COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.     

mPGES-1 deletion potentiates urine concentrating capability after water deprivation

PGE(2) plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE(2) output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V(2) receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE(2) reduces urine concentrating ability through suppression of renal medullary expression of V(2) receptors and AQP2 but may enhance it by mediating the central AVP response.     

Defective renal water handling in transgenic mice over-expressing human CD39/NTPDase1

Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders.     

Effect of high and low sodium intake on urinary aquaporin-2 excretion in healthy humans

The degree of water transport via aquaporin-2 (AQP2) water channels in renal collecting duct principal cells is reflected by the level of the urinary excretion of AQP2 (u-AQP2). In rats, the AQP2 expression varies with sodium intake. In humans, the effect of sodium intake on u-AQP2 and the underlying mechanisms have not previously been studied. We measured the effect of 4 days of high sodium (HS) intake (300 mmol sodium/day; 17.5 g salt/day) and 4 days of low sodium (LS) intake (30 mmol sodium/day; 1.8 g salt/day) on u-AQP2, fractional sodium excretion (FE(Na)), free water clearance (C(H2O)), urinary excretion of PGE(2) (u-PGE(2)) and cAMP (u-cAMP), and plasma concentrations of vasopressin (AVP), renin (PRC), ANG II, aldosterone (Aldo), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) in a randomized, crossover study of 21 healthy subjects, during 24-h urine collection and after hypertonic saline infusion. The 24-h urinary sodium excretion was significantly higher during HS intake (213 vs. 41 mmol/24 h). ANP and BNP were significantly lower and PRC, ANG II, and Aldo were significantly higher during LS intake. AVP, u-cAMP, and u-PGE(2) were similar during HS and LS intake, but u-AQP2 was significantly higher during HS intake. The increases in AVP and u-AQP2 in response to hypertonic saline infusion were similar during HS and LS intake. In conclusion, u-AQP2 was increased during HS intake, indicating that water transport via AQP2 was increased. The effect was mediated by an unknown AVP-independent mechanism.     

Tumor necrosis factor-alpha is an endogenous inhibitor of Na+-K+-2Cl- cotransporter (NKCC2) isoform A in the thick ascending limb

The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ～40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ～60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ～30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.     

Sleep deprivation induces excess diuresis and natriuresis in healthy children

Urine production is reduced at night, allowing undisturbed sleep. This study was undertaken to show the effect of sleep deprivation (SD) on urine production in healthy children. Special focus was on gender and children at an age where enuresis is still prominent. Twenty healthy children (10 girls) underwent two 24-h studies, randomly assigned to either sleep or SD on the first study night. Diet and fluid intake were standardized. Blood samples were drawn every 4 h during daytime and every 2 h at night. Urine was fractionally collected. Blood pressure and heart rate were noninvasively monitored. Blood was analyzed for plasma antidiuretic hormone (AVP), atrial natriuretic peptide (ANP), angiotensin II, aldosterone, and renin. Urine was analyzed for aquaporin-2 and PGE(2). Successful SD was achieved in all participants with a minimum of 4 h 50 min, and full-night SD was obtained in 50% of the participants. During SD, both boys and girls produced markedly larger amounts of urine than during normal sleep (477 ± 145 vs. 291 ± 86 ml, P < 0.01). SD increased urinary excretion of sodium (0.17 ± 0.05 vs. 0.10 ± 0.03 mmol·kg(-1)·h(-1)) whereas solute-free water reabsorption remained unchanged. SD induced a significant fall in nighttime plasma AVP (P < 0.01), renin (P < 0.05), angiotensin II (P < 0.001), and aldosterone (P < 0.05) whereas plasma ANP levels remained uninfluenced (P = 0.807). Nighttime blood pressure and heart rate were significantly higher during SD (mean arterial pressure: 78.5 ± 8.0 vs. 74.7 ± 8.7 mmHg, P < 0.001). SD leads to natriuresis and excess diuresis in healthy children. The underlying mechanism could be a reduced nighttime dip in blood pressure and a decrease in renin-angiotensin-aldosterone system levels during sleep deprivation.     

Adenosine A1-receptor knockout mice have a decreased blood pressure response to low-dose ANG II infusion

Adenosine, acting on A(1)-receptors (A(1)-AR) in the nephron, increases sodium reabsorption, and also increases renal vascular resistance (RVR), via A(1)-ARs in the afferent arteriole. ANG II increases blood pressure and RVR, and it stimulates adenosine release in the kidney. We tested the hypothesis that ANG II-infused hypertension is potentiated by A(1)-ARs' influence on Na(+) reabsorption. Mean arterial pressure (MAP) was measured by radiotelemetry in A(1)-AR knockout mice (KO) and their wild-type (WT) controls, before and during ANG II (400 ng·kg(-1)·min(-1)) infusion. Baseline MAP was not different between groups. ANG II increased MAP in both groups, but on day 12, MAP was lower in A(1)-AR KO mice (KO: 128 ± 3 vs. 139 ± 3 mmHg, P < 0.01). Heart rates were significantly different during days 11-14 of ANG II. Basal sodium excretion was not different (KO: 0.15 ± 0.03 vs. WT: 0.13 ± 0.04 mmol/day, not significant) but was higher in KO mice 12 days after ANG II despite a lower MAP (KO: 0.22 ± 0.03 vs. WT: 0.11 ± 0.02 mmol/day, P < 0.05). Phosphate excretion was also higher in A(1)-AR KO mice on day 12. Renal expression of the sodium-dependent phosphate transporter and the Na(+)/glucose cotransporter were lower in the KO mice during ANG II treatment, but the expression of the sodium hydrogen exchanger isoform 3 was not different. These results indicate that the increase in blood pressure seen in A(1)-AR KO mice is lower than that seen in WT mice but was increased by ANG II nonetheless. The presence of A(1)-ARs during a low dose of ANG II-infusion limits Na(+) and phosphate excretion. This study suggests that A(1)-AR antagonists might be an effective antihypertensive agent during ANG II and volume-dependent hypertension.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Enhanced central response to dehydration in mice lacking angiotensin AT(1a) receptors

The objective was to determine the central nervous system (CNS) responses to dehydration (c-Fos and vasopressin mRNA) in mice lacking the ANG AT(1a) receptor [ANG AT(1a) knockout (KO)]. Control and AT(1a) KO mice were dehydrated for 24 or 48 h. Baseline plasma vasopressin (VP) was not different between the groups; however, the response to dehydration was attenuated in AT(1a) KO (24 +/- 11 vs. 10.6 +/- 2.7 pg/ml). Dehydration produced similar increases in plasma osmolality and depletion of posterior pituitary VP content. Neuronal activation was observed as increases in c-Fos protein and VP mRNA. The supraoptic responses were not different between groups. In the paraventricular nucleus (PVN), c-Fos-positive neurons (57.4 +/- 10.7 vs. 98.4 +/- 7.4 c-Fos cells/PVN, control vs. AT(1a) KO) and VP mRNA levels (1.0 +/- 0.1 vs. 1.4 +/- 0.1 microCi, control vs. AT(1a) KO) were increased with greater responses in AT(1a) KO. A comparison of 1- to 2-day water deprivation showed that plasma VP, brain c-Fos, and VP mRNA returned toward control on day 2, although plasma osmolality remained high. Data demonstrate that AT(1a) KO mice show a dichotomous response to dehydration, reduced for plasma VP and enhanced for PVN c-Fos protein and VP mRNA. The results illustrate the importance of ANG AT(1a) receptors in the regulation of osmotic and endocrine balance.     

Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause

Oxidative stress is implicated in menopause-associated hypertension and cardiovascular disease. The role of antioxidants in this process is unclear. We questioned whether the downregulation of thioredoxin (TRX) is associated with oxidative stress and the development of hypertension and target-organ damage (cardiac hypertrophy) in a menopause model. TRX is an endogenous antioxidant that also interacts with signaling molecules, such as apoptosis signal-regulated kinase 1 (ASK-1), independently of its antioxidant function. Aged female wild-type (WT) and follitropin receptor knockout (FORKO) mice (20-24 wk), with hormonal imbalances, were studied. Mice were infused with ANG II (400 ng x kg(-1) x min(-1); 14 days). Systolic blood pressure was increased by ANG II in WT (166+/-8 vs. 121+/-5 mmHg) and FORKO (176+/-7 vs. 115+/-5 mmHg; P<0.0001; n=9/group) mice. In ANG II-infused FORKO mice, cardiac mass was increased by 42% (P<0.001). This was associated with increased collagen content and augmented ERK1/2 phosphorylation (2-fold). Cardiac TRX expression and activity were decreased by ANG II in FORKO but not in WT (P<0.01) mice. ASK-1 expression, cleaved caspase III content, and Bax/Bcl-2 content were increased in ANG II-infused FORKO (P<0.05). ANG II had no effect on cardiac NAD(P)H oxidase activity or on O(2)(*-) levels in WT or FORKO. Cardiac ANG II type 1 receptor expression was similar in FORKO and WT. These findings indicate that in female FORKO, ANG II-induced cardiac hypertrophy and fibrosis are associated with the TRX downregulation and upregulation of ASK-1/caspase signaling. Our data suggest that in a model of menopause, protective actions of TRX may be blunted, which could contribute to cardiac remodeling independently of oxidative stress and hypertension.     

Attenuated renal vascular responses to acute angiotensin II infusion in smooth muscle-specific Na+/Ca2+ exchanger knockout mice

Recent studies in smooth muscle-specific Na(+)/Ca(2+) exchanger-1 knockout (NCX1(sm-/-)) mice reveal reduced arterial pressure and impaired myogenic responses compared with heterozygous littermates. In this study, we determined renal function in male anesthetized NCX1(sm-/-) mice and NCX1 heterozygous (NCX1(+/-)) littermates before and during acute ANG II infusions. Systolic blood pressure in awake mice was lower in NCX1(sm-/-) mice compared with NCX1(+/-) mice (119 ± 4 vs. 131 ± 3 mmHg, P < 0.05). Acute ANG II infusions (5 ng·min(-1)·g(-1) body wt) increased mean arterial pressure in anesthetized NCX1(+/-) (109 ± 2 to 134 ± 3 mmHg, P < 0.001, n = 8) and NCX1(sm-/-) (101 ± 8 to 129 ± 8 mmHg, P < 0.01, n = 6) mice to a similar extent (Δ25 ± 1 vs. Δ28 ± 4 mmHg, P > 0.05). In response to ANG II infusions, PAH clearance (C(PAH)) decreased from 1.39 ± 0.27 to 0.98 ± 0.22 ml·min(-1)·g(-1) (P < 0.05) and glomerular filtration rate (GFR) was reduced from 0.50 ± 0.09 to 0.32 ± 0.06 ml·min(-1)·g(-1) (P < 0.05) in NCX1(+/-) mice. In contrast, the NCX1(sm-/-) did not exhibit significant reductions in either C(PAH) (1.16 ± 0.30 to 1.22 ± 0.34 ml·min(-1)·g(-1), P > 0.05) or GFR (0.48 ± 0.08 to 0.41 ± 0.05 ml·min(-1)·g(-1), P > 0.05) during acute ANG II infusions. Using flometry to measure renal blood flow continuously, NCX1(sm-/-) mice had significantly attenuated responses to ANG II infusions (-34.2 ± 3.9%, P < 0.05) compared with those in NCX1(+/-) mice (-48 ± 2%) or in wild-type mice (-69 ± 7%). These data indicate that renal vascular responses to ANG II are attenuated in NCX1(sm-/-) mice compared with NCX1(+/-) mice and that NCX1 contributes to the renal vasoconstriction response to acute ANG II infusions.     

Intrarenal transfer of an intracellular fluorescent fusion of angiotensin II selectively in proximal tubules increases blood pressure in rats and mice

The present study tested the hypothesis that intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases blood pressure by activating AT(1) (AT(1a)) receptors. Intrarenal transfer of ECFP/ANG II was induced in the superficial cortex of rat and mouse kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter was used to drive ECFP/ANG II expression selectively in proximal tubules. Intrarenal transfer of ECFP/ANG II induced a time-dependent, proximal tubule-selective expression of ECFP/ANG II in the cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG II expression was low in the glomeruli and the entire medulla and was absent in the contralateral kidney or extrarenal tissues. At its peak of expression in proximal tubules at day 14, ANG II was increased by twofold in the kidney (P < 0.01) and more than threefold in proximal tubules (P < 0.01), but remained unchanged in plasma or urine. Systolic blood pressure was increased in ECFP/ANG II-transferred rats by 28 ± 6 mmHg (P < 0.01), whereas fractional sodium excretion was decreased by 20% (P < 0.01) and fractional lithium excretion was reduced by 24% (P < 0.01). These effects were blocked by losartan and prevented in AT(1a) knockout mice. Transfer of a scrambled ECFP/ANG IIc had no effects on blood pressure, kidney, and proximal tubule ANG II, or sodium excretion. These results provide evidence that proximal tubule-selective transfer of an intracellular ANG II fusion protein increases blood pressure by activating AT(1a) receptors and increasing sodium reabsorption in proximal tubules.     

Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice

Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.     

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.     

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ～10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.     

Genetic deletion of the P2Y2 receptor offers significant resistance to development of lithium-induced polyuria accompanied by alterations in PGE2 signaling

Lithium (Li)-induced polyuria is due to resistance of the medullary collecting duct (mCD) to the action of arginine vasopressin (AVP), apparently mediated by increased production of PGE(2). We previously reported that the P2Y(2) receptor (P2Y(2)-R) antagonizes the action of AVP on the mCD and may play a role in Li-induced polyuria by enhancing the production of PGE(2) in mCD. Hence, we hypothesized that genetic deletion of P2Y(2)-R should ameliorate Li-induced polyuria. Wild-type (WT) or P2Y(2)-R knockout (KO) mice were fed normal or Li-added diets for 14 days and euthanized. Li-induced polyuria, and decreases in urine osmolality and AQP2 protein abundance in the renal medulla, were significantly less compared with WT mice despite the lack of differences in Li intake or terminal serum or inner medullary tissue Li levels. Li-induced increased urinary excretion of PGE(2) was not affected in KO mice. However, prostanoid EP(3) receptor (EP3-R) protein abundance in the renal medulla of KO mice was markedly lower vs. WT mice, irrespective of the dietary regimen. The protein abundances of other EP-Rs were not altered across the groups irrespective of the dietary regimen. Ex vivo stimulation of mCD with PGE(2) generated significantly more cAMP in Li-fed KO mice (130%) vs. Li-fed WT mice (100%). Taken together, these data suggest 1) genetic deletion of P2Y(2)-R offers significant resistance to the development of Li-induced polyuria; and 2) this resistance is apparently due to altered PGE(2) signaling mediated by a marked decrease in EP3-R protein abundance in the medulla, thus attenuating the EP3-mediated decrease in cAMP levels in mCD.     

Loss of renal medullary endothelin B receptor function during salt deprivation is regulated by angiotensin II

We have recently demonstrated that chronic infusion of exogenous ANG II, which induces blood pressure elevation, attenuates renal medullary endothelin B (ET(B)) receptor function in rats. Moreover, this was associated with a reduction of ET(B) receptor expression in the renal inner medulla. The aim of this present work was to investigate the effect of a physiological increase in endogenous ANG II (low-salt diet) on the renal ET system, including ET(B) receptor function. We hypothesized that endogenous ANG II reduces renal medullary ET(B) receptor function during low-salt intake. Rats were placed on a low-salt diet (0.01-0.02% NaCl) for 2 wk to allow an increase in endogenous ANG II. In rats on normal-salt chow, the stimulation of renal medullary ET(B) receptor by ET(B) receptor agonist sarafotoxin 6c (S6c) causes an increase in water (3.6 ± 0.4 from baseline vs. 10.5 ± 1.3 μl/min following S6c infusion; P < 0.05) and sodium excretion (0.38 ± 0.06 vs. 1.23 ± 0.17 μmol/min; P < 0.05). The low-salt diet reduced the ET(B)-dependent diuresis (4.5 ± 0.5 vs. 6.1 ± 0.9 μl/min) and natriuresis (0.40 ± 0.11 vs. 0.46 ± 0.12 μmol/min) in response to acute intramedullary infusion of S6c. Chronic treatment with candesartan restored renal medullary ET(B) receptor function; urine flow was 7.1 ± 0.9 vs. 15.9 ± 1.7 μl/min (P < 0.05), and sodium excretion was 0.4 ± 0.1 vs. 1.1 ± 0.1 μmol/min (P < 0.05) before and after intramedullary S6c infusion, respectively. Receptor binding assays determined that the sodium-depleted diet resulted in a similar level of ET(B) receptor binding in renal inner medulla compared with rats on a normal-salt diet. Candesartan reduced renal inner medullary ET(B) receptor binding (1,414 ± 95 vs. 862 ± 50 fmol/mg; P < 0.05). We conclude that endogenous ANG II attenuates renal medullary ET(B) receptor function to conserve sodium during salt deprivation independently of receptor expression.     

Excitatory amino acids in rostral ventrolateral medulla support blood pressure during water deprivation in rats

Water deprivation is associated with regional increases in sympathetic tone, but whether this is mediated by changes in brain stem regulation of sympathetic activity is unknown. Therefore, this study tested the hypothesis that water deprivation increases excitatory amino acid (EAA) drive of the rostral ventrolateral medulla (RVLM), by determining whether bilateral microinjection of kynurenate (Kyn; 2.7 nmol) into the RVLM decreases arterial pressure more in water-deprived than water-replete rats. Plasma osmolality was increased in 48-h water-deprived rats (313 +/- 1 mosmol/kgH2O; P < 0.05) compared with 24-h water-deprived rats (306 +/- 2 mosmol/kgH2O) and water-replete animals (300 +/- 2 mosmol/kgH2O). Kyn decreased arterial pressure by 28.1 +/- 5.2 mmHg (P < 0.01) in 48-h water-deprived rats but had no effect in water-replete rats (-5.9 +/- 1.3 mmHg). Variable depressor effects were observed in 24-h water-deprived animals (-12.5 +/- 2.4 mmHg, not significant); however, in all rats the Kyn depressor response was strongly correlated to the osmolality level (P < 0.01; r2 = 0.47). The pressor responses to unilateral microinjection of increasing doses (0.1, 0.5, 1.0, and 5.0 nmol) of glutamate were enhanced (P < 0.05) during water deprivation, but the pressor responses to intravenous phenylephrine injection were smaller (P < 0.05). These data suggest that water deprivation increases EAA drive to the RVLM, in part by increasing responsiveness of the RVLM to EAA such as glutamate.     

Absence of PKC-Alpha Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus

Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.