bluuebunny/crossref_metadata_2025 huggingface dataset:
Exaggerated tubuloglomerular feedback activity in genetic hypertension is mediated by ANG II and AT1 receptors
Authors: Brannstrom K. , Morsing P. , Arendshorst W. J.
Published: December 2017

The purpose of the present study was to determine the role of endogenous angiotensin II in exaggerated tubuloglomerular feedback (TGF) in young euvolemic spontaneously hypertensive rats (SHR). TGF was characterized by measuring proximal tubular stop-flow pressure (Psf) responses to loop of Henle perfusion before and during losartan infusion in 7-wk-old SHR and Wistar-Kyoto rats (WKY). In the control period, TGF responses were exaggerated in SHR compared with WKY. This was evidenced by a larger flow-induced maximum decrease in Psf (19 vs. 13 mmHg), lower turning point (8 vs.12 nl/min), and higher reactivity (-6.4 vs. -3.0 mmHg.nl-1.min-1) in SHR. Losartan (DuP-753) was infused into the renal artery to antagonize angiotensin AT1 receptors in the experimental period. This was verified by losartan inhibiting > 90% of the decrease in whole kidney and superficial cortical blood flow produced by exogenous angiotensin II in both strains. Losartan infusion significantly attenuated TGF activity in SHR but not in WKY. In SHR losartan reduced the maximum Psf response (from 19 to 10 mmHg) and increased the turning point (from 8 to 11 nl/min). SHR values during losartan administration were similar to those obtained in WKY. WKY values were unaffected by losartan. The lack of change in maximum TGF responses after losartan treatment was not unique to WKY, inasmuch as similar results were obtained in euvolemic Munich-Wistar rats (-2.0 +/- 0.7 and -1.1 +/- 1.0 mmHg vs. -8.4 +/- 0.7 mmHg in SHR). Thus angiotensin II does not appear to play an essential role in basal TGF activity during euvolemia in normotensive animals when there is minimal stimulation of the renin-angiotensin system. In contrast, our observations indicate that the exaggerated TGF in young euvolemic SHR represents a functional resetting that is dependent on angiotensin II and losartan-sensitive AT1 receptors during the development of genetic hypertension.
Luminal adenosine receptors regulate amiloride-sensitive Na+ channels in A6 distal nephron cells
Authors: Ma H. , Ling B. N.
Published: December 2017

To investigate the effects of luminal adenosine on amiloride-sensitive Na+ channels, we applied the cell-attached patch-clamp technique to A6 distal nephron cells. Exposure to luminal 30 nM adenosine increased number of channels x open probability (NP0) from 0.38 +/- 0.08 to 0.77 +/- 0.09 (means +/- SE; P < 0.01, n = 17). Luminal exposure to an A1-receptor antagonist (30 nM 8-cyclopentyl-1,3-dipropylxanthine) abolished (P = 0.17, n = 11), whereas an A1 agonist (30 nM N6-cyclohexyladenosine) reproduced (P < 0.02, n = 6) the stimulatory effect of 30 nM adenosine. In contrast, higher concentrations of luminal adenosine (1 or 10 microM) decreased NP0 from 0.65 +/- 0.09 to 0.24 +/- 0.10 (P < 0.02, n = 11) and from 0.80 +/- 0.11 to 0.19 +/- 0.03 (P < 0.01, n = 8), respectively. Channel inhibition by high-dose luminal adenosine was abolished by an A2 antagonist (30 microM 3,7-dimethyl-1-propargylxanthine; P = 0.2, n = 10) and mimicked by an A2 agonist (100 nM CGS-21680 hydrochloride; P < 0.0005, n = 8). We conclude that 1) purinergic regulation of distal nephron Na+ channels is mediated by stimulatory apical A1 receptors and inhibitory apical A2 receptors; 2) basal urinary adenosine concentrations (in nM) would stimulate Na+ reabsorption, whereas higher urinary concentrations (in microM), e.g., renal ischemia and elevations in filtered NaCl load, would increase Na+ excretion; and 3) urinary adenosine may be involved in feedback regulation of distal nephron Na+ transport.
Demethylation of 3-O-methyldopa in the kidney: a possible source for dopamine in urine
Authors: Ibarra F. R. , Aguirre J. , Nowicki S. , Barontini M. , Arrizurieta E. E. , Armando I.
Published: December 2017

The possibility that demethylation of 3-O-methyldopa (OM-dopa) in the kidney could provide a source for dopamine in the urine was explored in male Wistar rats aged 60-90 days, using in vivo and in vitro approaches. The results showed that endogenous OM-dopa is filtered, reabsorbed and extensively metabolized in the kidney. Infusion of OM-dopa into anesthetized rats increased significantly urinary excretion of Na+, dopa, dopamine, and 3,4 dihydroxyphenylacetic acid. Whole kidney homogenates, slices from renal cortex, and microdissected proximal tubules produced significant amounts of both dopa and dopamine when incubated with OM-dopa. Renal cortex slices produced dose-dependent amounts of dopa and dopa-mine when incubated with 1-100 microM OM-dopa. Incubation of microdissected proximal tubule segments with 1 microM OM-dopa produced a fourfold (P < 0.025) increment in dopa and a twofold (P < 0.05) increment in dopamine (an effect similar to that observed with 1 microM L-dopa). One micromolar OM-dopa or 1 microM L-dopa decreased (P < 0.05) Na(+)-K(+)-adenosinetriphosphatase activity measured at maximal velocity condition in proximal tubules. In conclusion, these experiments show that in vitro the kidney is able to produce dopamine by demethylation of OM-dopa, while the results of the OM-dopa infusion suggest that this conversion may also occur in vivo.
Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt
Authors: Singh I. , Grams M. , Wang W. H. , Yang T. , Killen P. , Smart A. , Schnermann J. , Briggs J. P.
Published: December 2017

Experiments were performed to examine the effect of changes in dietary salt intake on the neuronal form of the constitutive nitric oxide synthase (ncNOS, type I NOS), renin, and angiotensinogen mRNA expression in the kidney. Three groups of Sprague-Dawley rats were studied as follows: rats maintained on a 3% Na diet plus 0.45% NaCl in the drinking fluid for 7 days (high salt), rats given a single injection of furosemide (2 mg/kg i.p.) and a 0.03% Na diet for 7 days (low salt), and rats on a diet containing 0.2% Na (control). mRNA expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR) methods using cDNA prepared from samples of renal cortex and microdissected tubular segments. ncNOS PCR products were quantified by comparison with a dilution series of a mutant deletion template. Compared with their respective control, ncNOS mRNA levels in renal cortical tissue were elevated in rats on a low-salt diet and reduced in rats on a high-salt diet. Similar changes were seen in the expression of renin and angiotensinogen mRNA. Dietary salt intake did not alter the mRNA levels for ncNOS from the inner medulla or for endothelial constitutive NOS (ecNOS, type III NOS) and inducible NOS (iNOS, type II NOS) in the renal cortex. ncNOS mRNA was found in glomeruli dissected with the macula densa-containing segment (MDCS), but only at marginal levels in glomeruli without MDCS. Furthermore, a low-salt diet stimulated ncNOS mRNA in glomeruli with MDCS by 6.2-fold compared with a high-salt diet. There was no effect of salt diet on ncNOS mRNA in glomeruli without MDCS or in inner medullary collecting ducts. These results suggest that ncNOS expression in macula densa cells is inversely regulated by salt intake, thus following the known response of the renin-angiotensin system to changes in salt balance.
Cl- channels in basolateral renal medullary vesicles XI. rbClC-Ka cDNA encodes basolateral MTAL Cl- channels
Authors: Zimniak L. , Winters C. J. , Reeves W. B. , Andreoli T. E.
Published: December 2017

The present experiments examined whether rbClC-Ka, a CIC family Cl-channel cDNA from rabbit outer medulla, encodes a basolateral membrane Cl- channel mediating net medullary thick ascending limb (MTAL) Cl- absorption. MTAL cells contain a Cl- channel having certain properties that make it a plausible candidate for the basolateral Cl- channel in that segment. Especially pertinent among properties is the fact that cytosolic Cl- increases in the range 2-25 mM activated these Cl- channels. Cultured mouse MTAL cells were grown in the presence of an antisense oligonucleotide specific for rbCIC-Ka or a random oligonucleotide with no complementarity to rbCIC-Ka. The abundance of Cl- channels was assessed by the frequency of incorporation of Cl- channels from membrane vesicles prepared from these cells into lipid bilayers and by Western blot analysis using an antiserum to the COOH terminus of the rbClC-ka protein. With the use of vesicles from untreated cells or cells treated with the random oligonucleotide, Cl- channels were incorporated into bilayers in 17% and 16% of trials, respectively. However, when vesicles were prepared from cells pretreated with antisense oligonucleotide, there was a virtual abolition of Cl- channel incorporation into bilayers but no effect on the frequency of K+ channel incorporation. In parallel with the reduction in Cl- channel incorporation, the abundance of rbClC-Ka protein was reduced approximately 50% on Western blots. Finally, exposure of Cl- channels in lipid bilayers to the rbClC-Ka antiserum resulted in a block in channel activity. These results support the contention that the basolateral Cl- channel mediating net Cl- absorption in the MTAL is encoded by rbClC-Ka.
Cellular localization of low-molecular-weight kininogen and bradykinin B2 receptor mRNAs in human kidney
Authors: Song Q. , Wang D. Z. , Harley R. A. , Chao L. , Chao J.
Published: December 2017

Kininogen is the precursor of the kinin peptide, which binds to kinin receptors and mediates a broad spectrum of physiological effects. To understand the function of kinin in the kidney, we have identified the cellular localization of the human low-molecular-weight (LMW) kininogen and bradykinin B2 receptor mRNAs in the human kidney by in situ hybridization histochemistry. Kininogen mRNA was found in the juxtaglomerular cells, mesangial areas, epithelium of parietal and visceral (podocytes) layers of Bowman's capsule, proximal and distal tubules, thin and thick segments of Henle's loop, collecting ducts, and the endothelial cells of the blood vessels. B2 receptor mRNA was colocalized with kininogen mRNA in the kidney except the podocytes. The most intense signals were observed in the distal tubules and collecting ducts for both kininogen and B2 receptor mRNAs. No signals were observed in the interstitial cells and macula densa. Control sections did not stain with either the kininogen or B2 receptor sense riboprobe. A Northern blot showed that the expression of LMW kininogen is in the liver and the kidney. Reverse transcription-polymerase chain reaction Southern blot showed expression of B2 receptor mRNA in the endothelial cells, renal proximal tubular cells, and kidney. Our results show the sites of action of kinin in the human kidney and provide further insight into the physiological role of the kallikrein-kinin system on renal function.
Collagen type I enhances endothelin-mediated contraction and induces nonproliferating phenotype in mesangial cells
Authors: Miralem T. , Whiteside C. I. , Templeton D. M.
Published: December 2017

Accumulation of glomerular extracellular matrix is a characteristic accompaniment of mesangial cell proliferation in progressive renal disease. We examined how growth on several matrices affected the proliferative phenotype of cultured rat mesangial cells. Compared with growth on plastic, Matrigel, or mesangial matrix, collagen type I caused a decreased cell number at 72 h, decreased total DNA per culture, and a decrease in the incorporation of [3H]thymidine during S phase in cells released from quiescence. These antiproliferative and antimitogenic effects of collagen type I required growth on a collagen gel; soluble collagen or collagen fragments were without effect. Because a number of agents elicit both proliferative and contractile responses in mesangial cells, we examined the effect of growth on collagen on contractility. Compared with plastic, cells grown on collagen type I were more contractile, showed a higher Ca2+ signal in response to endothelin, and responded to endothelin with a more rapid myosin light-chain kinase-dependent phosphorylation of myosin light chain. We conclude that growth on a collagen type I gel uncouples contractility from a proliferative response in mesangial cells, suppressing proliferation while enhancing contraction and Ca2+ signaling in response to endothelin.
Antisense oligodeoxynucleotides to inducible NO synthase rescue epithelial cells from oxidative stress injury
Authors: Peresleni T. , Noiri E. , Bahou W. F. , Goligorsky M. S.
Published: December 2017

Until recently, the lack of specific inhibitors of various forms of nitric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. Phosphorothioate derivatives of iNOS antisense and control sense or scrambled oligodeoxynucleotides (S-ODNs) were synthesized, and their effect on epithelial cell viability was examined under oxidant stress. Exposure of BSC-1 kidney tubular epithelial cells to H2O2 resulted in elevation of NO release, accompanied by a significant decrease in the population of viable cells (from 97.4 +/- 1.7% to 72.4 +/- 2.4% population). Nitrite production by BSC-1 cells exposed to H2O2 increased almost 10-fold compared with control. Pretreatment of the cells with 10 microM antisense ODNs significantly blunted this response, whereas sense or scrambled ODNs did not modify it. Pretreatment of BSC-1 cells with 10 microM antisense ODNs virtually prevented lethal cell damage in response to H2O2, whereas sense ODNs were ineffective. Lipopolysaccharide induction of iNOS, also preventable by the antisense construct, resulted in a lesser compromise to cell viability. Immunocytochemistry of iNOS in cells pretreated with antisense ODNs showed minimal cytoplasmic staining, as opposed to the untreated or sense ODN-treated positively stained cells. Staining with antibodies to nitrotyrosine was conspicuous in stressed cells but undetectable in antisense ODN-treated cells. In conclusion, oxidant stress is accompanied by the induction of iNOS, increased production of NO, and impaired cell viability; selective inhibition of iNOS using the designed antisense ODNs dramatically improved BSC-1 cell viability after oxidant stress.
Effect of dietary protein and enalapril on proximal tubular delivery and absorption of albumin in nephrotic rats
Authors: Fitzgibbon W. R. , Webster S. K. , Imamura A. , Ploth D. W. , Hutchison F. N.
Published: December 2017

In passive Heymann nephritis (PHN), angiotensin-converting enzyme inhibition (ACEI) or a low dietary protein intake decreases albuminuria (UAlbV). Although this reduction in albuminuria appears to result from an improvement in glomerular permselectivity, the effect of these treatments on albumin permeation and absorption by the nephron has not been clarified. This study used micropuncture techniques to examine the effect of these two treatments on albumin permeation (by measuring the delivery of albumin to the proximal tubule) and the tubular absorption of albumin. PHN rats (12-18 days after injection of FX1A) were switched from 23% to either 40% protein diet (HP), 40% protein diet and concomitantly treated with enalapril (40 mg.kg-1.day-1) (HPE), or to 8% (LP) protein diet for 4-6 days. Although left kidney glomerular filtration rate (GFR) did not differ among the groups, UAlbV from the left kidney in LP and HPE was only 20-40% of that observed for the HP group. In protocol 1, the fractional recovery of albumin (FRAlb) in urine was calculated following injection of artificial tubular fluid containing [14C]inulin and 125I-labeled albumin into the earliest identifiable proximal loops. There were no differences in FRAlb among the three groups. In protocol 2, timed quantitative collections of tubular fluid were obtained from proximal tubular loops. The rate of albumin delivery to the earliest accessible loops of the proximal tubule was significantly lower for the LP and HPE groups compared with the HP group. For each group, albumin concentration corrected for water absorption was not altered along the proximal tubule. The data indicate that alterations of dietary protein intake or ACEI treatment results in large changes in the delivery of albumin at the proximal tubule that could singularly account for the changes in urinary albumin excretion.
Insulin resistance but IGF-I sensitivity in chronic renal failure
Authors: Mak R. H.
Published: December 2017

The effects of insulin-like growth factor I (IGF-I) and insulin on glucose metabolism were compared in awake, chronically catheterized rats with chronic renal failure (CRF) and sham-operated, pair-fed controls. In control rats, IGF-I (5 micrograms.kg-1.min-1) and insulin (2 mU.kg-1.min-1) infusions produced similar twofold increases in total body glucose uptake from fasting values under euglycemic conditions (euglycemic clamps). Total body glucose uptake during euglycemic IGF-I clamps at 5 and 10 micrograms.kg-1.min-1 was not different between CRF and control rats. Total body glucose uptake during euglycemic insulin clamps at 2 and 4 mU.kg-1.min-1 was significantly lower in CRF rats compared with corresponding values in control rats. Hepatic glucose production was suppressed by insulin equally but not by IGF-I in both groups. Correction of metabolic acidosis by NaHCO3 partially improved insulin resistance in rats with CRF, whereas an equimolar amount of NaCl had no effect. Thus the capacity of IGF-I infusion to stimulate total body glucose uptake is maintained in CRF rats that are insulin resistant.
Transforming growth factor-beta modulation of the alpha 1(IV) collagen gene in murine proximal tubular cells
Authors: Kuncio G. S. , Alvarez R. , Li S. , Killen P. D. , Neilson E. G.
Published: December 2017

We have examined the expression of the alpha 1(IV) collagen gene in murine proximal tubular cells (MCT) to better understand how it is regulated in parenchymal cells. Transcriptional activity was examined using luciferase reporters driven by the alpha 1(IV) promoter and varying lengths of 5'-flanking sequences. The minimal bidirectional promoter showed low intrinsic activity in MCT cells, but addition of upstream sequences increased luciferase expression. Maximal activity resided within the first 1,200 bp upstream. A minigene construct was generated by placing a portion of the alpha 1(IV) first intron downstream from the promoter region. The intronic sequences significantly decreased activity of the promoter in MCT cells and 3T3 fibroblasts but greatly enhanced expression in murine parietal yolk sac (PYS) endodermal cells. Addition of transforming growth factor-beta (TGF-beta) to MCT cultures elevated the levels of secreted type IV collagen. Treatment of either transiently or stably transfected MCT cells with TGF-beta produced an increase in the levels of expression of all of the reporters tested. These data support the hypothesis that cell-specific regulation of alpha 1(IV) collagen is dependent upon downstream sequences, which act to decrease the expression of type IV collagen in tubular epithelium. The activity of the alpha 1(IV) collagen gene in proximal tubular cells is increased by TGF-beta, which acts on the domain(s) embedded within the intergenic bidirectional promoter.
Alpha 2-adrenergic-mediated inhibition of water and urea permeability in the rat IMCD
Authors: Rouch A. J. , Kudo L. H.
Published: December 2017

These studies were conducted to determine whether the alpha 2-agonists epinephrine and dexmedetomidine inhibit osmotic water permeability (Pf) and urea permeability (Pu) in the rat inner medullary collecting duct (IMCD). Wistar rat IMCD segments were perfused via standard methods, and Pf and Pu were determined in separate studies. The control period was followed by adding 220 pM arginine vasopressin (AVP) or 10(-4) M dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) to the bath. Epinephrine or dexmedetomidine, both at 1 microM, was then added to the bath, and this period was followed by adding 1 microM atipamezole, a selective alpha 2-antagonist. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was present in all experiments with DBcAMP. Epinephrine inhibited AVP- and DBcAMP-stimulated Pf by 90% and 80%, respectively. Dexmedetomidine inhibited AVP- and DBcAMP-stimulated Pf by 98% and 97%, respectively. Epinephrine inhibited AVP- and DBcAMP-stimulated Pu by 70% and 60%, respectively. Dexmedetomidine failed to affect Pu. Atipamezole reversed all inhibitory effects. These data confirm an alpha 2-mediated mechanism in the IMCD that modulates Pf and Pu, and they indicate that inhibition occurs via post-cAMP cellular events.
Coupling of entry to exit by peritubular K+ permeability in a mathematical model of rat proximal tubule
Authors: Weinstein A. M.
Published: December 2017

In the proximal tubule in vivo, glomerulotubular balance requires that tubule epithelial cells accommodate a twofold variation in Na+ reabsorption through the Na+/H+ exchanger of the luminal membrane. In a mathematical model of proximal tubule, in which permeability coefficients are fixed, doubling flux through the Na+/H+ antiporter produces a substantial increase in cell volume and cytosolic HCO3-. In this model, it is possible to vary peritubular K+ permeability with changes in luminal Na+ entry, so that cell volume is constrained to be constant. In these calculations, the model predicts that peritubular hyperpolarization and nearly constant cytosolic HCO3- will accompany increases in luminal Na+ entry. Realistic models of variable peritubular K+ permeability might include a functional dependence on flux through the Na(+)-K(+)-adenosinetriphosphatase, cytosolic pH, or cell volume. When K+ permeability is represented as a function of any of these variables, homeostatic control of cell volume and pH can be obtained over a physiological variation of Na+/H+ flux. However, when luminal Na+ entry is via Na(+)-glucose cotransport, volume homeostasis is best when peritubular K+ permeability depends on the rate of active Na+ transport. For any modulator of K+ permeability, realistic constraints on the value of this parameter suggest that peritubular K+ permeability is, by itself, not sufficient to maintain cell volume within narrow limits. Parallel activation of another exit pathway, such as peritubular Na(+)-3 HCO3- cotransport, may be required to achieve the necessary homeostasis.
Renal reserve during human pregnancy
Authors: Sturgiss S. N. , Wilkinson R. , Davison J. M.
Published: December 2017

Pregnancy in healthy women is associated with increments in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). We hypothesized that the hyperfiltration of normal pregnancy attenuates or exhausts renal reserve. In 21 healthy females studied serially in early and late pregnancy and then on average 15 wk postpartum, GFR and ERPF were determined by inulin and p-aminohippurate clearances, respectively, before and during either an amino acid infusion (Vamin 9; Kabi Pharmacia) (n = 14) or a control infusion of Hartman's solution (n = 7), both infused at 4 ml/min for 210 min. In early and late pregnancy, GFR increased significantly in response to amino acid infusion [from 137 +/- 29 to 162 +/- 35 ml/min (P < 0.001) and from 134 +/- 29 to 148 +/- 40 ml/min (P < 0.01), respectively], with the increments (18 and 10%, respectively) not significantly different from postpartum (non-pregnant) when GFR increased by 12% from 94 +/- 22 to 105 +/- 23 ml/min (P < 0.002). Amino acid infusion significantly increased ERPF from 874 +/- 188 to 980 +/- 215 ml/min in early pregnancy (P < 0.01), from 684 +/- 135 to 773 +/- 181 ml/min in late pregnancy (P < 0.01), and from 507 +/- 121 to 560 +/- 141 ml/min postpartum (P < 0.006), increments of 12, 13, and 10%, respectively. GFR did not change in response to control infusion. We conclude that, despite gestational increments in renal hemodynamics of > 40%, pregnancy does not attenuate the renal response to amino acid infusion.
Expression of aquaporins-1 and -2 during nephrogenesis and in autosomal dominant polycystic kidney disease
Authors: Devuyst O. , Burrow C. R. , Smith B. L. , Agre P. , Knepper M. A. , Wilson P. D.
Published: December 2017

Aquaporin-1 (AQP1), located in proximal tubules (PT) and descending thin limbs of Henle (DTL), and aquaporin-2 (AQP2), located in collecting ducts (CD), are channels involved in water transport across renal tubule epithelia. Using antibodies against AQP1 and AQP2, we here show expression of AQP1 and AQP2 in normal human developing and adult kidneys and in autosomal dominant polycystic kidney disease (ADPKD). Unlike in rats, AQP1 and AQP2 are expressed early during human nephrogenesis (12-wk gestation). AQP1 was first seen in developing PT epithelia, predominantly in apical cell membranes, and, at 15 wk, was also detected in DTL. AQP2 was seen in apical cell membranes of the branching ureteric bud and CD system from 12 wk and throughout development. In adult normal kidneys, AQP1 was localized to apical and basolateral membrane domains of PT and DTL, whereas AQP2 was restricted to principal cells of CD. This distribution of AQP1 and AQP2 was also seen in early stage ADPKD, except that AQP1 was mostly located in the apical membrane region of expanded PT. In end-stage ADPKD, two-thirds of the cysts expressed either AQP1 or AQP2, but these two water channels were never colocalized in the same cyst. Western blot analysis showed maximal expression of AQP1 and AQP2 in normal adult kidneys, lower levels in fetal kidneys, and decreases associated with degree of cystic progression in ADPKD. These data 1) demonstrate specific, mutually exclusive localization of AQP1 and AQP2 in human fetal and adult kidneys; 2) show that both channels are expressed early during nephrogenesis; and 3) show that the mutual exclusivity of localization is maintained even into end-stage ADPKD.
Abnormal postpartum renal development and cystogenesis in the bcl-2 (-/-) mouse
Authors: Sorenson C. M. , Padanilam B. J. , Hammerman M. R.
Published: December 2017

Mice deficient for B cell leukemia/lymphoma gene 2 [bcl-2(-/-) mice] manifest congenital renal hypoplasia and develop multicystic kidney disease and renal failure postnatally. To characterize postpartum renal development, to identify the cellular origin of the cysts, and to provide insight into the role that bcl-2 deficiency plays in the cystogenic process, we examined the morphology of kidneys from bcl-2 (-/-) mice and wild-type littermates [bcl-2 (+/+)] from birth (P0) to postpartum day 28 (P28), determined whether abnormalities of cellular proliferation and apoptosis accompany cyst development, and characterized expression of the bcl-2-related protein, bax. Between P0 and P7, kidneys from bcl-2 (-/-) and bcl-2 (+/+) mice undergo a comparable increase in weight and have similar histological appearances. However, during the next 2 wk of life, weight gain in kidneys from bcl-2 (-/-) mice is reduced compared with that in kidneys from bcl-2 (+/+) animals, and cysts develop in tubules with staining characteristics of proximal tubule, distal tubule/medullary thick ascending limb of Henle's loop, and collecting duct. Unaffected glomeruli and proximal tubules in kidneys of bcl-2 (-/-) mice undergo compensatory growth. Cystogenesis is accompanied by enhanced incorporation of 5-bromo-2'-deoxyuridine in cells within cortex and medulla and apoptosis of cells within cysts and in the renal interstitium. Bax protein is expressed in the distal tubule in kidneys of bcl-2 (+/+) and bcl-2 (-/-) mice and in some, but not all cysts. We conclude that abnormal regulation of DNA synthesis and apoptosis accompany cystogenesis in bcl-2 (-/-) mice during postpartum kidney development. Continued expression of bax could enhance apoptotic cell death.0
Pharmacokinetics and renal metabolism of atrial natriuretic factor during rat pregnancy
Authors: Omer S. , Varma D. R. , Cernacek P. , Mulay S.
Published: December 2017

The present studies were done to determine if the attenuation of the natriuretic and diuretic effects of atrial natriuretic factor (ANF) during rat pregnancy was caused by an increase in its metabolism. It was found that the plasma half-life (min), plasma clearance (ml.kg-1.min-1), and volume of distribution (ml/kg) of ANF were, respectively, 2.5 +/- 4, 115 +/- 19, and 371 +/- 44 in pentobarbital-anesthetized virgin rats (n = 6) and not different from the corresponding values of 3.1 +/- 0.5, 124 +/- 26, and 526 +/- 120 in 20-day gravid animals (n = 6). Rates of metabolism of ANF (pmol.min-1.microgram protein-1) by renal cortical membranes from virgin (n = 5) and gravid (n = 5) rats were, respectively, 45 +/- 0.6 and 45 +/- 0.5; likewise, cortical membrane neutral endopeptidase activities in virgin and 20-day gravid rats (n = 7) did not differ. It is concluded that the attenuation of the renal effects of ANF during pregnancy is not caused by changes in its systemic or renal metabolism but might be due to a decrease in guanylate cyclase-linked renal ANF receptors and/or receptor-mediated effects.
Extracellular chloride regulates mesangial cell calcium response to vasopressor peptides
Authors: Stevanovic Z. S. , Salter M. W. , Whiteside C. I.
Published: December 2017

The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, the components of vasopressor-stimulated calcium signaling were defined in rat mesangial cells cultured on coverslips and preloaded with fura 2. By spectrofluorometry, manganese uptake (reflecting divalent cation channel activation) was observed by quenching of fura 2, or intracellular cytosolic calcium concentration was calculated by dual-excitation ratiometric measurement. In cells depolarized with KCl (45 mM), enhanced manganese uptake or increased cytosolic calcium were inhibited with verapamil (10 microM). Pretreatment of mesangial cells with verapamil reduced the sustained calcium level in response to endothelin-1 (0.1 microM) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 microM) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement confirmed that endothelin-1 stimulated a sustained increase in cytosolic calcium or divalent cation entry only in the presence of simultaneous depolarization. In chloride-free buffer (chloride replaced with impermeant anions), sustained calcium response to endothelin-1 was reduced by 72 +/- 8 (n = 8) and by 65 +/- 4% (n = 8) in the presence of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 microM). In chloride-free buffer, cytosolic calcium (unstimulated) increased to > 200 nM by 30 min. These data indicate that reduced extracellular chloride increases mesangial cell basal cytosolic calcium and decreases the transient and sustained cytosolic calcium response to vasopressor peptides.
Abnormal renal development in the Os/+ mouse is intrinsic to the kidney
Authors: Sorenson C. M. , Rogers S. A. , Hammerman M. R.
Published: December 2017

The oligosyndactylism (Os/+) mouse, is a genetic model for oligomeganephronic congenital renal hypoplasia. To define the abnormality in renal development and to determine whether the abnormality is kidney autonomous, we examined kidneys from newborn and 21- and 63-day-old Os/+ and wild-type (+/+) mice, obtained metanephric kidneys from embryonic day 12 (E12) Os/+ and +/+ embryos, and compared growth and development of the metanephroi in vitro. Kidneys from newborn Os/+ mice were smaller than those from newborn +/+ mice and contained fewer glomeruli per midsagittal section. Following birth, kidneys from Os/+ mice manifest compensatory growth of glomeruli and proximal tubules. Metanephroi from E12 Os/+ and +/+ embryos were comparable in size. However, during 4 days in culture, growth and development of metanephroi from Os/+ embryos were visibly reduced compared with metanephroi from +/+ embryos. Expression of B cell leukemia/lymphoma gene 2 (bcl-2), the absence of which is known to result in congenital renal hypoplasia, was detected in the Os/+ mouse kidneys. We conclude that the renal abnormality in Os/+ mice is intrinsic to the kidney and does not result from the absence of bcl-2 expression.
Effect of atrial natriuretic peptide on renal hemodynamics and sodium excretion during human pregnancy
Authors: Irons D. W. , Baylis P. H. , Davison J. M.
Published: December 2017

The effect of infused atrial natriuretic peptide (ANP) on sodium excretion (UNa), glomerular filtration rate (GFR), and effective renal plasma flow (ERPF) was studied in 12 normotensive primigravidae at 32 wk gestation [late pregnancy (LP)] and again 4 mo postpartum [nonpregnant (NP)]. Three 20-min steady-state (renal) clearances of inulin and p-aminohippurate were used to measure GFR and ERPF, respectively, before and after infusion of ANP at 2 pmol.kg-1.min-1. Basal plasma ANP (pANP) was increased in LP compared with NP [7.8 +/- 0.6 vs. 3.3 +/- 0.4 pmol/l (P < 0.0001), respectively]. In LP, infusion of ANP increased pANP from 7.8 +/- 0.6 to 21.8 +/- 1.4 pmol/l (P < 0.00001), which produced a natriuresis [UNa of 0.18 +/- 0.02 vs. 0.25 +/- 0.03 mmol/min (P = 0.03), respectively], with no change in GFR (153 +/- 13 vs. 142 +/- 8 ml/min, P = 0.16) but a significant reduction in ERPF (766 +/- 52 vs. 660 +/- 31 ml/min, P = 0.002). In NP, ANP infusion increased pANP from 3.3 +/- 0.4 to 27.7 +/- 2.5 pmol/l (P < 0.00001), which produced no significant natriuresis [UNa of 0.22 +/- 0.07 vs. 0.26 +/- 0.09 mmol/min (P = 0.15), respectively] and no change in GFR (87 +/- 3 vs. 89 +/- 3 ml/min), but again a reduction in ERPF (486 +/- 17 vs. 414 +/- 9 ml/min, P < 0.001).
P2 purinoceptor stimulation attenuates PTH inhibition of phosphate uptake by a G protein-dependent mechanism
Authors: Lederer E. D. , McLeish K. R.
Published: December 2017

Pages F309'F316: E. D. Lederer and K. R. McLeish. “P2 purinoceptor stimulation attenuates PTH inhibition of phosphate uptake by a G protein-dependent mechanism.” The American Heart Association of Kentucky should have been acknowledged for their support of E. D. Lederer.
Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis
Authors: Zhang Y. , Mircheff A. K. , Hensley C. B. , Magyar C. E. , Warnock D. G. , Chambrey R. , Yip K. -P. , Marsh D. J. , Holstein-Rathlou N. -H. , McDonough A. A.
Published: December 2017

Pages F1004'F1014: Y. Zhang, A. K. Mircheff, C. B. Hensley, C. E. Magyar, D. G. Warnock, R. Chambrey, K.-P. Yip, D. J. Marsh, N.-H. Holstein-Rathlou, and A. A. McDonough. “Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis.” The immunoblot panels in Figures 2 and 5–7 were inadvertently printed from low-resolution copies of the original artwork; in addition, the panels in Fig. 6 were incorrectly labeled. The correct figures are reproduced on the following pages.

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A ureteric bud cell line induces nephrogenesis in two steps by two distinct signals
Authors: Barasch J. , Pressler L. , Connor J. , Malik A.
Published: December 2017

Nephrons develop from mesenchymal cells that have contacted the ureteric bud (UB). To determine whether cell associated or secreted ureteric molecules induce the mesenchyme, we have isolated UB cell lines from mice transgenic for T antigen. These cells express epithelial and ureteric (Dolichos lectin staining, c-ret, c-met without hepatocyte growth factor) specific markers, which identifies them as authentic UB cells. Medium conditioned by our cells rescues mesenchyme from apoptosis without inducing the appearance of epithelial aggregates. The same was found by culturing mesenchymes upon the apical surface of a UB monolayer. In contrast, tubules were induced in mesenchymes contacting trypsinized pellets of UB cells. As revealed by staining for T antigen and Dolichos lectin or by prelabeling UB cells with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), we found that our cells encapsulated the mesenchyme but did not incorporate in the tubules. These data demonstrate that nephrogenesis is stimulated by two distinct ureteric signals, secreted molecules rescue the mesenchyme from apoptosis, whereas diffusion-limited basolateral molecules trigger mesenchymal/epithelial conversion.
Transcriptional and posttranscriptional mechanisms regulate human renin gene expression in Calu-6 cells
Authors: Lang J. A. , Ying L. H. , Morris B. J. , Sigmund C. D.
Published: December 2017

We have recently identified a human pulmonary carcinoma cell line (Calu-6) that expresses human renin (hREN) mRNA endogenously, and we use it herein as a model to examine the regulation of the hREN gene. Transfection analysis of a deletion series (-2750 to -149) of hREN promoter-luciferase fusion constructs revealed the presence of multiple weak regulatory elements within the first 1,301 bp of the 5'-flanking region and a classic silencer element within the first intron (intron A) of the gene. The 5'-flanking regulatory domain consisted of three closely linked elements, two negative and one positive, each contributing a cell-specific threefold modulation of transcriptional activity. Treating Calu-6 cells with forskolin caused a 100-fold increase in steady-state endogenous hREN mRNA but no increase in hREN promoter activity in transient transfections or in nuclear runoff transcription assays. Nevertheless, de novo transcription and translation were necessary for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated induction. Our results suggest that multiple regulatory elements regulate basal transcriptional activity of the hREN gene and the increase in hREN mRNA by cAMP may be mediated by posttranscriptional mechanisms.
Low permeabilities of apical membranes of barrier epithelia: what makes watertight membranes watertight?
Authors: Zeidel M. L.
Published: December 2017

Several epithelia, including those of collecting duct (in the absence of antidiuretic hormone), thick ascending limb of Henle, and mammalian bladder exhibit extremely low permeabilities for water, small nonelectrolytes like urea, and protons. This brief review describes how apical membrane structure might account for these low permeabilities by incorporating recent biophysical and molecular modeling results.
Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding
Authors: Lucking K. , Nielsen J. M. , Pedersen P. A. , Jorgensen P. L.
Published: December 2017

For understanding the regulation of sodium reabsorption, it is important to know whether the alpha 2- or alpha 3-isoform of Na-K-adenosinetriphosphatase (Na-K-ATPase) is expressed in mammalian kidney in addition to the predominant alpha 1 beta 1-isozyme. Here we applied competitive polymerase chain reaction (PCR) for estimation of mRNA in parenchymal zones of rat kidney for comparison to high-affinity [3H]ouabain binding. The alpha 3-isoform mRNA was demonstrated to form 0.04-0.05% of the amount of alpha 1-isoform mRNA in the cortex, medulla, and papilla of rat kidney. The alpha 2-mRNA was demonstrated in all three zones and constituted 0.03% of the amount of alpha 1-mRNA in cortex. The abundance of the alpha 1 truncated mRNA was 0.1-0.8% of that of the alpha 1-mRNA. The upper limit for expression of Na-K-ATPase isozyme with high ouabain affinity (dissociation constant, 69-141 nM) was 0.096-0.14% of the concentration of alpha 1 beta 1-Na-K-ATPase. Thus a small but well-defined pool of alpha 2- and alpha 3-isoforms constitutes < or = 0.1% of the amount of alpha 1-isoform at both the mRNA and protein level in rat kidney.
cAMP activation of phosphoenolpyruvate carboxykinase transcription in renal LLC-PK1-F+ cells
Authors: Liu X. , Curthoys N. P.
Published: December 2017

Phosphoenolpyruvate carboxykinase (PCK) is a key regulatory enzyme in renal ammoniagenesis and gluconeogenesis. LLC-PK1-F+ cells are porcine renal proximal tubule-like cells that express significant levels of the cytosolic PCK. Treatment of subconfluent LLC-PK1-F+ cells with 0.1 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) for 8 h causes a 21-fold increase in PCK mRNA. This response is very rapid and is not inhibited by 0.5 mM cycloheximide, indicating that ongoing protein synthesis is not required. Similarly, cells transfected with PCK(-490)CAT exhibit an 8- to 10-fold increase in chloramphenicol acetyltransferase (CAT) activity when treated with cAMP for 24 h. The addition of okadaic acid, a protein phosphatase inhibitor, both stimulated the CAT activity and potentiated the cAMP effect by twofold, suggesting that phosphorylation may contribute to the transcriptional activation. Assays using a series of PCK-CAT constructs containing specific deletions or block mutations established that the CRE-1 the P3(II) elements are required for the cAMP response. Cotransfection experiments using dominant negative expression vectors indicated that a CCAAT enhancer binding protein (C/EBP) transcription factor, and not CREB, mediates cAMP activation of transcription in LLC-PK1-F+ cells.
Basal nitric oxide production curtails arteriolar vasoconstrictor responses to ANG II in rat kidney
Authors: Ikenaga H. , Fallet R. W. , Carmines P. K.
Published: December 2017

Experiments were performed to test the hypothesis that renal arteriolar vasoconstrictor responses to angiotensin II (ANG II) are curtailed through a mechanism that involves stimulation of endogenous nitric oxide (NO) synthesis. The in vitro blood-perfused juxtamedullary nephron technique was exploited to monitor arteriolar lumen diameter responses to exogenous ANG II before and during treatment with the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA). Under control conditions, 1 nM ANG II reduced afferent and efferent arteriolar diameters by 13 and 11%, respectively. In the presence of L-NNA, 1 nM ANG II decreased afferent diameter by 26% and efferent diameter by 35%. This augmentation could not be attributed to the L-NNA-induced decrease in baseline diameter. L-NNA also augmented vasopressin responses, indicating a lack of agonist specificity in this interaction. ANG II reactivity during L-NNA treatment was not enhanced when tissue NO activity was fixed at basal levels (exposure to 1 microM sodium nitroprusside). These results indicate that endogenous NO modulates the vasoconstrictive impact of ANG II on both afferent and efferent arterioles of juxtamedullary nephrons and that this process does not require stimulation of NO synthesis.
Dopamine inhibits AVP-dependent Na+ transport and water permeability in rat CCD via a D4-like receptor
Authors: Sun D. , Schafer J. A.
Published: December 2017

We studied the receptor responsible for dopamine action in isolated perfused cortical collecting ducts (CCD) from rats treated with deoxy-corticosterone. (Critical experiments were repeated in CCD from untreated rats with the same results.) At doses > or = 1 microM, dopamine inhibited arginine vasopressin (AVP)-dependent Na+ and water transport (measured by the unidirectional lumen-to-bath 22Na+ flux and the transepithelial voltage) and osmotic water permeability (Pf). The effects of dopamine were not reversed by the dopamine-1 (D1) antagonist SCH-23390, and no inhibition was produced by the D1 agonists fenoldopam or SKF-81247. When Na+ transport and Pf were stimulated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, dopamine did not inhibit, suggesting a "D2-type" receptor. However, the D2 agonist quinpirole had no effect on the AVP-dependent transepithelial voltage (VT), and the D2 and D3 antagonists domperidone and pimozide did not reverse dopamine inhibition of VT. The only agent tested that reversed the effects of dopamine was the D4-specific antagonist clozapine. We conclude that dopamine inhibition of salt and water transport in the CCD is mediated by a D4-like receptor.
Fluorescence imaging study of organic anion transport from renal proximal tubule cell to lumen
Authors: Miller D. S. , Letcher S. , Barnes D. M.
Published: December 2017

The mechanisms driving organic anion transport from cell to lumen were studied in intact killifish proximal tubules using fluorescence microscopy. Three fluorescent substrates were used as follows: 1) fluorescein (FL); 2) carboxyfluorescein (CF), generated intracellularly from carboxyfluorescein diacetate (CFDA); and 3) bimane-S conjugates, generated intracellularly by conjugation of monochlorobimane (MCB) with glutathione (GSH) and subsequent metabolism. The latter two substrates bypassed the basolateral uptake mechanism, allowing direct study of luminal transport mechanisms. At steady state, for all three substrates, luminal fluorescence was two to three times higher than cellular fluorescence. With FL as substrate, addition of p-aminohippurate (PAH) or probenecid to the incubation medium reduced cellular and luminal fluorescence to roughly the same extent. With CFDA or MCB as substrate, PAH and probenecid only slightly reduced cellular fluorescence but greatly reduced luminal fluorescence. MCB blocked transport of FL from cell to lumen; CFDA blocked transport of bimane-S conjugates from cell to lumen. Finally, depolarizing tubule cells with high-potassium medium did not affect the steady-state lumen-to-cell distribution of FL, CF, or bimane-S conjugates. These results show that organic anion transport from cell to lumen is mediated and uphill but not sensitive to the electrical potential difference across the luminal membrane.
Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors
Authors: Volk K. A. , Zhang C. , Husted R. F. , Stokes J. B.
Published: December 2017

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.