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PubMed1999

CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line.

Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle's loop (MTAL) cells and other cell types. The 5'-flanking region of the NOS2 gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at -150 to -142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-gamma (IFN-gamma) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-gamma compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-gamma activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPbeta stimulated basal NOS2 promoter activity. Thus C/EBPbeta appears to be an important trans-activator of the NOS2 gene in the MTAL.

Animals↗
PubMed1999

Thromboxane A2 contributes to the enhanced tubuloglomerular feedback activity in young SHR.

We performed micropuncture studies to determine the role of thromboxane A2 in the exaggerated tubuloglomerular feedback (TGF) activity in young spontaneously hypertensive rats (SHR). Glomerular function was assessed by changes in proximal tubular stop-flow pressure (SFP) produced by different rates of orthograde perfusion through Henle's loop. Seven-week-old SHR exhibited an exaggerated TGF activity compared with Wistar-Kyoto rats (WKY) during euvolemia, confirming earlier studies. During control periods, the feedback-induced maximal SFP response (DeltaSFP) was greater in SHR (18-19 vs. 12-13 mmHg in WKY), whereas basal SFP and proximal tubular free-flow pressure were similar in both strains. In one series, the thromboxane A2 agonist U-46619 was added to the tubular perfusate for a final concentration of 10(-6) M. In WKY, DeltaSFP was increased by 100% to 26 mmHg. In contrast, DeltaSFP in young SHR was unaffected by the thromboxane A2 agonist. In other animals, the thromboxane synthase inhibitor pirmagrel (50 mg/kg) was injected intravenously to inhibit thromboxane production. In SHR, pirmagrel decreased DeltaSFP by 8.5 mmHg and reduced reactivity. Less attenuation was observed in WKY; DeltaSFP was reduced by 3 mmHg, whereas reactivity was unchanged. In other studies, tubular perfusion with the thromboxane receptor inhibitor SQ-29548 (10(-6) M) reduced DeltaSFP more in SHR (7 vs. 3 mmHg in WKY) and also decreased reactivity more in SHR (2.3 vs. 0.5 mmHg. nl-1. min-1). Coperfusion of SQ-29548 and U-46619 resulted in an 85% block of the effect of U-46619 on DeltaSFP. Tubular perfusion with the agonist U-46619 during thromboxane synthase inhibition markedly enhanced DeltaSFP in both strains, with a greater effect in WKY. These results suggest that elevated levels of thromboxane A2 in young SHR contribute to the exaggerated TGF control of glomerular function in SHR during the developmental phase of hypertension.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2001

CaR-mediated COX-2 expression in primary cultured mTAL cells.

Primary cultures of medullary thick ascending limb (mTAL) cells retain the capacity to express calcium-sensing receptor (CaR) mRNA and protein. Increases in cyclooxygenase-2 (COX-2) mRNA accumulation, protein expression, and PGE(2) synthesis were observed in a dose- and time-dependent manner after exposure of these cells to extracellular calcium (Ca(o)(2+)). Moreover, transfection of mTAL cells with a CaR overexpression vector significantly enhanced COX-2 expression and PGE(2) production in response to calcium compared with cells transfected with an empty vector. Challenge with the CaR-selective agonist poly-L-arginine (PLA) also increased COX-2 mRNA accumulation, protein expression, and PGE(2) synthesis. Furthermore, Ca(o)(2+)- and PLA-mediated PGE(2) production was abolished in the presence of NS-398 or nimesulide, two different COX-2-selective inhibitors. These data suggest that intracellular signaling mechanisms initiated via activation of CaR contribute to COX-2-dependent PGE(2) synthesis in the mTAL. Because Ca(o)(2+) concentration varies along Henle's loop, calcium may contribute to salt and water balance via a COX-2- and CaR-dependent mechanism. Thus novel calcimimetics might be useful in conditions such as hypertension in which manipulation of extracellular fluid volume provides beneficial effects.

Animals↗
PubMed1984

Studies on the nephron segment with reduced sodium reabsorption during starvation natriuresis.

The segment of the nephron where carbohydrate deprivation depresses Na transport leading to natriuresis was sought by a new clearance technique designed to measure segmental reabsorption in each portion of the human renal tubule. Experiments were performed during maximal water diuresis before and 4 days after carbohydrate withdrawal. Proximal reabsorption had fallen from 70 +/- 4 to 60 +/- 5 ml X min-1, p less than 0.05, by the 4th day of sugar deprivation, accounting for the natriuresis and the associated weight loss of 1.8 kg. By the 4th day of fasting, when Na excretion had returned to control levels, GFR had fallen nonsignificantly from 99 +/- 6 to 95 +/- 5 ml X min-1, while Na reabsorption along distal segments had risen. In fact, Na transport, expressed by the equivalent volumes of solute free-water generated, rose from 17.4 +/- 3.4 to 23.6 +/- 2.1 along the ascending limb of Henle's loop, and from 8.1 +/- 0.8 to 9.2 +/- 1.3 ml X min-1 X GFR-1 X 100 along the distal tubule. Thus, analysis of segmental Na transport by this method discloses that starvation natriuresis is a proximal tubular event, progressively counterbalanced by enhanced Na reclamation in more distal sites. Volume contraction and the attendant fall in GFR concur to curb delivery out of the proximal tubule which is matched by enhanced distal Na reabsorption till a new steady-state excretion is attained.

Absorption↗
PubMed1982

The renal tubular defect of Bartter's syndrome.

The site of reduced electrolyte transport in Bartter's syndrome (BS) was studied with a new technique whereby resorption can be separately measured as equivalent volumes of free water generated along the ascending limb of Henle's loop (CH2O-HL) and cortical distal tubule (CH2O-DT): the fractional proximal resorption (FPR) and the volume of free water dissipated along collecting ducts (CD) by back diffusion (CH2O-BD) in the absence of ADH are also measured during maximal water diuresis. Data are expressed as ml . min-1 GFR-1 . 100. The studies were performed on 2 brothers with all clinical and laboratory features of BS. They achieved external Na balance within 3 days when placed on either 10, 100, or 230 mEq Na daily. With the 100-mEq-Na diet indomethacin caused a stable 1.5 kg weight gain. FPR was 0.69 in normal controls (NC), 0.77 in BS; CH2O-HL 16.7 vs. 16.4; CH2O-DT 9.7 vs. 3.9; CH2O-BD 13.8 vs. 13.8; CH2O (free water excretion) 12.5 vs. 6.1; urine flow rate (V) 17.6 vs. 9.9. Thus, BS is characterized by a slight fall in proximal delivery, normal HLNa transport, a striking impairment of DTNa transport and preserved interstitial hypertonicity which drives a normal osmotic flow of CH2O-BD. Aspirin injected intravenously during water load affected CH2O and V in proportion to the change in GFR, which fell from 145 +/- 19 to 114 +/- 12 ml . min-1, p less than 0.01). Thus, the primary abnormality of BS is impaired Na transport along the early portion of the distal tubule. This is compensated by volume contraction attended by reduced proximal delivery and full activation of renin-angiotensin-aldosterone system. Consequently, more distal cation exchange sites reclaim Na+ at the expenses of excessive K+ and H+ losses, trapping NH4Cl within tubular lumen and generating hypokalemic metabolic alkalosis. The excess angiotensin is counterbalanced by increased prostaglandin (PG) secretion, which brings renal vascular resistances toward normal and causes tachyphylaxis to angiotensin. Inhibition of PG synthesis leads to a fall in GFR and proximal delivery: this causes distal delivery to fall below reabsorptive capacity for Na: therefore both Na and K retention ensues causing partial volume reexpansion till a new balance is established. PGs do not affect either Na or Cl resorption in BS except by a purely hemodynamic action.

Adult↗
PubMed2004

Urinary potassium excretion and sodium sensitivity in blacks.

Based on racial differences in urinary potassium excretion and responses to diuretics, we present a model suggesting that a major cause of sodium sensitivity in blacks is an augmented activity of the Na-K-2Cl cotransport in the thick ascending limb of Henle's loop. This would result in an increased ability to conserve not only sodium but also water, and an upward and rightward shift in the operating point of tubuloglomerular feedback, which may cause an increase in the glomerular capillary hydraulic pressure and predilection to glomerular injury with and without hypertension. In this sense, the biological implication of sodium sensitivity in blacks and in humans in general has ramifications above and beyond salt-evoked increase in blood pressure.

Animals↗
PubMed1983

Insulin binding sites in various segments of the rabbit nephron.

Insulin binds specifically to basolateral renal cortical membranes and modifies tubular electrolyte transport, but the target sites of this hormone in the nephron have not been identified. Using a microassay that permits measurement of hormone binding in discrete tubule segments we have determined the binding sites of 125I-insulin along the rabbit nephron. Assays were performed under conditions that minimize insulin degradation, and specific binding was measured as the difference between 125I-insulin bound in the presence or absence of excess (10(-5) M) unlabeled hormone. Insulin monoiodinated in position A14 was used in all assays. Specific insulin binding (attomol . cm-1 +/- SE) was highest in the distal convoluted tubule (180.5 +/- 15.0) and medullary thick ascending limb of Henle's loop (132.9 +/- 14.6), followed by the proximal convoluted and straight tubule. When expressed per milligram protein, insulin binding capacity was highest along the entire thick ascending limb (medullary and cortical portions) and the distal convoluted tubule, i.e., the "diluting segment" (congruent to 10(-13) mol . mg protein-1), and was lower (congruent to 4 X 10(-14) mol . mg protein-1), and remarkably similar, in all other nephron segments. Binding specificity was verified in competition studies with unlabeled insulin, insulin analogues (proinsulin and desoctapeptide insulin), and unrelated hormones (glucagon, 1-34 parathyroid hormone, prolactin, follicle-stimulating hormone). In addition, serum containing antiinsulin receptor antibody from two patients with type B insulin resistance syndrome markedly inhibited insulin binding to isolated tubules. Whether calculated per unit tubule length or protein content, insulin binding is highest in the thick ascending limb and the distal convoluted tubule, the same nephron sites where a regulatory role in sodium transport has been postulated for this hormone.

Animals↗
PubMed1983

Chlorpropamide action on renal concentrating mechanism in rats with hypothalamic diabetes insipidus.

To determine vasopressin (VP)-potentiating effect of chlorpropamide (CPMD), we studied the effect of CPMD in vivo and in vitro in kidneys and in specific tubule segments of rats with hypothalamic diabetes insipidus, homozygotes of the Brattleboro strain (DI rats). Rats on ad lib. water intake were treated with CPMD (20 mg/100 g body wt s.c. daily) for 7 d. While on ad lib. water intake, the urine flow, urine osmolality, urinary excretion of Na +, K +, creatinine, or total solute excretion did not change. However, corticopapillary gradient of solutes was significantly increased in CPMD-treated rats. Higher tissue osmolality was due to significantly increased concentration of Na +, and to a lesser degree urea, in the medulla and papilla of CPMD-treated rats. Consequently, the osmotic gradient between urine and papillary tissue of CPMD-treated rats (delta = 385 +/- 47 mosM) was significantly (P less than 0.001) higher compared with controls (delta = 150 +/- 26 mosM). Minimum urine osmolality after water loading was higher in CPMD-treated DI rats than in controls. Oxidation of [14C]lactate to 14CO2 coupled to NaCl cotransport was measured in thick medullary ascending limb of Henle's loop (MAL) microdissected from control and CPMD-treated rats. The rate of 14CO2 production was higher (delta + 113% +/- 20; P less than 0.01) in CPMD-treated MAL compared with controls, but 14CO2 production in the presence of 10(-3) M furosemide did not differ between MAL from control and from CPMD-treated rats. These observations suggest that CPMD treatment enhances NaCl transport in MAL. Cyclic AMP metabolism was analyzed in microdissected MAL and in medullary collecting tubule (MCT). MCT from control and from CPMD-treated rats did not differ in the basal or VP-stimulated accumulated of cAMP. The increase in cAMP content elicited by 10(-6) M VP in MAL from CPMD-treated rats (delta + 12.0 +/- 1.8 fmol cAMP/mm) was significantly (P less than 0.02) higher compared with MAL from control rats (delta + 5.1 +/- 1.0 fmol cAMP/mm). Preincubation of MAL dissected from Sprague-Dawley rats with 10(-4) M CPMD in vitro increased cAMP accumulation in the presence of VP, but no such enhancement was found in preincubated MCT. Adenylate cyclase activity, basal or stimulated by VP, 5'guanylimidodiphosphate, or by NaF, assayed in isotonic medium did not differ between MAL or MCT from control rats and MAL or MCT from CPMD-treated rats. When assayed in hypertonic medium (800 mosM), the adenylate cyclase activity in the presence of 10(-6) M VP was significantly higher in MAL of CPMD-treated rats. MAL and MCT from control and CPMD-treated rats did not differ in the activities of cAMP phosphodiesterase. The rate of [(14)C]prostaglandin E2 by medullary and papillary microsomes was not different between the control and CPMD-treated rats; likewise, there was no difference in accumulation of immunoreactive prostaglandin E2 in the medium of in vitro incubated medullary or papillary slices prepared from control and CPMD-treated rats. Based on the findings recounted above, we propose a hypothesis that CPMD administration enhances the antidiuretic effect of VP, primarily by increasing medullary and papillary tonicity dye to increased NaCl reabsorption in MAL. There is no evidence that CPMD sensitizes collecting tubules to the action of VP, at least at the camp-generation step. Therefore, increased antidiuretic response to VP in the kidneys of CPMD-treated DI rats is due to enhanced osmotic driving force for water reabsorption (lumen-to-interstitium osmotic gradient) in collecting tubules, rather than due to increased VP-dependent water permeability of tubular epithelium.

Animals↗
PubMed1990

Effects of endothelin on peptide-dependent cyclic adenosine monophosphate accumulation along the nephron segments of the rat.

We investigated the tubular action of endothelin in rat nephron segments. The effects of endothelin on arginine vasopressin (AVP)-, parathyroid hormone-, glucagon-, calcitonin-, and isoproterenol-dependent cAMP accumulation were studied. The following nephron segments were microdissected: glomerulus (Gl), proximal convoluted tubule (PCT), cortical and medullary thick ascending limbs of Henle's loop (cTAL and mTAL, respectively), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). Endothelin dose dependently (10(-8)-10(-10)M) inhibited AVP-dependent cAMP accumulation in CCD, OMCD, and IMCD. This effect was independent of the presence or absence of phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, Ca channel blocker nicardipine, or indomethacin, but was abolished in the presence of protein kinase C inhibitor H-7. Protein kinase C stimulator dioctanoyl glycerol mimicked the effect of endothelin. On the other hand, endothelin had no inhibitory effect on AVP-dependent cAMP accumulation in cTAL or mTAL, parathyroid hormone-dependent cAMP accumulation in Gl and PCT, or glucagon-, calcitonin-, and isoprotereol-dependent cAMP accumulation in OMCD. We conclude that endothelin specifically inhibits AVP-dependent cAMP accumulation in CCD, OMCD, and IMCD through activating protein kinase C. This effect possibly has a role in maintaining urine volume to counteract the decrease in GFR caused by endothelin itself.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Cyclic guanosine monophosphate is the mediator of platelet-activating factor inhibition on transport by the mouse kidney thick ascending limb.

Since we have previously shown a direct inhibitory effect of platelet-activating factor (PAF) on Cl reabsorption in the medullary thick ascending limb of Henle's loop (TAL), the aim of this study was to extend this effect to the whole TAL and to further investigate the signaling pathway involved. In microperfused cortical TALs, PAF significantly decreased Cl reabsorption by 50.3 +/- 6.5%. On the one hand, this effect was not modified in the presence of staurosporine and was not mimicked by phorbol ester; chelating cytosolic Ca by BAPTA/AM failed to suppress the inhibitory effect of PAF on Cl reabsorption; moreover, no significant increase in intracellular Ca concentration could be observed in the presence of PAF on isolated tubules. On the other hand, 8-bromo cyclic GMP mimicked the PAF effect on Cl reabsorption and prevented a further effect of this agent; the PAF effect was significantly reduced by H-8, a cyclic GMP-dependent protein kinase inhibitor; in medullary TALs, PAF significantly increased by twofold cyclic GMP content, an effect inhibited by the PAF antagonist BN 50730, whereas PAF did not significantly modify cAMP content in basal or stimulated conditions. Finally, inhibition of nitric oxide production by NAME or NMMA failed to prevent the effect of PAF on Cl reabsorption. It is concluded that the PAF-induced inhibition of Cl reabsorption in the TAL was mediated by cyclic GMP, likely independent of a nitric oxide synthesis.

Amino Acid Oxidoreductases↗
PubMed1996

Expression of the Na-K-2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron.

Sodium and chloride transport by the macula densa and thick ascending limb of Henle's loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.

Animals↗
PubMed2006

Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of Claudin-16.

Claudin-16 (Cldn16) is selectively expressed at tight junctions (TJs) of renal epithelial cells of the thick ascending limb of Henle's loop, where it plays a central role in the reabsorption of divalent cations. Over 20 different mutations in the CLDN16 gene have been identified in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a disease of excessive renal Mg2+ and Ca2+ excretion. Here we show that disease-causing mutations can lead to the intracellular retention of Cldn16 or affect its capacity to facilitate paracellular Mg2+ transport. Nine of the 21 Cldn16 mutants we characterized were retained in the endoplasmic reticulum, where they underwent proteasomal degradation. Three mutants accumulated in the Golgi complex. Two mutants were efficiently delivered to lysosomes, one via clathrin-mediated endocytosis following transport to the cell surface and the other without appearing on the plasma membrane. The remaining 7 mutants localized to TJs, and 4 were found to be defective in paracellular Mg2+ transport. We demonstrate that pharmacological chaperones rescued surface expression of several retained Cldn16 mutants. We conclude that FHHNC can result from mutations in Cldn16 that affect intracellular trafficking or paracellular Mg2+ permeability. Knowledge of the molecular defects associated with disease-causing Cldn16 mutations may open new venues for therapeutic intervention.

Amino Acid Sequence↗
PubMed2005

Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice.

BACKGROUND: Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. RESULTS: The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. CONCLUSION: The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals.

Animals↗
PubMed1986

Parathyroid hormone binding in vivo to renal, hepatic, and skeletal tissues of the rat using a radioautographic approach.

We have examined in vivo binding of bovine (b) PTH-(1-84) and the analog [Nle8,18, Tyr34]bPTH-(1-34) amide to hepatic, skeletal, and renal rat tissues. Bioactive 125I-labeled bPTH-(1-84) or the 125I-labeled bPTH-(1-34) analog was injected alone into experimental animals or with either unlabeled PTH or unlabeled unrelated hormone into control animals. Corresponding tissues from experimental and control animals were then processed for light and electron microscope radioautography and analyzed quantitatively and qualitatively. Binding of both PTH forms occurred on hepatocytes and sinusoidal cells in liver, and hepatocyte binding was clearly specific and competitive. Intact PTH-(1-84), but not the amino-terminal fragment, bound to Kupffer cells, indicating a sequence-specific interaction. In bone, specific competitive PTH binding was seen over osteoblasts, but not osteoclasts. Skeletal PTH binding was also seen over connective tissue mononuclear cells, and sinusoidal endothelial cells. In kidney, specific competitive PTH binding was seen over glomerular podocytes, and over the antiluminal surface of cells of the proximal tubule, the thick ascending limb of Henle's loop, and the distal tubule. Noncompetitive binding was seen on the luminal surface of proximal convoluted tubule cells. We have, therefore, distinguished specific competitive binding sites, probably related to hormone action, from noncompetitive binding sites, presumably associated with hormone metabolism, on discrete cell types or cell regions within several tissues. Our approach provides morphological correlates to the biochemical interaction of PTH with its target tissues and should enhance our understanding of the relationship of target cell structure and function.

Animals↗
PubMed2002

Localization and regulation of a functional GHRH receptor in the rat renal medulla.

To provide information about the kidney GHRH receptor (GHRH-R), we assessed its tissue and cellular localization, defined its pattern of expression in developing and aging rats, and studied the effects of GHRH on the regulation of GHRH-R mRNA levels and receptor internalization. In situ hybridization and ribonuclease protection assay demonstrated that GHRH-R mRNA is restricted to the Henle's loop (HL). GHRH-R mRNA levels were low in the medulla from 3- and 12-d-old male rats, increased significantly in that from 30- to 70-d-old rats, and decreased in that from 12- and 18-month-old animals. Compared with the GHRH-R mRNA profile obtained in the pituitary, these data support the concept of a tissue-specific regulation of GHRH-R. In HL cell cultures from 70-d-old rats, a 4-h incubation with 1-100 nM rat GHRH-(1-29)NH(2) reduced GHRH-R mRNA levels significantly. As anti-GHRH-R- (392-404) immunoreactivity was demonstrated in HL cells, internalization of [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2),Ala(8), Ala(15),Lys(22)]hGHRH-(1-29)NH(2) in a time- and temperature-dependent manner and inhibition of this process by phenyl arsine oxide indicate that desensitization to GHRH involves both GHRH-R internalization and down-regulation of GHRH-R mRNA levels. Localization of a functional GHRH-R in HL and its regulation during development and aging suggest roles associated with cellular proliferation, differentiation, and/or water/electrolyte transport.

Animals↗
PubMed2001

Altered expression of prostacyclin synthase in a subset of the thick ascending limb cells and mesangial cells in 5/6-nephrectomized rats.

The aim of this study was to evaluate the possible role of prostacyclin (PGl2) in the onset and development of hypertension and chronic renal failure in 5/6-nephrectomized rats (5/6NX). We measured the systolic blood pressure, 24-h urinary excretion levels of 6-keto-PGF1alpha, which was a stable metabolite of PGI2, and levels of PGI2 synthase (PCS) mRNA in the kidneys. Immunostaining for PCS in the kidneys was also evaluated. Systolic blood pressure was higher in 5/6NX than in sham-operated rats. The 24-h urinary excretion levels of 6-keto-PGF1alpha in 5/6NX at 1 week postsurgery were lower than in sham-operated rats. In renal morphology, tubulointerstitial injury was observed at 2 weeks postsurgery, and glomerulosclerosis at 4 weeks. Levels of PCS mRNA in 5/6NX decreased significantly at 1 and 2 weeks postsurgery compared with those in sham-operated rats, but at 8 weeks these levels showed a tendency to increase. Immunostaining for PCS was positive in a subset of the cortical thick ascending limb of Henle's loop cells, including macula densa in both groups. Moreover, in 5/6NX at 8 weeks postsurgery, mesangial cells also stained positive for PCS. In conclusion, our findings suggest that PCS might play an important role in mitigating glomerular hemodynamic changes associated with reduction of renal mass.

6-Ketoprostaglandin F1 alpha↗
PubMed2005

Effects of vasopeptidase inhibition on renal function and tubuloglomerular feedback in spontaneously hypertensive rats.

Vasopeptidase inhibitors are a novel class of antihypertensive agents that concomitantly inhibit angiotensin converting enzyme and neutral endopeptidase. Our purpose was to investigate the effects of omapatrilat, a vasopeptidase inhibitor, on renal function and tubuloglomerular feedback (TGF) response in anesthetized 9-10-week-old spontaneously hypertensive rats (SHR). Intravenous injection of omapatrilat at 10 micromol/kg decreased systemic blood pressure and renal vascular resistance. Renal plasma flow was unchanged, whereas glomerular filtration rate (GFR) and filtration fraction (FF) were reduced. Increased urinary sodium excretion of tubular origin was observed. These parameters remained unaltered with vehicle treatment. Micropuncture study revealed that the maximal reduction of early proximal flow rate (EPFR) induced by orthograde perfusion of Henle's loop with artificial tubular fluid (ATF) was significantly reduced by omapatrilat treatment (28.5+/-3.1% vs. 72.0+/-2.8% of control) and was not significantly changed in the vehicle-treated group (vehicle 70.8+/-1.7% vs. control 71.0+/-2.1%). EPFR at zero perfusion was comparable between omapatrilat and vehicle treatment (29.7+/-2.2 vs. 31.3+/-2.1 nl/min, respectively). Luminal perfusion of 10(-4) mol/l 7-nitroindazole in ATF abrogated the blunting of TGF response by omapatrilat but elicited no change in the vehicle-treated group. The suppression of the TGF mechanism and the reduction in FF suggest that omapatrilat respectively dilates the afferent and efferent arterioles. Under such conditions, reduction of GFR may indicate a fall in intraglomerular pressure. The restoration of nitric oxide signaling in the juxtaglomerular apparatus of SHR seems to participate in the inhibition of TGF by omapatrilat. These findings suggest that omapatrilat may provide a novel approach to the treatment of systemic and glomerular hypertension.

Animals↗
PubMed1996

Determination of choline dehydrogenase activity along the rat nephron.

A radioenzymatic microassay was developed to quantitate choline dehydrogenase activity in single microdissected nephron segments. This enzyme is the rate limiting step in the biosynthesis of betaine, which serves as an intracellular osmoregulatory organic solute in mammalian kidney. The enzyme localized in renal mitochondrial inner membrane forms betaine aldehyde, which in the assay is converted to betaine by oxidative treatment. A histochemical procedure based on the formazan detection of tetranitroblue tetrazolium chloride was applied in parallel. The results show that activities in proximal convoluted and straight tubules are more than 5 times higher (21 to 25 pmol h-1 mm tubule-1) compared to distal nephron segments with no significant differences along the proximal tubule. Along the osmotic gradient from the outer medullary towards the papillary structures enzyme activities increased in ascending limbs of Henle's loop and collecting tubules. Collecting ducts showed two times higher activities than ascending loop segments when corrected for tubular cell volumes. The quantitative data were confirmed by the histochemical procedure. The results allow for the conclusion that betaine synthesis is sufficient to build up renal betaine, but cannot explain the distribution pattern of betaine along the corticopapillary axis. Additional mechanisms like intrarenal and tubular transport have to be postulated.

Alcohol Oxidoreductases↗