bluuebunny/crossref_metadata_2025 huggingface dataset:
ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop
Authors: Wolf G. , Ziyadeh F. N. , Helmchen U. , Zahner G. , Schroeder R. , Stahl R. A.
Published: December 2017

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.
Effect of nitric oxide on renin secretion. II. Studies in the perfused juxtaglomerular apparatus
Authors: He X. R. , Greenberg S. G. , Briggs J. P. , Schnermann J. B.
Published: December 2017

To examine the possible role of NO in macula densa control of renin secretion, we examined the effects of varying NO availability on renin release in the isolated perfused rabbit juxtaglomerular apparatus (JGA). Gradual increments of luminal Na/Cl concentration ratio (mM/mM) from 26/7 over 46/27, 66/47, to 86/67 caused a progressive decrease in renin secretion from (as log of nano-Goldblatt hog units vs. time, i.e., log nGU/min) 1.09 +/- 0.34 to 0.46 +/- 0.24 log nGU/min, with the greatest change occurring at the first concentration step. The presence of 0.7 mM N omega-nitro-L-arginine (NNA), an NO synthase inhibitor, in the luminal fluid significantly reduced renin secretion at the lowest Na/Cl concentration ratio to 0.65 +/- 0.32 log nGU/min (P < 0.01 compared with control). Renin secretion at the higher Na/Cl concentration ratios was not significantly affected by NNA compared with control. In contrast to these results, the addition of the NO donor nitroprusside (1 mM) to the bath caused a reduction in renin secretion from 1.0 +/- 0.39 to 0.47 +/- 0.46 log nGU/min (P < 0.05), an effect that was reversed by bath addition of 0.01 mM methylene blue. Similarly, addition of L-arginine (0.7 mM) to the bath reduced renin secretion from 0.99 +/- 0.37 to 0.81 +/- 0.38 log nGU/min (P < 0.01), whereas addition of L-arginine to the luminal fluid increased renin secretion from 0.85 +/- 0.43 to 1.94 +/- 0.46 log nGU/min (P < 0.05). The stimulatory effect of luminal L-arginine was reversed by the luminal addition of NNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of barium ions on tubuloglomerular feedback
Authors: Schnermann J.
Published: December 2017

The furosemide sensitivity of the tubuloglomerular feedback (TGF) response has suggested an important role for the Na-2Cl-K cotransporter in the mechanism by which increased luminal NaCl concentration causes afferent arteriolar vasoconstriction. The present experiments in anesthetized rats were performed to evaluate the effect of K channel blockade with Ba on TGF, since Ba has been shown to inhibit NaCl transport in the thick ascending limb. The presence of either 1.5 or 2 mM BaCl2 during retrograde perfusion with a 135 mM NaCl solution reduced the decrease of early proximal flow rate (VEP) by 2.7 +/- 0.76 (P < 0.02) and 4.2 +/- 0.8 nl/min (P < 0.01) compared with perfusion without BaCl2. Retrograde perfusion with 38 mM NaCl + 5 mM KCl reduced VEP by 10.4 +/- 1.3 nl/min, whereas 40 mM NaCl + 1.5 mM BaCl2 caused a reduction by only 6.1 +/- 1.4 nl/min (P < 0.001). In contrast to the inhibition caused by retrograde perfusion with low concentrations of BaCl2, increased vasoconstriction was seen during retrograde perfusion with 5 mM BaCl2 or during orthograde perfusion with 10 mM BaCl2. The addition of 10(-4) M furosemide to a solution containing 5 mM BaCl2 largely blocked the increased vasoconstrictor response. Peritubular perfusion with a solution containing 5 mM BaCl2 caused a fall in stop-flow pressure in an adjacent nephron by 10.7 +/- 1.5 mmHg (P < 0.001). These results indicate that under our experimental conditions Ba ions exert a dual effect on vascular responses to changes in luminal NaCl concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
A novel method for infusing drugs continuously into the renal artery of rats
Authors: Parekh N.
Published: December 2017

A method is described to achieve a homogeneous intravascular distribution of drugs infused into the renal artery of anesthetized rats. The device for intrarenal infusion consisted of a multiple-catheter system with a cannula inserted into the renal artery, which was connected to different lines for drug infusions and to one line for oscillating blood back and forth in the renal cannula with a magnetic membrane pump. The blood oscillation served to mix the drugs with renal arterial blood. To verify the usefulness of this procedure, Lissamine green was infused into the renal artery; without the mixing pump the dye was located on a small portion of the kidney surface, whereas the dye could be visualized evenly distributed and less concentrated over the entire kidney with the pump. With the mixing device, intrarenal infusion of angiotensin II, 5 pmol.kg-1.min-1, or norepinephrine, 150 pmol.kg-1.min-1, reduced renal blood flow by approximately 25% without affecting blood pressure. Tenfold higher doses given intravenously had comparable renal effects, but these increased systemic pressure. Without the mixing pump, vasoactive drugs given into the renal artery had a distinctly smaller effect on renal blood flow than with the pump (angiotensin II, 39%; norepinephrine, 49%; and acetylcholine, at 5 nmol.kg-1.min-1, 33%). The results show that intrarenally infused drugs, without a mixing device, have access to an unpredictably small kidney volume, and estimation of their effects on kidney functions can be equivocal. The present device ensures an adequate mixing of drugs with renal blood.
Glomerular and tubular interactions between renal adrenergic activity and nitric oxide
Authors: Gabbai F. B. , Thomson S. C. , Peterson O. , Wead L. , Malvey K. , Blantz R. C.
Published: December 2017

Endothelium-dependent nitric oxide (EDNO) exerts control over the processes of glomerular filtration and tubular reabsorption. The importance of the renal nerves to the tonic influence of EDNO in the glomerular microcirculation and proximal tubule was tested by renal micropuncture in euvolemic adult male Munich-Wistar rats. The physical determinants of glomerular filtration and proximal reabsorption were assessed before and during administration of the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), in control animals and in animals 5–9 days after either ipsilateral surgical renal denervation (DNX) or after either sham surgery (SHX). L-NMMA caused single-nephron glomerular filtration rate to decline in control and SHX animals but not in DNX rats. L-NMMA caused a reduction in proximal reabsorption in control and SHX rats, which was prevented by prior DNX. DNX did not alter urinary guanosine 3',5'-cyclic monophosphate excretion, and, although DNX upregulates glomerular angiotensin II (ANG II) receptors, prior DNX did not alter intrarenal ANG II content as evaluated by radioimmunoassay. Some component of renal adrenergic activity is required for the full expression of the glomerular and tubular effects of blockade of nitric oxide synthase.
A decreased tubular uptake of dopa results in defective renal dopamine production in aged rats
Authors: Armando I. , Nowicki S. , Aguirre J. , Barontini M.
Published: December 2017

A major proportion of urinary dopamine derives from the renal decarboxylation of circulating dopa. This study evaluates the effects of aging on renal production of dopamine using 3- and 12-mo-old male Wistar rats. Urinary excretion of Na+, norepinephrine (NE), 3,4-dihydroxyphenylglycol, and dopa were similar in the two groups. Urinary dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were lower in older animals (dopamine, 20 +/- 6 vs. 47 +/- 7 nmol/24 h, P < 0.001; DOPAC, 142 +/- 36 vs. 304 +/- 56 nmol/24 h, P < 0.03). Urinary 3-O-methyldopa (OM-dopa) was higher in 12-mo-old rats (6.2 +/- 2.0 vs. 3.3 +/- 0.20 nmol/24 h, P < 0.03). Levels of dopa and NE in renal cortex from 12-mo-old rats were higher (P < 0.001) than in younger animals. Dopamine content in renal cortex from 3-mo-old rats was 295 +/- 64 pmol/g, whereas it was undetectable in 12-mo-old animals. Aromatic-L-amino-acid decarboxylase and monoamine oxidase activities were higher (P < 0.001) in renal cortex from 12-mo-old animals. Catechol-O-methyltransferase activity was similar in both groups. The uptake of dopa by the luminal membrane was explored using brush-border membrane vesicles. The Na(+)-gradient-driven (100 mM) uptake of dopa into vesicles from 3-mo-old animals showed at 10 s an overshoot threefold greater than the equilibrium uptake. The overshoot was blunted in 12-mo-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)
ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments
Authors: Lee W. S. , Hebert S. C.
Published: December 2017

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.
Expression cloning of an AVP-activated, calcium-mobilizing receptor from rabbit kidney medulla
Authors: Burnatowska-Hledin M. A. , Spielman W. S. , Smith W. L. , Shi P. , Meyer J. M. , Dewitt D. L.
Published: December 2017

Arginine vasopressin (AVP) is a nonapeptide that regulates body fluid and blood pressure homeostasis. We have used expression cloning in the Xenopus laevis oocyte system to identify cDNA clones from a rabbit renal medullary expression library encoding an AVP receptor linked to Ca2+ mobilization. cRNA generated from positive clones conferred upon oocytes the capacity to mobilize intracellular Ca2+ in response to AVP. A cDNA clone encoding a protein of 780 amino acids was isolated, sequenced, and subcloned into an SV40-based expression vector. Expression of the cloned protein [designated the vasopressin-activated, calcium-mobilizing (VACM-1) protein] in COS-1 cells, resulted in increased 125I-labeled AVP binding [dissociation constant (Kd) of approximately 2 nM] and increased AVP-induced mobilization of Ca2+. Importantly, 125I-AVP could be immunoprecipitated both from detergent-solubilized membranes from COS-1 cells expressing VACM-1 protein and from an in vitro translation system, in which VACM-1 protein was synthesized, using antibodies prepared against a synthetic peptide derived from the NH2-terminal sequence of VACM-1. Interestingly, immunohistochemical staining of rabbit kidney sections with this antibody showed specific staining of collecting tubule epithelia. The deduced amino acid sequence is not homologous with any nucleic acid or amino acid sequences reported to date, including those of the V1 and V2 AVP receptors. The VACM-1 protein may represent a novel AVP receptor.
RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb
Authors: Borensztein P. , Froissart M. , Laghmani K. , Bichara M. , Paillard M.
Published: December 2017

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.
Function and structure of H-K-ATPase in the kidney
Authors: Wingo C. S. , Smolka A. J.
Published: December 2017

The present review summarizes recent functional and structural evidence indicating that the kidney possesses at least one and probably more than one isoform of a proton- and potassium-activated adenosinetriphosphatase (H-K-ATPase). Functional studies have examined in detail the mechanism of luminal acidification and K/Rb absorption by the outer medullary collecting duct (OMCD) from the inner stripe, a high-capacity distal site of urinary acidification. These studies indicate that the mechanism of proton secretion in this segment is similar to a model proposed for gastric acid secretion. Specifically, the profound effect of H-K-ATPase inhibitors or luminal K removal on net bicarbonate (HCO3) absorption indicates a major role for an H-K pump in luminal acidification by the OMCD. The importance of an H-K-ATPase is further supported by the finding that nanomolar concentrations of bafilomycin A1, which specifically inhibit vacuolar-type H-ATPase, have significantly smaller effects on net HCO3 absorption than do H-K-ATPase inhibitors. Studies on the perfused inner medullary collecting duct (IMCD) and cultured IMCD cells also suggest a significant role for H-K-ATPase in luminal acidification by the IMCD. Evidence has accrued from studies in the cortical CD and OMCD that the mechanism of H-K-ATPase-mediated luminal proton secretion differs under K-replete and K-restricted conditions. In K repletion, luminal K ions transported by the pump recycle back into the lumen by a Ba-sensitive mechanism. However, in K restriction, the mechanism of the H-K-ATPase involves luminal proton secretion and K absorption that is insensitive to luminal Ba and, by inference, apical K recycling. Moreover, in K restriction, K/Rb absorption is inhibited by basolateral Ba, indicating that the pump operates to reabsorb K/Rb across the epithelium. The structural evidence reviewed here indicates the presence of mRNA within the mammalian kidney that is either identical or highly homologous to mRNAs for gastric and putative colonic H-K-ATPase alpha-subunits and gastric H-K-ATPase beta-subunit. Localization of these transcripts by in situ hybridization demonstrates gastric alpha- and beta-subunit mRNAs in intercalated cells of both the cortical and medullary CD, principal cells of the CD, and IMCD cells. Additional studies in transgenic mice indicate that regulatory sequences upstream to the H-K-ATPase beta-subunit gene direct transcription in both gastric parietal cells and the renal CD.(ABSTRACT TRUNCATED AT 400 WORDS)
Endogenous ANP in postischemic acute renal allograft failure
Authors: Vinot O. , Bialek J. , Canaan-Kuhl S. , Scandling J. D. , Dafoe D. , Alfrey E. , Myers B. D.
Published: December 2017

Circulating atrial natriuretic peptide (ANP) levels and glomerular binding sites for ANP were examined in 23 subjects undergoing renal transplantation. Subjects were divided into two groups, group 1 (n = 12) with prompt and group 2 (n = 11) with delayed allograft function. Sixty to 180 min after graft reperfusion, renovascular resistance was threefold higher and glomerular filtration rate (GFR) depressed by 79% in group 2 vs. group 1. Corresponding median plasma ANP (114 vs. 140 pg/ml) and guanosine 3',5'-cyclic monophosphate (cGMP) levels (22 vs. 28 pmol/ml) were similarly elevated in the two groups [P = not significant (NS)]. Autoradiographic analysis of glomeruli in an allograft biopsy revealed the median density of total receptors (24 vs. 28 fmol/mm3), A receptors (15 vs. 19 fmol/mm3), and C receptors (6 vs. 9 fmol/mm3) for ANP to also be similar in group 2 vs. group 1, respectively (P = NS). By postoperative day 3, allograft GFR averaged only 6 +/- 2 in group 2 vs. 59 +/- 4 ml/min in group 1. Median plasma ANP levels doubled in each group to 262 and 251 pg/ml, respectively (P = NS). However, median values for plasma levels (38 vs. 17 pmol/ml) and the fractional clearance of cGMP (1.9 vs. 1.2) were significantly higher in group 2 than group 1. We conclude that, despite an adequate density of glomerular ANP receptors and enhanced cGMP generation, neither renal vasoconstriction nor hypofiltration is alleviated by a progressive elevation of plasma ANP levels in renal transplant recipients with sustained postischemic injury. We infer that constricted afferent arterioles are unresponsive to the vasorelaxant action of endogenous ANP in this form of postischemic, acute renal failure.
Diffusive transport of solute in the rat medullary microcirculation
Authors: Pallone T. L. , Nielsen S. , Silldorff E. P. , Yang S.
Published: December 2017

Outer medullary descending vasa recta (OMDVR) permeability to sodium (PNa) is much lower than to urea (Purea). Based on these findings, we hypothesized that sodium and urea diffuse across the OMDFR wall by separate routes. To further test this, we simultaneously perfused OMDVR with 22Na and 36Cl, [14C]urea, [3H]raffinose, or tritiated water. The permeability of OMDVR to 22Na and [3H]raffinose was found to increase markedly and reversibly with perfusion rate. PNa was highly correlated with the permeability to Cl (PCl) and to [3H]raffinose (Praf) (R = 0.90 and 0.95, respectively) but not with Purea (R = 0.23). Praf was also correlated with inulin permeability (PIn) (R = 0.93). The intercepts for the regressions of PNa with PCl and Praf and for Praf with PIn were zero. In contrast, OMDVR with low PNa retained very high diffusional water permeability (PD) and Purea, a finding consistent with separate routes for permeation of those tracers. We previously established that thiourea is a competitive inhibitor of OMDVR urea transport. In the presence of 100 mM thiourea, OMDVR PNa and Purea were correlated (R = 0.71) but retained an intercept much > 0. We conclude that Na, Cl, raffinose, and inulin are likely to traverse the OMDVR wall through a common pathway, whereas specific mechanisms exist to regulate the permeation by urea and water.
Mechanisms of iron-induced proximal tubule injury in rat remnant kidney
Authors: Harris D. C. , Tay Y. C. , Chen J. , Chen L. , Nankivell B. J.
Published: December 2017

The proposition that proximal tubule (PT) iron accumulation may cause PT injury by lysosomal destabilization or reactive oxygen species generation in human and animal chronic renal disease was examined in partially nephrectomized [remnant kidney (RK)] and sham-operated (SO) Wistar rats. Electron microscopic histochemistry with horseradish peroxidase indicated iron uptake into and release from lysosomes. PT cytoplasmic iron was seen in RK but not in SO by energy-dispersive X-ray spectrometry. Total (9.66 +/- 1.89 vs. 3.30 +/- 0.31 nmol/mg protein; P < 0.01), low-molecular-weight (1.39 +/- 0.09 vs. 0.91 +/- 0.07; P < 0.001), and catalytic iron (1.88 +/- 0.27 vs. 1.28 +/- 0.09; P = 0.05) were higher in RK cytoplasm than in SO. Lysosomal enzyme activity was greater in RK than in SO [e.g., N-acetyl-beta-D-glucosaminidase (NAG): 0.75 +/- 0.05 vs. 0.57 +/- 0.06 mumol p-nitrophenol.h-1.mg protein-1; P < 0.05] and was increased further by chronic iron loading (e.g., RK and NAG: 0.84 +/- 0.04 vs. 0.60 +/- 0.07; P < 0.05). There was no enzymatic evidence of lysosomal fragility, and chronic iron loading of RK decreased fragility as assessed by NAG release (1.36 +/- 0.14 vs. 2.17 +/- 0.14; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular analysis of spontaneous glomerulosclerosis in Os/+ mice, a model with reduced nephron mass
Authors: He C. , Zalups R. K. , Henderson D. A. , Striker G. E. , Striker L. J.
Published: December 2017

Oligosyndactyly mice (ROP Os/+) are a radiation-induced mutant strain with reduced glomerular number and increased glomerular size. We found that they develop glomerulosclerosis. At 3 mo, ROP Os/+ mice had diffuse mesangial expansion by light microscopy, whereas their +/+ littermates did not. Electron microscopic morphometry revealed a twofold increase in mesangial areas but no changes in the thickness of glomerular basal laminae. Mean glomerular volume was increased 1.8-fold. Cell number and thymidine labeling index were increased 1.3- and 2.4-fold, respectively. The amount of glomerular type IV collagen and tenascin but not laminin was increased by immunofluorescence microscopy. mRNA levels in microdissected glomeruli were measured by competetive reverse transcription-polymerase chain reaction and corrected for cell number. alpha 1-Chain type IV collagen and tenascin mRNAs were increased 3.2-fold and 1.8-fold, whereas laminin B1 mRNA levels were not. The levels of 72-kDa collagenase mRNA were increased 1.6-fold. Transforming growth factor-beta 1 mRNA levels were elevated 1.8-fold, but platelet-derived growth factor-B mRNA levels remained normal. This is the first analysis of glomerular molecular and cellular changes in a model of congenital nephron reduction.
Effect of pH on potassium and proton conductance in renal proximal tubule
Authors: Gu X. , Sackin H.
Published: December 2017

The effect of intracellular and extracellular pH on potassium conductance (GK) was examined in isolated amphibian (Rana pipiens) proximal tubule cells under whole cell voltage clamp conditions. Internal perfusion of the patch pipette was used to precisely control intracellular pH. In the region of normal resting potential (-51 +/- 3 mV), raising cell pH from 6.5 to 8.0 did not significantly increase GK (1.1 +/- 0.3 vs. 1.3 +/- 0.3 nS; P > 0.08, n = 8). Similar elevations in external (bath) pH had even less of an effect on GK. In contrast, when cells were voltage clamped to 30 mV more negative than the resting potential, raising internal pH from 6.5 to 8.0 did increase GK from 1.05 +/- 0.3 to 1.8 +/- 0.5 nS (P < 0.04; n = 8). These results suggest that modest changes in pH have little effect on GK, except at large negative potentials. In the process of examining the pH dependence of GK, a slowly activating, voltage-dependent conductance of 7.5 +/- 1 nS (n = 20; for 20 microns cells) was observed during cell depolarization. Although the instantaneous current-voltage relation of this conductance was linear, its marked voltage dependence produced an apparent steady-state rectification, with Gm = 0.5 +/- 0.2 nS and Gout = 9.0 +/- 1 nS (n = 11). Outward current was reversibly blocked by 3 mM Cu, Cd, or Co. In the absence of Na, K, and Ca (and only trace amounts of Cl), rapid changes in bath pH from 6.5 to 8.0 shifted the steady-state reversal potential (Erev) by -37 +/- 4 mV (n = 9) and the instantaneous Erev by -56 +/- 4 mV (n = 9). These shifts in Erev were consistent with a hydrogen ion conductance (GH), similar to what has been reported for snail neuron, neutrophils, alveolar epithelial cells, and phagocytes. Since the magnitude of this GH would be insignificant at resting cell pH and membrane potential, its role in renal proximal tubule under normal conditions is somewhat obscure. Nonetheless, in pathological situations, GH could function to prevent acid overload during any process that depolarizes the cell, such as low temperature or metabolic inhibition.
P2 purinoceptor stimulation attenuates PTH inhibition of phosphate uptake by a G protein-dependent mechanism
Authors: Lederer E. D. , McLeish K. R.
Published: December 2017

Purinergic P2 receptors are present on proximal renal tubules, but their function is unknown. Because P2 agonists antagonize vasopressin-stimulated water transport in the distal tubule by inhibiting activation of adenylyl cyclase, we postulated that P2 receptor activation blocks parathyroid hormone (PTH) inhibition of phosphate uptake in proximal tubule by preventing PTH-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation. PTH inhibition of sodium-dependent phosphate uptake was attenuated by alpha,beta-methylene-ATP (AMP-CPP), a P2x receptor agonist, but not by 2-methyl-thio-ATP, a P2y receptor agonist, in a dose-dependent manner. AMP-CPP did not attenuate inhibition of phosphate uptake produced by direct activation of adenylyl cyclase with forskolin, by addition of the cAMP analogue 8-bromo-cAMP, or by inhibition of cAMP phosphodiesterase with RO-20-1724. Additionally, AMP-CPP had no effect on basal or PTH-stimulated cAMP production. As PTH also stimulates protein kinase C activation, the effect of AMP-CPP on inhibition of phosphate uptake stimulated by phorbol 12-myristate 13-acetate (PMA) was tested. AMP-CPP had no effect on PMA-induced inhibition of phosphate uptake. Pretreatment with pertussis toxin abolished the attenuating effect of AMP-CPP on PTH inhibition of sodium-dependent phosphate uptake. We conclude that activation of purinergic P2 receptors attenuates the inhibitory effect of PTH on sodium-dependent phosphate uptake by a G protein-dependent mechanism that is independent of cAMP generation protein kinase A activation, or protein kinase C activation.
Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction
Authors: Quigley R. , Kennerly D. A. , Sheu J. N. , Baum M.
Published: December 2017

The present study investigated the signal-transduction pathway responsible for the epidermal growth factor (EGF) stimulation of phosphate transport (JPhos) in the rabbit proximal convoluted tubule (PCT). Genistein, 10(-4) M, bath and lumen, an inhibitor of EGF receptor tyrosine kinase activity, blocked the EGF effect on JPhos, consistent with a role for tyrosine kinase in the signal-transduction pathway. Both staurosporine (5 x 10(-8) M) and calphostin C (10(-8) M), inhibitors of protein kinase C, blocked the EGF stimulation of JPhos, indicating that protein kinase C is involved in EGF signaling. Intracellular calcium (Ca2+i) concentrations were measured in perfused tubules using fura PE3 to determine whether changes in Ca2+i were also part of the signaling pathway. After addition of 3 nM EGF, there was no change in Ca2+i, suggesting that stimulation of protein kinase C is not from phosphatidylinositol hydrolysis by phospholipase C-gamma. To determine whether phospholipase A2 (PLA2) is involved, the inhibitor mepacrine was used. Mepacrine (5 x 10(-5) M) had no direct effect on PCT transport but blocked the stimulatory effect of EGF on JPhos. PLA2 activity, assessed as free arachidonic acid release from proximal tubules in suspension, increased by 18.8% with 3 nM EGF. Thus the stimulation of JPhos by EGF is mediated via a signal-transduction pathway involving tyrosine kinase, protein kinase C, and PLA2.
Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell
Authors: Kubokawa M. , McNicholas C. M. , Higgins M. A. , Wang W. , Giebisch G.
Published: December 2017

The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel "rundown") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca(2+)-dependent PP (PP-2B), and okadaic acid (OA, 1 microM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP-free solution maintained channel activity at levels of approximately 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 microM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory-effects of PP-1 (1 U/ml) and PP-2B (20 micrograms/ml) were minor. These results suggest that rundown of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential expression of multiple glutaminase mRNAs in LLC-PK1-F+ cells
Authors: Porter D. , Hansen W. R. , Taylor L. , Curthoys N. P.
Published: December 2017

LLC-PK1-F+ cells are porcine proximal tubule-like cells that have been used to model the renal ammoniagenic response to metabolic acidosis. A 3.2-kb porcine glutaminase (GA) cDNA (pGA201) containing 528 bp of coding sequence and 2.7 kb of 3'-untranslated region was cloned and sequenced. Probes derived from both porcine and rat GA cDNAs were used to characterize the expression of putative GA mRNAs in LLC-PK1-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb in length) were resolved and a smaller 2.5-kb species was also observed. The level of the 5.0-kb mRNA is detectable in freshly split LLC-PK1-F+ cells and increases as the cells reach confluence. In contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly split cells and decreases gradually as the cells approach confluence. The levels of the 5.0- and 2.5-kb mRNAs are also affected by refeeding the cells, and the 2.5-kb mRNA accumulates to high levels if cells are retained in the same media for 4 days. Exposure to acidic media had little or no effect on the levels of GA mRNAs expressed in confluent or postconfluent cells, whereas, in growing and undifferentiated cells, this treatment did affect the level of the 4.5-kb mRNA. Thus the putative GA mRNA species are differentially expressed. Given this complexity, a careful assessment of GA mRNA species, of basal expression, and of growth conditions are essential for a meaningful analysis of GA mRNA levels in cultured cells.
Biphasic effect of angiotensin II on intracellular sodium concentration in rat proximal tubules
Authors: Reilly A. M. , Harris P. J. , Williams D. A.
Published: December 2017

Intracellular Na+ concentration ([Na+]i) was determined using ratiometric measurement of the Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). Angiotensin II (ANG II, 10(-11)-10(-7) M), applied to the basolateral membrane of rat isolated proximal convoluted tubules, induced a rapid and reversible dose-dependent increase in [Na+]i, which was initiated within 300 ms. A maximal response was observed over the range 10(-9)-10(-7) M ANG II, with an average increase in [Na+]i of 7.4 +/- 1.0 mM. At higher concentrations (10(-6)-10(-5) M) ANG II decreased [Na+]i compared with control (14.2 +/- 0.6 mM). The increase in [Na+]i induced by 10(-9) M ANG II was attenuated by inhibiting the Na+/H+ antiporter with clonidine, whereas HOE-694, a specific blocker of the NHE-1 isoform of the Na+/H+ exchanger, had no effect. The increase in [Na+]i induced by 10(-9) M ANG II was enhanced by inhibition of the Na(+)-HCO3- cotransporter with hydrogen-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, with an average increase in [Na+]i of 17.1 +/- 6.6 mM. The data provide direct, high time resolution measurements of the effects of ANG II on [Na+]i in the proximal tubule and support the proposition that an increase in transepithelial Na+ reabsorption by ANG II involves stimulation of both an Na+/H+ exchanger and the Na(+)-HCO3- cotransporter.
Decreased transcription of the sodium-phosphate transporter gene in the hypophosphatemic mouse
Authors: Collins J. F. , Scheving L. A. , Ghishan F. K.
Published: December 2017

Recently, it has been hypothesized that the proximal tubular Na(+)-Pi transporter may play a role in murine X-linked hypophosphatemic vitamin D-resistant rickets. In the present investigation, Western blot analysis of renal brush-border membrane proteins, utilizing polyclonal antisera raised against the mouse Na(+)-Pi transporter, revealed a predominant band at 87 kDa in normal and hypophosphatemic (Hyp) mice. The intensity of this band was reduced in the Hyp mouse by 4.5-fold (Hyp/normal = 0.22 +/- 0.04, n = 3, P < 0.05). Additionally, immunohistochemical analysis of kidney cortex in both mice localized the protein to the apical membrane of the proximal tubules. Relative transcription rates of the Na(+)-Pi transporter gene in the normal and Hyp mouse were then investigated. Nuclear run-on assays showed a 51 +/- 0.02% decreased rate of transcription of the Na(+)-Pi transporter gene in the Hyp mice (n = 3). Thus abnormal transcriptional control of this gene in the Hyp mouse likely plays a role in X-linked hypophosphatemia.
Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney
Authors: Brosius F. C. , Nguyen K. , Stuart-Tilley A. K. , Haller C. , Briggs J. P. , Alper S. L.
Published: December 2017

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.
NH3 permeability of principal cells and intercalated cells measured by confocal fluorescence imaging
Authors: Yip K. P. , Kurtz I.
Published: December 2017

The cortical collecting duct (CCD) is an important site for NH3 secretion in mammalian nephron. However, given the cellular heterogeneity of this epithelium, the transcellular sites for NH3 secretion are unknown. In the present study, a dual-excitation confocal microscope was designed and optimized to have sufficient temporal resolution to measure the permeability of ammonia (PNH3) across the basolateral and apical membrane of principal cells (PCs) and intercalated cells (ICs) in perfused rabbit CCDs. The rate of cellular NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein after 20 mM NH4Cl was added to the bath or luminal perfusate. The time course of increase in pHi was calculated from 488/442 image pairs stored at a rate of 4 Hz. The apparent basolateral and apical PNH3 values of PCs were 36 +/- 5 and 113 +/- 11 microns/s, respectively. The values were 5.0 +/- 0.7 and 34 +/- 3 microns/s after membrane folding correction. The apparent basolateral and apical PNH3 values of ICs were 38 +/- 6 and 132 +/- 15 microns/s. Corrected for membrane folding, the values were 9.0 +/- 1.0 and 47 +/- 5 microns/s, respectively. The results demonstrate that the apical surface was more permeable than the basolateral surface in both cell types. In addition, ICs were more permeable to NH3 than PCs across both membranes. The transcellular PNH3 of PCs and ICs were 27.3 and 29.5 microns/s, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of colonic H-K-ATPase mRNA in cortical collecting duct: regulation by acid/base balance
Authors: Fejes-Toth G. , Rusvai E. , Longo K. A. , Naray-Fejes-Toth A.
Published: December 2017

In addition to the gastric isoform of H-K-ATPase, the colonic isoform is also expressed in the kidney, but its intrarenal localization and exact function are not known. The goal of this study was to determine whether the colonic H-K-ATPase is expressed in the rabbit cortical collecting duct (CCD) and whether it is regulated by changes in acid/base balance. With quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (approximately 430 bp) was amplified. The amplified DNA was further characterized by nested PCR and sequencing. Direct sequencing of the 434-bp PCR product revealed 83% identity at the nucleotide level and an 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. With the same primers and cDNA originating from rabbit distal colon, a DNA fragment with a size and nucleotide sequence identical to that originating from CCD cells was amplified. Furthermore, using PCR screening, we isolated and sequenced a 1.5-kb cDNA clone from a rabbit CCD library. The predicted amino acid sequence of the protein encoded by this cDNA is 85 and 82% identical to the corresponding regions of the guinea pig and rat colonic H-K-ATPase, respectively, and 70% identical to the H-K-ATPase recently cloned from Bufo marinus, whereas it shows only 45 and 42% homology to the rat Na-K-ATPase alpha 1-subunit and the rat gastric H-K-ATPase, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of calcineurin activity and alpha-subunit isoforms in specific segments of the rat nephron
Authors: Tumlin J. A. , Someren J. T. , Swanson C. E. , Lea J. P.
Published: December 2017

Calcineurin activity and alpha-subunit expression were studied in microdissected proximal tubules (S2), medullary thick ascending limbs (MTAL), cortical collecting ducts (CCD), connecting tubules (CNT), and outer medullary collecting ducts (OMCD). We have shown that cyclosporin A (CsA) and FK-506 inhibit sodium-potassium-adenosinetriphosphatase (Na-K-ATPase) activity in CCD, OMCD, and MTAL but did not uncover the mechanism for resistance of proximal tubule segments to these drugs. Because cells expressing high calcineurin activity are relatively resistant to the biological effects of CsA and FK-506, we hypothesized that the resistance of proximal tubules may be linked to increased calcineurin expression. Consequently, we measured calcineurin activity in microdissected tubules using a calcineurin-specific substrate. Calcineurin activity in S2 proximal tubule segments was 10-fold higher than in CCD, CNT, OMCD, or MTAL. FK-506 (6.0 ng/ml) inhibited calcineurin activity in CCD, CNT, and MTAL but not S2; 250 ng/ml FK-506 inhibited S2 calcineurin activity by 50%. Likewise, high concentrations of CsA (25 micrograms/ml) and FK-506 (250 ng/ml) inhibited S2 Na-K-ATPase activity by 77 and 73%, respectively. To investigate whether the resistance of S2 segments might be due to differential expression of calcineurin alpha-subunit isoforms, we determined the isoform expression by Western blot analysis using isoform-specific antibodies against the alpha 1-, alpha 2-, and alpha 3-isoforms. We found that alpha 1 expression in S2 was significantly greater than in the CCD and MTAL, whereas alpha 2 expression in the S2 was significantly less than in CCD and MTAL. No alpha 3 was detected in any nephron segment tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Transgenic animal models of renal development and pathogenesis
Authors: Kopp J. B. , Klotman P. E.
Published: December 2017

The use of transgenic animals represents a powerful tool with which to address the role of particular gene products in vivo. Recent technical and biological advances have simplified the process of creating both transgenic mice and null-mutation mice. Increasing numbers of genetic control elements are available to direct transgene expression to particular renal cell types and to enhance the consistency of expression. These approaches have contributed significantly to our understanding of renal development and pathogenesis, in particular in the following areas: the roles of various oncogenes, homeobox genes, and growth factors in renal development and the pathogenesis of cystic renal diseases; the contribution of systemic and local expression of the renin-angiotensin system to blood pressure control; the role of growth factors and cytokines in progressive glomerular disease; the role of viral proteins in the pathogenesis of glomerular and tubular disease; and mechanisms of immune-mediated renal disease.
Quantitative RT-PCR analysis of calcitonin receptor mRNAs in the rat nephron
Authors: Firsov D. , Bellanger A. C. , Marsy S. , Elalouf J. M.
Published: December 2017

A quantitative assay based on the method of reverse transcription and polymerase chain reaction (RT-PCR) was developed to study the expression of calcitonin (CT) receptors in microdissected rat nephron segments. Steady-state mRNA levels of two CT-receptor spliced variants (CT1a and CT1b) were measured using a mutant cRNA as internal standard. CT1a, but not the CT1b isoform, was detected in the kidney cortex, outer medulla, and papilla. Among the tested segments, predominant expression of CT1a mRNA was found in the cortical thick ascending limb of Henle's loop (754 +/- 87 mRNA molecules/mm tubular length; n = 8). Lower expression levels were measured in the medullary thick ascending limb (460 +/- 62 molecules/mm tubule length; n = 7) and in the cortical collecting duct (327 +/- 61 molecules/mm tubule length; n = 6). A weak expression was also detected in the outer medullary collecting duct and the glomerulus. No expression was found in the proximal convoluted tubule, pars recta, and thin descending and thin ascending limb of Henle's loop. We conclude that only the CT1a-receptor mRNA is present in the rat kidney, with a significant level of expression in the cortical and medullary thick ascending limb and in the cortical collecting duct.
Regulation of luminal alkalinization and acidification in the cortical collecting duct by angiotensin II
Authors: Weiner I. D. , New A. R. , Milton A. E. , Tisher C. C.
Published: December 2017

Angiotensin II (ANG II) regulates whole kidney ion transport, yet its effects in the collecting duct are unknown. The purpose of these studies was to determine whether ANG II regulates luminal alkalinization and acidification in the rabbit cortical collecting duct (CCD). The rate of luminal alkalinization or acidification was measured as the rate of change of luminal fluid pH under stop-flow conditions using in vitro microperfused CCD segments. Outer CCD alkalinized the luminal fluid, consistent with net HCO3- secretion. Addition of ANG II, 10(-7) M, to the peritubular solution for 30 min significantly stimulated luminal alkalinization. The stimulatory effect of ANG II was not due to time-dependent effects and was blocked by peritubular addition of the ANG II type 1 (AT1) receptor antagonist, losartan, at 10(-6) M. Losartan, 10(-6) M, when added to the peritubular solution, did not alter the rate of luminal alkalinization independent of ANG II. In contrast, peritubular ANG II, 10(-7) M, did not alter inner CCD luminal acidification. Addition of ANG II to the peritubular solution at the lower concentration of 10(-10) M did not alter the rates of luminal alkalinization and acidification in the outer and inner CCD, respectively. Peritubular ANG II, 10(-7) M, but not vehicle, stimulated B cell apical HCO3- secretion occurring in response to peritubular Cl- removal. These studies demonstrate that ANG II acts through a basolateral AT1 receptor to stimulate outer CCD luminal alkalinization via, at least in part, B cell stimulation.
Homeostatic efficiency of tubuloglomerular feedback is reduced in established diabetes mellitus in rats
Authors: Vallon V. , Blantz R. C. , Thomson S.
Published: December 2017

We tested the hypothesis that the ability of the tubuloglomerular feedback (TGF) to stabilize renal function is impaired in rats with 7-8 wk of insulin-treated streptozotocin-diabetes. Proximal tubular flow was measured in free-flowing nephrons using a noninvasive optical technique. The homeostatic efficiency of TGF was determined from the fractional compensation for perturbations in ambient flow. Fractional compensation was substantially reduced in diabetic rats. To assess the roles of the proximal tubule and loop of Henle as determinants of TGF efficiency, we tested the effect of diabetes on proximal tubular reabsorption as determined by standard micropuncture and on the ionic content of early distal tubular fluid by employing a microelectrode for on-line measurement of electrical conductivity (TED). Diabetes caused glomerular hyperfiltration and increased fractional proximal tubular reabsorption (FPR), such that late proximal tubular flow (VLP) and early distal tubular flow were unaffected. The increase in FPR was a minor contributor to the overall effect on fractional compensation. Diabetes decreased the ambient TED without affecting the slope of the relationship between VLP and TED. These results demonstrate that the homeostatic, efficiency of the TGF system is reduced in diabetes and that this cannot be fully accounted for by changes in tubular reabsorption. Impaired TGF efficiency renders the diabetic glomerular microvasculature more susceptible to impact from fluctuations in systemic hemodynamics.