bluuebunny/crossref_metadata_2025 huggingface dataset:
Thiazide-sensitive sodium chloride cotransport in early distal tubule
Authors: Ellison D. H. , Velazquez H. , Wright F. S.
Published: December 2017

At least two pathways mediate sodium absorption across the luminal membrane of the renal distal tubule. One pathway is a conductive channel and the other appears to be a coupled Na-Cl cotransport pathway. The distal tubule comprises three segments: the distal convoluted tubule, the connecting tubule, and the initial collecting duct. To provide information about cellular locations of the proposed sodium transport pathways, we perfused early (14-38% of whole distal length) and late (61-83% of whole distal length) segments of whole distal tubules separately in vivo in anesthetized rats. When perfused with a solution that resembles fluid normally arriving at the distal tubule (75 mM Na, 68 mM Cl), rates of sodium absorption were similar in early and late segments (early 68 +/- 29.6, late 67 +/- 27.5 pmol X min-1 X mm-1). When perfused with a solution that resembles interstitial fluid (148 mM Na, 110 mM Cl), sodium transport was significantly higher in early than in late segments (276 +/- 28.4 vs. 113 +/- 29.7 pmol X min-1 X mm-1). Chlorothiazide (10(-3) M), which blocks sodium and chloride absorption in whole distal tubules, reduced sodium and chloride transport to zero in early distal tubules but had no significant effect in late distal tubules. Removing all chloride from perfusion solutions reduced sodium transport in early but not late distal segments.(ABSTRACT TRUNCATED AT 250 WORDS)
Roles and mechanisms of urinary buffer excretion
Authors: Hamm L. L. , Simon E. E.
Published: December 2017

Excretion of acid (or generation of bicarbonate) by the kidneys is necessary for acid-base homeostasis. Most of this acid is excreted in the form of ammonia and titratable acid, the latter representing the amount of acid required to titrate the urine buffers from the plasma pH to urine pH. The transport of ammonia in the kidney is now recognized to entail more than simple nonionic diffusion of NH3 and trapping of NH4+. NH4+ transport in the kidney probably occurs by passive diffusion and by transport on the Na+-H+ exchanger, the Na+-K+-2Cl- transporter and on Na+-K+-ATPase. NH3 diffusion is stimulated by an acid disequilibrium pH in various nephron segments. Also, diffusion equilibrium of NH3 in various regions of the kidney has now been disproved. These various mechanisms of ammonia transport are considered in terms of their possible changes with acid-base disturbances. Phosphate is the most predominant urine buffer; its urinary excretion increases with acidosis. The mechanisms probably involve a decrease in the preferentially transported species, HPO4(2-), and a direct effect of pH on proximal tubule apical phosphate transport. With chronic acidosis, changes in the activity of the apical Na+-phosphate transporter occur. These effects of systemic acid-base balance interact with parathyroid hormone and dietary phosphate status to alter phosphate reabsorption. Citrate transport in the kidney is analyzed because of its sensitivity to systemic pH and because of the possible influence on systemic acid-base status in certain circumstances. Alterations in citrate excretion with acid-base disturbances depend on changes in the concentration of the transported species, citrate2-, and on changes in renal metabolism.
Evidence for the transport function of uricase, an oxidative enzyme
Authors: Pordy W. T. , Lipkowitz M. S. , Abramson R. G.
Published: December 2017

[2-14C]urate uptake was examined in proteoliposomes prepared with phosphatidylcholine and either pig liver uricase or albumin, and in protein-free liposomes. Urate uptake was only evident in proteoliposomes that contained active uricase. Uptakes were indistinguishable in the presence and absence of inwardly directed gradients of sodium, potassium, or choline chloride or outwardly directed hydroxyl gradients. Both urate and allantoin accumulated within proteoliposomes during urate uptake; however, [2–14C]allantoin was not taken up by proteoliposomes. Urate uptake was accelerated in the presence of unlabeled urate in the trans position, saturable, and competitively inhibited by oxonate, findings consistent with carrier-mediated transport. Finally, the kinetics of urate uptake and oxidation were virtually identical, implying that the transporter is uricase. Thus, these studies provide evidence that uricase can function as a transport protein for urate when inserted in a lipid bilayer: transport via uricase is neither cation dependent (not a cotransporter) nor dependent on an exchangeable anion (not a urate/anion exchanger). Additionally, these studies demonstrate that neither urate nor allantoin cross lipid bilayers by simple or nonionic diffusion.
Release of renin and angiotensin II into plasma and lymph during hyperchloremia
Authors: Wilcox C. S. , Peart W. S.
Published: December 2017

Renal vascular resistance (RVR) was related to intrarenal release of renin (RR) and angiotensin II (ANG II) during increases in plasma osmolality (Posmol) or chloride (PCl). Intrarenal infusions of 1.232 M solutions given to in situ kidneys increased Posmol by 7-18%. Hypertonic dextrose (n = 8) reduced RVR (P less than 0.005) but increased RR into plasma (P less than 0.01) and increased lymph renin (LR) (P less than 0.05). The response to hypertonic Na acetate (n = 5) was similar, but hypertonic NaCl (n = 9) increased RVR (P less than 0.02) without changing RR. During infusion of arachidonic acid (AA), hypertonic NaCl increased RVR (P less than 0.02) but decreased RR into plasma and decreased LR (P less than 0.05). Overall group mean changes in RVR were inversely related to LR (r = -0.96; P less than 0.01). An increase in PCl of 12+/- 1 mmol/l was induced in denervated kidneys by changing an intrarenal 0.616 M infusion of Na acetate to NaCl. Hyperchloremia increased RVR from 14+/- 2 to 19+/- 2 mmHg . ml-1 . min-1 . kg-1 (P less than 0.01), reduced GFR from 1.4+/- 0.1 to 1.1+/- 0.1 ml . min-1 . kg-1 (P less than 0.01), and reduced ANG II (pg/ml) in renal venous plasma (55+/- 6 to 35+/- 6; P less than 0.05) and lymph (366+/- 111 to 209+/- 53; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Passive, one-dimensional countercurrent models do not simulate hypertonic urine formation
Authors: Wexler A. S. , Kalaba R. E. , Marsh D. J.
Published: December 2017

Simulations were performed to test the ability of the countercurrent hypothesis to predict measured concentrations of NaCl and urea in the interstitium of the renal medulla. The simulations included one-dimensional representations of loops of Henle, distal tubules, collecting ducts, and vasa recta, and recent estimates of descending limb, thick ascending limb, and collecting duct transport parameters. The nonlinear two-point boundary value problem was solved numerically via quasi-linearization. The simulations failed to predict measured concentrations or concentration gradients of NaCl in the inner medulla. Including countertransport of urea and NaCl in thin ascending limbs added minimally to the performance of the system. The single most effective change in the model was the inclusion of a coefficient to permit preferential solute exchange among vasa recta. This result suggests that the three-dimensional ordering of blood vessels and tubules is an essential construct in the concentrating mechanism.
Alpha 1-adrenergic stimulation of renal Na reabsorption requires glucose metabolism
Authors: Baines A. D. , Drangova R. , Ho P.
Published: December 2017

The effect of glucose on renal responses to alpha 1-adrenergic stimulation was examined in isolated rat kidneys perfused with 5 mM lactate, 5 mM alanine, 0.5 mM pyruvate, and 0.2 mM mercaptopicolinic acid. Adding 5 mM glucose to this perfusate vasodilated the kidneys and raised the glomerular filtration rate (GFR) and free water clearance without altering fractional sodium excretion. alpha 1-Adrenergic actions were evaluated with norepinephrine (NE, 60 nM), methoxamine (600 nM), and prazosin (1 microM). Glucose altered neither the vasoconstriction nor the reduction in GFR produced by NE and methoxamine but increased the antinatriuretic responses (P less than 0.001). Glucose was required for prazosin to inhibit the effect of NE on sodium and lithium excretion. The nonmetabolized glucose analogue, alpha-methyl-D-glucoside (5 mM), did not increase the antinatriuretic response to NE, therefore glucose metabolism and not Na cotransport stimulated the alpha 1-adrenergic response. Glucose did not alter alpha 1-adrenoceptor number or binding affinity for prazosin in renal cortical homogenates. We conclude that glucose metabolism in the proximal tubule is involved in the stimulation of sodium reabsorption by alpha 1-adrenoceptor agonists.
Binding and functional effects of atrial natriuretic factor in isolated rat kidney
Authors: Suzuki M. , Almeida F. A. , Nussenzveig D. R. , Sawyer D. , Maack T.
Published: December 2017

A new methodological approach was developed to study the relationship between specific binding and dose-response curves of the renal effects of atrial natriuretic factor (ANF) in isolated perfused rat kidneys (IK). IK were perfused with 125I-labeled and unlabeled ANF 1-28 (4 pM to 1 microM) to determine the following: 1) distribution, capacity (Cmax), and apparent affinity (S50) of specific binding of ANF 1-28 in cortex, outer medulla, and papilla and 2) dose-response curves of the effects of ANF 1-28 on renal hemodynamics and excretion of fluid and electrolytes. The kidney had a very high density of high-affinity binding sites for ANF. Cortex had greater than 90% of total binding sites (Cmax = 6.8 pmol/g tissue; S50 = 54 pM), whereas papilla had less than 2% of total binding sites with a 10-fold lower apparent affinity (S50 = 860 pM) than in cortex. ANF-induced increases in glomerular filtration rate and excretion of fluid and electrolytes were detectable at 10-100 pM and maximal effects occurred at 1-10 nM ANF. Below 1 nM there was no dissociation between the renal hemodynamic and natriuretic effects of ANF. There was a close agreement between dose-response and binding curves of ANF to cortex. Results demonstrate that binding site occupancy in kidney cortex and renal effects of ANF occur at near physiological concentrations of the hormone.
Lack of neural control of atrial natriuretic peptide release in conscious dogs
Authors: Nishida Y. , Miyata A. , Morita H. , Uemura N. , Kangawa K. , Matsuo H. , Hosomi H.
Published: December 2017

To determine whether or not cardiac nerves control the release of atrial natriuretic polypeptide (ANP), we examined the effects of intrapericardial cardiac denervation on circulating ANP in conscious dogs. Base-line level of circulating ANP in six cardiac-denervated (CD) dogs (191 +/- 24 pg/ml) was not significantly different from that in seven intact (INT) dogs (196 +/- 18 pg/ml). Three percent dextran-40 in saline (18 ml/kg, 20% of estimated blood volume) was infused over 5 min, and the elevated left atrial pressure (LAP; approximately 8 mmHg) was maintained by supplemental infusion over 40 min. The increase in circulating ANP after infusion in the CD dogs (462 +/- 61 pg/ml) was not significantly different from that in the INT dogs (435 +/- 73 pg/ml). The regression line relating circulating ANP (normalized with regard to the control value) with the change in LAP in the INT dogs was not significantly different from that in the CD dogs. These results suggest that the cardiac nerves do not control the tonic release of ANP or the volume expansion-induced release of ANP.
Ovine fetal urachus--physiological and hormonal control of its contractile activity
Authors: Lye S. J. , Freitag C. L. , Challis J. R.
Published: December 2017

We have investigated whether the ovine fetal urachus near term has the potential to regulate the passage of urine from the bladder into the allantoic cavity. Experiments were performed primarily on longitudinal urachal muscle. In Krebs bicarbonate buffer (pH 7.4 bubbled with 95% O2-5% CO2 at 37 degrees C) the urachus spontaneously developed a prolonged elevated resting tension (RT, 1.1–1.5 g). Intra-abdominal urachus showed a greater development of tension than extra-abdominal urachus, which was associated with the amount of smooth muscle observed in histological specimens. Isoproterenol (1 microM) produced a significant reversible reduction in RT (1.36 +/- 0.15 to 0.67 +/- 0.08 g, P less than 0.01, mean +/- SE). Replacement of normal-O2 buffer (PO2 420.4 +/- 19.5 mmHg) with a low-O2 buffer (PO2 101.3 +/- 4.7 mmHg), or very low-O2 buffer (PO2 38.2 +/- 2.2 mmHg) while maintaining PCO2 and pH constant, produced a reduction in RT (from 1.35 +/- 0.21 to 0.52 +/- 0.13 g and from 1.20 +/- 0.17 to 0.46 +/- 0.04 g, respectively; both P less than 0.01). This effect was reversed when high PO2 was reinstated. Circular muscle showed a similar response when PO2 was reduced. The urachus showed a significant increase in RT (0.74 +/- 0.08 to 1.71 +/- 0.28 g, P less than 0.01) when prostaglandin F2 alpha (PGF2 alpha; 10 microM) was added to the low-O2 buffer. These results demonstrate that the urachus can develop spontaneous contractile activity and responds to alterations in O2, a beta-agonist, and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of peritubular [Ca] or ionomycin on hydrosmotic response of CCTs to ADH or cAMP
Authors: Jones S. M. , Frindt G. , Windhager E. E.
Published: December 2017

To evaluate in the mammalian kidney the effect of maneuvers thought to alter intracellular [Ca2+] on the hydraulic conductivity (Lp) response to vasopressin (VP) or 8-(p-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (ClPheS-cAMP), water flow was measured in isolated perfused rabbit cortical collecting tubules (CCTs) exposed to either varying bath [Ca] or ionomycin or quin 2-acetoxymethyl ester (AM). The development of the response to VP (20 microU/ml) was enhanced 28% by lowering Ca from 1.0 to 0.1 mM, but was not altered by increasing Ca from 1.0 to 3.75 mM. When measured in the same tubule, the maintenance phase of a previously established hormone response was inhibited by acutely raising peritubular Ca from 1.0 to 3.75 mM. Exposing the tubules to 0.1 mM Ca and 65 microM quin 2-AM, inhibited by 68% the development of the response to VP compared with that observed in tubules bathed in 1.0 mM Ca Ringer without quin 2-AM. In contrast, quin 2-AM and low peritubular Ca added during the maintenance phase enhanced the VP response by 50%. The Ca ionophore ionomycin (1.0 microM) reduced the development of the VP-elicited Lp by 65% and reversibly decreased by 42% the maintenance phase of the VP-stimulated Lp in a Ca-dependent manner. A longer exposure to the ionophore was required to inhibit the development and maintenance phases of the response to 10(-4) M ClPheS-cAMP. These results are consistent with the view that transient changes in intracellular [Ca2+] may be required for the development of the hormone response but that sustained increases in cytoplasmic Ca2+ levels inhibit the development as well as the maintenance phase of the hydrosmotic response to VP or cAMP in CCTs.
A rapid method for isolating rabbit renal microvessels
Authors: Chaudhari A. , Kirschenbaum M. A.
Published: December 2017

A technique has been described to isolate rabbit renal preglomerular microvessels using an infusion of a magnetized iron oxide suspension into the renal artery. An initial separation of the vascular tissue was performed with a magnet and a final separation was performed using a nylon sieve allowing nonvascular, tubular, and glomerular contaminants to be filtered free from the microvessels. Light and electron microscopy showed morphology consistent with interlobular arteries and afferent arterioles. The purity of the preparation was greater than 95%. In the absence of exogenous arachidonic acid, the microvessels were able to synthesize prostacyclin (7.5 +/- 1.2 ng.mg protein-1.min-1) in excess of prostaglandin E2 (2.2 +/- 0.7 ng.mg protein-1.min-1). The tissue exhibited a time-dependent increase in prostanoid biosynthesis up to 60 min and responded to stimulators (e.g., melittin and arachidonic acid) and inhibitors (e.g., meclofenamate and indomethacin) of prostanoid biosynthesis. These studies indicate that a high-yielding and versatile preparation of renal preglomerular microvessels can be rapidly obtained. The microvessels are viable and may represent an excellent model for studying a variety of biochemical interactions under in vitro conditions.
Evidence suggesting a role for hydroxyl radical in passive Heymann nephritis in rats
Authors: Shah S. V.
Published: December 2017

We examined the effect of scavengers of reactive oxygen metabolites on proteinuria in the passive Heymann nephritis model of membranous nephropathy. Passive Heymann nephritis was induced by a single intravenous injection of anti-Fx1A IgG in a dose of 10 mg/100 g body weight. Superoxide dismutase, a scavenger of superoxide or catalase which destroys hydrogen peroxide, did not affect the proteinuria. In contrast, dimethylthiourea (DMTU, 500 mg/kg followed by 125 mg/kg ip twice a day), a scavenger of hydroxyl radical, markedly reduced the proteinuria (day 5: anti-Fx1A 53 +/- 13, n = 18; anti-Fx1A + DMTU, 21 +/- 6 mg/24 h, n = 15, P less than 0.001). Experiments with 125I-labeled anti-Fx1A antibody demonstrated that DMTU did not affect the amount of antibody deposited in the kidney. Semiquantitative estimation of IgG and complement deposition in the kidney showed no differences between the DMTU-treated and control rats. A second hydroxyl radical scavenger, sodium benzoate (150 mg/kg ip twice a day), also resulted in marked reduction in proteinuria (day 5: anti-Fx1A 56 +/- 7, n = 9; anti-Fx1A + benzoate, 14 +/- 4 mg/24 h, n = 8, P less than 0.01). Because of the participation of iron in biological systems to generate hydroxyl radical, we also examined the effect of deferoxamine (DFO, 35 mg/day), an iron chelator, on the anti-Fx1A-induced proteinuria. There was a significant reduction in proteinuria in rats treated concurrently with DFO (day 5: anti-Fx1A 67 +/- 13, n = 15; anti-Fx1A + DFO, 29 +/- 4 mg/24 h, n = 15, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Micropuncture study of the effect of ANP on the papillary collecting duct in the rat
Authors: van de Stolpe A. , Jamison R. L.
Published: December 2017

Micropuncture collections were obtained from the terminal collecting duct (CD) at base and tip of the renal papilla of the rat. Group 1 was studied before and during infusion with atrial natriuretic peptide (ANP), group 2 was administered the vehicle only, and group 3 received acetazolamide to increase sodium delivery to the base to a similar extent as after ANP. ANP caused a decrease in blood pressure, a slight increase in GFR, natriuresis, and diuresis. Sodium delivery to the collecting duct at the base of the papilla increased. Between base and tip, sodium reabsorption was inhibited. Tubule fluid sodium concentration (TFNa) was increased at the base and remained high at the tip; in contrast TFNa fell between base and tip in control and acetazolamide groups. After acetazolamide, sodium reabsorption in the terminal CD was not inhibited. These results demonstrate that in vivo ANP 1) increases the delivery of sodium to the terminal CD and 2) inhibits sodium reabsorption in the terminal CD. The findings for chloride were similar to those for sodium. ANP also increased delivery of H2O, K, Ca, and Mg to the CD at the papillary base but did not significantly affect their transport by the terminal CD.
Sodium restriction corrects hyperfiltration of diabetes
Authors: Bank N. , Lahorra G. , Aynedjian H. S. , Wilkes B. M.
Published: December 2017

The effect of a low-sodium diet on the abnormal glomerular hemodynamics of early diabetes was studied in rats. Starting 5 to 7 days after onset of streptozotocin-induced insulin-dependent diabetes mellitus (IDDM), rats were fed a low-sodium diet for 4-5 days. Normal rats fed the same diet served as controls. Micropuncture measurements were made during a control period, followed by a second period when saralasin was infused into the left renal artery. During the first period, single-nephron glomerular rate (SNGFR), glomerular plasma flow (QA), glomerular blood flow (SNBF), filtration fraction (SNFF), and glomerular hydraulic pressure (PG) in the diabetic rats were not significantly different from the normal controls. Saralasin infusion resulted in striking increases in SNGFR, QA, SNBF, and Kf, and significant decreases in SNFF, PG, and delta P. The responses to saralasin imply that the low-sodium diet resulted in ANG II-mediated vascular constriction at pre- and postglomerular sites, and probably the glomerular mesangial cells as well. Our observations suggest that the abnormally elevated glomerular blood flow and filtration rate of early IDDM can be corrected by a low-sodium diet via stimulation of endogenous ANG II.
Effects of sodium intake and aldosterone on the renal pressure-natriuresis
Authors: Harrap S. B. , Clark S. A. , Fraser R. , Towrie A. , Brown A. J. , Lever A. F.
Published: December 2017

To study the effects of dietary sodium and plasma aldosterone on pressure-natriuresis (PN) we examined six groups of adult, male Sprague-Dawley rats. Group 1 received normal-sodium diet, group 2 high-sodium diet, and group 3 low-sodium diet for 3 wk; group 4 was given low sodium for 3 wk then high sodium for 3 wk; groups 5 and 6 received high sodium for 3 wk but during the 3rd wk were also given aldosterone by subcutaneous infusion to mimic the plasma aldosterone seen in groups 1 and 3, respectively. After the diets, rats were killed, and urinary sodium excretion, glomerular filtration rate (GFR), and calculated tubular sodium reabsorption (FRNa) were measured during stepwise increases in perfusion pressure in isolated perfused kidneys from each group. No significant differences in blood pressure were seen between any of the groups. The PN curves for groups 2 and 3 were significantly different (P less than 0.001) and shifted to the left and right of group 1, respectively. These shifts appeared to be the result of significant (P less than 0.001) differences in FRNa rather than changes in GFR. PN was not significantly different in groups 4 and 2, indicating that the effects of low-sodium diet were reversible. The infusion of aldosterone in groups 5 and 6 was associated with modest and significant (P less than 0.001) shifts, respectively, of the PN curve to the right of the curve of rats in group 2. In group 6 this shift appeared to be due to significant (P less than 0.001) changes in FRNa, so as to resemble that seen in low-sodium rats of group 3.(ABSTRACT TRUNCATED AT 250 WORDS)
Lysophospholipids are natriuretic and diuretic in rats
Authors: Rauch A. L. , Buckalew V. M.
Published: December 2017

The effects of lysophosphatidylcholine (LPC) and some of its analogues on renal sodium (UNaV) and water excretion (V) were examined in the pentobarbital sodium-anesthetized rat. LPC caused the increase in UNaV and V with a steep dose response. The natriuretic effect began within 20 min after a bolus injection and lasted for 7 h. The natriuresis and diuresis were not affected when the phospholipid was given as an LPC-albumin complex. Natriuretic activity was eliminated by substituting unsaturated bonds in the 1-acyl group and by removing the choline group on position 3. Natriuretic activity was not affected by substitution of 1-alkyl for 1-acyl groups and was stereospecific. These results demonstrate a unique, previously unrecognized effect of lysophospholipids and suggest that they may account for some of the previously demonstrated natriuretic activity of plasma.
SITS-sensitive basolateral anion current in rabbit proximal convoluted tubules
Authors: Kuwahara M. , Rector F. C. , Berry C. A.
Published: December 2017

To assess the presence and nature of steady-state anion current across the basolateral membrane in in vitro rabbit proximal convoluted tubules bathed and perfused with a high-chloride, low-bicarbonate solution simulating late proximal tubular fluid, steady-state basolateral cell membrane potential difference (Vb1) was measured by conventional microelectrodes. The mean value of Vb1 was -52 mV. Addition of 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to the bath solution hyperpolarized Vb1 by 30 mV, suggesting the presence of basolateral anion current. Total chloride removal did not change Vb1 significantly, and formate, a presumptive stimulant of electroneutral sodium chloride transport, depolarized Vb1 both in the presence and absence of chloride, suggesting that the formate-stimulated change in Vb1 was chloride independent. In the total absence of chloride and bicarbonate, 1 mM bath SITS and 0.1 mM lumen and bath acetazolamide hyperpolarized Vb1 by 27–35 and 23 mV, respectively. These results suggest that the SITS-sensitive change in Vb1 is independent of chloride and associated with a basolateral anion current that is predominantly due to bicarbonate exit. In the absence of exogenous CO2, cell-to-bath HCO3-dependent anion current can be derived from metabolic CO2.
Plasma clearance and effects of alpha-hANP infused in patients with end-stage renal failure
Authors: Tonolo G. , McMillan M. , Polonia J. , Pazzola A. , Montorsi P. , Soro A. , Glorioso N. , Richards M. A.
Published: December 2017

The effects and clearance of synthetic atrial natriuretic peptide (alpha-hANP) were investigated in eight patients with end-stage renal failure and six normal volunteers. ANP or vehicle was infused for 1 h at 10 pmol.kg-1.min-1 in random order on two separate occasions. During ANP infusions in end-stage renal patients, microhematocrit rose by 9.8 +/- 2% (P less than 0.005, n = 8), from base-line values of 0.24 +/- 0.02. Serum protein and albumin rose consistently. In contrast, during placebo infusions, no significant changes were seen. Blood pressure, heart rate, plasma renin concentration, serum creatinine, and electrolytes did not change significantly during either study phase. In end-stage renal failure patients, metabolic clearance rate of infused ANP was 1.04 +/- 0.095 l/min and its plasma half-life was 4 min 34 s. In normal volunteers, metabolic clearance rate was 2.6 l/min and its plasma half-life 3 min 30 s. The data suggest that ANP promotes contraction of plasma volume via a mechanism independent of renal function and also indicate that the kidney is not the only organ involved in the ANP metabolism.
cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH
Authors: Gapstur S. M. , Homma S. , Dousa T. P.
Published: December 2017

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanism of alpha 2-adrenoceptor agonist-induced diuresis
Authors: Gellai M. , Edwards R. M.
Published: December 2017

In vivo and in vitro studies were performed to assess the mechanism of the diuretic effect of B-HT 933, a selective alpha 2-adrenoceptor agonist. In conscious Sprague-Dawley rats whose plasma vasopressin (AVP) levels were increased by infusion of hypertonic NaCl, B-HT 933 had no effect on AVP secretion. In Brattleboro homozygous (DI) rats, the antidiuretic dose response to AVP was shifted to the right by B-HT 933. In addition, a sustained antidiuresis induced in rats by infusion of 10 pg/min AVP was attenuated by B-HT 933 in a concentration-dependent manner. Pretreatment of DI rats with pertussis toxin (2 micrograms/kg iv) 4-5 days before testing abolished the inhibitory effect of B-HT 933 on AVP-induced antidiuresis. In outer medullary collecting ducts of DI rats, norepinephrine and B-HT 933 produced significant inhibition of AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. In contrast, the selective alpha 1-adrenoceptor agonist cirazoline had no effect on AVP-induced cAMP formation. The inhibitory effect of norepinephrine was antagonized by the selective alpha 2-adrenoceptor antagonist rauwolscine but not by prazosin, a selective alpha 1-antagonist. In outer medullary collecting ducts dissected from the pertussis toxin-treated DI rats used in the in vivo studies, the inhibitory effect of norepinephrine and B-HT 933 on AVP-stimulated cAMP accumulation was abolished. The results indicate that the hydrosmotic action of AVP is inhibited by alpha 2-agonists via a pertussis toxin-sensitive mechanism.
Characterization of ANP receptors in rabbit inner medullary collecting duct cells
Authors: Gunning M. E. , Ballermann B. J. , Silva P. , Brenner B. M. , Zeidel M. L.
Published: December 2017

The final urinary Na+ concentration is determined in the inner medullary collecting duct (IMCD) and is under hormonal control. In suspensions of IMCD cells we have previously shown that atrial natriuretic peptide (ANP) inhibits Na+ transport-dependent O2 consumption and causes an increase in cellular guanosine 3',5'-cyclic monophosphate (cGMP) content. In this study we sought to identify and characterize the receptor for ANP in these cells. Equilibrium binding studies revealed a single class of cell surface ANP receptors of high affinity (Kd = 66.2 pM) with a total number of 3,000 sites/cell. Specificity of these receptors was shown by the rank order of binding affinities for ANP analogues: ANP-(1–28) = ANP-(4–28) greater than ANP-(5–28) much greater than ANP-(5–25). We have further defined this receptor in a solubilized cell preparation and found it to be of molecular mass 130 kDa by affinity cross linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. This is the first characterization of an epithelial cell receptor for ANP; as in other systems this receptor appears to be linked to transport processes via the production of cGMP.
Effects of adenosine on intrarenal oxygenation
Authors: Dinour D. , Brezis M.
Published: December 2017

Although generally a vasodilator, adenosine vasoconstricts cortical vessels in the kidney, reduces glomerular filtration rate (GFR), and increases medullary blood flow, effects likely to improve the medullary O2 deficiency characteristic of mammalian kidneys. To evaluate a possible role of adenosine in medullary O2 balance, we investigated the effect of adenosine upon cortical and medullary tissue PO2. Adenosine was infused into renal interstitium through chronically implanted capsules. Cortical and medullary PO2 were measured using sensitive Clark-type O2 microelectrodes inserted into kidneys of anesthetized rats at the respective depths of 1.8 and 3.7 mm. Infusion of adenosine (0.1-0.5 mumol/min) increased medullary PO2 from 17 +/- 3 (SE) to 40 +/- 5 mmHG (P less than 0.001) and decreased cortical PO2 from 64 +/- 4 to 47 +/- 3 mmHg (P less than 0.001). After the infusion was stopped, PO2 returned to baseline at both sites. Coadministration of adenosine receptor antagonist 8-phenyltheophylline (0.01 mumol/min) prevented both cortical and medullary effects of adenosine. We concluded that adenosine could play an important protective and regulatory role in renal medullary O2 balance.
Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH
Authors: Bindels R. J. , Hartog A. , Timmermans J. , Van Os C. H.
Published: December 2017

Rabbit connecting tubules and cortical collecting ducts, which represent 79 +/- 5% of the calbindin-D28k-containing kidney cells, were isolated by immunodissection from the rabbit kidney superficial cortex and seeded on permeable filters. After 6 days in culture the monolayers developed a potential difference (PD) of -24 +/- 3 mV (lumen negative) and a transepithelial resistance (R) of 284 +/- 19 omega.cm2. Addition of 10(-6) M amiloride to or removal of Na+ from the mucosal side reversed the PD to +6 +/- 4 mV and concomitantly increased R to 660 +/- 122 omega.cm2. The cells developed functional parathyroid hormone (PTH) and arginine vasopressin receptors, but calcitonin receptors were absent. The monolayer actively absorbed Ca2+ against an electrochemical gradient with a rate of 121 +/- 13 nmol.h-1.cm-2. Removal of serosal Na+ inhibited Ca2+ absorption by 63 +/- 8%. Exposure to 10(-7) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] for 48 h or 10(-7) M bovine PTH (bPTH)-(1-34) for 1 h, increased transcellular Ca2+ absorption by 53 +/- 13% or 24 +/- 8%, respectively. The effects of 1,25(OH)2D3 and PTH in combination were neither additive nor potentiating. In addition, the cultured cells expressed calbindin-D28k, and, after exposure to 10(-7) M 1,25(OH)2D3 for 48 h, the calbindin-D28k content increased fourfold.
Bifurcation analysis of TGF-mediated oscillations in SNGFR
Authors: Layton H. E. , Pitman E. B. , Moore L. C.
Published: December 2017

Recent micropuncture studies in rats have demonstrated the existence of oscillatory states in nephron filtration mediated by tubuloglomerular feedback (TGF). We develop a minimal mathematical model of the TGF system, consisting of a first-order hyperbolic partial differential equation describing thick ascending limb (TAL) NaCl reabsorption and an empirical feedback relation. An analytic bifurcation analysis of this model provides fundamental insight into how oscillatory states depend on the physiological parameters of the model. In the special case of no solute backleak in the TAL, the emergence of oscillations explicitly depends on two nondimensional parameters. The first corresponds to the delay time of the TGF response across the juxtaglomerular apparatus, and the second corresponds to the product of the slope of the TGF response curve at the steady-state operating point and the space derivative of the steady-state NaCl concentration profile in the TAL at the macula densa. Numerical calculations for the case without TAL backleak are consistent with this result. Numerical simulation of the more general case with TAL backleak shows that the bifurcation analysis still provides useful predictions concerning nephron dynamics. With typical parameter values, the analysis predicts that the TGF system will be in oscillatory state. However, the system is near enough to the boundary of the nonoscillatory region so that small changes in parameter values could result in nonoscillatory behavior.
cAMP-induced potassium channel activity in apical membrane of cultured A6 kidney cells
Authors: Hamilton K. L. , Eaton D. C.
Published: December 2017

In the present study, we report an adenosine 3',5'-cyclic monophosphate (cAMP)-induced potassium channel in the apical membrane of cultured A6 kidney cells grown on impermeable supports. The channel is present in approximately 10% of untreated cell-attached patches. After treatment with 1 mM dibutyryl-cAMP, the channel is present in greater than 70% of the same patches. The characteristics of this channel are 1) the channel is highly selective for potassium; 2) the channel has a unit conductance of 13 +/- 2 pS; 3) the probability of a channel opening increases in the presence of membrane permeable analogues of cAMP and with increasing depolarization of the cell interior; 4) channels are blocked by Ba2+; 5) the channel loses activity rapidly in excised patches; and 6) the channel has at least one open and two closed states. The mean open time is 3.5 +/- 1.0 ms, whereas the mean durations of the closed states are 3.2 +/- 1.4 and 29.4 +/- 3.4 ms. The channels could mediate potassium secretion in A6 cells, if the channels are normally present under transporting conditions; however, surface expression of the channels appears to depend on growth substrate and the state of cellular differentiation, since the channels are not observed in cells grown on permeable supports.
RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney
Authors: Terada Y. , Moriyama T. , Martin B. M. , Knepper M. A. , Garcia-Perez A.
Published: December 2017

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.
cDNA cloning and immunolocalization of a Na(+)-H+ exchanger in LLC-PK1 renal epithelial cells
Authors: Reilly R. F. , Hildebrandt F. , Biemesderfer D. , Sardet C. , Pouyssegur J. , Aronson P. S. , Slayman C. W. , Igarashi P.
Published: December 2017

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na(+)-H+ exchangers that can be distinguished by their different sensitivities to the amiloride analogue, N-ethyl-N-isopropylamiloride. In the present study, the polymerase chain reaction (PCR) and library screening were used to clone a cDNA for one of the exchangers, based on homology with the recently isolated cDNA for a human growth factor-activatable Na(+)-H+ exchanger. There proved to be significant homology between the LLC-PK1 and human sequences, with nucleotide identities of 75, 93, and 85% in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. The LLC-PK1 cDNA encodes a predicted protein of 818 amino acids with a relative molecular mass of 90,999, consisting of an amino-terminal hydrophobic region and a carboxy-terminal hydrophilic region; its deduced amino acid sequence shows 95% identity with that of the human protein. To investigate the localization of the encoded protein, antisera were generated against a synthetic oligopeptide from the hydrophobic region and a fusion protein from the carboxy-terminal hydrophilic domain. Indirect immunofluorescence and confocal microscopy revealed that the antisera labeled the basolateral but not the apical membrane of confluent LLC-PK1 cells. Labeling by the antipeptide antibody was specifically blocked by preincubation with the synthetic peptide and coincided exactly with the pattern produced by a monoclonal antibody against Na(+)-K(+)-ATPase. Thus, the LLC-PK1 cDNA encodes the basolateral Na(+)-H+ exchanger, which must differ structurally from the apical form, at least in the region of the oligopeptide and the fusion protein.
Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures
Authors: Ling B. N. , Hinton C. F. , Eaton D. C.
Published: December 2017

Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low-selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels.
Effect of inhibitors of oxidative phosphorylation on erythropoietin mRNA in isolated perfused rat kidneys
Authors: Tan C. C. , Ratcliffe P. J.
Published: December 2017

We have used RNase protection to measure oxygen-dependent changes in erythropoietin (EPO) mRNA in isolated perfused kidneys and to compare the effect of hypoxia with the response to inhibitors of oxidative phosphorylation. In well-oxygenated kidneys perfused for 2 h at 12 ml/min, with hematocrit of 0.09 +/- 0.005 and PO2 of 443 +/- 67 mmHg, EPO mRNA levels were similar to the baseline levels measured in nonperfused contralateral kidneys from the same animals. When perfusions were performed under identical conditions but at a PO2 of 32 +/- 4 mmHg, EPO mRNA increased approximately 16-fold. In contrast, graded concentrations of cyanide (10, 100, and 300 microM and 1 mM), antimycin (0.01, 0.1, 0.5, and 1 microM), and oligomycin (0.01, 0.1, and 1 microM) did not alter EPO mRNA in well-oxygenated perfused kidneys. However, in kidneys perfused at low PO2 with a high concentration of each inhibitor, EPO mRNA levels were increased, demonstrating that the ability to respond to hypoxia was retained. Thus inhibitors of oxidative phosphorylation did not mimic the effects of hypoxia, indicating that oxygen-dependent expression of the EPO gene in the kidney is not effected through hypoxic compromise of oxidative phosphorylation.
Production and polarized secretion of basement membrane components by glomerular epithelial cells
Authors: Natori Y. , O'Meara Y. M. , Manning E. C. , Minto A. W. , Levine J. S. , Weise W. J. , Salant D. J.
Published: December 2017

To study the formation of basement membrane by glomerular epithelial cells (GECs), production and secretion of type IV collagen and laminin by rat GECs in culture were evaluated. GECs produced two chains of type IV collagen (180 and 170 kDa) in the ratio of approximately 2 to 1, when immunoprecipitated with antibody to type IV collagen of mouse Engelbreth-Holm-Swarm (EHS) sarcoma. GECs also produced proteins that were precipitated by antibody to EHS laminin, i.e., two bands each in the positions of the A and B chains of mouse laminin. On enzyme-linked immunosorbent assay (ELISA), type IV collagen and laminin were found mainly in the cell-associated fraction and in the subepithelial culture medium. Confluent GECs on membrane filters formed a tight barrier against the flux of macromolecules. Under these conditions, 80% of newly synthesized and secreted matrix proteins were detected in the basolateral medium. Moreover, treatment with ammonium chloride, which is known to affect polarized secretion, caused both type IV collagen and laminin to be secreted via the basolateral and apical surfaces in similar amounts. These results indicate that cultured GECs are polarized and that they produce and secrete basement membrane components via the basolateral side.
Isolation of putative voltage-gated epithelial K-channel isoforms from rabbit kidney and LLC-PK1 cells
Authors: Desir G. V. , Hamlin H. A. , Puente E. , Reilly R. F. , Hildebrandt F. , Igarashi P.
Published: December 2017

Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.