Yushi Nakayama

Vasopressin regulates the renin-angiotensin-aldosterone system via V1a receptors in macula densa cells

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in V1a receptor-deficient (V1aR-/-) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mori T. Proc Natl Acad Sci USA 103: 7807-7812, 2006). In this study, we investigated the role of V1aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using V1aR-/- mice. Urine volume of V1aR-/- mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in V1aR-/- mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in V1aR-/- mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in V1aR-/- mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-/- mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system.

Aldosterone Requires Vasopressin V1a Receptors on Intercalated Cells to Mediate Acid-Base Homeostasis

Both aldosterone and luminal vasopressin may contribute to the maintenance of acid-base homeostasis, but the functional relationship between these hormones is not well understood. The effects of luminal vasopressin likely result from its interaction with V1a receptors on the luminal membranes of intercalated cells in the collecting duct. Here, we found that mice lacking the V1a receptor exhibit type 4 renal tubular acidosis. The administration of the mineralocorticoid agonist fludrocortisone ameliorated the acidosis by restoring excretion of urinary ammonium via increased expression of Rhcg and H-K-ATPase and decreased expression of H-ATPase. In a cell line of intercalated cells established from transgenic rats expressing the mineralocorticoid and V1a receptors, but not V2 receptors, knockdown of the V1a receptor gene abrogated the effects of aldosterone on H-K-ATPase, Rhcg, and H-ATPase expression. These data suggest that defects in the vasopressin V1a receptor in intercalated cells can cause type 4 renal tubular acidosis and that the tubular effects of aldosterone depend on a functional V1a receptor in the intercalated cells.

Endothelin Converting Enzyme-1 Gene Expression in the Kidney of Spontaneously Hypertensive Rats

Abnormal renal handling of water and sodium is implicated in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). Alteration of renal endothelin-1 synthesis is also reported in SHR. Endothelin-1, a potent vasoconstrictor and regulator of sodium reabsorption in the nephron, has a pathophysiological potential in the development of hypertension. Because synthesis of bioactive endothelin-1 requires endothelin converting enzyme-1 (ECE-1), we investigated whether renal ECE-1 gene expression is altered in the kidney of SHR. Kidneys from both 4- and 12-week-old SHR and age-matched Wistar-Kyoto rats (WKY) were studied. ECE-1 mRNA in microdissected nephron segments was assessed by reverse transcription-competitive polymerase chain reaction, and ECE-1 protein level by Western blot. In 4-week-old SHR, ECE-1 mRNA was significantly increased in the proximal straight tubule, medullary thick ascending limb, cortical thick ascending limb, and inner medullary collecting duct. ECE-1 protein level was increased in both the outer and inner medulla. In 12-week-old SHR, ECE-1 gene expression was significantly increased in the proximal straight tubule, medullary thick ascending limb, and also in the glomeruli. Glomerular preproendothelin-1 mRNA expression was not different between the two strains at both 4 and 12 weeks. We conclude that high ECE-1 gene expression in the nephron, via increase of endothelin-1 synthesis, may promote sodium retention that contributes to the development and/or maintenance of hypertension in SHR.

Acute and chronic metabolic acidosis interferes with aquaporin-2 translocation in the rat kidney collecting ducts

Renal aquaporin-2 (AQP2) expression plays a key role in urine concentration. However, it is not known whether metabolic acidosis affects urine-concentrating ability through AQP2 expression in the kidney and urine. We examined urinary excretion and renal expression of AQP2 in control and acidosis rats, using RT-competitive PCR, immunoblot and immunocytochemistry. Urinary excretion of AQP2 is decreased by 92% even with the increase in AQP2 mRNA and protein expressions in the collecting ducts by metabolic acidosis in rats. Urine osmolality in control rats was 1670±198 mOsm per kg H2O, and immunocytochemistry revealed the presence of AQP2 in the apical plasma membrane of the principal cells in the collecting ducts. Urine osmolality in acidosis rats was lower than that in control (1397±243 mOsm per kg H2O), and immunocytochemistry showed the diffuse presence of AQP2 in the cytoplasm of the principal cells. Differential centrifugation-coupled immunoblot showed a significant decrease in the ratio of AQP2 in plasma membrane-enriched fraction to that in intracellular vesicle-enriched fraction by metabolic acidosis. In summary, AQP2 translocation is largely decreased by metabolic acidosis even with increased expression in the collecting ducts. A disorder of AQP2 translocation in the collecting ducts with acidosis may be responsible for the diuresis in patients with chronic renal failure.

Regulation of V2R transcription by hypertonicity and V1aR-V2R signal interaction.

Arginine vasopressin (AVP) and hypertonicity in the renal medulla play a major role in the urine concentration mechanism. Previously, we showed that rat vasopressin V2 receptor (rV2R) promoter activity was increased by vasopressin V2R stimulation and decreased by vasopressin V1a receptor (V1aR) stimulation in a LLC-PK1 cell line stably expressing rat V1aR (LLC-PK1/rV1aR). In the present study, we investigated the effects of hypertonicity on the rV2R promoter activity and on the suppression of rV2R promoter activity by V1aR stimulation in LLC-PK1/rV1aR cells. rV2R promoter activity was increased in NaCl- or mannitol-induced hypertonicity. The hypertonicity-responsive site in the rV2R promoter region was limited to 10 bp, including the Sp1 motif. The increase of V2R promoter activity by hypertonicity was significantly inhibited by a JNK inhibitor (SP600125) and PKA inhibitor (H89). In contrast, rV2R promoter activity was remarkably suppressed by V1aR stimulation in the hypertonic condition rather than in the isotonic condition. The AVP-stimulated intracellular Ca2+ concentration was increased in the hypertonic condition, suggesting the functional activation of V1aR by hypertonicity. In conclusion, 1) V2R promoter activity is increased by hypertonicity via the JNK and PKA pathways, 2) suppression of V2R expression by the V1aR-Ca2+ pathway is enhanced by hypertonicity, and 3) hypertonicity enhances the V1aR-Ca2+ pathway. The counteractivity of V2R and V1aR could be required to maintain minimum urine volume in the dehydrated state.

Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Different Mechanisms for the Progression of CKD with ACE Gene Polymorphisms.

Background/Aims: The blockade of the renin-angiotensin-aldosterone system is the major target of efforts to prevent the progression of chronic kidney disease (CKD). Dual blockade with angiotensin-converting enzyme (ACE) inhibitor and angiotensin II receptor blocker has been reported to show additive renoprotection. However, three types of insertion/deletion (I/D) polymorphism have been reported, and it is unclear whether the dual blockade is effective for all the ACE genotypes. Methods: We treated 93 CKD patients with or without dual blockade and analyzed the effects on blood pressure (BP), proteinuria, progression of CKD and the relationship to I/D ACE polymorphisms. Results: After long-term medication (average 33 ± 2 months), BP decreased in all the genotype groups. However, urinary protein excretion decreased only in the II and DI groups (II: –27.1%, DI: –20.5%, DD: +0.8%). In the II and DI groups, amelioration of the progression of renal failure was correlated with reductions in BP and urinary protein excretion. However, the progression rate of renal failure was not correlated with proteinuria in the DD group. Conclusion: Proteinuria and BP are key factors for the progression of CKD in II/DI patients, while controlling the BP rather than reducing the proteinuria appears to be crucial in DD patients.

Angiotensin-converting enzyme inhibitor withdrawal and ACE gene polymorphism

AIMS Withdrawal of angiotensin-converting enzyme (ACE) inhibitors may affect the progression of chronic renal failure and an insertion/deletion (I/D) polymorphism of the ACE gene may influence it. METHODS We retrospectively collected patients with chronic glomerulonephritis and benign nephrosclerosis who discontinued ACE inhibitor use. The relationship between the decline of renal function after the withdrawal and the influencing factors such as ACE gene polymorphism, blood pressure and proteinuria were evaluated using multiple regression analysis. RESULTS Forty-two patients (initial serum creatinine 0.5 - 6.5 mg/dl) had been treated and discontinued ACE inhibitor use. Only patients with the II or DI genotypes of the ACE gene developed the deterioration of renal function, starting at 2 months after the withdrawal. Stepwise regression analysis revealed that the level of proteinuria after the withdrawal, presence of the insertion of ACE gene and serum creatinine level at the time of withdrawal mainly influenced the decline of renal function after the withdrawal (adjusted R2 = 0.48). CONCLUSION Withdrawal of ACE inhibitor causes the deterioration of renal function in patients with the II or DI genotypes, high proteinuria after the withdrawal, and high serum creatinine level at the withdrawal, which probably causes the rebound increase in serum ACE activity.

Vasopressin and hyperosmolality regulate NKCC1 expression in rat OMCD

Secretory-type Na-K-2Cl cotransporter (NKCC1) is known to play roles in both acid and sodium excretion, and is more abundant in dehydration. To determine the mechanisms by which dehydration stimulates NKCC1 expression, the effects of vasopressin, oxytocin and hyperosmolality on NKCC1 mRNA and protein expressions in the outer medullary collecting duct (OMCD) of rats were investigated using RT-competitive PCR and western blot analysis. Microdissected OMCD was incubated in isotonic or hypertonic solution, or with AVP or oxytocin for 60 min at 37°C. Hyperosmolality induced by NaCl, mannitol or raffinose increased NKCC1 mRNA expression in OMCD by 130–240% in vitro. The stimulation of NKCC1 mRNA expression by NaCl was highest at 690 mosmol kg−1 H2O and gradually decreased at higher osmolalities. The incubation of OMCD with AVP (10−7 M) for 60 min increased NKCC1 mRNA expression by 100%. The administration of AVP to rats for 4 days using an osmotic mini-pump also increased NKCC1 mRNA and protein expressions in OMCD by 130%. In contrast, oxytocin (10−7 M) did not stimulate the NKCC1 mRNA expression in OMCD in vitro. Chronic injection of oxytocin increased the NKCC1 mRNA expression by 36%. These data showed that hyperosmolality and vasopressin stimulate NKCC1 mRNA and protein expressions in rat OMCD. It is concluded that NKCC1 expression is regulated directly and indirectly by vasopressin.

A novel method for dry weight assessment in hemodialysis patients: Utilization of inferior vena cava flat ratio to correct for individual variations in vessel diameter

Abstract:  Measurement of the inferior of vena cava (IVC) diameter by ultrasonography (US) has been shown to be useful for assessing dry weight (DW) in hemodialysis (HD) patients. We have previously observed some cases in which the IVC diameter differed depending on whether measurements were obtained from sagittal or cross‐sectional images. Thus, in the present study we introduce a new concept, the flat ratio, to define the magnitude of IVC deflation. The flat ratio of the IVC (F‐IVC) is determined from cross‐sectional ultrasonographic images of the IVC. The present study showed that F‐IVC is significantly correlated with changes in body weight (ΔBW(%)). Similarly, the change in F‐IVC (ΔF‐IVC) during HD is correlated with ΔBW(%) but not with changes in systolic or diastolic blood pressure during HD. The results of the present study suggest that F‐IVC measurement can serve as a useful tool for DW assessment.