bluuebunny/crossref_metadata_2025 huggingface dataset:
Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes
Authors: Rivers Rickey , Blanchard Anne , Eladari Dominique , Leviel Francois , Paillard Michel , Podevin Rene-Alexandre , Zeidel Mark L.
Published: December 2017

The medullary thick ascending limb (MTAL) reabsorbs solute without water and concentrates [Formula: see text] in the interstitium without a favorable pH gradient, activities which require low water and NH3permeabilities. The contributions of different apical and basolateral membrane structures to these low permeabilities are unclear. We isolated highly purified apical and basolateral MTAL plasma membranes and measured, by stopped-flow fluorometry, their permeabilities to water, urea, glycerol, protons, and NH3. Osmotic water permeability at 20°C averaged 9.4 ± 0.8 × 10−4cm/s for apical and 11.9 ± 0.5 × 10−4cm/s for basolateral membranes. NH3permeabilities at 20°C averaged 0.0023 ± 0.00035 and 0.0035 ± 0.00080 cm/s for apical and basolateral membranes, respectively. These values are consistent with those obtained in isolated perfused tubules and can account for known aspects of MTAL function in vivo. Because the apical and basolateral membrane unit permeabilities are similar, the ability of the apical membrane to function as the site of barrier function arises from its very small surface area when compared with the highly redundant basolateral membrane.
B2kinin receptor upregulation by cAMP is associated with BK-induced PGE2production in rat mesangial cells
Authors: Castaño Maria E. Marin , Schanstra Joost P. , Hirtz Christophe , Pesquero João B. , Pecher Christiane , Girolami Jean-Pierre , Bascands Jean-Loup
Published: December 2017

In the rat mesangial cell (MC), activation of the bradykinin B2receptor (B2R) by bradykinin (BK) is associated with both phospholipase C (PLC) and A2(PLA2) activities and with inhibition of adenosine 3′,5′-cyclic monophosphate (cAMP) formation leading to cell contraction. Because cAMP plays an important role in the regulation of gene expression in general, we investigated the effect of increasing the intracellular cAMP concentration ([cAMP]i) in mesangial cells on the B2mRNA expression, on the density of B2receptor binding sites, on the BK-induced increase in both the free cytosolic Ca2+concentration ([Ca2+]i), and in the prostaglandin E2(PGE2) production. Forskolin, PGE2, and cAMP analog, 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP), were used to increase [cAMP]i. Twenty-four-hour treatment with forskolin, PGE2, and 8-BrcAMP resulted in significant increases in B2receptor binding sites, which were inhibited by cycloheximide. The maximum B2receptor mRNA expression (160% above control) was observed in cells treated during 24 h with forskolin and was prevented by actinomycin D. In contrast, thed- myo-inositol 1,4,5-trisphosphate (IP3) formation and the BK-induced increase in [Ca2+]i, reflecting activation of PLC, were not affected by increased levels of [cAMP]i. However, the BK-induced PGE2release, reflecting PLA2activity, was significantly enhanced. These data bring new information regarding the dual signaling pathways of B2receptors that can be differentially regulated by cAMP.
Development of pH regulatory transport in glomerular mesangial cells
Authors: Ganz Michael B. , Saksa Brett A.
Published: December 2017

Developmental changes in activity or expression of transporters may account for alterations in cell behavior as the nonpolarized cell matures. We sought to ascertain whether there is a maturational change in each of the major acid-base transporters in the developing mesangial cell (MC), the Na/H exchanger, Na-dependent Cl/HCO3exchanger, and the Cl/HCO3exchanger. Intracellular pH (pHi) was determined by the use of the fluorescent pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We assessed transporter activity by studying recovery from an acid load (NH4/NH3) in CO2/HCO3. In adult MCs, Na/H exchanger was responsible for 35.2 ± 4.3% of steady-state pHi, whereas the Na-dependent Cl/HCO3exchanger contributed 58.7 ± 6.1 ( n= 14). In term MCs (tMCs), from days 1–3 after birth, the Na/H exchanger contributes 62.9 ± 7.8% ( n = 11, P < 0.001 vs. adult), whereas the Na-dependent Cl/HCO3exchanger contributes 34.0 ± 5.7% ( n = 12, P < 0.001 vs. adult), to the rate of recovery from an acid load in these cells. However, in tMCs ( days 4–6), the Na/H contributes 47.2 ± 5.9% ( n = 8, P < 0.05 vs. adult), whereas the Na-dependent Cl/HCO3exchanger contributes 48.7 ± 7.3% ( n = 13, P < 0.05 vs. adult), to the rate of recovery. tMCs ( days 6–12) yielded transporter activity that was not statistically different than adult MCs (37.8 ± 4.9 and 54.3 ± 10.2% for Na/H and Na-dependent Cl/HCO3, respectively). The magnitude of the stimulated response to angiotensin II by Na/H and Na-dependent Cl/HCO3exchanger in adult and tMCs is unchanged throughout development. The Na/H exchanger appears to play a greater role in pHihomeostasis earlier on in development, and this may reflect developmental needs of the maturing cell.
Extrarenal resistance to atrial natriuretic peptide in rats with experimental nephrotic syndrome
Authors: Valentin Jean-Pierre , Ying Wei-Zhong , Couser William G. , Humphreys Michael H.
Published: December 2017

Nephrotic syndrome is associated with resistance to the renal actions of atrial natriuretic peptide (ANP). We performed experiments in anesthetized, acutely nephrectomized rats 21–28 days after injection of adriamycin (7–8 mg/kg iv) or 9–14 days after injection of anti-Fx1A antiserum (5 ml/kg ip) (passive Heymann nephritis; PHN) to test whether extrarenal resistance also occurred. Proteinuria was significantly elevated in both models compared with controls before study. ANP infusion (1 μg ⋅ kg−1⋅ min−1) caused arterial pressure to decrease similarly in control rats, adriamycin-treated rats, and rats with PHN (by 8.2 ± 1.0, 9.4 ± 2.3, and 9.0 ± 2.0%, respectively; all P < 0.05 vs. both baseline and vehicle-infused control rats). In control rats, hematocrit increased progressively to a maximal value 9.5 ± 0.9% over baseline as a result of the infusion, an increase corresponding to a reduction in plasma volume of 16.1 ± 0.9%. The ANP-induced increase in hematocrit was preserved in adriamycin-treated rats (9.2 ± 1.3%) but was markedly blunted in rats with PHN (2.4 ± 1.3%; P < 0.0001 vs. ANP infusion in control rats). ANP infusion increased plasma ANP levels to the same extent in the three groups, whereas plasma guanosine 3′,5′-cyclic monophosphate was significantly lower in rats with PHN compared with both control and adriamycin-treated rats. Infusion of a subpressor dose of angiotensin II (ANG II, 2.5 ng ⋅ kg−1⋅ min−1) fully restored the ANP-induced increase in hematocrit in rats with PHN. This study demonstrates that 1) the hemoconcentrating and hypotensive actions of ANP are preserved in adriamycin-treated rats, 2) the effect of ANP on hematocrit and fluid distribution is blunted in rats with PHN while its hypotensive action is preserved, and 3) low-level ANG II infusion normalizes the hemoconcentrating effect of exogenously infused ANP in rats with PHN. Thus deficient ANG II generation in rats with PHN, but not adriamycin nephrosis, may contribute to extrarenal ANP resistance.
K depletion stimulates in vivo HCO3reabsorption in surviving rat distal tubules
Authors: Levine David Z. , Iacovitti Michelle , Buckman Susan , Luck Brian , Hincke Maxwell T. , Burns Kevin D. , Fryer James N.
Published: December 2017

To evaluate whether K depletion enhances in vivo bicarbonate reabsorption ([Formula: see text]in surviving distal tubules (DT), we compared DT[Formula: see text]in five-sixths nephrectomized rats (Nx) with and without dietary K depletion (Nx-K). Furthermore, to identify possible mechanisms of increased[Formula: see text], we perfused inhibitors of proton secretion in both Nx and Nx-K rats.[Formula: see text](102 ± 8 pmol ⋅ min−1⋅ mm−1) was significantly increased in Nx-K vs. Nx rats (65 ± 7 pmol ⋅ min−1⋅ mm−1, P < 0.05) but unaffected by 10−6M losartan perfusion (94 ± 6 pmol ⋅ min−1⋅ mm−1, P = not significant). Although 10−5M Sch-28080 also had no significant effect, 5 × 10−9M concanamycin A perfusion significantly decreased[Formula: see text]in Nx-K rats to 65 ± 8 pmol ⋅ min−1⋅ mm−1( P < 0.05). Morphometric evaluation and H+-ATPase immunogold labeling of Nx-K A-type intercalated cells revealed cellular hypertrophy, elaborated apical microplicae, and enhanced H+-ATPase apical polarization. Accordingly, these combined studies confirm that K depletion enhances[Formula: see text]in surviving DT by stimulating H+-ATPase activity, independent of the AT1receptor.
Blood pressure variability and urine flow in the conscious dog
Authors: Nafz Benno , Ehmke Heimo , Wagner Claus D. , Kirchheim Hartmut R. , Persson Pontus B.
Published: December 2017

Pressure-dependent urine production is considered to be a major factor in long-term blood pressure control. The phenomenon has been well characterized for fixed levels of renal perfusion pressure (RPP), but the influence of physiological fluctuations in RPP and spontaneous variations in renal blood flow (RBF) on short-term urine flow (UV) remain unclear. To clarify this issue, we studied the interdependence of RPP, RBF, and UV in 13 conscious foxhounds during a single-step pressure reduction, under normal conditions, and with induced pressure changes. Reducing RPP in a single step to ∼80 mmHg revealed short response times of RBF (0.4 ± 0.1 s, n = 7) as well as of UV (8.1 ± 0.8 s, n = 7). Under control conditions, UV was coupled with spontaneous variations of RBF ( r = 0.94, P < 0.001), in contrast to RPP, which showed no significant correlation with UV ( r = 0.09, P = NS). To discern the pressure and blood flow dependency of UV at a reduced RPP, we induced 0.9-mHz blood pressure oscillations (80 ± 10 mmHg), which phase shifted RPP and RBF. Conversely, under these conditions, UV was dependent on RPP ( r = 0.95, P < 0.001). These results suggest that spontaneous fluctuations in RBF around a normal baseline level lead to concomitant changes in urine production, in contrast to physiological short-term oscillations in RPP, which are not correlated to changes in UV. However, during induced oscillations of perfusion pressure, the blood flow dependence was no longer observed and UV was entirely pressure dependent.
Direct assessment of renal microvascular responses to P2-purinoceptor agonists
Authors: Inscho Edward W. , Cook Anthony K. , Mui Vy , Miller Jason
Published: December 2017

Studies were performed to determine the responsiveness of rat juxtamedullary afferent arterioles to receptor-selective P2-purinoceptor agonists. Experiments were performed in vitro using the blood perfused juxtamedullary nephron technique, combined with videomicroscopy. Renal perfusion pressure was set at 110 mmHg and held constant. Basal afferent arteriolar diameter averaged 22.0 ± 0.6 μm ( n = 69). Stimulation with 0.1, 1.0, 10, and 100 μM ATP ( n = 10) elicited a concentration-dependent vasoconstriction averaging 8 ± 2, 17 ± 2, 21 ± 4, and 23 ± 5%, respectively. A nearly identical afferent arteriolar vasoconstriction was observed in response to the P2X-selective agonist β,γ-methylene ATP ( n = 10); however, another P2X agonist, α,β-methylene ATP, evoked marked receptor desensitization ( n = 10). Vessel diameter decreased by ∼7 ± 2, 16 ± 2, 23 ± 3, and 22 ± 3%, respectively, over the same concentration range. The P2Y-selective agonist, 2-methylthio-ATP, evoked only a modest vasoconstriction, whereas UTP and adenosine 5′- O-(3-thiotriphosphate) (ATPγS) reduced afferent diameter markedly at concentrations >1.0 μM. Afferent arteriolar diameter decreased by 5 ± 4, 31 ± 8, and 72 ± 8% during UTP administration ( n = 7) at concentrations of 1.0, 10, and 100 μM, respectively. Similarly, ATPγS ( n = 6) decreased afferent diameter by 16 ± 2, 58 ± 8, and 98 ± 3%, respectively, over the same concentration range. Nitric oxide synthesis inhibition with Nω-nitro-l-arginine did not significantly alter the afferent arteriolar response to ATP but did potentiate ATP-mediated arcuate artery vasoconstriction. The following data suggest the presence of multiple P2 receptors on juxtamedullary afferent arterioles and are consistent with classification of those receptors as members of the P2X- and P2Y2(P2U)-receptor subtypes.
Adenosine-stimulated Ca2+reabsorption is mediated by apical A1receptors in rabbit cortical collecting system
Authors: Hoenderop Joost G. J. , Hartog Anita , Willems Peter H. G. M. , Bindels René J. M.
Published: December 2017

Confluent monolayers of immunodissected rabbit connecting tubule and cortical collecting duct cells, cultured on permeable supports, were used to study the effect of adenosine on net apical-to-basolateral Ca2+transport. Apical, but not basolateral, adenosine increased this transport dose dependently from 48 ± 3 to 110 ± 4 nmol ⋅ h−1⋅ cm−2. Although a concomitant increase in cAMP formation suggested the involvement of an A2receptor, the A2agonist CGS-21680 did not stimulate Ca2+transport, while readily increasing cAMP. By contrast, the A1agonist N6-cyclopentyladenosine (CPA) maximally stimulated Ca2+transport without significantly affecting cAMP. Adenosine-stimulated transport was effectively inhibited by the A1antagonist 1,3-dipropyl-8-cyclopenthylxanthine but not the A2antagonist 3,7-dimethyl-1-propargylxanthine, providing additional evidence for the involvement of an A1receptor. Both abolishment of the adenosine-induced transient increase in intracellular Ca2+concentration by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid and downregulation of protein kinase C (PKC) by prolonged phorbol ester treatment were without effect on adenosine-stimulated Ca2+transport. The data presented suggest that adenosine interacts with an apical A1receptor to stimulate Ca2+transport via a hitherto unknown pathway that does not involve cAMP formation, PKC activation, and/or Ca2+mobilization.
Effect of modifying O2diffusivity and delivery on glomerular and tubular function in hypoxic perfused kidney
Authors: Baines A. D. , Adamson G. , Wojciechowski P. , Pliura D. , Ho P. , Kluger R.
Published: December 2017

Is O2 diffusivity within renal capillaries rate limiting for O2 delivery to hypoxic renal tubules? Equations based on diffusion theory and developed here predict that soluble hemoglobin (Hb) increases O2 diffusivity by a factor of 1 + [442 Hb%/(P50 +[Formula: see text])], where P50 is the partial pressure of O2 at which the Hb is half saturated. To examine the effect of P50 and Hb concentrations on renal function, we perfused isolated rat kidneys with Hb-P35(P50 = 35 mmHg) and Hb-P11(P50 = 11 mmHg). Venous[Formula: see text] was lower with Hb-P11 (10 ± 1 vs 16 ± 1 mmHg with arterial [Formula: see text] = 35 mmHg and 28 ± 2 vs. 40 ± 2 mmHg with arterial[Formula: see text] =140 mmHg; P < 0.001). Perfusate P50 did not influence vascular resistance, glomerular filtration rate, O2 consumption, Na reabsorption, protein excretion, or free water clearance. Percent glucose and phosphate excretion were lower with Hb-P11 than with Hb-P35( P < 0.001). Urine glucose was 0.17 mmol/l with Hb-P11 and 0.77 mmol/l with Hb-P35( P < 0.001). Hb-P35 (2%) doubled O2 delivery and lowered glucose and phosphate excretion to the level obtained with 1% Hb-P11. Thus Hb-P11 delivered O2 twice as effectively as Hb-P35 to high-affinity sodium glucose and phosphate cotransporters in the late proximal tubule (S3 segment). Hb-P11 may also have shunted O2 from the outer cortex to the outer medulla and facilitated O2 diffusion where[Formula: see text] was low. We conclude that diffusivity is a limiting factor in delivery of O2 to hypoxic tubules.
Isolation and characterization of the lower portion of the thin limb of Henle in primary culture
Authors: Grupp Clemens , Begher Michael , Cohen David , Raghunath Michael , Franz Hans-Eduard , Müller Gerhard A.
Published: December 2017

To further characterize cells of the lower portion of the thin limb of Henle (TLHlp) under defined conditions in vitro, we developed a technique to enrich this cell population in suspension. TLHlpcells were isolated by enzymatic digestion of rat inner medulla, elimination of collecting ducts by lectin-coated beads, and differential centrifugation. Immunohistochemical staining of primary cultures of TLHlpcells with various markers revealed the preparations to be >90% pure. The hormonal stimulation pattern of PGE2and cAMP production by arginine vasopressin, angiotensin II, and dopamine in the isolated cells also argued against significant contamination by other cell types. Staining with an antibody against the aquaporin-1 water channel showed the distribution of cells from the ascending and descending limbs to be approximately equal in the isolated population. This technique allows the enrichment of cells from the lower portion of the thin limb of Henle in suspension to a very high degree of purity with the option to start primary cultures. Because these segments of the tubular system in particular are relatively inaccessible for microdissection, the presented method renders the possibility of addressing new questions regarding these tubular segments under defined conditions in vitro.
Volume repletion after exercise-induced volume depletion in humans: replacement of water and sodium losses
Authors: Shirreffs Susan M. , Maughan Ronald J.
Published: December 2017

Sodium and water loss during, and replacement after, exercise-induced volume depletion was investigated in six volunteers volume depleted by 1.89 ± 0.17% (SD) of body mass by intermittent exercise in a warm, humid environment. Subjects exercised in a large, open plastic bag, allowing collection of all sweat secreted during exercise. For over 60 min beginning 40 min after the end of exercise, subjects ingested drinks containing 0, 25, 50, or 100 mmol/l sodium ( trials 0, 25, 50, and 100) in a volume (ml) equivalent to 150% of the mass lost (g) by volume depletion. Body mass loss and sweat electrolyte (Na+, K+, and Cl−) loss were the same on each trial. The measured sweat sodium concentration was 49.2 ± 18.5 mmol/l, and the total loss (63.9 ± 38.7 mmol) was greater than that ingested on trials 0 and 25. Urine production over the 6-h recovery period was inversely related to the amount of sodium ingested. Subjects were in whole body negative sodium balance on trials 0 (−104 ± 48 mmol) and 25 (−65 ± 30 mmol) and essentially in balance on trial 50(−13 ± 29 mmol) but were in positive sodium balance on trial 100 (75 ± 40 mmol). Only on trial 100 were subjects in positive fluid balance at the end of the study. There was a large urinary loss of potassium over the recovery period on trial 100, despite a negligible intake during volume repletion. These results confirm the importance of replacement of sodium as well as water for volume repletion after sweat loss. The sodium intake on trial 100 was appropriate for acute fluid balance restoration, but its consequences for potassium levels must be considered to be undesirable in terms of whole body electrolyte homeostasis for anything other than the short term.
Assessment of the charge selectivity of glomerular basement membrane using Ficoll sulfate
Authors: Bolton Glen R. , Deen William M. , Daniels Barbara S.
Published: December 2017

The extent to which the glomerular basement membrane (GBM) contributes to the charge selectivity of the glomerular capillary wall has been controversial. To reexamine this issue, the size and charge selectivity of filters made from isolated rat GBM were assessed, using polydisperse Ficoll and Ficoll sulfate as test macromolecules. Ficoll sulfate, a novel tracer with spherical shape synthesized for this purpose, exhibited little or no binding to serum albumin, thereby avoiding a major difficulty that has been reported with dextran sulfate. The sieving coefficients of Ficoll sulfate were not different from those of Ficoll at physiological ionic strength, although the values for Ficoll sulfate were depressed at low ionic strength. These results confirm that the GBM possesses fixed negative charges but suggest that its charge density is insufficient to confer significant charge selectivity under physiological conditions, where electrostatic interactions are relatively well screened. The sieving coefficients of Ficoll sulfate and Ficoll were elevated significantly and by similar amounts when bovine serum albumin (BSA) was present in the retentate at 4 g/dl. This could be explained as the combined effect of two nonspecific physical factors, namely, the reduction in filtration velocity due to the osmotic pressure of BSA and the effect on macromolecular partitioning of repulsive solute-solute interactions. The view that BSA does not affect the intrinsic properties of the GBM is supported also by the absence of an effect on the hydraulic permeability of isolated GBM. The sieving coefficient of BSA was roughly half that of Ficoll or Ficoll sulfate of similar Stokes-Einstein radius. Given the finding of negligible charge selectivity, this difference may be attributed to the nonspherical shape of albumin. The results suggest that, to the extent that isolated GBM is similar to GBM in vivo, the charge selectivity of the glomerular capillary wall must be due to the endothelial and/or epithelial cell layers.
Developmental expression of human angiotensinogen in transgenic mice
Authors: Yang Gongyu , Sigmund Curt D.
Published: December 2017

Transgenic mice containing the human angiotensinogen ( HAGT) gene were utilized to determine the developmental regulation of HAGT expression. RNase protection assay on total RNA obtained from whole transgenic fetuses revealed that HAGT expression was first detected at embryonic day 8.5( E8.5) and was abundant from E9.5 onward. The earliest expression of the HAGT transgene appeared to precede the earliest expression of the endogenous mouse AGT gene by 1–2 days. Northern blot analysis revealed moderate levels of HAGT mRNA in liver and kidney and low levels of HAGT mRNA in heart and brain from E16.5 ( day 16.5 of gestation) onward. HAGT mRNA in liver, although abundant during late gestation and in 2-wk-old and adult mice, decreased transiently around birth. In situ hybridization performed on sections from whole fetuses revealed that HAGTmRNA was restricted to the developing liver and heart between E9.5 and E11.5 but became more widespread to include the developing aorta, brain, subcutaneous tissues, and vertebra at E13.5. In situ hybridization analysis on fetal kidneys from late gestation, newborn, and 2-wk-old mice demonstrated a progressive restriction of HAGT mRNA to developing cortical proximal tubular cells. These data illustrate the developmental tissue-specific regulation of HAGTexpression and demonstrate that sequences present in the transgene can confer an appropriate developmental expression profile.
Estradiol reverses TGF-β1-stimulated type IV collagen gene transcription in murine mesangial cells
Authors: Silbiger Sharon , Lei Jun , Ziyadeh Fuad N. , Neugarten Joel
Published: December 2017

We have previously shown that estradiol suppresses types I and IV collagen synthesis by mesangial cells grown in the presence of serum. In the present study, we examined the interaction between estradiol and transforming growth factor-β (TGF-β) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen promoter and α1-chain regulatory sequences, we found that TGF-β1 (2 ng/ml) stimulated α1-collagen IV gene transcription in serum-free media (140.5 ± 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed the stimulatory effects of TGF-β1 on reporter gene transcription in a dose-dependent manner [for 2.5 × 10−9M, 114.2 ± 0.2, P < 0.002 vs. TGF-β1; for 10−7M, 89.5 ± 4.0, P < 0.001 vs. TGF-β1 and P = not significant (NS) vs. control]. Using immunoprecipitation techniques, we found that estradiol (10−7M) reversed TGF-β1-stimulated type IV collagen synthesis (175.3 ± 14.7 vs. 111.6 ± 7.1, expressed as a percent of control untreated cells, P < 0.001) but did not affect TGF-β1-stimulated type I collagen synthesis (166.9 ± 18.8 vs. 162.2 ± 16.2, P = NS). These results were confirmed with Western blotting. Nuclear extracts from mesangial cells treated with TGF-β1 showed increased binding to a Sp1 consensus binding sequence oligonucleotide and to an Sp1 binding site in the collagen IV promoter. Estradiol reversed this enhanced binding. These data suggest that estradiol antagonizes TGF-β1-stimulated type IV collagen synthesis at a transcriptional level and that this effect may be mediated by interactions with the transcription factor Sp1.
Identification of the synthetic surfactant nonylphenol ethoxylate: a P-glycoprotein substrate in human urine
Authors: Charuk Jeffrey H. M. , Grey Arthur A. , Reithmeier Reinhart A. F.
Published: December 2017

P-glycoprotein (Mdr1p) is an ATP-dependent drug efflux pump that is overexpressed in multidrug-resistant cells and some cancers. Mdr1p is also expressed in normal tissues like the kidney, where it can mediate transepithelial drug transport. A human urinary compound that reverses multidrug resistance and blocks [3H]azidopine photolabeling of P-glycoprotein was purified to homogeneity and identified by1H-NMR and mass spectrometry as the synthetic surfactant nonylphenol ethoxylate (NPE). Multidrug-resistant Chinese hamster ovary (CHO) C5 cells accumulated less [3H]NPE than parental drug-sensitive Aux-B1 cells, and Mdr1p substrates, verapamil and cyclosporin A, increased this surfactant’s accumulation in C5 cells. NPE blocked the net transepithelial transport (basolateral to apical) of [3H]cyclosporin A in epithelia formed by Madin-Darby canine kidney (MDCK) cells. Net transepithelial transport (basal to apical) of [3H]NPE was demonstrated in MDCK cells and was inhibited by cyclosporin A. These findings show NPE is a Mdr1p substrate excreted into urine by kidney P-glycoprotein. NPE is a widely used surfactant and a known hormone disrupter that is readily absorbed orally or topically. The current findings indicate the function of kidney Mdr1p may be to eliminate exogenous compounds from the body.
Gene targeting in physiological investigations: studies of the renin-angiotensin system
Authors: Coffman Thomas M.
Published: December 2017

Gene targeting using homologous recombination in embryonic stem cells provides an avenue for the direct application of precise molecular genetic interventions to the study of complex systems in whole animals. As such, it represents a powerful approach for physiological investigation. Although its applications in physiology were initially limited because of technical difficulties in performing whole animal experiments in mice, these difficulties have been rapidly overcome, and gene targeting has been used productively in physiological experimentation. Studies have been performed using mice in which genes in the renin-angiotensin system (RAS) have been altered by gene targeting, and these studies illustrate both the feasibility and the utility of this technique for addressing physiological issues. These studies have demonstrated novel roles for the RAS in the development and maintenance of kidney structure and have added to the understanding of how RAS gene products regulate blood pressure and renal function. Finally, these experiments may contribute to understanding how naturally occurring mutations in RAS genes cause hypertension.
Activation of kininogen expression during distal nephron differentiation
Authors: El-Dahr Samir S. , Dipp Susana , Yosipiv Igor V. , Carbini Luis A.
Published: December 2017

Previous studies have shown that the epithelial precursors of the connecting tubule and collecting duct express tissue kallikrein and bradykinin B2receptors, respectively, suggesting the presence of a local kinin-producing/responsive system in the maturing distal nephron. However, evidence for the existence of kininogen in the developing nephron is still lacking. This study examined the spatiotemporal relationships between segmental nephron differentiation and the ontogeny of kininogen and kinins in the rat. Kininogen immunoreactivity is detectable in the metanephros as early as embryonic day 15. In the nephrogenic zone, the terminal ureteric bud branches are the main kinin-expressing segments. Kininogen is also observed in the stromal mesenchyme. In contrast, proximal ureteric bud branches, metanephrogenic mesenchyme, and pretubular aggregates express little or no kininogen. After completion of nephrogenesis, kininogen distribution assumes its classic “adult” pattern in the collecting ducts. Peak kininogen mRNA and protein expression occur perinatally, corresponding to the period of active nephrogenesis in the rat, and declines gradually thereafter. Estimations made by RT-PCR, Western blotting, and radioimmunoassays indicate that renal kininogen mRNA and protein levels are at least 20-fold higher in newborn than adult rats. Likewise, immunoreactive tissue kinin levels are 2.3-fold higher in newborn than adult kidneys ( P < 0.05). In summary, the present study demonstrates the activation of kininogen gene expression and kinin production in the developing kidney. The terminal ureteric bud branches and their epithelial derivatives are the principal kinin-producing segments in the maturing nephron. The results suggest an autocrine/paracrine role for the kallikrein-kinin system in distal nephron maturation.
Expression of a hypotonic swelling-activated Cl conductance during ontogeny of collecting duct epithelium
Authors: Huber Stephan M. , Horster Michael F.
Published: December 2017

Developmental expression of ion channels possibly participating in regulatory volume decrease was studied in rat embryonic ( day E17) and perinatal ( days P1–6) ureteric bud and in postnatal ( P9–14) cortical collecting duct cells in primary monolayer culture. In isotonic bath solution, whole cell conductance (in nS/10 pF) was highest in E17 (4.0 ± 0.5, n = 31) compared with P1–6 (2.0 ± 0.1, n = 16) and P9–14 (1.3 ± 0.2, n = 12) due to a decreasing contribution of a DIDS-sensitive Cl conductance, from E17 (2.8 ± 0.7, n = 12) to P1–6 (0.53 ± 0.07, n = 9) and P9–14 (0.05 ± 0.1, n = 7). Cl conductance in E17 exhibited a permselectivity of I ≈ Cl ≈ Br ≫ gluconate, and it activated time dependently. Hypotonic bath solution induced a large increase of whole cell conductance in P1–6 and in P9–14 but not in E17 (by 20.0 ± 3.7, 21.5 ± 5.5, and 4.9 ± 1.7; n = 11, 12, and 25, respectively) due to the activation of a time-dependently inactivating Cl conductance with a permselectivity of I ≥ Br > Cl ≫ gluconate. In conclusion, the expression of Cl channels, as studied in vitro, appears to shift from an apparently constitutively active embryonic to a hypotonic swelling-activated type during late embryonic development of the collecting duct.
Endogenous angiotensin II modulates rat proximal tubule transport with acute changes in extracellular volume
Authors: Quan Albert , Baum Michel
Published: December 2017

In the present study, we examined whether the effect of endogenously produced angiotensin II on proximal tubule transport in the male Sprague-Dawley rat is regulated by acute changes in extracellular volume. We measured the magnitude of endogenous angiotensin II-mediated stimulation of transport by sequentially perfusing proximal tubules in vivo, first with an ultrafiltrate-like solution, then by reperfusion of the same tubule with an ultrafiltrate-like solution containing 10−8 M losartan (angiotensin II receptor antagonist). During volume contraction, 10−8 M losartan decreased volume reabsorption from 4.20 ± 0.50 to 1.70 ± 0.30 nl ⋅ mm−1 ⋅ min−1( P < 0.05), a decrease of 58.0 ± 7.0%. In contrast, after acute volume expansion, 10−8 M losartan decreased volume reabsorption from 1.84 ± 0.20 to 1.31 ± 0.20 nl ⋅ mm−1 ⋅ min−1( P < 0.05), a decrease of 29.6 ± 9.0%. In hydropenic rats, addition of exogenous luminal angiotensin II had no effect on transport. However, in volume-expanded rats, addition of 10−8 M angiotensin II increased volume reabsorption from 2.10 ± 0.34 to 4.38 ± 0.59 nl ⋅ mm−1 ⋅ min−1( P < 0.005). These data are consistent with endogenously produced angiotensin II augmenting proximal tubule transport to a greater degree during volume contraction than after volume expansion.
Vascular and glomerular expression of endothelin-1 in normal human kidney
Authors: Herman William H. , Emancipator Steven N. , Rhoten R. L. Patrick , Simonson Michael S.
Published: December 2017

To understand better the function of endothelin-1 (ET-1) in renal physiology, we examined vascular and glomerular expression of ET-1 in normal human kidney and in lupus nephritis. Immunohistochemical analysis revealed that renal endothelium of glomeruli, arteries, veins, and capillaries expressed ET-1. Endothelial cells were the principal source of glomerular ET-1; positive immunostaining was detected only rarely in mesangial cells and vascular smooth muscle cells from normal kidney. However, mesangial staining for ET-1 was elevated in patients with lupus nephritis, suggesting that under certain conditions mesangial cells elaborate ET-1. Indeed cultured human mesangial cells from normal subjects secreted ET-1 peptide. ET-1 secretion was augmented by the protein kinase C activator phorbol ester and by transforming growth factor-β1 (TGF-β1), a cytokine implicated in the development of glomerulosclerosis. Transient transfection of cultured mesangial cells with a preproET-1 reporter construct showed that the preproET-1 promoter is transcriptionally active in mesangial cells and is stimulated by TGF-β1, phorbol ester, or ectopic expression of protein kinase β1. Cultured human mesangial cells have both ETAand ETBreceptors that contribute to ET-1-stimulated mitogenesis. Taken together, these results demonstrate that ET-1 is expressed at sites where paracrine or autocrine signaling by ET-1 might control renal vasoconstriction, glomerular filtration rate, and remodeling of the glomerulus in renal disease.
Low-salt diet downregulates plasma but not tissue kallikrein-kinin system
Authors: Hilgenfeldt U. , Puschner T. , Riester U. , Finsterle J. , Hilgenfeldt J. , Ritz E.
Published: December 2017

The kallikrein-kinin system (KKS) is involved in the regulation of blood pressure and in the sodium and water excretion. In humans, the KKS is divided functionally into a plasma KKS (pKKS) generating the biologically active peptide bradykinin and into the tissue (glandular) KKS (tKKS) generating the active peptide kallidin. The objective of this study was to examine the effect of a low-NaCl diet on the concentration of both pKKS and tKKS in plasma and urine in 10 healthy volunteers. After a 4-day low-NaCl diet, the urinary sodium and chloride excretions had decreased from 234 to 21.2 mmol/24 h and from 198 to 14.6 mmol/24 h, respectively. The plasma levels of ANG I, aldosterone, and angiotensin converting enzyme (ACE) significantly increased from 50.4 to 82.8 pg/ml, from 129 to 315 pg/ml, and from 46.4 to 59.8 U/ml, respectively, demonstrating the physiological adjustment to the low-salt diet. In plasma, the levels of bradykinin and plasma kallikrein had significantly decreased from 13.7 to 7.57 pg/ml and 14.4 to 7.13 U/ml, respectively. However, the levels of high-molecular-weight kininogen (HMW kininogen) remain unchanged (101 vs. 112 μg/ml, not significant). Contrary to plasma kallikrein, the plasma levels of tissue kallikrein increased (0.345 vs. 0.500 U/ml; P < 0.01). The plasma kallidin levels, however, did not change (64.7 vs. 68.6 pg/ml, not significant). This can be explained by a simultaneous decrease in the plasma low-molecular-weight kininogen (LMW kininogen) levels (89.9 vs. 44.4 μg/ml; P < 0.05). As in plasma, we find increased urinary concentrations of renal (tissue) kallikrein (23.3 to 42.8 U/24 h; P < 0.05) that contrast with, and are presumably counterbalanced by, urinary LMW kininogen levels (77.0 vs. 51.8 μg/24 h; P < 0.05). Consequently, in urine low-NaCl diet caused no significant change in either bradykinin or kallidin (9.2 vs. 10.8 μg/24 h, and 10.9 vs. 10.3 μg/24 h). It is concluded that the stimulation of the renin-angiotensin system on a low-NaCl diet is associated with a decrease in pKKS (bradykinin and plasma kallikrein) but not in tissue and renal KKS. Although tissue kallikrein is increased, there is no change in kallidin, as LMW kininogen in plasma and urine is decreased. These data suggest a difference in the regulation of pKKS and tKKS by low-salt diet.
Phenotypic plasticity in the intercalated cell: the hensin pathway
Authors: Al-Awqati Qais , Vijayakumar S. , Hikita C. , Chen J. , Takito J.
Published: December 2017

The collecting duct of the renal tubule contains two cell types, one of which, the intercalated cell, is responsible for acidification and alkalinization of urine. These cells exist in a multiplicity of morphological forms, with two extreme types, α and β. The former acidifies the urine by an apical proton-translocating ATPase and a basolateral Cl/HCO3exchanger, which is an alternately spliced form of band 3. This kidney form of band 3, kAE1, is present in the apical membrane of the β-cell, which has the H+-ATPase on the basolateral membrane. We had suggested previously that metabolic acidosis leads to conversion of β-types to α-types. To study the biochemical basis of this plasticity, we used an immortalized cell line of the β-cell and showed that these cells convert to the α-phenotype when plated at superconfluent density. At high density these cells localize a new protein, which we term “hensin,” to the extracellular matrix, and hensin acts as a molecular switch capable of changing the phenotype of these cells in vitro. Hensin induces new cytoskeletal proteins, makes the cells assume a more columnar shape and retargets kAE1 and the H+-ATPase. These recent studies suggest that the conversion of β- to α-cells, at least in vitro, bears many of the hallmarks of terminal differentiation.
Decreased vasopressin-mediated renal water reabsorption in rats with compensated liver cirrhosis
Authors: Jonassen Thomas E. N. , Nielsen Søren , Christensen Sten , Petersen Jørgen Søberg
Published: December 2017

Experiments were performed to investigate vasopressin type 2 receptor (V2)-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in cirrhotic rats with sodium retention but without ascites. In addition, the expression of the furosemide-sensitive type 1 Na-K-2Cl cotransporter (BSC-1) and the natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) during acute V2-receptor blockade was measured. Acute V2-receptor blockade with the selective nonpeptide antagonist OPC-31260 (800 μg ⋅ kg−1⋅ h−1) was performed during conditions in which volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 150 mM glucose. OPC-31260 produced a significantly smaller increase in urine flow rate (−26%) and free water clearance (−18%) in cirrhotic rats than in control rats. The natriuretic response to an intravenous test dose furosemide (7.5 mg/kg) was significantly increased in cirrhotic rats (+52%), but pretreatment with OPC-31260 did not affect the natriuretic response to furosemide in neither cirrhotic nor in control rats. Semiquantitative immunoblotting showed a significant downregulation of AQP-2 in the renal cortex (−72%) and in the outer medulla (−44%). The relative expression of BSC-1 in the outer medulla was unchanged in cirrhotic rats. The corticopapillary gradient of Na was significantly increased in cirrhotic rats. Since daily urine flow rate was similar in cirrhotic and sham-operated rats, we suggest that non-vasopressin-mediated water reabsorption is increased in cirrhotic rats probably as a result of an increased corticomedullary gradient due to exaggerated NaCl reabsorption in the thick ascending limb of Henle’s loop.
Chronic administration of furosemide augments renal weight and glomerular capillary pressure in normal rats
Authors: Lane Pascale H. , Tyler Larry D. , Schmitz Paul G.
Published: December 2017

Angiotensin II (ANG II) is believed to promote progressive renal injury via augmented glomerular capillary hydraulic pressure (PGC). Acute volume reduction secondary to diuretic administration increases circulating ANG II and augments PGC, yet the hemodynamic effects of sustained diuretic administration are unknown. Therefore, glomerular micropuncture studies were performed in male Munich-Wistar rats after 6–8 wk of treatment with daily furosemide (F, 40 mg/day), furosemide plus the AT1receptor antagonist, losartan (F + L, 5 mg/day), or no therapy (C, control). Renal weight was increased in F rats (1.23 ± 0.7 g) vs. C (1.00 ± 0.06 g) or F + L (0.97 ± 0.01 g). In addition, PGCwas elevated in F animals (52.1 ± 1.5 mmHg) vs. C (43.7 ± 1.5) or F + L-treated rats (41.3 ± 1.7). F-treated rats were also characterized by a relative increase in efferent arteriolar resistance and filtration fraction. The latter was markedly attenuated in F + L-treated animals. Collectively, these findings are consistent with an ANG II-mediated alteration in intrarenal hemodynamics. In contrast to acute volume manipulations, however, chronic furosemide augmented renal growth, whereas losartan administration completely arrested this phenomenon. Further studies are warranted to determine whether the hemodynamic and growth adaptations elicited by chronic F administration induce or accelerate renal injury.
Differential regulation of ROMK expression in kidney cortex and medulla by aldosterone and potassium
Authors: Wald H. , Garty H. , Palmer L. G. , Popovtzer M. M.
Published: December 2017

This study explores the role of K+ and aldosterone in the regulation of mRNA of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, in the rat kidney. K+ deficiency downregulated ROMK mRNA in cortex to 47.1 ± 5.1% of control ( P < 0.001) and in medulla to 56.1 ± 3.4% ( P < 0.001). High-K+ diet slightly increased ROMK mRNA in medulla to 122 ± 9% ( P < 0.05 vs. control). Adrenalectomy (Adx) downregulated cortical ROMK mRNA to 30.7 ± 6.8% ( P < 0.001 vs. control), and increased it in medulla to 138 ± 12.9% ( P < 0.02 vs. control). In Adx rats, K+ deficiency decreased ROMK mRNA in cortex and medulla similar to intact rats. The α1- and β1-Na-K-ATPase subunits were regulated in parallel to that of ROMK. In medulla, ROMK mRNA correlated with serum K+ concentration at R = 0.9406 ( n = 6, P < 0.001) and α1-Na-K-ATPase mRNA at R = 0.9756 ( n = 6, P < 0.001). ROMK2 also correlated with serum K+ concentration ( R = 0.895; n = 6, P < 0.01). These results show that cortical ROMK expression is regulated by aldosterone and K+, whereas the medullary ROMK mRNA is regulated by serum K+.
An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro
Authors: Tomita Naruya , Horiuchi Masatsugu , Tomita Sawako , Gibbons Gary H. , Kim John Y. S. , Baran Dana , Dzau Victor J.
Published: December 2017

The transcription factor E2F controls expression of several genes involved in cell proliferation including c- myc, c- myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.
Functional and molecular characterization of luminal and basolateral Cl−/ HCO 3 − exchangers of rat thick limbs
Authors: Eladari Dominique , Blanchard Anne , Leviel Françoise , Paillard Michel , Stuart-Tilley Alan K. , Alper Seth L. , Podevin René-Alexandre
Published: December 2017

Cl−/[Formula: see text]exchange was measured in luminal (LMV) and basolateral (BLMV) membrane vesicles purified from rat medullary thick ascending limb (MTAL). Cl−/[Formula: see text]exchange in BLMV and LMV was inhibited by DIDS, with respective IC50values of 3.2 ± 0.9 and 15.2 ± 5.2 μM, whereas Cl−conductances were DIDS insensitive. At constant external pH, BLMV36Cl−/[Formula: see text]and36Cl−/Cl−exchanges exhibited a sigmoidal pattern of activation as internal pH (pHi) increased from 6.1 to 8.0, whereas LMV36Cl−/Cl−exchange was unchanged between pHi6.7 and 7.8. The 165-kDa AE2 polypeptide and ∼115-kDa AE1-related polypeptide were present only in BLMV. In contrast, AE1-related polypeptides of ∼90 and 95 kDa were present not only in BLMV but also (in variable abundance) in LMV. We conclude that rat MTAL BLMV and LMV express distinct anion exchange activities and distinct sets of AE polypeptides. AE2 (and perhaps AE1) in BLMV likely contribute to[Formula: see text] absorption. In contrast, LMV exchangers may contribute to NaCl absorption via parallel coupling with the luminal Na+/H+antiporters and/or may provide negative feedback regulation of[Formula: see text] absorption.
Contribution of 20-HETE to the vasodilator actions of nitric oxide in renal arteries
Authors: Alonso-Galicia Magdalena , Sun Cheng-Wen , Falck John R. , Harder David R. , Roman Richard J.
Published: December 2017

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10−5, 10−4, and 10−3 M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 ± 2, 43 ± 5, and 7 ± 1% of control, respectively ( n = 4). In other experiments, the vasodilator response to SNP (10−7 to 10−3 M) was examined in rat renal interlobular arteries (<90 μm ID), preconstricted with phenylephrine (1 μM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3- a]quinoxalin-1-one (ODQ) (10 μM, n = 6) or of cGMP-dependent protein kinase with 8 R,9 S,11 S-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7 b,11 a-trizadibenzo-( a, g)-cycloocta-( c, d, e)-trinden-1-one (KT-5823, 1 μM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 μM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 μM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM ( n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP ( n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.