bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular analysis of beta-adrenergic receptor subtypes in rat collecting duct: effects on cell cAMP and Ca2+ levels
Authors: Mandon B. , Siga E. , Champigneulle A. , Imbert-Teboul M. , Elalouf J. M.
Published: December 2017

Expression and functional properties of beta-adrenergic receptors (beta-ARs) were studied in rat collecting tubules isolated by microdissection. Reverse transcription-polymerase chain reaction experiments demonstrated that the beta 1- and beta 2-AR mRNAs, but not the beta 3-subtype, are expressed in the cortical collecting duct (CCD). Quantitation of mRNAs, carried out using mutant RNAs as internal standards, further showed that beta 1- and beta 2-ARs transcripts are present at comparable amounts in CCD (3,000–4,000 copies/mm of tubular length), but reach 6–8 times lower levels in the outer medullary collecting duct (OMCD: beta 1, 480 +/- 180; beta 2, 590 +/- 110 copies/mm of tubular length). Functional studies, carried out in CCD, corroborated the expression of these two receptor subtypes. The rank order of potency of beta-agonists for stimulating adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was isoproterenol > norepinephrine = epinephrine, and similar efficiencies were found for a beta 1- and a beta 2-antagonist to inhibit isoproterenol-dependent cAMP formation. Fura 2 fluorescence measurements revealed that isoproterenol (10 microM) induces a biphasic rise of intracellular free Ca2+ concentration ([Ca2+]i), consisting of an initial fast increase (delta [Ca2+]i = 122 nM) followed by a plateau phase (delta [Ca2+]i = 58 nM). In the absence of basolateral Ca2+, the initial peak was still observed, suggesting intracellular Ca2+ release. Norepinephrine and epinephrine, as well as selective beta 1- and beta 2-agonists, also increased [Ca2+]i in CCD. Only slight [Ca2+]i variations were produced by isoproterenol in the OMCD (delta [Ca2+]i = 21 nM) and the cortical thick ascending limb (delta [Ca2+]i = 25 nM). These results show that both beta 1- and beta 2-ARs are expressed in the collecting tubule and that they predominate in the CCD. The two receptor subtypes contribute to cAMP accumulation induced by beta-agonists. They also trigger [Ca2+]i variations, indicating their possible coupling to several transduction pathways in the rat CCD.
Luminal prostaglandin E2 modulates sodium and water transport in rabbit cortical collecting ducts
Authors: Ando Y. , Asano Y.
Published: December 2017

We have previously found that arginine vasopressin (AVP) acts not only from the basolateral side but also from the luminal side of the rabbit cortical collecting duct (CCD). In the present study, we examined whether prostaglandin E2 (PGE2), another classic and potent modulator of the collecting duct functions, exerts luminal actions in the rabbit CCD perfused in vitro. Although luminal prostaglandin I2 was inert, luminal PGE2 (> 1 nM) induced transient hyperpolarization of transepithelial voltage followed by sustained depolarization in a dose-dependent manner. This action was preserved in the presence of basolateral PGE2, luminal AVP, or luminal BaCl2, but abolished by basolateral ouabain or luminal amiloride. Furthermore, unlike luminal AVP, luminal PGE2 suppressed Na transport and increased osmotic water permeability. The present study suggests that PGE2, similar to AVP but in a different fashion, modulates transepithelial transports from both luminal and basolateral sites in the CCD in vivo.
Interaction of alpha-KG with basolateral organic anion transporter in isolated rabbit renal S3 proximal tubules
Authors: Shpun S. , Evans K. K. , Dantzler W. H.
Published: December 2017

To understand the basolateral p-aminohippurate (PAH) transporter in the S3 segment of rabbit proximal tubules and its relationship to the transporter in the S2 segment, we measured the 30-s uptake and efflux of PAH across the basolateral membrane of single isolated S3 segments at 37 degrees C in bicarbonate-buffered media. Kinetic analysis of uptake data revealed a concentration of PAH at one-half Jmax of approximately 107 microM (same as in the S2 segment) but a Jmax of 600 fmol.min-1.nl-1 (one-tenth that of S2 segment). The coefficient for efflux across the basolateral membrane was also only one-sixth to one-tenth of that in the S2 segment. These data suggest that the basolateral PAH transporter is the same in both segments but that there are fewer transporters in the S3 than in the S2 segment. However, the apparent inhibitor constant values for cis-inhibition by probenecid (approximately 29 microM in S3, approximately 15 microM in S2) and by alpha-ketoglutarate (alpha-KG) in the presence of LiCl (approximately 40 microM in S3, approximately 160 microM in S2) suggest that the transporters may not be identical in the two segments. In bicarbonate-buffered medium, preloading the tubules with 100 microM alpha-KG did not trans-stimulate PAH uptake across the basolateral membrane, whereas preloading with 1.0 microM alpha-KG caused a significant stimulation of 43%. However, in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered medium, preloading the tubules with 100 microM alpha-KG caused a twofold increase in PAH uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolic effects of fatty acid-bearing albumin on a proximal tubule cell line
Authors: Thomas M. E. , Morrison A. R. , Schreiner G. F.
Published: December 2017

In glomerular disease, fatty acids carried on albumin are taken up by the proximal tubule with filtered albumin. We postulate that the fatty acids carried on filtered albumin could contribute to the deleterious effects of proteinuria. The effects of fatty acid-albumin complexes on lipid metabolism have been studied in opossum kidney (OK) cells, a proximal tubule cell line. OK cells transported two-thirds of [14C]palmitate-albumin (5 mg/ml) intracellularly within 16 h. [14C]palmitate-albumin was distributed into phosphatidylcholines, phosphatidylinositols, and tri- and diglycerides. 14C-labeled unsaturated fatty acid albumins (oleate, linoleate, and arachidonate) showed preferential incorporation into triglycerides, with lesser incorporation into phospholipids. Studies of total lipid pools showed that fatty acid-albumin uptake produced a particularly marked increase in total triglyceride levels (approximately 10-fold). Oil red O staining of OK cells cultured with oleate-albumin showed a marked increase in intracellular lipid droplets, compared with cells cultured with delipidated albumin, consistent with triglyceride accumulation. Less than 1% of [14C]palmitate taken up was isolated as intracellular free fatty acid. Less than 5% of [14C]palmitate internalized was oxidized to 14CO2. Different fatty acids, when taken up by the OK cell, have distinct metabolic fates. Each fatty acid is incorporated in a characteristic fashion into certain complex lipids, possibly dependent on the presence or absence of double bonds. We propose that this may have functional consequences for the proximal tubule in the human nephrotic syndrome.
Urea transport in initial IMCD of rats fed a low-protein diet: functional properties and mRNA abundance
Authors: Ashkar Z. M. , Martial S. , Isozaki T. , Price S. R. , Sands J. M.
Published: December 2017

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844–847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)
Oxytocin as an antidiuretic hormone. II. Role of V2 vasopressin receptor
Authors: Chou C. L. , DiGiovanni S. R. , Luther A. , Lolait S. J. , Knepper M. A.
Published: December 2017

We conducted this study to determine what receptor mediates the effect of oxytocin to increase osmotic water permeability (Pf) in the rat inner medullary collecting duct (IMCD). Reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that mRNA for both the oxytocin receptor and the V2 receptor is present in the rat terminal IMCD. In isolated perfused IMCD segments, we found that the V2 vasopressin receptor antagonist [d(CH2)5(1),D-Ile2,Ile4,Arg8]vasopressin, but not oxytocin receptor antagonists, blocked the hydrosmotic response to 200 pM oxytocin. The selective oxytocin receptor agonist [Thr4,Gly7]oxytocin did not increase water permeability. Oxytocin also increased urea permeability in IMCD segments. Studies in IMCD suspensions showed that oxytocin increases adenosine 3',5'-cyclic monophosphate production in a dose-dependent fashion with a half-maximal (EC50) response at 5.2 nM. The dose-response curves were virtually identical for IMCD suspensions from Sprague-Dawley rats and Brattleboro rats. The oxytocin dose-response curve was displaced to the right of the vasopressin dose-response curve (EC50, 0.44 nM). From these results, we conclude that the V2 receptor mediates the hydrosmotic action of oxytocin in rat IMCD.
Inner medullary external osmotic driving force in a 3-D model of the renal concentrating mechanism
Authors: Thomas S. R. , Wexler A. S.
Published: December 2017

The mechanism by which the renal medulla establishes and maintains a gradient of osmolarity along the corticomedullary axis, especially in the inner medulla, where there is no active transmural flux out of the ascending limbs of Henle, remains a source of controversy. We show here that, if realistic values of urea permeability in the inner medullary descending limbs and water permeability in the upper inner medullary section of the collecting ducts are taken into account, even a model including the three-dimensional vascular bundle structures [A. S. Wexler, R. E. Kalaba, and D. J. Marsh. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F368-F383, 1991] fails to explain the experimentally observed inner medullary osmolality gradient. We show here that this failure can be overcome by application of an external osmotic driving force, an idea recently revived by J. F. Jen and J. L. Stephenson (Bull. Math. Biol. 56: 491-514, 1994) in the context of a single-solute, single-loop central core model. We show that inclusion of such an external driving force with a value equivalent to at least 100 mosM of inner medullary interstitial osmolytes in the three-dimensional model of Wexler et al. accounts for a physiological osmolality gradient, even in the face of realistic permeability values. Furthermore, inclusion of the external driving force makes the model less dependent on the positions of descending and ascending limbs of Henle with respect to the collecting ducts. In an effort to assess whether there is any experimental basis for osmolytes, we show that a significant amount of extra inner medullary interstitial osmolytes is plausible, based on extrapolation from existing experimental data.
Subcellular distribution and membrane association of Rho-related small GTP-binding proteins in kidney cortex
Authors: Boivin D. , Beliveau R.
Published: December 2017

We have examined the subcellular distribution of Rho-related small GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP-binding proteins were found to be expressed at high levels in rat outer kidney cortex. Western blot analysis showed that these proteins were predominantly associated with brush-border and basolateral plasma membranes, with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a small fraction was associated with cytoskeletal elements. A GDP-dissociation inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionation of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with the RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of these proteins with RhoGDI at an estimated size of approximately 45 kDa after gel-filtration chromatography. However, a second peak of RhoA eluted as a 20-kDa protein, indicating that not all RhoA is complexed to RhoGDI. Addition of RhoA- and CDC42-enriched fractions to purified membranes from kidney cortex resulted in their translocation to the membranes and their carboxyl methylation. Both processes were stimulated by guanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effect on the translocation of RhoA to membranes, suggesting that this covalent modification is not required for association to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of osmotic perturbation on [Ca2+]i and pHi in rabbit proximal tubular cells in primary culture
Authors: Raat N. J. , van Os C. H. , Bindels R. J.
Published: December 2017

The effects of anisosmotic media on intracellular Ca2+ and H+ concentrations ([Ca2+]i and pHi, respectively) were studied to investigate whether these changes play a role in epithelial cell volume regulation. [Ca2+]i and pHi were measured in rabbit proximal tubular cells in primary culture using the fluorescent ratio probes fura 2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Changing medium osmolarity from 300 to 150 mosmol resulted in a rapid transient increase in fura 2 ratio from 0.89 +/- 0.02 to 1.15 +/- 0.03, which lasted for several minutes and returned to base line within 10 min. The source of Ca2+ was extracellular as well as intracellular. Simultaneous with this increase in [Ca2+]i, cells slowly acidified from pHi of 7.51 +/- 0.03 to 6.86 +/- 0.02. This osmotic swelling-induced acidification could not be explained by a decrease in the rate of Na+/H+ exchange or increase in the rate of Cl-/HCO3- exchange. Subsequently increasing medium osmolarity from 150 to 500 mosmol decreased the fura 2 ratio below the initial level observed in isotonic media, while pHi increased from 6.96 +/- 0.02 to 7.37 +/- 0.03. This decrease in [Ca2+]i was due to inhibition of Ca2+ influx and to an increase in Ca2+ efflux. The osmotic shrinkage-induced alkalinization was slightly inhibited by ethylisopropylamiloride, indicative of activation of Na+/H+ exchange. To test whether an increase in [Ca2+]i causes a decrease in pHi or vice versa, pHi and [Ca2+]i were manipulated at isotonic conditions. Surprisingly, a decrease in [Ca2+]i was accompanied by a decrease in pHi, and an increase in pHi resulted in an increase in [Ca2+]i, in the absence of osmotic perturbation.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of glucagon on GFR and on urea and electrolyte excretion: direct and indirect effects
Authors: Ahloulay M. , Dechaux M. , Laborde K. , Bankir L.
Published: December 2017

Clearance experiments were performed in anesthetized male Wistar rats to determine the level of peripheral glucagon concentration required to elicit changes in glomerular filtration rate (GFR) and in solute excretion. Glucagon was intravenously infused at a rate of 1.25 (group G-1, n = 8), 3.75 (group G-3, n = 7), or 12.5 (group G-10, n = 7) ng.min-1.100 g body wt-1 for 100 min. Measurements were performed before, during, and after this infusion. Group G-10 resulted in a plasma concentration of glucagon severalfold higher than usually observed in peripheral blood after a protein meal but normal for the hepatic circulation. Group G-10 simultaneously increased GFR, plasma adenosine 3',5'-cyclic monophosphate (cAMP) concentration, and the excretion of water (i.e., urinary flow rate), Na, Cl, PO4, K, and urea. Some of the effects of glucagon on electrolyte excretion were also observed with group G-1 and/or G-3 and were fully reversible, suggesting a direct renal action of glucagon. The significant and reversible increase in K excretion in group G-3 suggests that glucagon exerts a direct stimulatory influence on K secretion in the distal nephron. Increases in urinary flow rate, PO4, Na, and urea fractional excretions were seen with group G-10 only and were not reversible, suggesting an indirect action of glucagon on the proximal tubule. Because glucagon stimulates cAMP formation in hepatocytes and because this cAMP is released in the blood and secreted by proximal tubule cells, cAMP of hepatic origin could induce a parathyroid hormone-like effect in this nephron segment. In summary, these experiments suggest that glucagon influences different aspects of renal function by a combination of direct and indirect (probably liver-dependent) effects.
GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation
Authors: Wu X. , Dolecki G. J. , Lefkowith J. B.
Published: December 2017

We recently observed that cytokine-induced neutrophil chemoattractant (CINC), a GRO chemokine, contributes to neutrophil migration into the inflamed glomerulus in rat. Therefore, we sought to clarify how expression of the GRO chemokines, CINC and macrophage inflammatory protein-2 (MIP-2), is regulated in mesangial cells in vitro and the kidney in vivo. Mesangial cells expressed both GRO chemokine mRNAs in response to mediators of acute renal inflammation [interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS)], but not chronic renal inflammation (transforming growth factor-beta 1), with CINC mRNA expression predominating over MIP-2. The kinetics of GRO chemokine mRNA expression in response to both IL-1 beta and TNF-alpha (but not LPS) paralleled those defined for polymorphonuclear leukocyte (PMN) migration during nephritis in vivo. IL-1 beta and TNF-alpha displayed nonparallel concentration-response relationships for GRO chemokine mRNA expression, and together were synergistic together rather than additive. Expression of GRO chemokine mRNAs in response to both cytokine agonists, however, was inhibited by genistein, a tyrosine kinase inhibitor. GRO chemokine mRNAs were rapidly expressed in inflamed glomeruli during immune complex glomerulonephritis with MIP-2 predominating over CINC. Expression of both chemokines was substantially inhibited by complement, leukocyte, and PMN depletion. In sum, GRO chemokines are expressed coordinately by mesangial cells and inflamed glomeruli and appear both to transduce the response to mediators of acute inflammation into a chemotactic signal and to amplify this response both temporally and quantitatively.(ABSTRACT TRUNCATED AT 250 WORDS)
Luminal prostaglandin E receptors regulate salt and water transport in rabbit cortical collecting duct
Authors: Sakairi Y. , Jacobson H. R. , Noland T. D. , Breyer M. D.
Published: December 2017

Prostaglandin E2 (PGE2) is the major renal cyclooxygenase metabolite of arachidonic acid. Urinary excretion of PGE2 is increased by dietary salt restriction, as well in cirrhosis and congestive heart failure. To determine whether urinary PGE2 affects transport along the nephron, the actions of luminal PGE2 were studied in the isolated perfused rabbit cortical collecting duct (CCD). Luminal PGE2 transiently hyperpolarized transepithelial voltage (Vt) in a dose-dependent manner (half-maximal effect approximately 10(-8) M) in contrast to a sustained depolarization of Vt produced by basolateral PGE2. Luminal PGE2 (0.1 microM) also significantly stimulated osmotic water permeability in the CCD. In CCDs cultured on semipermeable supports, apical PGE2 stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, suggesting the effects of luminal PGE2 are mediated by adenylyl cyclase-stimulating EP2 or EP4 receptors. Sulprostone, a PGE2 analogue selective for EP1 and EP3 receptors, affected Vt only when applied from the basolateral but not the luminal surface. Luminal application of the EP2 receptor agonist butaprost was also without effect. These results suggest that luminal PGE2 affects Vt via a butaprost-insensitive EP4 receptor. The Vt effect of luminal PGE2 was not blocked by pertussis toxin, also arguing against an EP3-mediated Gi-coupled effect. Finally, 1 microM luminal PGE2 only slightly increased CCD intracellular calcium concentration ([Ca2+]i), in contrast to the marked increase in [Ca2+]i produced by basolateral PGE2 (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Tubulointerstitial injury and impaired renal function after recovery from acute puromycin nephrosis
Authors: Baboolal K. , Meyer T. W.
Published: December 2017

Renal function was assessed at 2 and 8 wk after infusion of puromycin into the left renal artery of Munich Wistar rats. At 2 wk, albumin excretion averaged 90 +/- 12 micrograms/min in the left kidney and 4 +/- 1 microgram/min in the right kidney. Unilateral nephrosis was accompanied by reduction in the glomerular filtration rate (GFR) (left, 0.71 +/- 0.04; right, 1.31 +/- 0.02 ml/min) and by impaired excretion of sodium (FENa; left, 0.025 +/- 0.004; right, 0.064 +/- 0.006%). Reductions in GFR and FENa in the nephrotic kidney were not reversed by acute angiotensin II receptor blockade with losartan. At 8 wk, albumin excretion averaged 6 +/- 1 in the left kidney and 8 +/- 1 microgram/min in the right kidney. Recovery from nephrosis was accompanied by persistent reduction in GFR (left, 1.05 +/- 0.05; right, 1.41 +/- 0.05 ml/min) and impairment of sodium excretion in the previously nephrotic left kidney (left, 0.031 +/- 0.004; right, 0.051 +/- 0.004%). Losartan again did not return GFR and FENa toward normal. The reductions in GFR and FENa in the previously nephrotic left kidney were associated with structural changes, including intratubular casts, an increased fractional volume of the interstitium (left, 25 +/- 1; right, 15 +/- 1%), decreased fractional volume of tubules (left, 66 +/- 2; right, 77 +/- 1%), and glomerular collapse (left, 15 +/- 2; right, 1 +/- 1%). These findings suggest that tubulointerstitial injury can cause persistent reduction in GFR and impairment of sodium excretion after recovery from acute nephrosis.
Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter
Authors: Igarashi P. , Vanden Heuvel G. B. , Payne J. A. , Forbush B.
Published: December 2017

A full-length cDNA encoding the murine renal Na-K-Cl cotransporter (NKCC2) was cloned using library screening and anchored polymerase chain reaction. The deduced protein sequence contained 1,095 amino acids and was 93.5% identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two potential sites of phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase and seven potential sites of phosphorylation by protein kinase C, which were previously identified in the rabbit and rat sequences, were phylogenetically conserved in the mouse. The expression of NKCC2 in the mouse was examined with Northern blot analysis and in situ hybridization. Expression of NKCC2 was kidney specific in both adult and embryonic mice. In the developing metanephros, NKCC2 was induced at 14.5 days post coitus and was expressed in distal limbs of immature loops of Henle but was absent from the ureteric bud, S-shaped bodies, and earlier nephrogenic structures. Similar to the rabbit, isoforms of NKCC2 that differed in the sequence of a 96-bp segment were identified in the mouse. In situ hybridization revealed that the isoforms exhibited different patterns of expression in the mature thick ascending limb of the loop of Henle as follows: isoform F was most highly expressed in the inner stripe of outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the cortical thick ascending limb. To verify that the isoforms were generated by alternative splicing of mutually exclusive cassette exons, genomic clones encoding murine NKCC2 were characterized. Cassette exons were identified that corresponded to each of the three isoforms and were flanked by consensus splice donor and acceptor sequences.
1 alpha, 25-dihydroxyvitamin D3 receptor ontogenesis in fetal renal development
Authors: Johnson J. A. , Grande J. P. , Roche P. C. , Sweeney W. E. , Avner E. D. , Kumar R.
Published: December 2017

We used immunohistochemical techniques to examine the distribution of 1 alpha, 25-dihydroxyvitamin D3 receptors (VDR) in developing rat and mouse kidneys and murine metanephric organ culture. In vivo, the patterns of expression in the two species were similar despite the slight difference in gestational periods (rat, 22 days; mouse, 19 days). Starting at gestational day 15, epitopes for VDR were found in cells of branching ureteral buds and in surrounding mesenchyme and at later developmental stages in glomerular visceral and parietal epithelial cells and proximal and distal tubules (DT). Epitopes for the 28-kDa calcium-binding protein (calbindin D28k) were found exclusively in DTs starting at gestational day 19. The pattern of VDR expression during in vitro nephrogenesis in serum-free murine metanephric organ culture paralleled that seen in vivo. At the time of explantation into organ culture (gestational day 13), VDR epitopes were not detected. By 3 days of in vitro development, VDR expression was identical to that found in gestational day 15 metanephroi in vivo. VDR expression after 5 days of in vitro development mirrored the pattern of gestational day 17 metanephroi in vivo. No calbindin D28k epitopes were seen at any in vitro developmental stage studied. We demonstrate for the first time that VDR are present in specific areas of the developing rat and mouse kidney early in gestation. Calbindin D28k appears later in developing rat and mouse kidney and is distributed differently than the VDR. Metanephric organ culture may be a useful model for studying the regulation and function of VDR during early renal development.
Autoregulation of zonal glomerular filtration rate and renal blood flow in spontaneously hypertensive rats
Authors: Wang X. , Aukland K. , Ofstad J. , Iversen B. M.
Published: December 2017

Autoregulation of total and zonal glomerular filtration rate (GFR) in outer, middle and inner cortex was estimated in spontaneously hypertensive rats (SHR), from the tubular uptake of 125I-labeled aprotinin (125I-Ap), injected at control renal arterial pressure (RAP), and 131I-Ap, injected at reduced RAP in left kidney. Normotensive Wistar-Kyoto (WKY) rats were used as controls. Renal blood flow (RBF) autoregulation was reset to higher pressure levels in SHR. When RAP was lowered close to the lower pressure limit of RBF autoregulation, total GFR was reduced to 89.5 +/- 3.1 and 88.1 +/- 3.3% of control in 10- and 40-wk WKY and to 87.7 +/- 2.3 and 88.0 +/- 2.2% in 10- and 40-wk SHR. In WKY, the fall of GFR in the three cortical layers was not different during RAP reduction. In 10- and 40-wk-old SHR, however, GFR fell significantly less in inner than in middle and outer cortex (P < 0.05). We conclude that autoregulation of GFR is most efficient in the inner cortex of SHR. In all animals, GFR was less well autoregulated than RBF.
Increased sensitivity of the renal vasculature to adenosine in streptozotocin-induced diabetes mellitus rats
Authors: Pflueger A. C. , Schenk F. , Osswald H.
Published: December 2017

Adenosine (ADO) has been implicated as a pathophysiological factor in contrast media (CM)-induced acute renal failure, which has been encountered more often in patients with diabetes and impaired renal function. Therefore, we studied the renal vascular response to exogenous and endogenous ADO in streptozotocin-induced diabetic rats. We found that exogenous ADO (0.01–100 nmol), injected into the abdominal aorta, decreased renal blood flow (RBF) in a dose-dependent manner. The dose-response curve was shifted to the left by factor 30 in diabetic, compared with nondiabetic rats rats. Renal vascular response to endogenous ADO, assessed by postocclusive reduction of RBF after a 30-s renal artery occlusion, was significantly enhanced (P < 0.001) in diabetic rats (75.6 +/- 3.9%) compared with nondiabetic rats (36.5 +/- 2%). ADO A1-receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine attenuated exogenous and endogenous ADO-induced renal vasoconstriction in both groups. We conclude that the ADO A1-receptor signal-transduction chain is altered in diabetic animals and that the enhanced vasoconstrictive action of ADO could be involved in the kidney pathophysiology of diabetes mellitus.
Heterogeneity of NH+4 transport in mouse inner medullary collecting duct cells
Authors: Wall S. M. , Trinh H. N. , Woodward K. E.
Published: December 2017

Previous studies from our laboratory have demonstrated that NH+4 substitutes for K+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in rat terminal inner medullary collecting duct cells (tIMCD). To examine other NH+4 transport pathways, a transgenic mouse cell line, mIMCD-3, was employed. 86Rb+ was used as a K+ congener to explore NH+4/Rb+ (K+) competition on the extracellular K+ binding site of the Na(+)-K(+)-2Cl- cotransporter and the Na(+)-K(+)-ATPase. Addition of K+ or NH+4 reduced both bumetanide- and ouabain-sensitive Rb+ uptake. This reduction in Rb+ uptake with NH+4 addition was not due to intracellular pH-mediated changes in transporter activity. K+ and NH+4 are competitive inhibitors on both transporters. On the Na(+)-K(+)-2Cl- cotransporter, the Michaelis constant (Km) for K+ was 4.6 +/- 0.5 mM with an inhibitory constant (Ki) for NH+4 of 2.8 mM. In contrast, on the Na(+)-K(+)-ATPase, the apparent affinity for K+ was greater than for NH+4. To test Na(+)-K(+)-2Cl- cotransport-mediated NH+4 flux, bumetanide-sensitive NH+4/Rb+ exchange was measured. Bumetanide-sensitive Rb+ efflux was greater with extracellular K+ or NH+4 present relative to efflux with extracellular N-methyl-D-glucamine. This demonstrates both K+/Rb+ and NH+4/Rb+ countertransport by the Na(+)-K(+)-2Cl- cotransporter. In conclusion, NH+4 is transported in a bumetanide-sensitive Na(+)-NH+4-Cl- mode, and both NH+4 and Rb+ (K+) are competitive inhibitors for the extracellular K+ binding site. However, the kinetics of Na(+)-K(+)-2Cl(-)-mediated NH+4 transport differ from other K+ transport-mediated NH+4 pathways, such as the Na(+)-K(+)-ATPase.
Analysis of interaction between TGF and the myogenic response in renal blood flow autoregulation
Authors: Feldberg R. , Colding-Jorgensen M. , Holstein-Rathlou N. H.
Published: December 2017

The present study investigates the interaction between the tubuloglomerular feedback (TGF) response and the myogenic mechanism by use of a mathematical model. The two control mechanisms are implemented in a spatially distributed model of the rat renal juxtamedullary afferent arteriole. The model of the afferent arteriole is based on in vivo measurements of the stress-strain relation in muscle strips. Analysis of experimental data shows that the myogenic response can be modeled by a linear relation between the transmural pressure and the level of activation of the vascular smooth muscle cells. The contribution of TGF to smooth muscle activity is assumed to be a linear function of the glomerular capillary pressure. The results show that the myogenic response plays an important role in renal blood flow autoregulation. Without a myogenic response, mechanisms such as TGF that are localized in the distal segments of the microvasculature would not be able to achieve autoregulation because of passive, pressure-mediated effects in the upstream vascular segments. In addition, it is shown that a strong myogenic response may lead to both propagation and enhancement of vascular effects mediated through mechanisms located in the distal part of the afferent arteriole. An ascending myogenic response could enhance the regulatory efficiency of the TGF mechanism by increasing the open-loop gain of the system. However, such a synergistic interaction will only be observed when the two mechanisms operate on more or less separate segments of the afferent arteriole. In the case where they operate on common segments of the arteriole, the outcome of the interaction may well be antagonistic.
Cl- channels in basolateral renal medullary vesicles. IX. Channels from mouse MTAL cell patches and medullary vesicles
Authors: Reeves W. B. , Winters C. J. , Filipovic D. M. , Andreoli T. E.
Published: December 2017

The experiments reported herein compared Cl- channels fused into bilayers from rabbit outer medullary vesicles with Cl- channels in excised patches of basolateral membranes from cultured mouse medullary thick ascending limb (MTAL) cells and evaluated whether the latter were plausible candidates for the Cl- channels mediating net NaCl absorption in microperfused mouse MTAL segments. The unique signature characteristics of Cl- channels incorporated into lipid bilayers from outer medullary vesicles include activation of open probability (Po) by increases in the Cl- concentrations bathing intracellular faces; activation of Po by protein kinase A (PKA) + ATP, when the Cl- concentrations bathing intracellular faces are low; and no effect of PKA + ATP on Po with high cytoplasmic-face Cl- concentrations. These same properties were observed in Cl- channels studied using excised patches of basolateral membranes from mouse MTAL cells. Moreover, in both bilayers and in excised patches, the sharpest fractional increase in Cl- channel Po occurred with cytosolic-face Cl- concentration increases to values similar to the antidiuretic hormone (ADH)-dependent values of intracellular Cl- activity in microperfused mouse MTAL segments, and these fractional Po increases were adequate to account quantitatively for the ADH-dependent increase in basolateral membrane Cl- conductance in microperfused mouse MTAL segments. Thus the excised-patch basolateral Cl- channels reported here are reasonable candidates for those mediating net Cl- absorption in the MTAL.
Aquaporin-3 water channel localization and regulation in rat kidney
Authors: Ecelbarger C. A. , Terris J. , Frindt G. , Echevarria M. , Marples D. , Nielsen S. , Knepper M. A.
Published: December 2017

The aquaporins are a family of water channels expressed in several water-transporting tissues, including the kidney. We have used a peptide-derived, affinity-purified polyclonal antibody to aquaporin-3 (AQP-3) to investigate its localization and regulation in the kidney. Immunoblotting experiments showed expression in both renal cortex and medulla, with greatest expression in the base of the inner medulla. Subcellular fractionation of membranes, using progressively higher centrifugation speeds, revealed that AQP-3 is present predominantly in the 4,000 and 17,000 g pellets and, in contrast to AQP-2, is virtually absent in the high-speed (200,000 g) pellet that contains small intracellular vesicles. Immunocytochemistry and immunofluorescence studies revealed that labeling is restricted to the cortical, outer medullary, and inner medullary collecting ducts. Within the collecting duct, principal cells were labeled, whereas intercalated cells were unlabeled. Consistent with previous immunofluorescence studies (K. Ishibashi, S. Sasaki, K. Fushimi, S. Uchida, M. Kuwahara, H. Saito, T. Furukawa, K. Nakajima, Y. Yamaguchi, T. Gojobori, and F. Marumo. Proc. Natl. Acad. Sci. USA 91: 6269-6273, 1994; T. Ma, A. Frigeri, H. Hasegawa, and A. S. Verkman. J. Biol. Chem. 269: 21845-21849, 1994), the labeling was confined to the basolateral domain. Immunoelectron microscopy, using the immunogold technique in ultrathin cryosections, demonstrated a predominant labeling of the basolateral plasma membranes. In contrast to previous findings with AQP-2, there was only limited AQP-3 labeling of intracellular vesicles, suggesting that this water channel is not regulated acutely through vesicular trafficking. Immunoblotting studies revealed that thirsting of rats for 48 h approximately doubled the amount of AQP-3 protein in the inner medulla. These studies are consistent with a role for AQP-3 in osmotically driven water absorption across the collecting duct epithelium and suggest that the expression of AQP-3 is regulated on a long-term basis.
Protein phosphatase-1 in the kidney: evidence for a role in the regulation of medullary Na(+)-K(+)-ATPase
Authors: Li D. , Aperia A. , Celsi G. , da Cruz e Silva E. F. , Greengard P. , Meister B.
Published: December 2017

Previous studies of hormonal regulation of renal Na(+)-K(+)-ATPase have indicated that the activity of the sodium pump is regulated by phosphorylation-dephosphorylation reactions. Here we report that okadaic acid (OA) and calyculin A (CL-A), inhibitors of protein phosphatase (PP)-1 and PP-2A, inhibited Na(+)-K(+)-ATPase activity in cells from the rat thick ascending limb (TAL) of loop of Henle in a dose-dependent manner. CL-A was 10-fold more potent than OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. In situ hybridization studies with oligonucleotide probes revealed very strong PP-1 alpha and PP-1 gamma 1 mRNA labeling in the outer stripe of the outer medulla, strong labeling in the inner stripe of the outer medulla, and weak labeling in the inner medulla. Very weak labeling was demonstrated in the outer cortex. PP-1 beta mRNA labeling was very strong in the inner stripe of the outer medulla, whereas the outer stripe had weaker labeling, and the inner medulla had weak labeling. PP-1 alpha, PP-1 beta, and PP-1 gamma 1 mRNA were also demonstrated in the transitional epithelium of the ureter. The abundance of the PP-1 alpha and PP-1 gamma isoforms as measured by immunoblotting was very high in tissue from the outer medulla, which also has a high abundance of the endogenous dopamine-regulated PP-1 inhibitor, DARPP-32.(ABSTRACT TRUNCATED AT 250 WORDS)
Unique localization of mRNA encoding plasma membrane Ca2+ pump isoform 3 in rat thin descending loop of Henle
Authors: Caride A. J. , Chini E. N. , Yamaki M. , Dousa T. P. , Penniston J. T.
Published: December 2017

In our previous study of plasma membrane Ca2+ pump (PMCA) isozymes in the rat kidney, we found that the mRNA coding isoform PMCA3 was detected primarily in the outer medullary zone of rat kidney tissue. We now investigated the location of the mRNAs coding for isoforms PMCA3 and PMCA4 of Ca2+ pump in the nephron segments that are present in outer medullary parenchyma using the method of reverse transcription and polymerase chain reaction. In mRNA extracted from whole dissected outer medulla we detected mRNA encoding three splice variants (a, b, and c) of isoform PMCA3; isoform PMCA4 was found in the outer medulla almost exclusively as variant b. Analysis of mRNA from microdissected tubule segments show that proximal straight tubules (PST), medullary thick ascending limbs, outer medullary collecting ducts, and descending thin limb of Henle's loop (DTL) all contained mRNA for isoform 4b. In contrast, the mRNA encoding isoform 3 was detected exclusively in DTL and not in other nephron segments. The unique presence of isoform 3 in DTL is rather surprising, since the specific role of this nephron segment in vectorial Ca2+ transport or in intracellular Ca2+ signaling is not known. The data suggest that isoform 3 in cells of the DTL may have a hitherto unrecognized specific role in Ca2+ signaling or transport of Ca2+, which is distinct from the role of the isoforms of the PMCA in all other nephron segments.
Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling
Authors: Fern R. J. , Hahm M. S. , Lu H. K. , Liu L. P. , Gorelick F. S. , Barrett P. Q.
Published: December 2017

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.
Distribution of aquaporin-4 water channel expression within rat kidney
Authors: Terris J. , Ecelbarger C. A. , Marples D. , Knepper M. A. , Nielsen S.
Published: December 2017

The aquaporins are a family of transmembrane proteins that function as molecular water channels. Recently, a mercurial-insensitive water channel [MIWC or aquaporin-4 (AQP4)] has been cloned, and its mRNA was found to be expressed strongly in kidney inner medulla and several nonrenal tissues. We prepared affinity-purified polyclonal antipeptide antibodies to AQP4 to define the regional distribution and cellular location of this water channel within the kidney. Immunoblotting of membrane fractions from different regions of the kidney revealed strongest expression in the base of the renal inner medulla, with detectable levels also in the inner medullary tip, but little or no expression in the outer medulla or cortex. Immunocytochemistry (light microscopy) revealed renal AQP4 labeling exclusively in the collecting duct principal cells, chiefly in the proximal two-thirds of the inner medullary collecting duct (IMCD). Little or no expression was seen in the outer medullary and cortical collecting ducts. Immunoelectron microscopy demonstrated AQP4 labeling of the basolateral membrane of IMCD cells, with relatively little labeling of intracellular vesicles. Differential centrifugation of inner medullary homogenates also revealed a lack of distribution to the vesicle-enriched fraction, which contains the vasopressin-regulated water channel, aquaporin-2. In contrast to aquaporin-2 and aquaporin-3, water restriction of rats did not increase the level of AQP4 expression. These results suggest a possible role for AQP4 in the basolateral exit of water from the IMCD.
In vivo and in vitro osmotic regulation of HSP-70 and prostaglandin synthase gene expression in kidney cells
Authors: Cowley B. D. , Muessel M. J. , Douglass D. , Wilkins W.
Published: December 2017

As a function of the urinary concentrating mechanism, the cells of the renal medulla are exposed to elevated and constantly varying osmolalities and adapt to this environment by selectively expressing certain mRNAs. We evaluated the expression and regulation of two RNAs that may be important in adaptation of rental medullary cells to hyperosmolality. We demonstrate selective, modulated expression in the renal medulla of heat shock protein HSP-70 mRNA and prostaglandin synthase-1 mRNA, with the abundance of these two mRNAs regulated in vivo in concert with changes in medullary sodium and urea. We also determined the abundance of these mRNAs in cultured kidney cells (MDCK) in response to an increase in extracellular osmolality due to selected osmotic agents. HSP-70 and prostaglandin synthase-2 mRNA levels increased when extracellular osmolality was increased to 400-600 mosmol/kg by the addition of NaCl. At 500 mosmol/kg this response was evident at 6 h, was maximal near 24 h, and persisted for a total of 90 days. Prostaglandin synthase-1 mRNA levels in MDCK cells were also increased after chronic exposure to extracellular osmolality. Increased extracellular osmolality caused by agents to which cells are impermeable caused increased levels of HSP-70 and prostaglandin synthase-2 mRNAs, whereas increased extracellular osmolality caused by agents to which cells are permeable did not; thus osmotic regulation involved osmotic water movement. We conclude that the abundance of HSP-70 and prostaglandin synthase-1 mRNAs in the renal medulla is regulated in response to renal medullary osmolality and suggest that this may also be true for other medullary mRNAs yet to be described.
Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct
Authors: Kishore B. K. , Chou C. L. , Knepper M. A.
Published: December 2017

The P2u class of nucleotide receptors is linked to mobilization of intracellular Ca2+ in many cell types, including the renal collecting duct cells. In the present studies, we examined the effects of nucleotides (ATP, UTP, and ADP; 10 microM each) on the arginine vasopressin (AVP, 0.1 nM)-stimulated osmotic water permeability (Pf) in in vitro perfused terminal inner medullary collecting ducts (IMCD) of rat. ATP or UTP, when added to the bath, decreased the AVP-stimulated Pf by approximately 40%. These effects were reversible upon withdrawal of the nucleotides. However, addition of ADP to the bath or sham exchange of the bath had no significant effect on the Pf. Furthermore, ATP did not have any significant effect on the Pf stimulated either by a membrane-permeant, nonhydrolyzable adenosine 3',5'-cyclic monophosphate (cAMP) analogue [8-(4-chlorophenylthio)-cAMP, 0.1 mM] o by forskolin (1 microM). In line with these findings, ATP decreased the AVP-stimulated cAMP levels in IMCD suspensions to approximately 68%. In addition, ATP did not exert an inhibitory effect on the AVP-stimulated Pf in the presence of calphostin C (150 nM), an inhibitor of protein kinase C. These results lead us to conclude the following: 1) agonist occupancy of the putative nucleotide receptor in the terminal IMCD causes an inhibition of AVP-stimulated Pf; and 2) this effect is due to a decrease in cellular cAMP levels, most likely resulting from activation of the phosphoinositide signaling pathway.