bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of H+-K+-ATPase by CO2 requires a basolateral Ba2+-sensitive pathway during K restriction
Authors: Zhou Xiaoming , Lynch I. Jeanette , Xia Shen-Ling , Wingo Charles S.
Published: December 2017

We studied the activation of H+-K+-ATPase by CO2 in the renal cortical collecting duct (CCD) of K-restricted animals. Exposure of microperfused CCD to 10% CO2 increased net total CO2 flux ( J t CO2 ) from 4.9 ± 2.1 to 14.7 ± 4 pmol · mm−1· min−1 ( P < 0.05), and this effect was blocked by luminal application of the H+-K+-ATPase inhibitor Sch-28080. In the presence of luminal Ba, a K channel blocker, exposure to CO2 still stimulated J t CO2 from 6.0 ± 1.0 to 16.8 ± 2.8 pmol · mm−1 · min−1 ( P < 0.01), but peritubular application of Ba inhibited the stimulation. CO2substantially increased 86Rb efflux (a K tracer marker) from 93.1 ± 23.8 to 249 ± 60.2 nm/s ( P < 0.05). These observations suggest that during K restriction 1) the enhanced H+-K+-ATPase-mediated acidification after exposure to CO2 is dependent on a basolateral Ba-sensitive mechanism, which is different from the response of rabbits fed a normal-K diet, where activation of the H+-K+-ATPase by exposure to CO2 is dependent on an apical Ba-sensitive pathway; and 2) K/Rb absorption via the apical H+-K+-ATPase exits through a basolateral Ba-sensitive pathway. Together, these data are consistent with the hypothesis of cooperation between H+-K+-ATPase-mediated acidification and K exit pathways in the CCD that regulate K homeostasis.
l-Dopa uptake and dopamine production in proximal tubular cells are regulated by β2-adrenergic receptors
Authors: Carranza Andrea , Nowicki Susana , Barontini Marta , Armando Ines
Published: December 2017

This study assessed the role of adrenergic receptors on the regulation of the uptake of l-dopa and the production of dopamine by renal tubular cells. Scatchard analysis showed two l-dopa uptake sites with different affinities ( Km0.316 vs 1.53 μM). l-Dopa uptake was decreased by the nonselective adrenergic agonists epinephrine or norepinephrine (40%), by the β-selective agonist isoproterenol or the β2-selective agonist terbutaline (60%), but not by α-selective agonists (all 1 μM). The effect of norepinephrine, isoproterenol, or terbutaline was unaffected by addition of the β1-antagonist atenolol, abolished by ICI-118,551, a β2-antagonist (both 0.1 μM), and mimicked by the addition of dibutyryl-cAMP (1 μM). Preincubation with terbutaline decreased the number of high-affinity uptake sites ( Vmax= 1.10 ± 0.3 vs. 0.5 ± 0.1 pmol · mg protein−1· min−1) without changing their affinity. Norepinephrine or terbutaline decreased dopamine production by isolated cells, and this effect was abolished by ICI-118,551 (0.1 μM). In vivo administration of ICI-118,551 reduced the urinary excretion of l-dopa and increased the excretion of 3,4-dihydroxyphenylacetic acid without significant changes in plasma l-dopa concentrations. These results demonstrate that stimulation of β2-adrenergic receptors decreases the number of high-affinity l-dopa uptake sites in isolated tubular cells resulting in a reduction of the uptake of l-dopa and the production of dopamine and provide evidence for the presence of this mechanism in the intact animal.
Glomerular size and charge selectivity in the rat as revealed by FITC-Ficoll and albumin
Authors: Ohlson Maria , Sörensson Jenny , Haraldsson Börje
Published: December 2017

The fractional clearances (θ) for FITC-Ficoll and albumin were estimated in isolated perfused rat kidneys in which the tubular activity was inhibited by low temperature (8°C) and/or 10 mM NH4Cl. The Ficoll data were analyzed according to a two-pore model giving small and large pore radii of 46 Å and 80–87 Å, respectively. The estimated negative charge density was 35–45 meq/l at 8°C. Perfusion with erythrocyte-free solutions of kidneys at 37°C reduced glomerular size and charge permselectivity. Thus the large pore fraction of the glomerular filtrate (fL) was 1.64% at 37°C compared with 0.94% at 8°C. The θ for albumin was four times higher at 37°C than at 8°C (0.86% vs. 0.19%, respectively). NH4Cl caused further irreversible damage to the glomerular barrier. We conclude that there are no deleterious effects on the glomerular barrier of a reduction in temperature from 37°C to 8°C. Therefore our data seem to disprove the hypothesis of low glomerular permselectivity and transtubular uptake of intact albumin and support the classic concept of a highly selective glomerular barrier.
Nitric oxide modulates mechanical strain-induced activation of p38 MAPK in mesangial cells
Authors: Ingram A. J. , James L. , Thai K. , Ly H. , Cai L. , Scholey J. W.
Published: December 2017

Mesangial cells (MC), grown on extracellular matrix (ECM) protein-coated plates and stretched, proliferate and produce ECM, recapitulating in vivo responses to increased glomerular capillary pressure (Pgc). Transduction of strain involves mitogen-activated protein kinases (MAPK), and we have shown that p38 MAPK is activated by strain in MC. Because in vivo studies show that nitric oxide (NO) in the remnant kidney limits glomerular injury without reducing Pgc, we studied whether NO attenuated stretch-induced p38 activation in MC. Increasing p38 activation occurred with increasing stretch, maximally at 10 min at −27-kPa vacuum. Cyclic strain increased nuclear translocation of phosphorylated p38 by immunofluorescent microscopy and nuclear protein binding to nuclear factor-κB (NF-κB) consensus sequences by mobility shift assay. Both events were largely abrogated by the p38 inhibitor SB-203580. The NO donors 3-morpholinosydnonimine, S-nitroso- N-acetylpenicillamine, and 8-bromoguanosine 3′,5′-cyclic monophosphate, a stable cGMP analog, prevented p38 activation and nulcear translocation. Thus strain induces p38 activity and translocation to the nucleus and p38-dependent increases in nuclear protein binding to NF-κB. This pathway is attenuated by the NO donors or a cGMP analog.
Influence of nephron mass in development of chronic renal failure after prolonged warm renal ischemia
Authors: Cruzado Josep M. , Torras Joan , Riera Marta , Herrero Immaculada , Hueso Miguel , Espinosa Luís , Condom Enric , Lloberas Núria , Bover Jordi , Alsina Jeroni , Grinyó Josep M.
Published: December 2017

The present study examined the long-term consequences of warm renal ischemia (WRI) with or without renal ablation. Male Sprague-Dawley rats (250–300 g) were subjected to 60 min of complete WRI by pedicle clamping and then followed for 52 wk. Animals were organized into four groups: rats in which both kidneys were subjected to warm ischemia (2WIK); rats with left WRI and right nephrectomy (1WIK); uninephrectomized rats with a left nonischemic kidney (1NK); and sham-operated rats (2NK). Additional animals were studied at 24 h, 7 days, and 16 and 32 wk. In the first week after WRI, rats from the 2WIK and 1WIK groups displayed a similar degree of acute renal damage. After recovering from acute renal failure, 1WIK rats developed progressive and severe proteinuria, whereas it was mild in the 2WIK group, as well as in the 1NK and 2NK groups. Only animals from the 1WIK group developed severe chronic renal failure, glomerulosclerosis, interstitial fibrosis, and upregulation of transforming growth factor-β1(TGF-β1) gene, which was associated with increased TGF-β1protein expression in tubular epithelial cells, arterioles, and in areas of mononuclear interstitial cell infiltrate. On the contrary, long-term renal TGF-β1expression, function, and histology were similar in 2WIK and 2NK rats. The present study shows that prolonged bilateral WRI, when both kidneys are retained in place, induces very mild long-term renal lesions as opposed to the severe renal scarring observed when WRI is combined with contralateral nephrectomy.
Shear stress-mediated NO production in inner medullary collecting duct cells
Authors: Cai Zheqing , Xin Jingdong , Pollock David M. , Pollock Jennifer S.
Published: December 2017

Recent evidence suggests that nitric oxide (NO) within the inner medullary collecting duct (IMCD) functions to regulate sodium and water reabsorption. Because fluid shear stress has been shown to increase NO production in endothelial and vascular smooth muscle cells, experiments were designed to determine whether a similar mechanism exists in IMCD cells. Cultured IMCD-3 cells derived from murine IMCD were subjected to 60 min of pulsatile shear stress. Nitrite production (2,3-diaminonaphthalene fluorometric assay) increased 12-, 16-, and 23-fold at 3.3, 10, and 30 dyn/cm2, respectively, compared with static control cultures. Preincubation with the non-isoform-specific NO synthase inhibitor nitro-l-arginine methyl ester reduced nitrite production by 83% in response to 30 dyn/cm2. Western blotting and immunofluorescence analysis of static IMCD-3 cell cultures revealed the expression of all three NO synthase isoforms (NOS-1 or neuronal NOS, NOS-2 or inducible NOS, and NOS-3 or endothelial NOS) in IMCD-3 cultures. These results indicate that NO production is modulated by shear stress in IMCD-3 cells and that fluid shear stress within the renal tubular system may play a role in the regulation of sodium and water excretion by control of NO production in the IMCD.
Roles of ANG II and bradykinin in the renal regional blood flow responses to ACE inhibition in sodium-depleted dogs
Authors: Omoro Sophia A. , Majid Dewan S. A. , El Dahr Samir S. , Navar L. Gabriel
Published: December 2017

The relative contributions of ANG II and bradykinin (BK) to the renal regional blood flow responses during angiotensin-converting enzyme (ACE) inhibition remain unclear. This study was performed to evaluate renal cortical (CBF) and medullary blood flow (MBF) responses to intrarterial administration of enalaprilat (33 μg · kg−1 · min −1) after blockade of the ANG II AT1 receptors with candesartan (100 μg) in 7 dogs fed a low-salt diet (0.01%) for 5 days. Laser-Doppler flowmetry was used to measure relative changes in CBF and MBF. Candesartan alone increased CBF (+20 ± 2%) and MBF (+22 ± 7%). Enalaprilat infusion after candesartan administration resulted in further increases in both CBF (+21 ± 5%) and MBF (+41 ± 8%). However, the relative changes in MBF were significantly greater ( P < 0.01) than those in CBF. Administration of the BK B2 receptor blocker icatibant (300 μg) after enalaprilat returned CBF and MBF to values seen with candesartan alone. These data support a substantive role for BK potentiation during ACE inhibitor-induced renal vasodilation in dogs maintained on a low-sodium diet, with a relatively greater effect on MBF compared to CBF.
Role of PGE2in α2-induced inhibition of AVP- and cAMP-stimulated H2O, Na+, and urea transport in rat IMCD
Authors: Rouch Alexander J. , Kudo Lúcia H.
Published: December 2017

PGE2inhibits osmotic water permeability ( Pf) in the rat inner medullary collecting duct (IMCD) via cellular events occurring after the stimulation of cAMP, i.e., post-cAMP-dependent events. The α2-agonists also inhibit Pfin the rat IMCD via post-cAMP-dependent events. The purpose of this study was to determine whether PGE2plays a role in α2-mediated inhibition of Pf, Na+, and urea transport in the rat IMCD. Isolated terminal IMCDs from Wistar rats were perfused to measure, in separate experiments, Pf, lumen-to-bath22Na+transport ( Jlb), and urea permeability ( Pu). Transport was stimulated with 220 pM arginine vasopressin (AVP) or 0.1 mM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP). Indomethacin was used to inhibit endogenous prostaglandin synthesis, and the α2-agonists clonidine, oxymetazoline, and dexmedetomidine were used to test the role of PGE2in the α2-mediated mechanism that inhibits transport. All agents were added to the bath. Indomethacin at 5 μM significantly elevated CPT-cAMP-stimulated Pf, Jlb, and Pu, and subsequent addition of 100 nM PGE2reduced these transport parameters. Indomethacin reversed α2inhibition of CPT-cAMP-stimulated Pf, Jlb, and Pu, and subsequent addition of PGE2reduced transport in each case. Indomethacin partially reversed α2inhibition of AVP-stimulated Pf, Jlb, and Pu, and PGE2reduced transport back to the α2-inhibited level. These results indicate that PGE2is a second messenger involved in the mechanism of transport inhibition mediated by α2-adrenoceptors via post-cAMP-dependent events in the rat IMCD.
Use of serial analysis of gene expression to generate kidney expression libraries
Authors: El-Meanawy M. Ashraf , Schelling Jeffrey R. , Pozuelo Fatima , Churpek Matthew M. , Ficker Eckhard K. , Iyengar Sudha , Sedor John R.
Published: December 2017

Chronic renal disease initiation and progression remain incompletely understood. Genome-wide expression monitoring should clarify mechanisms that cause progressive renal disease by determining how clusters of genes coordinately change their activity. Serial analysis of gene expression (SAGE) is a technique of expression profiling, which permits simultaneous, comparative, and quantitative analysis of gene-specific, 9- to 13-bp sequence tags. Using SAGE, we have constructed a tag expression library from ROP-+/+ mouse kidney. Tag sequences were sorted by abundance, and identity was determined by sequence homology searching. Analyses of 3,868 tags yielded 1,453 unique kidney transcripts. Forty-two percent of these transcripts matched mRNA sequence entries with known function, 35% of the transcripts corresponded to expressed sequence tag (EST) entries or cloned genes, whose function has not been established, and 23% represented unidentified genes. Previously characterized transcripts were clustered into functional groups, and those encoding metabolic enzymes, plasma membrane proteins (transporters/receptors), and ribosomal proteins were most abundant (39, 14, and 12% of known transcripts, respectively). The most common, kidney-specific transcripts were kidney androgen-regulated protein (4% of all transcripts), sodium-phosphate cotransporter (0.3%), renal cytochrome P-450 (0.3%), parathyroid hormone receptor (0.1%), and kidney-specific cadherin (0.1%). Comprehensively characterizing and contrasting gene expression patterns in normal and diseased kidneys will provide an alternative strategy to identify candidate pathways, which regulate nephropathy susceptibility and progression, and novel targets for therapeutic intervention.
Structure, function, and regional distribution of the organic cation transporter OCT3 in the kidney
Authors: Wu Xiang , Huang Wei , Ganapathy Malliga E. , Wang Haiping , Kekuda Ramesh , Conway Simon J. , Leibach Frederick H. , Ganapathy Vadivel
Published: December 2017

We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.
Inner medullary lactate production and accumulation: a vasa recta model
Authors: Thomas S. Randall
Published: December 2017

Since anaerobic glycolysis yields two lactates for each glucose consumed and since it is reported to be a major source of ATP for inner medullary (IM) cell maintenance, it is a likely source of “external” IM osmoles. It has long been known that such an osmole source could theoretically contribute to the “single-effect” of the urine concentrating mechanism, but there was previously no suggestion of a plausible source. I used numerical simulation to estimate axial gradients of lactate and glucose that might be accumulated by countercurrent recycling in IM vasa recta (IMVR). Based on measurements in other tissues, anaerobic glycolysis (assumed to be independent of diuretic state) was estimated to consume ∼20% of the glucose delivered to the IM. IM tissue mass and axial distribution of loops and vasa recta were according to reported values for rat and other rodents. Lactate ( P LAC) and glucose ( P GLU) permeabilities were varied over a range of plausible values. The model results suggest that P LAC of 100 × 10−5 cm/s (similar to measured permeabilities for other small solutes) is sufficiently high to ensure efficient lactate recycling. By contrast, it was necessary in the model to reduce P GLU to a small fraction of this value (1/25th) to avoid papillary glucose depletion by countercurrent shunting. The results predict that IM lactate production could suffice to build a significant steady-state axial lactate gradient in the IM interstitium. Other modeling studies (Jen JF and Stephenson JL. Bull Math Biol 56: 491–514, 1994; and Thomas SR and Wexler AS. Am J Physiol Renal Fluid Electrolyte Physiol 269: F159–F171, 1995) have shown that 20–100 mosmol/kgH2O of unspecified external, interstitial, osmolytes could greatly improve IM concentrating ability. The present study gives several plausible scenarios consistent with accumulation of metabolically produced lactate osmoles, although only to the lower end of this range. For example, if 20% of entering glucose is consumed, the model predicts that papillary lactate would attain about 15 mM assuming vasa recta outflow is increased 30% by fluid absorbed from the nephrons and collecting ducts and that this lactate gradient would double if IM blood flow were reduced by one-half, as may occur in antidiuresis. Several experimental tests of the hypothesis are indicated.
Mechanism of increases inl-kynurenine and quinolinic acid in renal insufficiency
Authors: Saito Kuniaki , Fujigaki Suwako , Heyes Melvyn P. , Shibata Katsumi , Takemura Masao , Fujii Hidehiko , Wada Hisayasu , Noma Akio , Seishima Mitsuru
Published: December 2017

Marked increases in metabolites of thel-tryptophan-kynurenine pathway, l-kynurenine and quinolinic acid (Quin), were observed in serum and cerebrospinal fluid (CSF) of both the rat and human with renal insufficiency. The mechanisms responsible for their accumulation after renal insufficiency were investigated. In patients with chronic renal insufficiency, elevated levels of serum l-kynurenine and Quin were reduced by hemodialysis. In renal-insufficient rats, Quin andl-kynurenine levels in serum, brain, and CSF were also increased parallel to the severity of renal insufficiency. Urinary excretion of Quin (3.5-fold) and l-kynurenine (2.8-fold) was also increased. Liver l-tryptophan 2,3-dioxygenase activity (TDO), a rate-limiting enzyme of the kynurenine pathway, was increased in proportion to blood urea nitrogen and creatinine levels. Kynurenine 3-hydroxylase and quinolinic acid phosphoribosyltransferase were unchanged, but the activities of kynureninase, 3-hydroxyanthranilate dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) were significantly decreased. Systemic administrations of pyrazinamide (ACMSDase inhibitor) increased serum Quin concentrations in control rats, demonstrating that changes in body ACMSDase activities in response to renal insufficiency are important factors for the determination of serum Quin concentrations. We hypothesize the following ideas: that increased serum l-kynurenine concentrations are mainly due to the increased TDO and decreased kynureninase activities in the liver and increased serum Quin concentrations are due to the decreased ACMSDase activities in the body after renal insufficiency. The accumulation of CSFl-kynurenine is caused by the entry of increased seruml-kynurenine, and the accumulation of CSF Quin is secondary to Quin from plasma and/or Quin precursor into the brain.
Stretch-induced mesangial cell ERK1/ERK2 activation is enhanced in high glucose by decreased dephosphorylation
Authors: Dlugosz J. A. , Munk S. , Kapor-Drezgic J. , Goldberg H. J. , Fantus I. G. , Scholey J. W. , Whiteside C. I.
Published: December 2017

Glomerular hypertension and hyperglycemia are major determinants of diabetic nephropathy. We sought to identify the mechanisms whereby stretch-induced activation of mesangial cell extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) is enhanced in high glucose (HG). Mesangial cells cultured on fibronectin Flex I plates in normal glucose (NG; 5.6 mM) or HG (30 mM), were stretched by 15% elongation at 60 cycles/min for up to 60 min. In HG, a 5-min stretch increased ERK1/ERK2 phosphorylation by 6.4 ± 0.4/4.3 ± 0.3-fold ( P < 0.05 vs. NG stretch). In constrast, p38 phosphorylation was increased identically by stretch in NG and HG. Unlike many effects of HG, augmentation of ERK activity by HG was not dependent on protein kinase C (PKC) as indicated by downregulation of PKC with 24-h phorbol ester or inhibition with bisindolylmaleimide IV. In both NG and HG, pretreatment with arginine-glycine-aspartic acid peptide (0.5 mg/ml) to inhibit integrin binding or with cytochalasin D (100 ng/ml) to disassemble filamentous (F) actin, significantly reduced phosphorylation of ERK1/ERK2 and p38. To determine whether the rate of mitogen-activated protein kinase dephosphorylation is affected by HG, cellular kinase activity was inhibited by depleting ATP. Post-ATP depletion, phosphorylation of ERK1/ERK2 was reduced to 36 ± 9/51 ± 14% vs. 9 ± 5/7 ± 6% in NG ( P < 0.05, n = 5). Thus stretch-induced ERK1/ERK2 and p38 activation in both NG and HG is β1-integrin and F-actin dependent. Stretch-induced ERK1/ERK2 is enhanced in high glucose by diminished dephosphorylation, suggesting reduced phosphatase activity in the diabetic milieu. Enhanced mesangial cell ERK1/ERK2 signaling in response to the combined effects of mechanical stretch and HG may contribute to the pathogenesis of diabetic nephropathy.
Voltage clamping single cells in intact Malpighian tubules of mosquitoes
Authors: Masia R. , Aneshansley D. , Nagel W. , Nachman R. J. , Beyenbach K. W.
Published: December 2017

Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage ( Vpc) was −86.7 mV, and input resistance ( Rpc) was 388.5 kΩ ( n = 49 cells). In six cells, Ba2+(15 mM) had negligible effects on Vpc, but it increased Rpcfrom 325.3 to 684.5 kΩ ( P< 0.001). In the presence of Ba2+, leucokinin-VIII (1 μM) increased Vpcto −101.8 mV ( P < 0.001) and reduced Rpcto 340.2 kΩ ( P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652.0 kΩ; apical membrane resistance, 340.2 kΩ; shunt resistance ( Rsh), 344.3 kΩ; transcellular resistance, 992.2 kΩ. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and Rsh(3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized Vpcfrom −94.3 to −10.7 mV ( P< 0.001) and reversibly increased Rpcfrom 412 to 2,879 kΩ ( P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.
Importance of aquaporin-2 expression levels in genotype -phenotype studies in nephrogenic diabetes insipidus
Authors: Kamsteeg E. J. , Deen P. M. T.
Published: December 2017

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus. Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect (DNE) on wild-type (WT) AQP2 water permeability ( Pf) but only at low expression levels. Here, we determined the cell biological basis for this requirement. Injection of different amounts of WT-AQP2 cRNAs revealed that a correlation between AQP2 protein levels and Pfis only obtained with low expression levels. In coexpression studies of WT- and mutant AQP2 proteins, higher expression levels of AQP2-R187C also exerted a DNE on the Pfof WT-AQP2. Immunoblot and immunoprecipitation analysis revealed that this DNE was caused by competitive inhibition of WT-AQP2 expression and escape of AQP2-R187C from the endoplasmic reticulum, resulting in oligomerization with WT-AQP2. Because many disease-related mutants of multimeric renal membrane transporters and channels are likely to be identified, our data provide important information for studying the effects of such mutants on the activity of WT transporters and channels in oocytes.
Gentamicin traffics rapidly and directly to the Golgi complex in LLC-PK1cells
Authors: Sandoval Ruben M. , Dunn Kenneth W. , Molitoris Bruce A.
Published: December 2017

To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK1) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for ∼20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.
Matrix metalloproteinases and their inhibitors regulate in vitro ureteric bud branching morphogenesis
Authors: Pohl Martin , Sakurai Hiroyuki , Bush Kevin T. , Nigam Sanjay K.
Published: December 2017

Mammalian kidney development is initiated by the mutual interaction between embryonic metanephric mesenchyme (MM) and the ureteric bud (UB), leading to tightly controlled UB branching morphogenesis. In a three-dimensional cell culture model, which employs MM cell-derived conditioned medium (BSN-CM) to induce UB cell branching morphogenesis in extracellular matrix (ECM) gels (Sakurai H, Barros EJ, Tsukamoto T, Barasch J, and Nigam SK. Proc Natl Acad Sci USA 94: 6279–6284, 1997), branching morphogenesis was inhibited by both chemical agents (ilomastat and 1,10-orthophenanthroline) and a physiological protein factor [tissue inhibitor of metalloproteinases (TIMP)-2], known to act as matrix metalloproteinase (MMP) inhibitors. In addition, UB branching was inhibited in isolated UB culture (Qiao J, Sakurai H, and Nigam SK. Proc Natl Acad Sci USA96: 7330–7335, 1999) by TIMP-2 and ilomastat, suggesting a direct role for MMPs in UB branching. Gelatin zymography and enzymatic measurement of MMP activity revealed that MMPs could originate from at least three different sources: the conditioned medium, the ECM, and the UB cells themselves. In the UB cells, transcription of several MMPs [gelatinase A (MMP2) and B (MMP9), stromelysin (MMP3), MT1-MMP] and TIMPs was altered by BSN-CM and changed as more complex branching structures formed. The ECM appeared to serve as both a reservoir for MMPs and modulated their expression because different ECM compositions altered the total MMP activity as well as specific subsets of MMPs expressed by the UB cells (as determined by zymography and Northern analysis). In the context of UB branching morphogenesis during kidney development, our data suggest a complex model in which soluble factors produced by the MM, in the context of specific ECM components, modulate the expression of specific subsets of MMPs and TIMPs in the UB, which alter as structures develop and the matrix environment changes. This suggests distinct roles for different subsets of MMPs and their inhibitors during different phases of branching morphogenesis.
Melatonin attenuates acute renal failure and oxidative stress induced by mercuric chloride in rats
Authors: Nava Mayerly , Romero Freddy , Quiroz Yasmir , Parra Gustavo , Bonet Lizette , Rodríguez-Iturbe Bernardo
Published: December 2017

We evaluated the effect of melatonin (Mel), a potent scavenger of reactive oxygen species, in the course of HgCl2-induced acute renal failure. Rats received by gastric gavage 1 mg/kg of Mel ( n = 21) or vehicle ( n = 21), 30 min before the subcutaneous injection of HgCl2(2.5 mg/kg). Rats were killed at 24, 48, and 72 h, and plasma creatinine (Scr), renal histology, proliferative activity, apoptosis, and superoxide-producing cells were studied. We also determined the renal content of malondialdehyde (MDA) and glutathione (GSH) and the activities of glutathione peroxidase and catalase. Mel pretreatment (Mel plasma levels of 3.40 ± 3.15 μg/ml at the time of HgCl2injection) prevented the increment in Scrand reduced tubular necrosis from 41.0 ± 10.5 to 4.2 ± 5.1% of proximal tubules ( P < 0.01). Apoptosis and postnecrotic proliferative activity were twice more intense in the group untreated with Mel. Increment in renal content of MDA and decrease in GSH resulting from HgCl2toxicity were prevented by Mel. Mel also induced an important reduction in superoxide-positive cells. In contrast to the beneficial effects of pretreatment with Mel, the administration of Mel in conjunction with HgCl2had no effect on the oxidative damage and did not prevent nephrotoxicity. We conclude that the beneficial effects of pharmacological doses of Mel are due to its antioxidant properties.
Concerted action of dopamine on renal and intestinal Na+-K+-ATPase in the rat remnant kidney
Authors: Vieira-Coelho M. A. , Serrão P. , Guimarães J. T. , Pestana M. , Soares-Da-Silva P.
Published: December 2017

The present study evaluated renal and intestinal adaptations in sodium handling in uninephrectomized (Unx) rats and the role of dopamine. Two weeks after uninephrectomy, the remnant kidney in Unx rats weighed 33 ± 2% more than the corresponding kidney in sham-operated (Sham) animals. This was accompanied by increases in urinary levels of dopamine and major metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid] and increases in maximal velocity values (169 vs. 115 nmol · mg protein−1· 15 min−1) for renal aromatic l-amino acid decarboxylase, the enzyme responsible for the synthesis of renal dopamine. High salt (HS) intake increased ( P < 0.05) the urinary excretion of dopamine and DOPAC in Unx and Sham rats. However, the urinary levels ofl-3,4-dihydroxyphenylalanine, dopamine, and DOPAC in Sham rats during HS intake were lower than in Unx rats. Blockade of dopamine D1receptors (Sch-23390, 2 × 30 μg/kg) reduced the urinary excretion of sodium in Unx (31% decrease) more pronouncedly than in Sham (19% decrease) rats. However, inhibition of renal Na+-K+-ATPase activity by dopamine was of similar magnitude in Unx and Sham rats. In parallel, it was observed that uninephrectomy resulted in a significant reduction in jejunal sodium absorption and Na+-K+-ATPase activity in jejunal epithelial cells. In jejunal epithelial cells from Sham rats, dopamine (1 μM) failed to inhibit Na+-K+-ATPase activity, whereas in Unx rats it produced a significant reduction. It is concluded that uninephrectomy results in increased renal dopaminergic activity and dopamine-sensitive enhanced natriuresis. Furthermore, it is suggested that decreased jejunal absorption of sodium may take place in response to partial renal ablation, as an example of renal-intestinal cross talk.
Renal water handling in rats with decompensated liver cirrhosis
Authors: Jonassen Thomas E. N. , Christensen Sten , Kwon Tae-Hwan , Langhoff Susanna , Salling Nanna , Nielsen Søren
Published: December 2017

The present study was performed to investigate the renal handling of water in rats with decompensated liver cirrhosis. Liver cirrhosis was induced by intraperitoneal administration of carbon tetrachloride twice weekly for 16 wk. Control rats were treated with vehicle. The cirrhotic rats developed severe disturbances in water homeostasis: urine production was decreased and hyperosmotic, the rats had significantly decreased plasma sodium concentration and ascites, and the ability to excrete an intravenous water load was significantly impaired. Plasma concentrations of vasopressin and aldosterone were increased. Mean arterial pressure, glomerular filtration rate (GFR), and fractional lithium excretion were decreased. Acute vasopressin type 2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 μg · kg−1· h−1) was performed during conditions whereby volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. The aquaretic response to OPC-31260 was similar in cirrhotic and control rats. However, the OPC 31260-induced rises in fractional water excretion (ΔV/GFR; +24%) and fractional distal water excretion (ΔV/CLi; +46%) were significantly increased in the cirrhotic rats, where V is flow rate and Δ is change. This suggests that vasopressin-mediated renal water reabsorption capacity was increased in the cirrhotic rats. Semiquantitative immunoblotting revealed that the expression of the vasopressin-regulated water channel aquaporin-2 was unchanged in membrane fractions of both whole kidney and inner medulla from cirrhotic rats. Together, these results suggest a relative escape from vasopressin on collecting duct water reabsorption in rats with decompensated liver cirrhosis.
Neuronal nitric oxide synthase and systemic vasodilation in rats with cirrhosis
Authors: Xu Lieming , Carter Ethan P. , Ohara Mamiko , Martin Pierre-Yves , Rogachev Boris , Morris Kenneth , Cadnapaphornchai Melissa , Knotek Mladen , Schrier Robert W.
Published: December 2017

Cirrhosis is typically associated with a hyperdynamic circulation consisting of low blood pressure, low systemic vascular resistance (SVR), and high cardiac output. We have recently reported that nonspecific inhibition of nitric oxide synthase (NOS) with nitro-l-arginine methyl ester reverses the hyperdynamic circulation in rats with advanced liver cirrhosis induced by carbon tetrachloride (CCl4). Although an important role for endothelial NOS (eNOS) is documented in cirrhosis, the role of neuronal NOS (nNOS) has not been investigated. The present study was carried out to specifically investigate the role of nNOS during liver cirrhosis. Specifically, physiological, biochemical, and molecular approaches were employed to evaluate the contribution of nNOS to the cirrhosis-related hyperdynamic circulation in CCl4-induced cirrhotic rats with ascites. Cirrhotic animals had a significant increase in water and sodium retention. In the aorta from cirrhotic animals, both nNOS protein expression and cGMP concentration were significantly elevated compared with control. Treatment of cirrhotic rats for 7 days with the specific nNOS inhibitor 7-nitroindazole (7-NI) normalized the low SVR and mean arterial pressure, elevated cardiac index, and reversed the positive sodium balance. Increased plasma arginine vasopressin concentrations in the cirrhotic animals were also repressed with 7-NI in association with diminished water retention. The circulatory changes were associated with a reduction in aortic nNOS expression and cGMP. However, 7-NI treatment did not restore renal function in cirrhotic rats (creatinine clearance: 0.76 ± 0.03 ml · min−1· 100 g body wt−1in cirrhotic rats vs. 0.79 ± 0.05 ml · min−1· 100 g body wt−1in cirrhotic rats+7-NI; P NS.). Taken together, these results indicate that nNOS-derived NO contributes to the development of the hyperdynamic circulation and fluid retention in cirrhosis.
Extracellular adenosine modulates a volume-sensitive-like chloride conductance in immortalized rabbit DC1 cells
Authors: Rubera Isabelle , Barrière Hervé , Tauc Michel , Bidet Michel , Verheecke-Mauze Catherine , Poujeol Chantal , Cuiller Béatrice , Poujeol Philippe
Published: December 2017

Cl−currents induced by cell swelling were characterized in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule by the whole cell patch-clamp techniques and by125I−efflux experiments. Exposure of cells to a hypotonic shock induced outwardly rectifying Cl−currents that could be blocked by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM DIDS, and by 1 mM diphenylamine-2-carboxylate.125I−efflux experiments showed that exposure of the monolayer to a hypotonic medium increased125I−loss. Preincubation of cells with LaCl3or GdCl3prevented the development of the response. The addition of 10 μM adenosine to the bath medium activated outwardly rectifying whole cell currents similar to those recorded after hypotonic shock. This conductance was inhibited by the A1-receptor antagonist 8-cyclopentyl-1,3-diproxylxanthine (DPCPX), LaCl3, or GdCl3and was activated by GTPγS. The selective A1-receptor agonist N6-cyclopentyladenosine (CPA) mimicked the effect of hypotonicity on125I−efflux. The CPA-induced increase of125I−efflux was inhibited by DPCPX and external application of LaCl3or GdCl3. Adenosine also enhanced Mn2+influx across the apical membrane. Overall, the data show that DC1 cells possess swelling- and adenosine-activated Cl−conductances that share identical characteristics. The activation of both conductances involved Ca2+entry into the cell, probably via mechanosensitive Ca2+channels. The effects of adenosine are mediated via A1receptors that could mediate the purinergic regulation of the volume-sensitive Cl−conductance.
Role of basolateral carbonic anhydrase in proximal tubular fluid and bicarbonate absorption
Authors: Tsuruoka Shuichi , Swenson Erik R. , Petrovic Snezana , Fujimura Akio , Schwartz George J.
Published: December 2017

Membrane-bound carbonic anhydrase (CA) is critical to renal acidification. The role of CA activity on the basolateral membrane of the proximal tubule has not been defined clearly. To investigate this issue in microperfused rabbit proximal straight tubules in vitro, we measured fluid and HCO3−absorption and cell pH before and after the extracellular CA inhibitor p-fluorobenzyl-aminobenzolamide was applied in the bath to inhibit only basolateral CA. This inhibitor was 1% as permeant as acetazolamide. Neutral dextran (2 g/dl, molecular mass 70,000) was used as a colloid to support fluid absorption because albumin could affect CO2diffusion and rheogenic HCO3−efflux. Indeed, dextran in the bath stimulated fluid absorption by 55% over albumin. Basolateral CA inhibition reduced fluid absorption (∼30%) and markedly decreased HCO3−absorption (∼60%), both reversible when CA was added to the bathing solution. In the presence of luminal CA inhibition, which reduced fluid (∼16%) and HCO3−(∼66%) absorption, inhibition of basolateral CA further decreased the absorption of fluid (to 74% of baseline) and HCO3−(to 22% of baseline). CA inhibition also alkalinized cell pH by ∼0.2 units, suggesting the presence of an alkaline disequilibrium pH in the interspace, which would secondarily block HCO3−exit from the cell and thereby decrease luminal proton secretion (HCO3−absorption). These data clearly indicate that basolateral CA has an important role in mediating fluid and especially HCO3−absorption in the proximal straight tubule.
Functional role of sodium-calcium exchange in the regulation of renal vascular resistance
Authors: Schweda Frank , Seebauer Helga , Krämer Bernhard K. , Kurtz Armin
Published: December 2017

Our study aimed to assess a possible functional role of the Na+/Ca2+ exchanger in the regulation of renal vascular resistance (RVR). Therefore, we investigated the effects of an inhibition of the Na+/Ca2+ exchanger either by lowering the extracellular sodium concentration ([Na+]e) or, pharmacologically on RVR, by using isolated perfused rat kidneys. Graded decreases in [Na+]e led to dose-dependent increases in RVR to 4.3-fold (35 mM Na+). This vasoconstriction was markedly attenuated by lowering the extracellular calcium concentration, by the L-type calcium channel blocker amlodipine or by the chloride channel blocker niflumic acid. Further lowering of [Na+]e to 7 mM led to an increase in RVR to 7.5-fold. In this setting, amlodipine did not influence the magnitude but did influence the velocity of vasoconstriction. Pharmacological blockade of the Na+/Ca2+ exchanger with KB-R7943, benzamil, or nickel resulted in significant vasoconstriction (RVR 2.5-, 1.8-, and 4.2-fold of control, respectively). Our data suggest a functional role of the Na+/Ca2+ exchanger in the renal vascular bed. In conditions of partial replacement of [Na+]e, vasoconstriction is dependent on chloride and L-type calcium channels. A total replacement of [Na+]e leads to a vasoconstriction that is nearly independent of L-type calcium channels. This might be due to an active calcium transport into the cell by the Na+/Ca2+ exchanger.
Intrarenal expression and distribution of cyclooxygenase isoforms in rats with experimental heart failure
Authors: Abassi Zaid , Brodsky Sergey , Gealekman Olga , Rubinstein Irith , Hoffman Aaron , Winaver Joseph
Published: December 2017

The generation of PGs from arachidonic acid is mediated by cyclooxygenase (COX), which consists of a constitutive (COX-1) and an inducible (COX-2) isoform. The present study evaluated the relative expression and immunoreactive levels of COX-1 and COX-2, by means of RT-PCR, Western blot analysis, and immunohistochemistry, in the renal cortex and medulla of rats with congestive heart failure (CHF), induced by the placement of an aortocaval fistula. In addition, we examined the effects of a COX-1 inhibitor (piroxicam), COX-2 inhibitor (nimesulide), and nonselective COX inhibitor (indomethacin) at a dose of 5 mg/kg, on intrarenal blood flow by laser Doppler flowmetry. COX-1 and COX-2 mRNAs were abundantly expressed in the renal medulla of control and CHF rats and only minimally in the cortex. Moreover, both RT-PCR (32–36 cycles) and Western blot techniques revealed upregulation of medullary COX-2, but not of COX-1, in rats with advanced heart failure. In line with these findings, all three tested COX inhibitors provoked significant and sustained decreases (Δ ≈ −20%) in medullary blood flow (MBF), which were similar in magnitude and duration in control animals. However, in CHF rats, indomethacin produced a greater reduction in MBF than that obtained with either piroxicam or nimesulide. Taken together, these results indicate that 1) both COX-1 and COX-2 are predominantly expressed in the renal medulla and 2) experimental CHF is associated with selective overexpression of COX-2. The latter may represent a mechanism aimed at defending MBF in the face of a decrease in renal perfusion pressure during the development of CHF.
Increased AT1receptor expression and mRNA in kidney glomeruli of AT2receptor gene-disrupted mice
Authors: Saavedra Juan M. , Häuser Walter , Ciuffo Gladys , Egidy Giorgia , Hoe Kwang-Lae , Jöhren Olaf , Sembonmatsu Takaaki , Inagami Tadashi , Armando Inés
Published: December 2017

The proposed feedback between angiotensin II AT2and AT1receptors prompted us to study AT1receptor expression in kidneys of male AT2receptor-gene disrupted mice ( agtr2 −/y). In wild-type ( agtr2 +/y) mice, AT1receptor binding and mRNA is abundant in glomeruli, and AT1receptor binding is also high in the inner stripe of the outer medulla. AT2receptors are scarce, primarily associated to cortical vascular structures. In agtr2 −/y mice, AT1receptor binding and mRNA were increased in the kidney glomeruli, and AT1receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT2receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT1receptor expression. AT1upregulation alone may explain the AT2gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT1/AT2receptor feedback could have clinical significance because AT1antagonists are widely used in medical practice.
Renal effects of serine-7 analog of lymphoguanylin in ex vivo rat kidney
Authors: Fonteles Manasses C. , Carrithers Stephen L. , Monteiro Helena S. A. , Carvalho Andre F. , Coelho Gustavo R. , Greenberg Richard N. , Forte Leonard R.
Published: December 2017

Guanylin and uroguanylin compose a family of natriuretic, diuretic, and kaliuretic peptides that bind to and activate apical membrane receptor guanylyl cyclase signaling molecules in renal and intestinal epithelia. Recently, a complementary DNA encoding an additional member of the guanylin family of cGMP-regulating peptides was isolated from lymphoid tissues of the opossum and was termed lymphoguanylin (LGN). A peptide analog of opossum LGN was synthesized containing a single disulfide bond with the internal cysteine-7 replaced by a serine residue (LGNCys7→Ser7). The biological activity of LGNSerwas tested by using a cGMP bioassay with cultured T84 (human intestinal) cells and opossum kidney (OK) cells. LGNSerhas potencies and efficacies for activation of cGMP production in the intestinal and kidney cell lines that are 100- and 1,000-fold higher than LGN, respectively. In the isolated perfused rat kidney, LGNSerstimulated a maximal increase in fractional Na+excretion from 24.8 ± 3.0 to 36.3 ± 3.3% 60 min after administration and enhanced urine flow from 0.15 ± 0.01 to 0.24 ± 0.01 ml · g−1· min−1. LGNSer(0.69 μM) also increased fractional K+excretion from 27.3 ± 2.3 to 38.0 ± 3.0% and fractional Cl−excretion from 26.1 ± 0.8 to 43.5 ± 1.9. A ninefold increase in the urinary excretion of cGMP from 1.00 ± 0.04 to 9.28 ± 1.14 pmol/ml was elicited by LGNSer, whereas cAMP levels were not changed on peptide administration. These findings demonstrate that LGNSer, which contains a single disulfide bond like native LGN, activates guanylyl cyclase-C (GC-C) receptors in T84 and OK cells and may be very helpful in studying the physiological importance of activation of GC-C in vivo. LGNSeralso exhibits full activity in the isolated perfused kidney equivalent to that observed previously with opossum uroguanylin, suggesting a physiological role for LGN in renal function. Thus the single amino acid substitution enhances the activity and potency of LGN.