bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous telemetric blood pressure monitoring and glomerular injury in the rat remnant kidney model
Authors: Bidani A. K. , Griffin K. A. , Picken M. , Lansky D. M.
Published: December 2017

The pathogenesis of glomerular injury in the remnant kidney (RK) model remains controversial. Increased glomerular transmission of systemic hypertension has been postulated to be an important pathogenic mechanism, but the precise relationship between systemic pressures and glomerular injury has not been defined because of the limitations of the tail-cuff method. Systolic blood pressure (BP) was continuously recorded radiotelemetrically at 10-min intervals for 6 wk in rats after approximately 5/6 renal ablation (n = 16) or sham ablation (n = 7). Overall mean systolic BP in RK rats was significantly higher than sham (138 +/- 3.3 vs. 117 +/- 1.3 mmHg, P < 0.01). Additionally, marked lability of systolic BP was observed in RK rats as compared with sham rats. Glomerular injury was essentially confined to RK rats, but the percentage of injured glomeruli ranged between 1 and 55%. Glomerular injury in individual animals was strongly correlated (r = 0.88) with the mean systolic BP during the last approximately 4 wk and with the frequency of systolic BP readings of > 140 mmHg. These data strongly suggest that transmission of systemic hypertension to the renal microvasculature plays a predominant role in the pathogenesis of glomerular injury in the RK model and also support the potential usefulness of the radiotelemetric technique to investigate hypertensive target organ injury.
Regulation of intracellular pH in two cell populations of inner stripe of rabbit outer medullary collecting duct
Authors: Weiner I. D. , Wingo C. S. , Hamm L. L.
Published: December 2017

The inner stripe of the outer medullary collecting duct (OMCDis) is a major site of HCO3- reabsorption and urinary acidification. Whether this nephron segment consists of a single or multiple cell types remains unclear. Apical incubation of rabbit OMCDis via luminal perfusion with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester resulted in heterogeneous fluorescence, suggesting two cell types. This heterogeneity was not prevented by inhibition of either carbonic anhydrase or organic anion transport. Subsequent studies were directed at characterizing the major intracellular pH (pHi) regulatory transporters in these two cell populations. Both cell populations demonstrated similar rates of Na+/H+ exchange, as assessed by peritubular Na(+)-dependent, amiloride-sensitive pHi recovery from an intracellular acid load. In contrast, Na(+)-independent, HCO3(-)-independent pHi recovery from an acid load was present in both cell populations but had two to three times greater activity in a minority cell population. In vivo deoxycorticosterone acetate administration increases this rate in both populations but to a greater extent in the minority cell population. In CO2/HCO3(-)-containing solutions, Cl- removal from the peritubular solution caused 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive alkalinization of all cells. Again, the magnitude and rate of alkalinization were significantly greater in the minority cell population. These studies demonstrate that the OMCDis consists of qualitatively similar cells in different states of functional activity. Although they are similar in most characteristics, a minority of cells more actively secrete H+ (independent of Na+) and reabsorb HCO3-.
Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure
Authors: Schaudies R. P. , Nonclercq D. , Nelson L. , Toubeau G. , Zanen J. , Heuson-Stiennon J. A. , Laurent G.
Published: December 2017

The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic signals triggering regenerative hyperplasia after renal injury.
Inhibition by epinephrine of AVP- and cAMP-stimulated Na+ and water transport in Dahl rat CCD
Authors: Hawk C. T. , Kudo L. H. , Rouch A. J. , Schafer J. A.
Published: December 2017

We examined the effects of epinephrine in perfused cortical collecting ducts (CCD) isolated from inbred Dahl-Rapp salt-sensitive (SS) and salt-resistant (SR) rats and from Sprague-Dawley (SD) rats. Rats were treated with 2.5 mg deoxycorticosterone pivalate (DOC; depot injection 4-9 days before study), and the CCD were treated with 220 pM vasopressin (AVP) to maximize Na+ transport. In CCD from all three strains 10 microM epinephrine in the bathing solution completely inhibited net Na+ transport, osmotic water permeability (Pf), and transepithelial voltage. In the SS CCD, epinephrine increased the fractional resistance of the luminal membrane to the same extent as 10 microM amiloride, indicating that it blocked the amiloride-sensitive conductance of the luminal membrane. Even at 100 nM epinephrine inhibited 80-100% of Na+ and water transport, and 1 microM yohimbine reversed or prevented these effects. In SS CCD, 0.1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) plus 0.1 mM 3-isobutyl-1-methylxanthine in place of AVP increased lumen-to-bath Na+ flux (J1-->b) from 56 +/- 5 to 143 +/- 3 pmol.min-1 x mm-1 and Pf from 6 +/- 12 to 1067 +/- 152 microns/s, but 100 mM epinephrine still significantly inhibited cAMP-stimulated J1-->b and Pf by 40 +/- 5% and 31 +/- 9%, respectively. Similar results were observed in the SR and SD rat CCD; however, the ability of yohimbine to reverse the epinephrine effect on cAMP-dependent transport was variable among the rat strains. We conclude that epinephrine acts via an alpha 2-receptor to inhibit adenylate cyclase but that at least one additional intracellular second messenger system may be involved.
Regulation of Na(+)-3HCO3- cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor
Authors: Takeda M. , Yoshitomi K. , Imai M.
Published: December 2017

We investigated the role of adenosine A1-receptor in the regulation of basolateral Na(+)-3HCO3- cotransporter in the rabbit proximal convoluted tubule (PCT) microperfused in vitro by monitoring basolateral membrane potential and intracellular pH. FK-453, a highly specific A1 antagonist, inhibited basolateral HCO3- conductance in a concentration-dependent manner (10(-10)-10(-5) M). Other A1 antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 10(-5) M and theophylline at 10(-3) M, also had similar effects. N6-cyclohexyladenosine (CHA) at 10(-7) M attenuated the effect of low concentration (10(-8) M) of FK-453. Either enhancement of the degradation of adenosine by 0.1 U/ml adenosine deaminase (ADA) or inhibition of adenosine release from the cells by 10(-6) M S-(4-nitrobenzyl)-6-thioinosine (NBTI) mimicked the effects of A1 antagonists. These observations suggest that endogenous adenosine is released from PCT cells and stimulates Na(+)-3HCO3- cotransporter. Both 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 10(-6) M forskolin also inhibited basolateral HCO3- conductance. Both 10(-6) M FK-453 and 10(-4) M CPT-cAMP decreased the initial rate as well as the magnitude of intracellular acidification induced by reduction of peritubular HCO3- concentration from 25 to 0 mM. Neither 10(-6) M FK-453 nor 10(-7) M CHA changed intracellular Ca2+ concentration as measured by fura-2 fluorescence. These results indicate that adenosine might stimulate HCO3- exit across the basolateral membrane through Na(+)-3HCO3- cotransporter by decreasing intracellular cAMP via A1-receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelin-1 mRNA in glomerular and epithelial cells of kidney
Authors: Chen M. , Todd-Turla K. , Wang W. H. , Cao X. , Smart A. , Brosius F. C. , Killen P. D. , Keiser J. A. , Briggs J. P. , Schnermann J.
Published: December 2017

To examine the question of the tubular localization of renal endothelin-1 (ET-1) mRNA, cDNA generated by reverse transcription of isolated rat tubule RNA was amplified by polymerase chain reaction using rat ET-1-specific oligonucleotides. Product identity was determined by restriction enzyme digestion or direct product sequencing. ET-1 mRNA was found to increase in renal tissue in a corticomedullary direction. High levels of ET-1 mRNA were found in dissected glomeruli and in juxtaglomerular cells in short-term primary culture. Among tubule segments, ET-1 mRNA was most abundant in inner medullary collecting ducts (IMCD), but products were also found with cDNA derived from proximal convoluted and straight tubules, thick ascending limbs, and outer medullary collecting ducts. In kidneys of untreated, homozygous Brattleboro rats, the increase of ET-1 mRNA along the corticomedullary axis as well as the preponderance of tubular ET-1 mRNA in IMCD was not observed. Our data show that ET-1 mRNA is present in all nephron segments studied and that its expression may be dependent on the functional state of the kidney. Our results are consistent with the proposal that ET-1 modifies tubular function in an autocrine or paracrine fashion.
Stimulation of alpha 2-adrenergic receptors increases Na(+)-K(+)-ATPase activity in distal convoluted tubule cells
Authors: Gesek F. A.
Published: December 2017

The influence of alpha-adrenoceptor subtypes on Na+ transport in distal convoluted tubules (DCT) has not been examined due to the difficulty of isolating and quantifying responses in this segment. These experiments were designed to test the hypothesis that alpha-adrenergic receptors stimulate Na+ absorption in DCT cells. Norepinephrine and epinephrine increased 22Na+ uptake into immortalized mouse DCT cells by 49 and 55% compared with basal uptake. Selective alpha 2-agonists (guanabenz, clonidine, and B-HT 933) stimulated 22Na+ uptake by 39-45%, but alpha 1-agonists had no effect. alpha 2-Agonist-stimulated 22Na+ uptake was abolished with alpha 2-antagonists (yohimbine, idazoxan). The entry pathways for alpha 2-agonist-stimulated 22Na+ uptake were determined with the NaCl cotransport inhibitor, chlorothiazide (10(-4) M), and the Na+ channel blocker, amiloride (10(-6) M). Agonist-stimulated 22Na+ uptake was inhibited 42 +/- 5 with chlorothiazide and 47 +/- 7% with amiloride. These results suggested that alpha 2-receptors may activate Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), resulting in an increased driving force for luminal Na+ entry through both pathways. Ouabain-suppressible 86Rb uptake and intracellular Na+ activity ([Na+]i; measured in single cells on glass cover slips loaded with the fluorescent probe sodium-binding benzofuran isophthalate) were used to measure Na(+)-K(+)-ATPase activity. alpha 2-Agonists significantly increased 86Rb uptake within 30 s. After 2.5 min, epinephrine and B-HT 933 decreased [Na+]i from a control level of 13 +/- 1 to 5 +/- 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Intracellular Ca2+ and PKC activation do not inhibit Na+ and water transport in rat CCD
Authors: Rouch A. J. , Chen L. , Kudo L. H. , Bell P. D. , Fowler B. C. , Corbitt B. D. , Schafer J. A.
Published: December 2017

Experiments examined the effects of elevation of intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC) on Na+ and water transport in the rat cortical collecting duct (CCD). We measured the lumen-to-bath 22Na+ flux (J1-->b), transepithelial voltage (VT), and water permeability (Pf) in CCD from deoxycorticosterone (DOC)-treated rats. Ionomycin (0.5 and 1 microM) and thapsigargin (1 and 2 microM) were used to increase [Ca2+]i. Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 microM) and oleoyl-acetyl-glycerol (OAG; 100 microM) were used as activators of PKC. [Ca2+]i was measured in isolated perfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin (AVP) failed to affect [Ca2+]i, whereas 1 microM ionomycin increased [Ca2+]i by 103 +/- 15% and 2 microM thapsigargin increased [Ca2+]i by 24 +/- 4%. In flux studies, neither ionomycin nor thapsigargin affected J1-->b or Pf, although ionomycin caused marked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100 microM OAG nor 1 microM PMA affected J1-->b or Pf. OAG at 50 microM had no effect on VT or transepithelial resistance, indicating no inhibition of conductive Na+ transport. We conclude that increased [Ca2+]i and PKC activation do not affect J1--b or Pf in the rat CCD. These findings may account for the sustained increase in J1--b produced in the rat CCD by AVP.
Na/Ca exchanger isoforms expressed in kidney
Authors: Kofuji P. , Lederer W. J. , Schulze D. H.
Published: December 2017

The cardiac (versus retinal rod) Na/Ca exchanger gene has been cloned, sequenced and shown by RNA analysis to be present in diverse tissues. Analysis of published sequences shows that a single isoform is found in heart tissue from many species (NACA1 isoform). We provide evidence here by ribonuclease (RNase) protection assays and by reverse transcriptase-polymerase chain reaction (PCR) amplification with sequence analysis that a new isoform encoding the Na/Ca exchanger is present in renal tissue. This isoform (NACA3) reveals a 7-amino acid deletion in the tested region compared with the NACA2 isoform described by Reilly and Shugrue [Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F1105-F1109, 1992] and is the dominant exchanger transcript in kidney. Analysis of the sequence of all isoforms indicates that the differences in the isoforms reside in the large intracellular loop region of the protein. Alternative splicing of a single Na/Ca exchanger message may be responsible for these tissue-specific transcripts.
Segmentally distinct effects of depolarization on intracellular [Ca2+] in renal arterioles
Authors: Carmines P. K. , Fowler B. C. , Bell P. D.
Published: December 2017

Experiments were performed to determine the influence of depolarization on intracellular Ca2+ concentration ([Ca2+]i) in renal arterioles and the possible role of voltage-gated Ca2+ channels in these responses. Glomeruli with attached arterioles and thick ascending limb were dissected from rabbit kidney and loaded with fura 2. [Ca2+]i of nonperfused arterioles was monitored using a microscope-based dual-excitation wavelength spectrofluorometry system. Afferent arteriolar [Ca2+]i averaged 150 +/- 11 nM (n = 20) when bathed in Ringer solution containing 1.5 mM Ca2+ and 5 mM K+. Replacement of the normal Ringer solution with one containing 100 mM K+ significantly increased afferent arteriolar [Ca2+]i to 196 +/- 12 nM. This response was abolished in the absence of extracellular Ca2+. In the presence of 1 microM nifedipine, 100 mM K+ elicited a 10% decrease in afferent arteriolar [Ca2+]i (P < 0.05). Thus nifedipine reversed the afferent [Ca2+]i response to depolarization, implicating voltage-gated Ca2+ channels as the influx pathway. In contrast to the behavior of afferent arterioles, the 100 mM K+ solution reduced efferent arteriolar [Ca2+]i from 188 +/- 17 to 148 +/- 13 nM (n = 11, P < 0.01), an effect that was not influenced by nifedipine. These observations support a role for voltage-gated Ca2+ channels in eliciting depolarization-induced increases in afferent arteriolar [Ca2+]i while failing to provide evidence for operation of such a mechanism at efferent arteriolar sites.
Na(+)-K(+)-ATPase that redistributes to apical membrane during ATP depletion remains functional
Authors: Molitoris B. A.
Published: December 2017

We have previously demonstrated using immunocytochemical, histochemical, and biochemical techniques that ischemia in vivo and ATP depletion in vitro result in dissociation of Na(+)-K(+)-adenosinetriphosphatase (ATPase) from the actin cytoskeleton and redistribution to the apical domain in renal proximal tubule cells. To directly evaluate whether apical Na(+)-K(+)-ATPase retained Na+ pumping activity, a rapidly reversible model of cellular ATP depletion in confluent LLC-PK1 cells grown on semipermeable membranes was utilized. Tight-junction integrity, monitored by electrical resistance, was lost during ATP depletion and reestablished during 2 h of ATP repletion. Total cellular Na(+)-K(+)-ATPase activity and total surface membrane [3H]ouabain binding remained constant, but specific apical [3H]ouabain binding increased (7 vs. 26 fmol/filter, P < 0.01). Apical [3H]ouabain binding returned to baseline during 5 h of ATP repletion. Apically applied ouabain was then used to selectively inhibit apical Na(+)-K(+)-ATPase. It had no effect on apical-to-basolateral Na+ flux under physiological conditions (1.3 +/- 0.61 vs. 1.27 +/- 0.46 meq.filter-1.30 min-1), but it increased the apical-to-basolateral flux in ATP-depleted and then repleted monolayers (0.39 +/- 0.12 vs. 0.83 +/- 0.27 meq.filter-1.30 min-1, P < 0.01), implying that apical Na(+)-K(+)-ATPase retained Na+ pumping activity. Together, these data imply that ATP depletion induces loss of surface membrane polarity resulting in redistribution of functional proteins to the alternate domain.
H(+)-K(+)-ATPase of rat inner medullary collecting duct in primary culture
Authors: Kleinman J. G. , Tipnis P. , Pscheidt R.
Published: December 2017

pH recovery in response to addition of and removal from NH4Cl was examined using 2',7'-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein fluorescence in primary cultures of inner medullary collecting duct (IMCD) cells from rat kidneys. In 0 K+, pH recovery rate was 0.012 +/- 0.010 U/min; in 5 mM K+, the recovery rate was greater at 0.065 +/- 0.013 U/min (P = 0.026). The H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) inhibitors omeprazole and Sch-28080 and the P-type ATPase inhibitor vanadate significantly inhibited pH recovery at 100, 10, and 5 microM, respectively. The vacuolar H(+)-ATPase inhibitor bafilomycin failed to inhibit pH recovery, but N-ethylmaleimide (NEM) did. A range of Sch-28080 concentrations inhibited ouabain-resistant ATPase activity of microsomes from these cells in a reverse sigmoidal manner, with little inhibition < 1 microM, virtually 100% inhibition > 100 microM, and a 50% inhibitory concentration of approximately 20 microM. Bafilomycin only produced significant inhibition of activity at concentrations well in excess of those that are effective against the vacuolar H(+)-ATPase. The ouabain-resistant ATPase activity in cultured IMCD was also sensitive to vanadate (90% inhibition with 5 microM) but relatively resistant to N,N'-dicyclohexylcarbodiimide and NEM. These results indicate that pH regulation in primary cultures of IMCD cells, presumably reflecting H+ transport, is predominantly due to an H(+)-K(+)-ATPase.
Peritubular endothelial cell proliferation in mice during compensatory renal growth after unilateral nephrectomy
Authors: Kanda S. , Hisamatsu H. , Igawa T. , Eguchi J. , Taide M. , Sakai H. , Kanetake H. , Saito Y. , Yoshitake Y. , Nishikawa K.
Published: December 2017

The proliferation of peritubular endothelial cells during compensatory renal growth (CRG) following unilateral nephrectomy in mice was investigated using a labeling index. The labeling index of peritubular endothelial cells increased 6 h after uninephrectomy and decreased to the normal level within 72 h. Immunohistochemical study revealed that c-myc protein was expressed in the nuclei of both cortical tubular cells and peritubular endothelial cells. Furthermore, intravenous injection of anti-basic fibroblast growth factor (bFGF) neutralizing antibody just after uninephrectomy led to significant inhibition of proliferation of peritubular endothelial cells, but not tubular cells. These results indicate that peritubular endothelial cells proliferate transiently during CRG and that bFGF plays an important role for the growth regulation of that cell in the kidney.
Differential expression and cellular distribution of mRNAs encoding alpha- and beta-isoforms of Na(+)-K(+)-ATPase in rat kidney
Authors: Ahn K. Y. , Madsen K. M. , Tisher C. C. , Kone B. C.
Published: December 2017

We have used in situ hybridization histochemistry with isoform-specific, digoxigenin-labeled cRNA probes to characterize systematically the cellular distribution of mRNAs encoding alpha- and beta-subunit isoforms of the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the normal rat kidney. Transcripts encoding the alpha 1-, alpha 2-, alpha 3-, beta 1-, and beta 2-subunits were detected in virtually all of the nephron segments, with prominent hybridization signal in the S3 segment of the proximal tubule, the cortical and medullary thick ascending limb of Henle's loop, the distal convoluted tubule, the cortical collecting duct along its entire length, and the renal pelvic epithelium. Several differences in the cell-specific pattern of expression of the various isoforms were observed. Among the alpha-isoforms, the alpha 3-subunit appeared to be preferentially expressed in the glomerular podocytes and mesangial cells, papillary interstitial cells, and renal pelvic epithelium. The beta-isoforms also differed in their distribution pattern, with the beta 1-subunit expressed to a greater degree in the glomerulus and renal pelvic epithelium and the beta 2-subunit preferentially expressed in the papillary interstitial cells and papillary surface epithelium. The detection and expression pattern of alpha- and beta-subunit mRNAs in structures throughout the kidney is compatible with the possibility of six structurally unique Na(+)-K(+)-ATPase isozymes and suggests a potentially greater role for isozymes comprised of the alpha 2-, alpha 3-, and beta 2-subunits in renal sodium and potassium transport.
Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus
Authors: Kollert-Jons A. , Wagner S. , Hubner S. , Appelhans H. , Drenckhahn D.
Published: December 2017

Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.
Basic amino acid transport in renal papilla: microinfusion of Henle's loops and vasa recta
Authors: Dantzler W. H. , Silbernagl S.
Published: December 2017

To determine whether basic amino acids, like acidic and neutral amino acids, could be reabsorbed distal to tips of Henle's loops and recycled between loops and vasa recta in the renal papilla, we continuously microinfused ascending Henle's loops and vasa recta with 14C-labeled L-lysine (L-Lys; 1.28 mM) or L-arginine (L-Arg; 1.17 mM) and 3H-labeled inulin. We also determined percent of recovered radiolabel as intact amino acid. Like acidic and neutral amino acids, relative to inulin, approximately 30% of L-Lys and approximately 45% of L-Arg microinfused into Henle's loops were reabsorbed. However, whereas radiolabeled L-Lys reabsorption, like reabsorption of acidic and neutral amino acids, was not readily inhibited, radiolabeled L-Arg reabsorption was reduced to approximately 25% by addition of unlabled L-Arg (50 mM) or L-homoarginine (L-Homo-Arg) (50 mM) to infusate. This observation provides greater evidence for specific, carrier-mediated reabsorption for L-Arg than for acidic or neutral amino acids. About 36% (relative to inulin) of each of these amino acids microinfused into ascending vasa recta apparently was transferred directly into ipsilateral tubular structures (probably thin descending limbs of Henle's loops). Transfer of radiolabeled L-Arg was reduced to approximately 8% by the inclusion of unlabeled L-Arg (50 mM) in infusate. Transfer of unlabeled L-Lys was unaffected by inclusion of unlabeled L-Lys (50 mM) in infusate but was reduced to approximately 20% by inclusion of unlabeled L-Arg (50 mM) or L-Homo-Arg (50 mM) in infusate. (ABSTRACT TRUNCATED AT 250 WORDS)
Angiotensin receptor sites in renal vasculature of rats developing genetic hypertension
Authors: Chatziantoniou C. , Arendshorst W. J.
Published: December 2017

The purpose of the present study was to characterize angiotensin II (ANG II) receptors in renal resistance vessels of young spontaneously hypertensive rats (SHR) ANG II receptor subtypes were evaluated in biochemical and functional terms using nonpeptide ANG II antagonists of the types AT1 (Dup-753 and Dup-532) and AT2 (PD-123319 and CGP-42112). In vitro radiolabeled ligand binding studies were performed on preglomerular resistance vessels freshly isolated from kidneys of SHR and Wistar-Kyoto (WKY) rats. The method of isolation and purification of renal microvessels was based on iron oxide infusion into the kidneys and separation of the vessels with the aid of a magnetic field followed by successive passages through various sized sieves. Physiological receptor expression was evaluated in vivo by measuring renal blood flow responses to ANG II injected alone and in a mixture with a receptor antagonist into the renal artery of indomethacin-treated rats. Our results indicate the existence of at least two functional (vasoconstriction mediating) subtypes of ANG II receptors sites in the renal microcirculation. Eighth percent of the ANG II receptor sites displayed high affinity to Dup-753 and Dup-532 and low affinity to PD-123319 and CGP-42112, whereas the remaining 20% of sites showed low affinity to Dup-753 and Dup-532 and CGP-42112 and intermediate affinity to PD-123319. In addition, the renal vasculature of young SHR and WKY displays similar ANG II receptor characteristics and identical blood flow responses to ANG II and to mixtures of ANG II and its antagonists.
Segmental differences in angiotensin receptor subtypes in interlobular artery of hydronephrotic rat kidneys
Authors: Hayashi K. , Suzuki H. , Saruta T.
Published: December 2017

Recent studies demonstrate that angiotensin II (ANG II)-induced vascular action is mediated preferentially by AT1 receptors. Although autoradiographic studies indicate the presence of AT2 receptors in large preglomerular vessels, functional roles for AT2 receptors in ANG II-induced renal vasoconstriction remain undetermined. We examined the effects of DuP-753 and PD-123319 on ANG II-induced vasoconstriction of interlobular arteries (ILA) in isolated perfused hydronephrotic rat kidneys to directly assess the AT1- and AT2-mediated action of ANG II on renal microvessels. Both DuP-753 (0.1-10 microM) and PD-123319 (0.1-10 microM) elicited dose-dependent vasodilation of ANG II-induced ILA constriction, with 86 +/- 4% and 36 +/- 4% inhibition by 10 microM DuP-753 and PD-123319, respectively. The reversal by DuP-753 of ANG II-induced ILA vasoconstriction was greater in small-caliber segments than in large-caliber segments. In contrast, the ability of PD-123319 (10 microM) to inhibit the vasoconstriction was augmented as the vessel diameter increased (slope = +0.46, correlation coefficient = +0.68; P < 0.01). Thus, although AT1 predominantly mediates the ANG II-induced ILA vasoconstriction, PD-123319-sensitive ANG II receptors (e.g., AT2 or AT1B) may also participate partly in the ILA vasoconstriction, particularly at large-caliber segments. In conclusion, distribution of ANG II receptor subtypes may differ depending on the size of the renal microvasculature.
Calcium and free radicals in hypoxia/reoxygenation injury of renal epithelial cells
Authors: Greene E. L. , Paller M. S.
Published: December 2017

Hypoxia and reoxygenation (H/R) generate oxygen free radicals that result in renal cell injury. We tested the roles of calcium and calmodulin in mediating xanthine oxidase-derived oxygen free radical production during H/R. Lowering extracellular Ca2+ attenuated lethal cell injury. H/R increased superoxide radical production over basal levels, whereas removing extracellular Ca2+ before hypoxia decreased superoxide radical production to basal levels. Pretreatment with either 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or thapsigargin, to inhibit release or deplete stores of intracellular Ca2+, did not affect injury following H/R. Ionomycin increased lactate dehydrogenase release during H/R but did not increase superoxide radical to levels greater than that observed for H/R alone. The calmodulin inhibitors trifluoperazine, calmidazolium, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide decreased cell injury to varying degrees. Trifluoperazine also decreased superoxide radical production during H/R and was shown to inhibit the conversion of xanthine dehydrogenase to xanthine oxidase. Cell injury and superoxide radical production correlated with cytosolic free Ca2+ during H/R as determined with the Ca(2+)-sensitive fluoroprobe indo 1. Cytosolic free Ca2+ increased slightly during hypoxia and showed a dramatic increase as soon as cells were reoxygenated. Cells incubated in a Ca(2+)-free medium actually showed a small decrease in intracellular Ca2++ despite H/R. In summary, Ca2+ derived from extracellular sources promoted superoxide radical production and renal cell injury by a calmodulin-dependent conversion of xanthine dehydrogenase to xanthine oxidase, a major source of oxygen free radicals during H/R.
Functional and cytoskeletal changes induced by sublethal injury in proximal tubular epithelial cells
Authors: Kroshian V. M. , Sheridan A. M. , Lieberthal W.
Published: December 2017

Mouse proximal tubular (MPT) cells in culture were subjected to ATP depletion by incubating them with cyanide in the absence of dextrose for 1 h. This insult resulted in marked alterations in the actin cytoskeleton. These changes were not associated with a decrease in cell viability and thus reflected sublethal injury. The effect of sublethal injury on the functional integrity of the intercellular tight junction (TJ) was then examined in MPT cell monolayers grown on permeable supports. During chemical anoxia, monolayer permeability to the paracellular marker mannitol progressively increased to 297 +/- 62% of baseline after 1 h. Chemical anoxia also caused a reversible loss in cell-substrate adhesion when MPT cells were studied as confluent monolayers or as single cells. Thus disruption of the actin cytoskeleton in nonlethally injured cells results in important reversible alterations in renal epithelial function characterized by impairment of the “gate” function of the TJ as well as impaired cell-substrate adhesion. We hypothesize that sublethal epithelial cell injury without accompanying necrosis may contribute to the decrement in renal function characteristic of ischemic renal injury.
Intracellular processing of immune complexes formed on the surface of glomerular epithelial cells
Authors: Singh A. K. , Rahman M. A.
Published: December 2017

Immune complexes formed on the surface of glomerular epithelial cells (GEC) resolve slowly and therefore result in inflammation of the glomerular capillary wall, as in the case of human membranous glomerulonephritis. The metabolic defect in the processing of these complexes has not been identified. Immune complexes of cationic bovine gamma-globulin (BGG) and anti-BGG were formed on cultured GEC, and their intracellular processing was followed by tracing the fate of radioiodinated and colloidal gold-labeled anti-BGG in the endosomal, lysosomal, and extracellular compartments. It was determined that the complexes were rapidly internalized in endosomes (50% saturation achieved in 10 min). The rate of expulsion of complexes was much slower (50% of internalized complex exteriorized in approximately 90 min). Of the internalized anti-BGG, < 5% were proteolytically degraded, suggesting an inefficient lysosomal processing. This aspect was studied further by separating anti-BGG colloidal gold-loaded vesicles by low-speed centrifugation and quantitating the lysosomal enzymes acid phosphatase and beta-galactosidase. At equilibrium approximately 10% of total cellular enzymes was associated with the vesicles. It was concluded that immune complexes are rapidly internalized by the GEC. However, lysosomal processing of the complexes is slow and inefficient. The majority of accumulated endosomes route back to the plasma membrane and discharge their contents in the medium in the form of free antibody.
Effect of U-73,122, an inhibitor of phospholipase C, on actions of parathyroid hormone in opossum kidney cells
Authors: Martin K. J. , McConkey C. L. , Jacob A. K. , Gonzalez E. A. , Khan M. , Baldassare J. J.
Published: December 2017

The relative roles of the adenylate cyclase-protein kinase A system (AC-PKA), the phospholipase C-protein kinase C system (PLC-PKC), and increases in cytosolic calcium in mediating the final actions of parathyroid hormone (PTH) remain ill defined. Although an important role for the PLC-PKC system in the regulation of phosphate transport in response to PTH has been suggested, previous studies from our laboratory and others, in OK cells, have emphasized the major role of AC-PKA. The present studies were designed to dissociate the second messengers for PTH by using an inhibitor of PLC (U-73,122). Studies were performed in confluent cultures of OK cells with and without preincubation with U-73,122 (1 microM). This inhibitor did not alter adenosine 3',5'-cyclic monophosphate (cAMP) production or the activation of PKA in response to PTH. Preincubation with U-73,122, however, totally abolished PTH-stimulated increases in diglyceride mass, consistent with inhibition of PLC. Activation of particulate PKC was then examined in response to PTH in the absence and presence of U-73,122. Although PTH resulted in an increase in particulate PKC activity in control cultures, this effect was abolished in the presence of U-73,122 and actually decreased significantly. Therefore, having documented marked attenuation of PLC-PKC, we next examined the effects of PTH on phosphate transport. Basal phosphate uptake was not altered by 1 microM U-73,122. Dose-response curves of the inhibition of phosphate transport in response to PTH were identical in the presence or absence of U-73,122. Thus inhibition of PLC and PKC activities did not alter the effects of PTH on phosphate transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow
Authors: Zou A. P. , Imig J. D. , Kaldunski M. , Ortiz de Montellano P. R. , Sui Z. , Roman R. J.
Published: December 2017

The present study evaluated the role of endogenous P-450 metabolites of arachidonic acid (AA) on autoregulation of renal blood flow in rats. Whole kidney and cortical blood flows were well autoregulated when renal perfusion pressure was varied from 150 to 100 mmHg. Infusion of 17-octadecynoic acid (17-ODYA) into the renal artery (33 nmol/min) increased cortical and papillary blood flows by 12.6 +/- 2.5 and 26.5 +/- 4.6%, respectively. After 17-ODYA, autoregulation of whole kidney and cortical blood flows was impaired. Intrarenal infusion of miconazole (8 nmol/min) had no effect on autoregulation of whole kidney, cortical, or papillary blood flows. 17-ODYA (1 microM) inhibited the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) and 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) by renal preglomerular microvessels in vitro by 83.7 +/- 7.4% and 89.0 +/- 4.9%, respectively. Miconazole (1 microM) reduced the formation of EETs by 86.4 +/- 5.7%, but it had no effect on the production of 20-HETE. These results suggest that endogenous P-450 metabolites of AA, particularly 20-HETE, may participate in the autoregulation of renal blood flow.
Endothelin-1 production by rat inner medullary collecting duct: effect of nitric oxide, cGMP, and immune cytokines
Authors: Kohan D. E. , Padilla E.
Published: December 2017

Nitric oxide (NO), guanosine 3',5'-cyclic monophosphate (cGMP), and endothelin-1 (ET-1) inhibit collecting duct sodium reabsorption. Because the inner medullary collecting duct (IMCD) synthesizes NO and ET-1, we examined NO and cGMP regulation of IMCD ET-1 production. S-nitroso-N-acetylpenicillamine (SNAP, 6 h) increased NO and cGMP and modestly reduced ET-1 release in cultured rat IMCD. Atrial natriuretic peptide or dibutyryl cGMP (6 h exposure to each) also mildly decreased IMCD ET-1 release. In long-term exposure studies, IMCD cells were incubated with tumor necrosis factor (TNF) and interferon-gamma (IFN) up to 72 h. IFN/TNF increased NO and cGMP production while reducing ET-1 release by 84%; N-monomethyl-L-arginine inhibited this effect only marginally, suggesting NO was not primarily involved. IFN alone greatly reduced IMCD ET-1 release and ET-1 mRNA levels. These data indicate that short- and long-term increases in NO and cGMP modestly reduce IMCD ET-1 production. Additionally, IFN potently inhibits IMCD ET-1 release by an undetermined mechanism.
Characterization of human uroguanylin: a member of the guanylin peptide family
Authors: Kita T. , Smith C. E. , Fok K. F. , Duffin K. L. , Moore W. M. , Karabatsos P. J. , Kachur J. F. , Hamra F. K. , Pidhorodeckyj N. V. , Forte L. R. , et al.
Published: December 2017

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins (ST), is an endogenous activator of guanylate cyclase C (GC-C). We have initiated a search for other members of the guanylin peptide family and in the current study describe a "guanylin-like peptide" from human urine. Bioactivity was monitored by determining the effect of urine extracts on T84 cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. Purification yielded two bioactive peaks of peptides that, when sequenced by NH2-terminal analysis, possessed 15 and 16 amino acids. The sequence of the smaller peptide represented an NH2-terminal truncation of the larger peptide. We have termed the larger peptide human uroguanylin; it has the following amino acid sequence: NDDCELCVNVACTGCL. Human uroguanylin shares amino acid sequence homology with guanylin and ST. Synthetic uroguanylin increased cGMP levels in T84 cells, competed with 125I-labeled ST for receptors, and stimulated Cl- secretion as reflected by an increased short-circuit current. Thus we report the isolation from human urine of a unique peptide, uroguanylin, that behaves in a manner similar to guanylin and appears to be a new member of this peptide family.
Basolateral taurine transport system in reptilian renal cells
Authors: Benyajati S. , Bay S. M.
Published: December 2017

The characteristics of taurine transport across renal basolateral membranes were examined, using basolateral membrane vesicles (BLMV) isolated from garter snake (Thamnophis spp.) kidneys. BLMV fraction exhibited high enrichment for the basolateral marker enzyme, Na(+)-K(+)-adenosinetriphosphatase (23-fold), with approximately 10% contamination by brush-border membranes. Taurine uptake into BLMV was specifically stimulated by inwardly directed Na+ gradient in the presence of Cl-. Equilibrium NaCl condition and replacement of NaCl gradient by KCl, choline chloride, NaSCN, sodium gluconate, or mannitol inhibited taurine uptake. Unlike brush-border membrane vesicles (BBMV), taurine uptake into BLMV was not stimulated by a Cl- gradient. In further contrast to BBMV, BLMV taurine uptake was smaller in magnitude and not electrogenic. The stoichiometric relationship between Na+ and BLMV taurine uptake, determined by activation method, indicated a 1 Na+:1 taurine interaction, in contrast to the 3 Na+:1 taurine stoichiometry for BBMV taurine transport. Bromcresol green inhibited BBMV taurine transport but had no effect on BLMV taurine uptake. Efflux of taurine from BLMV was faster than that from BBMV. Unlike BBMV, the BLMV efflux was stimulated by external taurine. The observed characteristics of taurine transport on both membranes would integratively result in net transepithelial reabsorption of taurine across renal cells of the garter snake, a species that demonstrates both net reabsorption and secretion of taurine in vivo.
Effects of epidermal growth factor and transforming growth factor-alpha on rabbit proximal tubule solute transport
Authors: Quigley R. , Baum M.
Published: December 2017

The present in vitro microperfusion study examined the direct effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on rabbit proximal convoluted tubule (PCT) solute transport. Tubules were perfused with an ultrafiltrate-like solution and bathed in an ultrafiltrate-like solution containing albumin. Albumin binding studies showed that both growth factors were highly protein bound with a free fraction of EGF of 0.31 +/- 0.04% and TGF-alpha of 1.08 +/- 0.15%. EGF at concentrations from 3 x 10(-11) M to 3 x 10(-8) M stimulated phosphate transport (JPhos) in a dose-dependent fashion but did not affect volume absorption (Jv) or bicarbonate transport (JtCO2). At 3 x 10(-7) M, EGF stimulated PCT Jv and JtCO2 in addition to the stimulation in JPhos. TGF-alpha stimulated Jv, JtCO2, and JPhos, but its effects were seen at a concentration that was 100-fold lower than that where EGF affected PCT transport. At 3 x 10(-13) M, TGF-alpha stimulated JtCO2, and at 3 x 10(-12) M, TGF-alpha also stimulated Jv and JPhos. EGF receptor downregulation with 3 x 10(-8) M EGF was able to block the effect of 3 x 10(-10) M TGF-alpha on Jv and JtCO2. Neither luminal EGF nor TGF-alpha had an effect on PCT transport. PCT bicarbonate and mannitol permeabilities were also not affected by either growth factor. These results demonstrate that EGF and TGF-alpha have direct effects on PCT solute transport.
Bicarbonate dependency of betaine synthesis in cultured LLC-PK1 cells
Authors: Moeckel G. W. , Lien Y. H.
Published: December 2017

Betaine, one of the major renal organic osmolytes, is synthesized from choline by choline dehydrogenase (EC 1.1.99.1) and betaine-aldehyde dehydrogenase (EC 1.2.1.8) in the kidney. A recent in vitro study has shown that betaine synthesis by renal cortical homogenate is dependent on millimolar amounts of bicarbonate. The present study was aimed to investigate the bicarbonate dependency of betaine formation in cultured LLC-PK1 cells. The data show that betaine formation increases in accordance with a rise in extracellular bicarbonate levels. The measured quantities of [14C]betaine synthesis ranged from 13.4 +/- 1.5 (4.6 mM HCO3-) to 38.0 +/- 1.4 pmol.micrograms protein-1.h-1 (24 mM HCO3-). The carbonic anhydrase inhibitor acetazolamide, added to the incubation medium to block bicarbonate transport, reduced betaine synthesis from choline by 41-49%. We conclude that betaine synthesis in LLC-PK1 cells is dependent on extracellular bicarbonate levels and is reduced by the inhibition of carbonic anhydrase. Because betaine accumulates in renal medulla during antidiuresis, our observations suggest a possible link between acid-base homeostasis and concentration mechanisms in the kidney.
Coupling between transepithelial Na transport and basolateral K conductance in renal proximal tubule
Authors: Beck J. S. , Laprade R. , Lapointe J. Y.
Published: December 2017

A common feature of sodium-reabsorbing epithelia is their ability to match salt entry to salt exit. It is recognized that a key strategy to perform this feat involves the coupling between basolateral sodium pump and potassium conductance (pump-leak coupling). In the renal proximal tubule this coupling is of major importance, as regions of this nephron segment are faced with ever-changing reabsorptive loads. An understanding of this coupling can be facilitated by critically examining those studies that have looked at the problem from the point of view of the whole cell (macroscopic studies) and of single channels (microscopic studies). An overview of such work suggests that the transduction mechanisms which are likely to effect pump-leak coupling in the renal proximal tubule involve cell volume, ATP, and pH (but not calcium). Although the relationship between ATP and potassium conductance may be relatively straightforward, the involvement of pH is likely to be only transient and that of volume remains controversial, occurring either directly though stretch-activated channels in amphibian preparations or indirectly through an as yet unidentified second messenger system in mammalian preparations.