D-induced hypercalcemia in rats was associated with increased urine output and reduced urine osmolality, consistent with previous findings (Levi M, Peterson L, and Berl T. Kidney Int 23: 489–497, 1983). Semiquantitative immunoblotting revealed a significant reduction in the abundance of inner medullary AQP2 (52 ± 6% of control levels), consistent with previous studies, and of AQP2, which is phosphorylated at the PKA phosphorylation consensus site serine 256 (p-AQP2; 36 ± 8%). Moreover, AQP3 abundance was also significantly decreased (45 ± 7 and 61 ± 6% of control levels in inner medulla and whole kidney, respectively). Consistent with this, immunohistochemistry demonstrated reduced AQP3 immunolabeling along the entire collecting duct. AQP4 expression was not reduced. Surprisingly, total kidney AQP1 abundance was also reduced (60 ± 6%). AQP1 expression was reduced in the cortex and outer stripe of the outer medulla (48 ± 7%; i.e., in proximal tubules). In contrast, AQP1 levels were not changed in the inner stripe of the outer medulla or in the inner medulla (i.e., descending thin limbs and vasa recta). Treatment with the cAMP-PDE inhibitors rolipram and milrinone in combination (inhibiting PDE IV and PDE III isoenzymes) at day 2 and onward completely prevented the hypercalcemia-induced downregulation of AQP2 and AQP3 (but not AQP1) and completely prevented the development of polyuria. In conclusion, AQP3, AQP2, and p-AQP2 are downregulated and are likely to play critical roles in the development of polyuria associated with vitamin D-induced hypercalcemia. Moreover, PDE inhibitor treatment significantly prevented the reduced expression of collecting duct AQPs and prevented the development of polyuria.
Ca2+influx through the osteoclastic plasma membrane ryanodine receptor
Authors: Moonga Baljit S. , Li Sun , Iqbal Jameel , Davidson Robert , Shankar Vijai S. , Bevis Peter J. R. , Inzerillo Angela , Abe Etsuko , Huang Christopher L.-H. , Zaidi Mone
Published: March 2015

We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca2+influx channel and as a divalent cation (Ca2+) sensor. Cytosolic Ca2+measurements revealed Ca2+influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca2+change was, as expected, not seen in Ca2+-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (∼25 mV) ryanodine triggered extracellular Ca2+influx that was blocked by Ni2+. In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective ∼60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential ( Erev) ∼0 mV]. In the presence of a Ba2+gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ∼120 pS and Erevshifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca2+channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab34. To demonstrate that extracellular Ca2+sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca2+signals triggered by either Ni2+(5 mM) or Cd2+(50 μM). Finally, intracellular application of antiserum Ab34potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca2+influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates Ca2+influx but also functions as a sensor for extracellular divalent cations.
Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide
Authors: Kone Bruce C. , Kuncewicz Teresa , Zhang Wenzheng , Yu Zhi-Yuan
Published: March 2015

Nitric oxide (NO) is a potent cell-signaling, effector, and vasodilator molecule that plays important roles in diverse biological effects in the kidney, vasculature, and many other tissues. Because of its high biological reactivity and diffusibility, multiple tiers of regulation, ranging from transcriptional to posttranslational controls, tightly control NO biosynthesis. Interactions of each of the major NO synthase (NOS) isoforms with heterologous proteins have emerged as a mechanism by which the activity, spatial distribution, and proximity of the NOS isoforms to regulatory proteins and intended targets are governed. Dimerization of the NOS isozymes, required for their activity, exhibits distinguishing features among these proteins and may serve as a regulated process and target for therapeutic intervention. An increasingly wide array of proteins, ranging from scaffolding proteins to membrane receptors, has been shown to function as NOS-binding partners. Neuronal NOS interacts via its PDZ domain with several PDZ-domain proteins. Several resident and recruited proteins of plasmalemmal caveolae, including caveolins, anchoring proteins, G protein-coupled receptors, kinases, and molecular chaperones, modulate the activity and trafficking of endothelial NOS in the endothelium. Inducible NOS (iNOS) interacts with the inhibitory molecules kalirin and NOS-associated protein 110 kDa, as well as activator proteins, the Rac GTPases. In addition, protein-protein interactions of proteins governing iNOS transcription function to specify activation or suppression of iNOS induction by cytokines. The calpain and ubiquitin-proteasome pathways are the major proteolytic systems responsible for the regulated degradation of NOS isozymes. The experimental basis for these protein-protein interactions, their functional importance, and potential implication for renal and vascular physiology and pathophysiology is reviewed.
Peritubular AVP regulates bicarbonate reabsorption in cortical distal tubule via V1 and V2receptors
Authors: Musa-Aziz Raif , Barreto-Chaves Maria Luisa Morais , De Mello-Aires Margarida
Published: March 2015

10.1152/ajprenal.00056.2001. Peritubular arginine vasopressin (AVP) regulates bicarbonate reabsorption in the cortical distal tubule via V1 and V2 receptors. The dose-dependent effects of peritubular AVP on net bicarbonate reabsorption ( J HCO[Formula: see text] ) were evaluated by stationary microperfusion of in vivo early (ED; distal convoluted tubule) and late distal (LD; connecting tubule and initial collecting duct) segments of rat kidney, using double-barreled H+-sensitive, ion-exchange resin/reference (1 M KCl) microelectrodes. AVP (10−11 M) perfused into peritubular capillaries increased J HCO[Formula: see text] , compared with basal levels during intact capillary perfusion with blood, in ED and LD segments. AVP (10−9 M) also increased J HCO[Formula: see text] in both segments, but the effect of AVP (10−11 M) was significantly higher. A specificV1-receptor antagonist alone or with AVP (10−11 or 10−9 M) reduced J HCO[Formula: see text] below basal levels. A specific V2-receptor antagonist alone or plus AVP (10−11 M) did not affect J HCO[Formula: see text] but increased AVP (10−9 M)-mediated stimulation. 8-Bromoadenosine 3′,5′-cyclic monophosphate alone reduced J HCO[Formula: see text] below basal levels and also reduced AVP (10−11 M)-mediated stimulation. (Deamino-Cys1, d-Arg8) vasopressin (a V2-selective agonist) also reduced J HCO[Formula: see text] below basal levels. These results show that peritubular AVP stimulates J HCO[Formula: see text] in ED and LD segments via basolateral V1 receptors and that basolateral V2 receptors have a dose-dependent inhibitory effect mediated by cAMP. The data also indicate that endogenous AVP stimulates distal J HCO[Formula: see text] via basolateral V1 receptors.
Chronic hyperosmolarity mediates constitutive expression of molecular chaperones and resistance to injury
Authors: Santos Bento C. , Pullman James M. , Chevaile Alejandro , Welch William J. , Gullans Steven R.
Published: March 2015

Renal medullary cells are exposed to elevated and variable osmolarities and low oxygen tension. Despite the harsh environment, these cells are resistant to the effects of many harmful events. To test the hypothesis that this resistance is a consequence of these cells developing a stress tolerance phenotype to survive in this milieu, we created osmotically tolerant cells [hypertonic (HT) cells] by gradually adapting murine inner medullary collecting duct 3 cells to hyperosmotic medium containing NaCl and urea. HT cells have a reduced DNA synthesis rate, with the majority of cells arrested in the G0/G1phase of the cell cycle, and show constitutive expression of heat shock protein 70 that is proportional to the degree of hyperosmolarity. Unlike acute hyperosmolarity, chronic hyperosmolarity failed to activate MAPKs. Moreover, HT cells acquired protein translational tolerance to further stress treatment, suggesting that HT cells have an osmotolerant phenotype that is analogous to thermotolerance but is a permanent condition. In addition to osmotic shock, HT cells were more resistant to heat, H2O2, cyclosporin, and apoptotic inducers, compared with isotonic murine inner medullary duct 3 cells, but less resistant to amphotericin B and cadmium. HT cells demonstrate that in renal medullary cells, hyperosmotic stress activates biological processes that confer cross-tolerance to other stressful conditions.
Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin
Authors: Gburek Jakub , Birn Henrik , Verroust Pierre J. , Goj Boguslawa , Jacobsen Christian , Moestrup Søren K. , Willnow Thomas E. , Christensen Erik I.
Published: March 2015

Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kdvalues of 2.0 and 3 μM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced ( Kd= 5 μM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes ( Ki∼ 2 μM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.
Effect of flow and stretch on the [Ca2+]iresponse of principal and intercalated cells in cortical collecting duct
Authors: Liu Wen , Xu Shiyun , Woda Craig , Kim Paul , Weinbaum Sheldon , Satlin Lisa M.
Published: March 2015

An acute increase in tubular fluid flow rate in the microperfused cortical collecting duct (CCD), associated with a ∼20% increase in tubular diameter, leads to an increase in intracellular Ca2+concentration ([Ca2+]i)in both principal and intercalated cells (Woda CB, Leite M Jr, Rohatgi R, and Satlin LM. Am J Physiol Renal Physiol 283: F437-F446, 2002). The apical cilium present in principal but not intercalated cells has been proposed to be a flow sensor. To determine whether flow across the cilium and/or epithelial stretch mediates the [Ca2+]iresponse, CCDs from New Zealand White rabbits were microperfused in vitro, split-open (to isolate the effect of flow across cilia), or occluded (to examine the effect of stretch and duration/magnitude of the flow impulse), and [Ca2+]iwas measured using fura 2. In perfused and occluded CCDs, a rapid (<1 s) but not slow (>3 min) increase in luminal flow rate and/or circumferential stretch led to an approximately threefold increase in [Ca2+]iin both principal and intercalated cells within ∼10 s. This response was mediated by external Ca2+entry and inositol 1,4,5-trisphosphate-mediated release of cell Ca2+stores. In split-open CCDs, an increase in superfusate flow led to an approximately twofold increase in [Ca2+]iin both cell types within ∼30 s. These experimental findings are interpreted using mathematical models to predict the fluid stress on the apical membranes of the CCD and the forces and torques on and deformation of the cilia. We conclude that rapid increases in luminal flow rate and circumferential stretch, leading to shear or hydrodynamic impulses at the cilium or apical membrane, lead to increases in [Ca2+]iin both principal and intercalated cells.
Control of epithelial transport via luminal P2 receptors
Authors: Leipziger Jens
Published: March 2015

P2 membrane receptors are specifically activated by extracellular nucleotides like ATP, ADP, UTP, and UDP. P2 receptors are subdivided into metabotropic P2Y and ionotropic P2X receptors. They are expressed in all tissues and induce a variety of biological effects. In epithelia, they are found in both the basolateral and the luminal membranes. Their widespread luminal expression in nearly all transporting epithelia and their effect on transport are summarized. The P2Y2receptor is a prominent luminal receptor in many epithelia. Other luminal P2 receptors include the P2X7, P2Y4, and P2Y6receptors. Functionally, luminal P2Y2receptor activation elicits differential effects on ion transport. In nearly all secretory epithelia, intracellular Ca2+concentration-activated ion conductances are stimulated by luminal nucleotides to induce Cl−, K+, or HCO[Formula: see text] secretion. This encompasses respiratory and various gastrointestinal epithelia or tissues like the conjunctiva of the eye and the epithelium of sweat glands. In the distal nephron, all active transport processes appear to be inhibited by luminal nucleotides. P2Y2receptors inhibit Ca2+and Na+absorption and K+secretion. Commonly, in all steroid-sensitive epithelia (lung, distal nephron, and distal colon), luminal ATP/UTP inhibits epithelial Na+channel-meditated Na+absorption. ATP is readily released from epithelial cells onto their luminal aspect, where ecto-nucleotidases promote their metabolism. Adenosine generated by the action of 5′-nucleotidase may elicit further effects on ion transport, often opposite those of ATP. ATP release from epithelia continues to be poorly understood. Integrated functional concepts for luminal P2 receptors are suggested: 1) luminal P2 receptors are part of an epithelial “secretory” defense mechanism; 2) they may be involved in the regulation of cell volume when transcellular solute transport is out of balance; 3) ATP and adenosine may be important autocrine/paracrine regulators mediating cellular protection and regeneration after ischemic cell damage; and 4) ATP and adenosine have been suggested to mediate renal cyst growth and enlargement in polycystic kidney disease.
The mechanism of angiotensin II binding downregulation by high glucose in primary renal proximal tubule cells
Authors: Park Soo Hyun , Han Ho Jae
Published: March 2015

The renin-angiotensin system plays an important role in the development of diabetic nephropathy. However, the mechanism of ANG II receptor regulation in the renal proximal tubule in the diabetic condition has not been elucidated. Thus we investigated the signal pathways involved in high-glucose-induced downregulation of ANG II binding in primary cultured renal proximal tubule cells. Twenty-five millimolar glucose, but not mannitol andl-glucose, induced downregulation of the AT1receptor (AT1R) because of a significant decline in maximal binding with no significant change in the affinity constant. Twenty-five millimolar glucose also decreased AT1R mRNA and protein levels. The 25 mM glucose-induced increase in the formation of lipid peroxides was prevented by antioxidants, protein kinase C (PKC) inhibitors, or L-type calcium channel blockers. These agents also blocked 25 mM glucose-induced downregulation of125I-ANG II binding. In addition, 25 mM glucose increased transforming growth factor (TGF)-β1 secretion, and anti-TGF-β antibody significantly blocked 25 mM glucose-induced downregulation of125I-ANG II binding. Furthermore, the 25 mM glucose-induced increase in TGF-β1 secretion was inhibited by PKC inhibitors, L-type calcium channel blockers, or antioxidants. In conclusion, high glucose may induce downregulation of125I-ANG II binding via a PKC-oxidative stress-TGF-β signal cascade in primary cultured rabbit renal proximal tubule cells.
Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na+ channel
Authors: McDonald Fiona J. , Western Andrea H. , McNeil John D. , Thomas Brittany C. , Olson Diane R. , Snyder Peter M.
Published: March 2015

The epithelial Na+ channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4–2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na+ transport in epithelia.
Localization of Mg2+-sensing shark kidney calcium receptor SKCaR in kidney of spiny dogfish,Squalus acanthias
Authors: Hentschel Hartmut , Nearing Jacqueline , Harris H. William , Betka Marlies , Baum Michelle , Hebert Steven C. , Elger Marlies
Published: March 2015

We recently cloned a homologue of the bovine parathyroid calcium receptor from the kidney of a spiny dogfish ( Squalus acanthias) and termed this new protein SKCaR. SKCaR senses alterations in extracellular Mg2+after its expression in human embryonic kidney cells (Nearing J, Betka M, Quinn S, Hentschel H, Elger M, Baum M, Bai M, Chattopadyhay N, Brown E, Hebert S, and Harris HW. Proc Natl Acad. Sci USA 99: 9231-9236, 2002). In this report, we used light and electron microscopic immunocytochemical techniques to study the distribution of SKCaR in dogfish kidney. SKCaR antiserum bound to the apical membranes of shark kidney epithelial cells in the following tubular segments: proximal tubules (PIa and PIIb), late distal tubule, and collecting tubule/collecting duct as well as diffusely labeled cells of early distal tubule. The highly specific distribution of SKCaR in mesial tissue as well as lateral countercurrent bundles of dogfish kidney is compatible with a role for SKCaR to sense local tubular Mg2+concentrations. This highly specific distribution of SKCaR protein in dogfish kidney could possibly work in concert with the powerful Mg2+secretory system present in the PIIa segment of elasmobranch fish kidney to affect recycling of Mg2+from putative Mg2+-sensing/Mg2+-reabsorbing segments. These data provide support for the possible existence of Mg2+cycling in elasmobranch kidney in a manner analogous to that described for mammals.
Activity of the basolateral K+ channels is coupled to the Na+-K+-ATPase in the cortical collecting duct
Authors: Muto Shigeaki , Asano Yasushi , Wang WenHui , Seldin Donald , Giebisch Gerhard
Published: March 2015

Microelectrode and patch-clamp techniques were used in the isolated cortical collecting duct to study the effects of stimulating Na+-K+-ATPase by raising bath K+ (Fujii Y and Katz AI. Am J Physiol Renal Fluid Electrolyte Physiol 257: F595–F601, 1989 and Muto S, Asano Y, Seldin D, and Giebisch. Am J Physiol Renal Physiol 276: F143–F158, 1999) on the transepithelial ( VT) and basolateral membrane ( VB) voltages and basolateral K+ channel activity. Increasing bath K+ from 2.5 to 8.5 mM resulted in an initial hyperpolarization of both VT and VB followed by a delayed depolarization. The effects of raising bath K+ on VT and VB were attenuated by decreasing luminal Na+ from 146.8 to 14.0 mM and were abolished by removal of luminal Na+, whereas those were magnified in desoxycorticosterone acetate (DOCA)-treated rabbits. Increasing bath K+ also led to a significant reduction of the intracellular Na+ and Ca2+ concentrations. The transepithelial conductance ( GT) or fractional apical membrane resistance (f RA) were unaltered during the initial hyperpolarization phase, whereas, in the late depolarization phase, there were an increase in GT and a decrease in f RA, both of which were attenuated in the presence of low luminal Na+ (14.0 mM). In tubules from DOCA-treated animals, bath Ba2+ not only caused a significantly larger initial hyperpolarization of VT and VB but also blunted the late depolarization by high bath K+. Nω-nitro-l-arginine methyl ester (l-NAME) partially mimicked the effect of Ba2+ and decreased the amplitude of the late depolarization. Patch-clamp experiments showed that raising bath K+ from 2.5 to 8.5 mM resulted in an increased activity of the basolateral K+ channel, which was absent in the presence of l-NAME. We conclude that stimulation of Na+-K+-ATPase increases the basolateral K+ conductance and that this effect involves suppression of nitric oxide-dependent inhibition of K+ channels.
Smad3 and PKCδ mediate TGF-β1-induced collagen I expression in human mesangial cells
Authors: Runyan Constance E. , Schnaper H. William , Poncelet Anne-Christine
Published: March 2015

Transforming growth factor (TGF)-β has been associated with fibrogenesis in clinical studies and animal models. We previously showed that Smad3 promotes COL1A2 gene activation by TGF-β1in human mesangial cells. In addition to the Smad pathway, it has been suggested that TGF-β1could also activate more classical growth factor signaling. Here, we report that protein kinase C (PKC)δ plays a role in TGF-β1-stimulated collagen I production. In an in vitro kinase assay, TGF-β1treatment specifically increased mesangial cell PKCδ activity in a time-dependent manner. Translocation to the membrane was detected by immunocytochemistry and immunoblot, suggesting activation of PKCδ by TGF-β1. Inhibition of PKCδ by rottlerin decreased basal and TGF-β1-stimulated collagen I production, mRNA expression, and COL1A2 promoter activity, whereas blockade of conventional PKCs by Gö 6976 had little or no effect. In a Gal4-LUC assay system, inhibition of PKCδ abolished TGF-β1-induced transcriptional activity of Gal4-Smad3 and Gal4-Smad4(266-552). Overexpression of Smad3 or Smad3D, in which the three COOH-terminal serine phosphoacceptor residues have been mutated, increased activity of the SBE-LUC construct, containing four DNA binding sites for Smad3 and Smad4. This induction was blocked by PKCδ inhibition, suggesting that rottlerin decreased Smad3 transcriptional activity independently of COOH-terminal serine phosphorylation. Blockade of PKCδ abolished ligand-independent and ligand-dependent stimulation of COL1A2 promoter activity by Smad3. These data indicate that PKCδ is activated by TGF-β1in human mesangial cells. TGF-β1-stimulated PKCδ activity positively regulates Smad transcriptional activity and is required for COL1A2 gene transcription. Thus cross talk among multiple signaling pathways likely contributes to the pathogenesis of glomerular matrix accumulation.
Superoxide stimulates NaCl absorption by the thick ascending limb
Authors: Ortiz Pablo A. , Garvin Jeffrey L.
Published: March 2015

The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide (O[Formula: see text]) stimulates Na and water reabsorption by the kidney. However, it is not known whether O[Formula: see text]regulates transport along the nephron in general or in the THAL specifically. We hypothesized that O[Formula: see text] stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular O[Formula: see text] stimulates Cl absorption. Addition of the O[Formula: see text]-generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 ± 12.0 to 146.2 ± 13.9 pmol · mm−1· min−1, a 33% increase ( P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 ± 13.8 vs. 102.5 ± 8.5 pmol · mm−1· min−1). Furthermore, adding 200 nM H2O2to the bath did not significantly affect Cl absorption (from 130.3 ± 13.7 to 125.3 ± 19.6 pmol · mm−1· min−1). Because extracellular O[Formula: see text] stimulated Cl absorption, we next tested whether endogenously produced O[Formula: see text] could stimulate transport. Under basal conditions, THALs produced detectable amounts of O[Formula: see text], as measured by lucigenin-enhanced chemiluminescence. Adding the O[Formula: see text] scavenger tempol to the bath decreased Cl absorption from 198.1 ± 35.4 to 132.4 ± 23.5 pmol · mm−1· min−1, a 31% decrease ( P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 ± 9.3 pmol · mm−1· min−1. Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 ± 9.1 pmol · mm−1· min−1( P < 0.05). We concluded that both exogenous and endogenous O[Formula: see text] stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of O[Formula: see text] on nephron transport.
Regulation of expression of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats
Authors: Karinch Anne M. , Lin Cheng-Mao , Wolfgang Christopher L. , Pan Ming , Souba Wiley W.
Published: March 2015

During chronic metabolic acidosis, renal glutamine utilization increases markedly. We studied the expression of the system N1 (SN1) amino acid transporter in the kidney during chronic ammonium chloride acidosis in rats. Acidosis caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold increase in the cortex. Acidosis increased Na+-dependent glutamine uptake into basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 ± 66 control vs. 651 ± 180 pmol · mg−1· min−1acidosis; BBMV, 1,112 ± 189 control vs. 1,652 ± 148 pmol · mg−1· min−1acidosis, both P < 0.05). Na+-independent uptake was unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase in Na+-dependent glutamine uptake was eliminated by histidine, confirming transport by system N. SN1 protein was detected only in BLMV and BBMV from acidotic rats. After recovery from acidosis, SN1 mRNA and protein and Na+-dependent glutamine uptake activity rapidly returned to control levels. These data provide evidence that regulation of expression of the SN1 amino acid transporter is part of the renal homeostatic response to acid-base imbalance.
Functional and molecular characterization of the shark renal Na-K-Cl cotransporter: novel aspects
Authors: Gagnon Édith , Forbush Biff , Flemmer Andreas W. , Giménez Ignacio , Caron Luc , Isenring Paul
Published: March 2015

The Na-K-Cl cotransporter isoform 1 (NKCC1) has been isolated from several species, including Squalus acanthias. A second kidney-specific isoform (NKCC2) has been cloned mainly from higher vertebrates. Here, we have isolated the S. acanthias NKCC2 and found that it is produced in at least four spliced variants (saNKCC2A, saNKCC2F, saNKCC2AF, and saNKCC2AFno8) of ∼1,090 residues. Expression of these transcripts in Xenopus laevis oocytes revealed that only the A and F variants are functional and that they are more active after incubation in low-Cl or hyperosmolar media. Rates of activation after exposure to these media were exceptionally rapid, demonstrating for the first time that the NKCC2 itself represents an important site of regulation by Cl and that extracellular domains are involved. Another remarkable finding in this study was the failure to identify NKCC2B, a variant found in the kidney of higher vertebrates and expressed specifically in macula densa cells. This result, in conjunction with the fact that the shark kidney lacks a well-developed juxtaglomerular apparatus, suggests that the B exon evolved as a result of selective pressure (presumably by exon duplication) and that a restricted relationship exists between NKCC2B and macula densa.
Regulation of protein synthesis by IGF-I in proximal tubular epithelial cells
Authors: Senthil Duraisamy , Choudhury Goutam Ghosh , Abboud Hanna E. , Sonenberg Nahum , Kasinath Balakuntalam S.
Published: March 2015

Protein synthesis is required for renal hypertrophy, and proximal tubular epithelial cells are an important cell type involved in this process. We examined IGF-I regulation of protein synthesis in murine proximal tubular epithelial (MCT) cells. We focused on initial events in protein translation and the signaling events involved. Translation of capped mRNAs is under the control of eukaryotic initiation factor 4E (eIF4E). In the resting cell, eIF4E is normally kept in an inactive state by binding to 4E-BP1, its binding protein. Phosphorylation of 4E-BP1 results in dissociation of the eIF4E-4E-BP1 complex allowing eIF4E to initiate peptide synthesis. IGF-I stimulated protein synthesis, augmented phosphorylation of 4E-BP1 and promoted the dissociation of eIF4E from 4E-BP1. IGF-I stimulated the activities of phosphatidylinositol (PI) 3-kinase, Akt, and ERK1/2-type MAPK in MCT cells. IGF-I-induced phosphorylation of 4E-BP1, dissociation of the 4E-BP1-eIF4E complex, and increase in protein synthesis required activation of both PI 3-kinase and ERK pathways. Furthermore, ERK activation by IGF-I was also PI 3-kinase dependent. Transfection with the Thr37,46→Ala37,46mutant of 4E-BP1 showed that phosphorylation of Thr37,46residues was required for IGF-I induction of protein synthesis in MCT cells. Our observations reveal the importance of initial events in protein translation in IGF-I-induced protein synthesis in MCT cells and identify the regulatory signaling pathways involved.