bluuebunny/crossref_metadata_2025 huggingface dataset:
Endocrine responses of fetal sheep to prolonged hypoxemia with and without acidemia: relation to urine production
Authors: Wlodek M. E. , Brace R. A. , Cock M. L. , Hooper S. B. , Harding R.
Published: December 2017

Our aim was to examine the endocrine changes associated with alterations in fetal urine production during 24 h of hypoxemia induced by either reduced uterine blood flow (RUBF) or maternal N2 inhalation (N2). In contrast to RUBF, which caused a diuresis, N2 caused a transient antidiuresis; during both posthypoxemia periods (RUBF and N2), fetal urine production was increased. RUBF, but not N2, was associated with a transient acidemia. Fetal plasma arginine vasopressin (AVP) and atrial natriuretic factor (ANF) concentrations increased during RUBF and were inversely correlated to pH; there were no detectable AVP or ANF responses to N2. Fetal prostaglandin E2 (PGE2) increased during the hypoxemia and posthypoxemia periods induced by both methods, but RUBF caused the greater increase. AVP and PGE2 concentrations were positively correlated with urine production. Fetal arterial blood pressure increased during RUBF but not N2. During RUBF, the increases in AVP and PGE2 concentrations and/or fetal arterial blood pressure may have contributed to the diuresis. During N2, we suggest that low, but increased, levels of AVP may have caused the transient antidiuresis, whereas the diuresis observed during both posthypoxemia periods may have been mediated by elevated PGE2 concentrations and/or increased fetal arterial blood pressure.
Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney
Authors: Bachmann S. , Bosse H. M. , Mundel P.
Published: December 2017

Nitric oxide (NO) is generated from L-arginine by NO synthase (NOS). We have investigated the localization of constitutive NOS isoforms in rat, mouse, guinea pig, rabbit, pig, and human kidney. NADPH diaphorase (NADPH-d) reaction was used for histochemical detection of NOS enzyme activity, neuronal NOS (NOS I) and endothelial NOS (NOS III) were identified by specific antibody, and in situ hybridization was applied for NOS I mRNA detection. Strong presence of NOS I in macula densa (MD), previously detected in rat, was found in all species including humans. Additional NOS I-positive cells of the thick ascending limb (TALH) were defined. A clear-cut distinction between Tamm-Horsfall-protein-positive cells of the TALH and NOS I-positive cells of the TALH was shown. Ultrastructurally, NOS I was located in the cytosol. Intimate spatial relation between NOS I-positive cells and renin-containing preglomerular afferent arteriole suggests an effect of MD-derived NO on the juxtaglomerular granular cells. In the renal vasculature, both NADPH-d and NOS III were located in the endothelium of cortical and medullary vessels, whereas the muscle layer was unreactive. The glomerular arterioles showed stronger labeling in the efferent than in the afferent endothelium, and efferent endothelium selectively contained both NOS I and NOS III. The unique morphology of efferent endothelial cells indicates a particular role for NO in this vessel segment. At the capillary level, only the glomerular tuft showed NOS-positive endothelia. A subpopulation of renal nerves containing NADPH-d and NOS I was found in perivascular connective tissue and near pelvic epithelium. These results demonstrate a wide distribution of two constitutive NOS isoforms in the kidney of various animal species including humans. The distinct location of both isoforms in the cortex confirms that NO plays a crucial role in local glomerular signaling events.
Glomerular handling of circulating glycated albumin in the normal mouse kidney
Authors: Londono I. , Ghitescu L. , Bendayan M.
Published: December 2017

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation. Endogenous albumin, as well as dinitrophenylated native BSA (DNP-BSA) and glycated albumins (DNP-gBSA), were localized within the capillary lumen, glomerular and peritubular basement membranes, and the mesangial matrix. Morphometric evaluation of the labeling over the glomerular basement membrane (GBM) revealed a peak of labeling in the endothelial side for either endogenous albumin or DNP-BSA. In contrast, the labeling distribution for DNP-gBSA showed a shift toward the epithelial side, suggesting a further penetration of the glycated probe into the GBM. When coinjected with gBSA, DNP-BSA was found to display a labeling distribution similar to that displayed by DNP-gBSA. These results indicate that the glycated tracer penetrates the normal glomerular wall deeper than the nonglycated one. Moreover, glycated albumin increases the infiltration of the nonglycated tracer through the normal glomerular wall. Circulating glycated serum proteins thus appear to play an important role in the onset of the glomerular dysfunction and proteinuria, which take place in long-term hyperglycemic states.
Expression of angiotensin II AT2 receptor mRNA during development of rat kidney and adrenal gland
Authors: Shanmugam S. , Llorens-Cortes C. , Clauser E. , Corvol P. , Gasc J. M.
Published: December 2017

The angiotensin II (ANG II) receptors have been pharmacologically classified into two major distinct types, designated AT1 and AT2. A high transient expression of AT2 receptors in the fetal tissues has been previously demonstrated. This study describes the cellular distribution of AT2 receptor mRNA in the developing rat kidney and adrenal gland by in situ hybridization with 35S-labeled cRNA probes. From day 12 of fetal life (F12) to day 15 postpartum (D15) AT2 mRNA was detected in the undifferentiated nephrogenic mesenchymal tissue but not in the immature and mature glomeruli and tubules of the kidney. No AT2 mRNA was observed in the kidney after D22. The adrenal gland also expressed AT2 receptor mRNA early during development from F12 but, unlike the kidney, continuously expressed the mRNA at high levels through to adulthood. The disappearance of AT2 mRNA in the kidney was synchronous with the completion of nephrogenesis and suggests that ANG II might act through this receptor as a differentiation/growth factor during nephron development. In the adrenal gland ANG II could act as a hormone and also as a differentiation/growth factor via the AT2 receptor.
Capillary network structure does not affect theoretical analysis of glomerular size selectivity
Authors: Remuzzi A. , Ene-Iordache B.
Published: December 2017

Anatomical studies have demonstrated that the glomerular capillaries are complex and heterogeneous networks. Conventional models of glomerular size selectivity, however, are based on the assumption of simplified geometries. We developed a theoretical model of glomerular size-selective function based on the geometric data obtained in a previous reconstruction of a glomerular network from a normal Munich-Wistar rat. This heterogeneous model was compared with the homogeneous model conventionally used to calculate membrane selective parameters from the fractional clearance of two test solutes, neutral dextran and Ficoll. For both models we assumed a hypothetical log-normal distribution of pore sizes and calculated optimal membrane pore-size parameters using previously published values of fractional clearances. The difference between the sieving coefficients calculated with the two models was negligible, never exceeding 5.5%. Since the homogeneous model does not consider the pressure drop along the glomerular capillary, we also computed fractional clearances with the homogeneous model, assuming the same pressure drop as in the heterogeneous one. The differences in computed fractional clearances using the homogeneous model with and without a pressure drop were less than 1.2%. We concluded that models based on identical capillary networks can therefore be used for interpreting sieving coefficients for macromolecules.
Effect of P(i) restriction on renal Na(+)-P(i) cotransporter mRNA and immunoreactive protein in X-linked Hyp mice
Authors: Tenenhouse H. S. , Martel J. , Biber J. , Murer H.
Published: December 2017

Although renal Na(+)-P(i) cotransporter gene expression is decreased in X-linked Hyp mice, the mutants do respond to P(i) restriction with an adaptive increase in Na(+)-P(i) cotransport maximal velocity in renal brush-border membrane vesicles. In the present study, we examined the mechanism for the adaptive increase in Na(+)-P(i) cotransport in P(i)-deprived Hyp mice and normal littermates, using a cDNA probe encoding a rat, renal-specific Na(+)-P(i) cotransporter (NaPi-2) and a rabbit polyclonal antibody raised against a synthetic NaPi-2-derived peptide. The low-P(i) diet elicited an increase in Na(+)-P(i) cotransport in normal (141 +/- 13 to 714 +/- 158) and Hyp mice (59 +/- 6 to 300 +/- 62 pmol.mg protein-1.6 s-1; means +/- SE, n = 3, P < 0.01) that was accompanied by an increase in brush-border membrane NaPi-2 protein, relative to ecto-5'-nucleotidase, in normal (1.0 +/- 0.1 to 7.6 +/- 1.5) and Hyp mice (0.3 +/- 0.1 to 7.7 +/- 1.4) (means +/- SE, n = 4; P < 0.01). The low-P(i) diet also elicited an increase in the abundance of NaPi-2 mRNA, relative to the 18S RNA, in normal (157 +/- 9% of control diet, P < 0.05) and Hyp mice (194 +/- 10% of control diet, P < 0.01). Immunohistochemistry revealed that NaPi-2 protein was localized to the brush-border membrane of the proximal tubule and that both intensity of the signal and number of immunostained proximal tubules were increased in renal sections from normal and Hyp mice fed the low-P(i) diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Nitric oxide alters cytosolic potassium in cultured glomerular mesangial cells
Authors: Ganz M. B. , Kasner S. E. , Unwin R. J.
Published: December 2017

Endothelium-derived relaxing factor is believed to be nitric oxide (NO). Evidence suggests that it has important functions in the regulation of mesangial cell (MC) tone and possibly in inflammation. As a vasodilator, its vasorelaxant effect depends, in part, on changes in cell membrane potential. We therefore tested the hypothesis that MCs in culture produce NO that results in cell relaxation through the opening of K+ conductance pathways. The K(+)-sensitive fluorescent dye, potassium-binding benzofuran isophthalate, was used to detect rapid changes in intracellular K+ concentration ([K+]i). The membrane potential-sensitive dye, 1,3-(sulfonatopropyl)-4-([beta-di-n- butylamino)-6-naphthyl]vinyl)pyridinium betaine (Di-4-AN-EPPS), was used to detect changes in membrane potential. Basal [K+]i was 92 +/- 9 mM (n = 46). In response to sodium nitroprusside (SNP), acetylcholine (ACh), and bradykinin (BK), [K+]i decreased to 72 +/- 7 (n = 5, P < 0.05), 70 +/- 8 (n = 7, P < 0.05), and to 69 +/- 13 mM (n = 6, P < 0.05), respectively. [K+]i rapidly returned to basal level in the continued presence of all three agonists. The SNP-, ACh-, and BK-induced decrease in [K+]i was significantly blunted by Ba2+ by 85, 92, and 89%, respectively (n > 4 for each agonist examined, P < 0.0001). NG-monomethyl-L-arginine (L-NMMA) and methylene blue inhibited the [K+]i-lowering effect of ACh and BK by 94 and 85% for L-NMMA (n = 5 for each agonist, P < 0.0001) and by 67 and 72% for methylene blue (n = 5 for each agonist, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of transmembrane-type protein tyrosine phosphatase mRNA along rat nephron segments
Authors: Kaneko T. , Moriyama T. , Imai E. , Akagi Y. , Arai M. , Inoue T. , Xia C. , Noguchi T. , Kamada T. , Ueda N.
Published: December 2017

Protein phosphorylation on tyrosine residues is one of the main cell signaling mechanisms. Cellular phosphotyrosyl levels are regulated by the activities of protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPase). We have previously reported cDNA cloning of several types of PTPase from rat kidney, including LRP (leukocyte common antigen-related protein; also known as the transmembrane-type tyrosine phosphatase, i.e., RPTP alpha). LRP mRNA was shown to be abundant in the kidney; however, our understanding of the functional role of LRP in the kidney is very limited. To gain keener insight into the function of LRP in the kidney, our first approach was to reveal its mRNA distribution along rat nephron segments. Large signals were found in inner medulla by Northern blot analysis. By using a reverse transcription and polymerase chain reaction assay of individual microdissected tubule segments along the nephron [proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD)] and glomeruli, we revealed intrarenal localization of LRP mRNA. LRP mRNA was detected in all nephron segments tested but was relatively rich in the IMCD. Rank order of the signal intensity was IMCD > PCT = OMCD > CCD > MTAL = glomeruli. Immunohistochemistry also revealed that LRP was abundant in IMCD. This pattern of expression gives rise to an interesting possibility that LRP might be involved in the specific renal tubule function, such as urinary concentrating mechanism; however, further study is required to describe the function of LRP in more detail.
Selective neuronal nitric oxide synthase inhibition blocks furosemide-stimulated renin secretion in vivo
Authors: Beierwaltes W. H.
Published: December 2017

The macula densa is a regulatory site for renin. It contains exclusively the neuronal isoform of nitric oxide synthase (NOS), suggesting NO could stimulate renin secretion through the macula densa pathway. To test whether neuronal NOS mediates renin secretion, renin was stimulated by either the renal baroreceptor or the diuretic furosemide (acting through the macula densa pathway). Renin secretion rate (RSR) was measured in 12 Inactin-anesthetized rats at normal (104 +/- 3 mmHg) and reduced renal perfusion pressure (65 +/- 1 mmHg), before and after selective blockade of the neuronal NOS with 7-nitroindazole (7-NI, 50 mg/kg ip). 7-NI had no effect on basal blood pressure (102 +/- 2 mmHg) or renal blood flow (RBF). Decreasing renal perfusion pressure doubled RSR from 11.8 +/- 3.3 to 22.9 +/- 5.7 ng ANG I.h-1.min-1 (P < 0.01) (ANG I is angiotensin I). Similarly, in 7-NI-treated rats, reduced perfusion doubled RSR from 8.5 +/- 1.8 to 20.5 +/- 6.2 ng ANG I.h-1.min-1 (P < 0.01). Renal hemodynamics and RSR were measured in response to 5 mg/kg iv furosemide in 12 control rats and 11 rats treated with 7-NI. Blocking neuronal NOS did not alter blood pressure (102 +/- 2 mmHg), RBF (5.8 +/- 0.4 ml.min-1.g kidney wt-1), or renal vascular resistance (18.7 +/- 1.4 mmHg.ml-1.min.g kidney wt).(ABSTRACT TRUNCATED AT 250 WORDS)
Differential interaction of human renal P-glycoprotein with various metabolites and analogues of cyclosporin A
Authors: Charuk J. H. , Wong P. Y. , Reithmeier R. A.
Published: December 2017

Interactions of P-glycoprotein with several analogues and metabolites of cyclosporin A were studied to gain a better understanding of this immunosuppressant's mechanism of excretion and nephrotoxicity. Incorporation of [3H]azidopine into human renal P-glycoprotein in the presence of various concentrations of different cyclosporins was quantitated. Competitive [3H]azidopine photolabeling and 3H drug transport assays of CHRC5 multidrug-resistant cells were also conducted to evaluate effects of cyclosporins on P-glycoprotein function. Cyclosporins A [half-maximal inhibition constant (K0.5) = 20 nM] and G (K0.5 = 40 nM) blocked [3H]azidopine photolabeling of renal P-glycoprotein at very low concentrations, whereas higher concentrations of cyclosporin C (K0.5 = 500 nM) and metabolites 1, 17, and 21 (K0.5 = 200 nM) were required to inhibit photolabeling. Metabolites H and 8 were ineffective in inhibition of [3H]azidopine photolabeling of human renal P-glycoprotein. Similarly, cyclosporins A, C, and G were the best inhibitors of [3H]azidopine photolabeling of P-glycoprotein in multidrug-resistant C5 cells; the various metabolites were less effective. Cyclosporins A, C, and G also enhanced cellular accumulation of [3H]cyclosporin A and several other 3H-labeled compounds known to be transported by P-glycoprotein in multidrug-resistant C5 cells. Differential affinities of cyclosporin A metabolites for P-glycoprotein suggest considerable drug-binding site specificity. Our current hypothesis is that cyclosporin A may be more nephrotoxic than its metabolites by virtue of its superior ability to bind to and competitively inhibit urinary excretion of an endogenous P-glycoprotein substrate. Our findings provide the basis for future design and testing of new cyclosporin derivatives that have immunosuppressive activity yet may be less nephrotoxic because of their poor interaction with renal P-glycoprotein.
Prostaglandin E2 regulation of ion transport is absent in medullary thick ascending limbs from SHR
Authors: Good D. W. , Caflisch C. R. , George T.
Published: December 2017

Regulation of HCO3- and Cl- absorption by arginine vasopressin (AVP) and prostaglandin E2 (PGE2) was examined in isolated, perfused medullary thick ascending limbs (MTAL) from 4- to 7-wk-old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. AVP inhibited HCO3- absorption by 50% at 10(-10) M and by 25% at 2 x 10(-12) M in MTAL from both WKY and SHR. Cholera toxin (10(-9) M) or forskolin (10(-6) M) in the bath also inhibited HCO3- absorption by 50% in the SHR. In MTAL from WKY, PGE2 (10(-6) M in the bath) increased HCO3- absorption from 7.1 +/- 0.4 to 12.0 +/- 0.4 pmol.min-1.mm-1 (P < 0.005) and decreased Cl- absorption from 65 +/- 7 to 47 +/- 6 pmol.min-1.mm-1 (P < 0.001) in the presence of 10(-10) M AVP. Under the same conditions, PGE2 had no effect on HCO3- or Cl- absorption in MTAL from SHR. PGE2 also reversed submaximal inhibition of HCO3- absorption by 2 x 10(-12) M AVP in WKY but not in SHR. With 10(-10) M AVP in the bath, phorbol 12-myristate 13-acetate (10(-6) M in the bath) increased HCO3- absorption from 6.6 +/- 0.5 to 12.3 +/- 0.4 pmol.min-1.mm-1 in MTAL from WKY and from 7.6 +/- 0.7 to 12.6 +/- 1.2 pmol.min-1.mm-1 in MTAL from SHR (P < 0.005). These results demonstrate that 1) the effects of PGE2 to stimulate HCO3- absorption and inhibit Cl- absorption in the presence of AVP are absent in MTAL from SHR, 2) the defect may involve an inability of PGE2 to stimulate protein kinase C, and 3) regulation of HCO3- absorption by AVP via adenosine 3',5'-cyclic monophosphate is similar in MTAL from WKY and SHR. The lack of PGE2 inhibition of NaCl absorption in the MTAL may contribute to renal salt retention during the development of hypertension in the SHR.
Effect of luminal angiotensin II on ammonia production and secretion by mouse proximal tubules
Authors: Nagami G. T.
Published: December 2017

Angiotensin II is an important regulator of acid-base and ammonia metabolism in the proximal tubule. Because angiotensin II receptors exist on the apical membrane and because luminal fluid angiotensin II concentrations may be substantial, the effects of luminal angiotensin II on ammonia production rates and net luminal total ammonia (tNH3) secretion rates were examined in dissected mouse S2 proximal tubule segments. Ammonia production rates reflected the total release of ammonia via the basolateral and luminal aspects of the tubule, whereas net luminal secretion rates reflected the rates at which ammonia left the tubule via the luminal fluid leaving the distal end of the perfused segment. The results demonstrated that 1) luminal angiotensin II affected tNH3 production in a concentration-dependent fashion, 2) luminal angiotensin II at concentrations that stimulated tNH3 production could counteract the effect of inhibitory basolateral concentrations of angiotensin II, 3) the stimulation of tNH3 production and the rise in intracellular calcium concentration induced by 10(-10) M luminal angiotensin II were blocked by the addition of an angiotensin II receptor inhibitor, saralasin, or the calcium channel blocker nifedipine to the luminal perfusion solution, and 4) in contrast to basolateral angiotensin II, which inhibited net luminal tNH3 secretion, luminal angiotensin II stimulated amiloride-sensitive net luminal tNH3 secretion in parallel with stimulation of luminal fluid acidification. Thus luminal angiotensin II at physiological and superphysiological concentrations has important effects on ammonia production and transport in the proximal tubule that in some ways differ from the effects of basolateral angiotensin II.
Chronic L-NAME hypertension in rats and autoregulation of juxtamedullary preglomerular vessels
Authors: Bouriquet N. , Casellas D.
Published: December 2017

The impact of chronic NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (20 mg.kg-1.day-1 po, for 25 days) on pressure responsiveness was assessed in vessels ranging from arcuate arteries (ArcA) to juxtaglomerular afferent arterioles (JAA), using videomicroscopy and blood-perfused juxtamedullary nephron (JMN) preparations. Respective tail-cuff pressures of control and L-NAME rats were 127 +/- 2 (n = 8) and 173 +/- 4 mmHg (n = 5). Corresponding vessels of both groups had similar calibers at 60 mmHg. Increasing blood perfusion pressure to 200 mmHg constricted control ArcA and JAA by 26 +/- 4% (n = 20) and 43 +/- 5% (n = 15), respectively. Instead, a respective 3 +/- 4% (n = 15) and 21 +/- 9% (n = 6) pressure-induced dilation occurred in L-NAME vessels, and 86 +/- 2% of glomeruli expressed alpha-smooth muscle actin. Responses to acetylcholine (1 microM) but not to nitroprusside (1 mM) were impaired by L-NAME. Maximal relaxation induced by Mn2+ (10 mM) revealed equal basal tone and similar passive viscoelastic properties in control and L-NAME vessels. No vascular hypertrophy was found in L-NAME vessels. Chronic L-NAME hypertension is therefore associated with a selective loss of vascular autoregulation in JMNs, which may contribute to glomerular injury.
TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells
Authors: Lau K. S. , Nakashima O. , Aalund G. R. , Hogarth L. , Ujiie K. , Yuen J. , Star R. A.
Published: December 2017

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.
Glyburide attenuates calmodulin antagonist-stimulated renin release from isolated mouse juxtaglomerular cells
Authors: Linseman D. A. , Lawson J. A. , Jones D. A. , Ludens J. H.
Published: December 2017

Previous reports have shown that K+ channel openers elevate plasma renin activity in vivo and stimulate renin release (RR) from juxtaglomerular (JG) cells in vitro. Therefore, we examined whether the K+ channel blocker, glyburide, inhibits basal RR or RR stimulated by elevating cAMP or by inhibiting Ca2+/calmodulin activity in cultures of isolated mouse JG cells. Glyburide treatment (10-300 microM) had no effect on basal RR, which measured approximately 10% or 30% of the total cellular renin activity after 4 or 24 h, respectively. RR stimulated by elevating cAMP with isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine was also unaffected by glyburide. In contrast, glyburide significantly attenuated RR stimulated by the calmodulin antagonists, calmidazolium, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Calmidazolium-stimulated RR returned to basal levels with 100 microM glyburide cotreatment. Blockade of voltage-gated calcium channels with verapamil or inhibition of calcium release from intracellular stores with 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) had no effect on the ability of glyburide to attenuate calmidazolium-stimulated RR. However, lowering of the extracellular calcium concentration by the addition of EGTA abolished the inhibitory effects of glyburide. We conclude that modulation of K+ channels may influence RR by affecting Ca2+/calmodulin-regulated secretion, but not cAMP-mediated secretion, from JG cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Nephrotoxicant inhibition of renal proximal tubule cell regeneration
Authors: Counts R. S. , Nowak G. , Wyatt R. D. , Schnellmann R. G.
Published: December 2017

Although nephrotoxicants have been shown to have direct lethal effects on renal proximal tubule cells (RPTC), little is known concerning their effects on the renal regenerative process. Additionally, the mechanisms of RPT regeneration are still not clear. To examine these issues, an in vitro model of mechanically induced injury to primary cultures of rabbit RPTC was developed, and the effects of epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-beta 1), and nephrotoxicants on the regenerative process were examined. Experiments demonstrated that confluent monolayers swiped with a 4-mm Teflon policeman regenerated with 77% closure of the swipe in 7 days. DNA content in swiped monolayers increased, reached a maximum on day 3 (1.4-fold), and remained constant through day 7. EGF accelerated regeneration and resulted in 96% swipe closure on day 7 and increased DNA content 2.3-fold. TGF-beta 1 inhibited regeneration and resulted in 22% swipe closure on day 7 but did not inhibit the increase in DNA content. 5-Fluorouracil inhibited regeneration and resulted in 27% swipe closure on day 3, compared with 46% in the controls, and inhibited the increase in DNA content. Mercuric chloride, fumonisin B1, and dichlorovinyl-L-cysteine, at concentrations < or = 50% of their lethal concentration, inhibited regeneration and resulted in swiped areas 3.7, 4.2, and 2.1 times larger, respectively, than controls on day 7. At concentrations < or = 50% of its lethal concentration, tert-butylhydroperoxide had no effect on swipe closure.(ABSTRACT TRUNCATED AT 250 WORDS)
NaCl injections in brain induce natriuresis and blood pressure responses sensitive to ANG II AT1 receptors
Authors: Rohmeiss P. , Beyer C. , Nagy E. , Tschope C. , Hohle S. , Strauch M. , Unger T.
Published: December 2017

In the present study we tested the hypothesis that the natriuretic and pressor effects of intracerebroventricularly (icv) injected hypertonic saline involve a central angiotensinergic pathway. All experiments were performed in conscious Wistar rats. Bolus injections of hypertonic saline (0.19, 0.23, 0.30, and 0.60 M icv; injection volume 5 microliters) induced a concentration-dependent increase of renal sodium excretion without affecting urinary flow. The increase in renal sodium excretion after the two highest saline concentrations was accompanied by significant increases in mean arterial blood pressure (MAP). Pretreatment with the angiotensin (ANG) AT1 receptor antagonist, losartan (5 micrograms icv), reduced the natriuretic effect of 0.23 and 0.30 M saline but did not affect the natriuresis induced by 0.60 M saline. The increase in MAP after 0.30 and 0.60 M saline icv was markedly attenuated by intracerebroventricular pretreatment with losartan. Our results demonstrate the involvement of a central angiotensinergic mechanism in the natriuretic and pressor responses to hypertonic saline. In addition to the ANG II-mediated natriuresis, an additional natriuretic mechanism, independent of ANG II and associated with the saline-induced pressor effect, seems to be recruited with increasing concentrations of saline in the cerebrospinal fluid.
In situ hybridization of H-K-ATPase beta-subunit mRNA in rat and rabbit kidney
Authors: Campbell-Thompson M. L. , Verlander J. W. , Curran K. A. , Campbell W. G. , Cain B. D. , Wingo C. S. , McGuigan J. E.
Published: December 2017

Through a variety of techniques, several investigators have demonstrated the presence of an H-K-adenosinetriphosphatase (H-K-ATPase) enzyme in the renal collecting duct, suggesting that this enzyme serves an important physiological role in the regulation of acid-base balance and potassium excretion by the kidney. The present study was designed to localize cells expressing H-K-ATPase beta-subunit mRNA in rat and rabbit kidney by nonradioactive in situ hybridization. A 570-bp DNA fragment of rabbit renal H-K-ATPase beta-subunit was used to produce digoxigenin-labeled riboprobes by in vitro transcription. Northern blot hybridization demonstrated transcripts in rat gastric oxyntic mucosa and kidney. In situ hybridization on kidney tissue sections demonstrated H-K-ATPase beta-subunit mRNA localization in epithelial cells, including intercalated cells in the connecting segment and cortical and medullary collecting duct, principal cells in the inner stripe of the outer medullary collecting duct, and inner medullary collecting duct cells in both the rat and the rabbit. These observations provide evidence that H-K-ATPase beta-subunit mRNA is present throughout the collecting duct of the kidney. The distribution of this message is consistent with a role for H-K-ATPase in bicarbonate absorption in both the outer and inner medullary collecting duct.
Renal expression of osteopontin and alkaline phosphatase correlates with BUN levels in aged rats
Authors: Liang C. T. , Barnes J.
Published: December 2017

Renal expression of alkaline phosphatase (AP) and osteopontin (OP) in rats of different age was examined. Northern blot hybridization showed that AP mRNA was reduced moderately, whereas OP mRNA was stimulated drastically in old rats. Dot-blot quantitation analysis showed that AP mRNA decreased 30% in 24-compared with 6-mo-old rats. In contrast, OP mRNA increased 3.1- and 9.1-fold, respectively, in 12- and 24-mo-old rats. beta-Actin mRNA did not change with age. Blood urea nitrogen (BUN) increased 47 and 187% in 12- and 24-mo-old rats, respectively. Correlation analysis showed that BUN correlated negatively with AP mRNA and positively with OP mRNA. No correlation was observed with beta-actin. The expression of these markers was also examined in femurs. AP and OP mRNAs were marginally reduced in old bones. To test whether the correlation also exists in other types of renal insufficiency, we examined these parameters in young rats infused with parathyroid hormone (PTH). BUN was elevated 3.5-fold, whereas AP mRNA decreased 48%, and OP mRNA increased 15.3-fold in kidneys of PTH-treated rats. To elucidate the possible mechanisms that lead to the overexpression of OP in kidney, we examined the expression of transforming growth factor-beta 1 (TGF-beta 1) mRNA. No significant differences in TGF-beta 1 expression were observed between young and old rats and control and PTH-treated young rats. Changes in the expression of OP were also visualized by immunostaining of renal sections. Alterations in the levels of OP and AP were validated by Western blot analysis and enzyme assay of homogenate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Restoration of urine concentrating ability and accumulation of medullary osmolytes after chronic diuresis
Authors: Sone M. , Ohno A. , Albrecht G. J. , Thurau K. , Beck F. X.
Published: December 2017

Restoration of urine osmolality (Uosm) and medullary osmolyte contents after chronic diuresis was studied in rats infused for 6 days with furosemide and subsequently given the vasopressin analogue, 1-desamino-8-D-arginine vasopressin (DDAVP). Papillary tip intra- and extracellular electrolyte concentrations were measured by electron microprobe analysis, tissue contents of methylamines (glycerophosphorylcholine, betaine), polyols (myo-inositol, sorbitol), and several amino acids in different kidney zones by high-performance liquid chromatography. Administering DDAVP continuously after diuresis increased Uosm from (means +/- SE) 348 +/- 8 to 1,265 +/- 127 after 1 day and 2,485 +/- 186 mosmol/kgH2O after 3 days. The sum of all osmolytes at the papillary tip rose from 309.2 +/- 28.9 to 690.9 +/- 105.8 and 1,282.8 +/- 21.0 mmol/kg protein after days 1 and 3, respectively. Although interstitial tonicity (sum of Na, Cl, and K concentrations) was increased by 116 and 223% after 1 and 3 days DDAVP, intracellular tonicity was similar in chronic diuresis and following 1 or 3 days DDAVP. Coadministration of DDAVP with betaine, myo-inositol, and choline (“osmolyte treatment”) did not accelerate the restoration of Uosm but caused significantly higher contents of osmolytes (except myo-inositol) in inner medulla and/or papilla after 3 days. In a minority of animals, restoration of Uosm and reaccumulation of medullary osmolytes were impeded in both DDAVP- and DDAVP/osmolyte-treated rats. These data indicate that, after chronic diuresis, accumulation of organic osmolytes and restoration of Uosm proceed in parallel. Capacity for transport and/or synthesis of organic osmolytes, rather than their availability, appear to limit reaccumulation on the first day of recovery. By the third day, delivery of some osmolytes or their precursors may limit the restoration of medullary osmolyte content. The failure of some rats to attain sufficient concentrating ability within this time period may be related to deficient reaccumulation of medullary osmolytes.
Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril
Authors: Burdmann E. A. , Andoh T. F. , Nast C. C. , Evan A. , Connors B. A. , Coffman T. M. , Lindsley J. , Bennett W. M.
Published: December 2017

The pathogenesis of renal scarring in chronic cyclosporin nephropathy is unknown. In this study, we evaluated the effects of renin-angiotensin system blockade by enalapril and losartan in a salt-dependent model of cyclosporin-associated chronic tubulointerstitial fibrosis (TIF). Rats kept on normal or low-salt diet were given cyclosporin, cyclosporin+enalapril, cyclosporin+losartan, cyclosporin+enalapril#losartan, or vehicle for 14 and 28 days. Cyclosporin reduced glomerular filtration rate (GFR) in rats fed either diet, but only salt-depleted animals developed significant TIF. Cyclosporin also impaired renal concentrating ability and caused tubular enzymuria. Renin-angiotensin system blockade decreased blood pressure (BP) and promoted afferent arteriolar vasodilatation. Losartan reduced plasma renin activity and prevented cyclosporin-induced increment of cortical alpha 1(I) procollagen mRNA. Renin-angiotensin blockade did not improve GFR and tubular function; however, it strikingly prevented TIF development, even in presence of very low BP. Rats treated with cyclosporin, hydralazine, and furosemide achieved BP values similar to losartan or enalapril groups, but there was no protection against interstitial fibrosis development. These results suggest that cyclosporin-related chronic interstitial injury is mediated by angiotensin II and that the mechanisms promoting the interstitial scarring can be dissociated from glomerular and tubular dysfunction in cyclosporin nephropathy.
Regulation of H(+)-K(+)-ATPase expression in kidney
Authors: DuBose T. D. , Codina J. , Burges A. , Pressley T. A.
Published: December 2017

It is now widely accepted that proton secretion by the collecting duct is mediated, in part, by an H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). Controversy persists regarding which H(+)-K(+)-ATPase isoform is expressed in kidney. Several laboratories have reported preliminarily the amplification from kidney of stomach and/or colon-identical products using gastric or colonic-specific primers in the polymerase chain reaction (PCR). We have developed highly specific probes for the catalytic subunit using reverse transcriptase-PCR with gastric- or colonic-specific primers. The resulting cDNAs were verified by sequencing and were then used in Northern analysis of whole kidney total RNA obtained from one of the following three groups of rats: 1) controls, 2) chronic hypokalemia, or 3) chronic metabolic acidosis. Probes for both the colonic and gastric alpha-subunit H(+)-K(+)-ATPase isoforms hybridized to whole kidney total RNA derived from potassium-replete control rats. A marked elevation of colonic mRNA abundance, but not gastric message, was observed in response to chronic hypokalemia induced by dietary potassium deprivation. Elevation of either gastric or colonic mRNA was not observed with chronic metabolic acidosis. Under the conditions of the present study, it appears that the mRNA encoding the colonic alpha-isoform of the H(+)-K(+)-ATPase in kidney is upregulated by chronic hypokalemia but not by chronic metabolic acidosis. The observation that the gastric H(+)-K(+)-ATPase alpha-isoform does not appear to be regulated in either condition suggests that this isoform is expressed constitutively in kidney.
Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland
Authors: Lehrich R. W. , Forrest J. N.
Published: December 2017

We used the specific tyrosine kinase inhibitor genistein to define the involvement of tyrosine phosphorylation in the regulation of chloride transport in the rectal gland of the dogfish shark, a model for chloride secretion via a cystic fibrosis transmembrane conductance regulator (CFTR)-like channel. In the perfused gland, genistein (100 microM) promptly increased chloride secretion from basal values of 159 +/- 36 to 966 +/- 49 mueq.h-1.g-1 (P < 0.0001). Bumentanide fully reversed genistein-induced secretion. In primary culture monolayers of rectal gland tubular cells, genistein, but not the inactive 7-glucoside form, genistin, increased short-circuit current in a dose-dependent manner, from basal values of 2.7 +/- 4.3 to 104 +/- 10 microA/cm2 (P < 0.0001). Apically applied genistein induced significantly greater chloride secretion than basolateral addition. Genistein did not increase the adenosine 3',5'-cyclic monophosphate (cAMP) content of either perfused glands or cultured monolayers. Using an anti-phosphotyrosine antibody, we observed phosphorylation of multiple proteins. Four peptides, with molecular masses of 250, 210, 55, and 53 kDa, responded to genistein treatment with a decrease in tyrosine phosphorylation. These data demonstrate the following: 1) genistein induces bumetanide-sensitive chloride secretion in both perfused rectal glands and cultured tubular cells; 2) these effects are not accompanied by an elevation of tissue cAMP, indicating that genistein-induced secretion is not mediated by the cAMP-protein kinase A pathway; and 3) genistein-sensitive peptides are present in the rectal gland cell and are candidates for involvement in the regulation of chloride secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Actin filaments stimulate the Na(+)-K(+)-ATPase
Authors: Cantiello H. F.
Published: December 2017

In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.
Angiotensin IV receptors and signaling in opossum kidney cells
Authors: Dulin N. , Madhun Z. T. , Chang C. H. , Berti-Mattera L. , Dickens D. , Douglas J. G.
Published: December 2017

The pharmacological properties and signaling of angiotensin IV (ANG IV) receptors were studied in opossum kidney cell line OK7A. Saturation binding experiments with 125I-labeled ANG IV demonstrated the presence of high-affinity ANG IV binding sites in OK7A cell membranes with a dissociation constant (Kd) of 0.40 +/- 0.08 nM and a maximal amount of binding sites (Bmax) of 180 +/- 50 fmol/mg protein. In competition experiments, unlabeled ANG IV inhibited 125I-ANG IV binding biphasically: 20% of binding sites had high affinity [inhibition constant (Ki) = 0.44 +/- 0.04 nM] and 80% had low affinity (Ki = 130 +/- 10 nM). ANG III displaced 125I-ANG IV from binding sites with low affinity (Ki = 205 +/- 10 nM), and ANG II did not compete with 125I-ANG IV at concentrations up to 10 microM. The binding of ANG IV to OK7A cell membranes was significantly enhanced in the presence of 5 mM EDTA and completely blocked by 5 mM dithiothreitol. Guanosine 5'-O-(3-thiotriphosphate) inhibited the binding of 125I-ANG IV, indicating the G protein coupling of ANG IV receptors in OK7A cells. In signaling studies, ANG IV induced transient increase in intracellular calcium concentration ([Ca2+]i) from 49 +/- 3 to 280 +/- 45 nM. ANG IV failed to influence phosphoinositol metabolism, indicating that Ca2+ mobilization is not linked to ANG IV signaling. Ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid completely abolished ANG IV-induced increase in [Ca2+]i, consistent with Ca2+ influx. The voltage-sensitive Ca2+ channel blocking agents verapamil and nifedipine attenuated the effect of ANG IV on [Ca2+]i to 133 +/- 33 and 174 +/- 32 nM, respectively. These data suggest that ANG IV induces Ca2+ influx in OK7A cells, at least partially, through the voltage-sensitive Ca2+ channels.
FGF-1 in normal and regenerating kidney: expression in mononuclear, interstitial, and regenerating epithelial cells
Authors: Ichimura T. , Maier J. A. , Maciag T. , Zhang G. , Stevens J. L.
Published: December 2017

The proximal tubule epithelium regenerates following nephrotoxic damage. To determine the role of fibroblast growth factors (FGFs) in the regeneration of rat proximal tubule epithelial (RPTE) cells, we investigated proliferation, differentiation, and FGF-1 expression in vivo in rat kidney before and after nephrotoxic damage to the proximal tubule epithelium caused by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine administration. In undamaged kidneys, FGF-1 was expressed in distal tubule elements, including cortical and medullary collecting ducts, as well as in blood vessels and glomeruli, but was absent in RPTE. One day after damage, there was an increase in proliferation of surviving proximal tubule epithelial cells and a coincident increase in FGF-1 expression in invading mononuclear cells. After this initial burst of proliferation, FGF-1 expression increased in poorly differentiated vimentin-positive regenerative epithelial cells, indicating that autocrine FGF-1 expression in the regenerative epithelium is a later event in the regeneration process. FGF-1 staining persisted in foci of macrophages, interstitial cells, and nephropathic tubules within areas of interstitial expansion 2 wk after damage. We concluded that transient paracrine and autocrine expression of FGF-1 could play mitogenic and/or morphogenic roles during tubular regeneration. Persistent expression in macrophages, fibroblasts, and nephropathic tubules may be associated with tubular degeneration. FGF-1 expression may be an important contributor to both tubular regeneration and degenerative disease following toxicant exposure.
Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells
Authors: Cybulsky A. V. , Monge J. C. , Papillon J. , McTavish A. J.
Published: December 2017

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.
Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice
Authors: Breton S. , Alper S. L. , Gluck S. L. , Sly W. S. , Barker J. E. , Brown D.
Published: December 2017

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.