Waichi Kitajima

Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney.

To examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD. Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884 +/- 245 mOsm/kg) was significantly higher than that in the control (938 +/- 91). In the V2R antagonist group, Uosm was significantly decreased to 249 +/- 29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor.

Changes in blood pressure, urinary kallikrein, and urinary prostaglandin E2 in rats with streptozotocin-induced diabetes

SummaryTo reveal the mechanism of hypertension in diabetes mellitus, changes in blood pressure were measured in streptozotocin (STZ)-induced diabetes mellitus in male Wistar rats. Development of diabetes mellitus and elevation of blood pressure were observed in rats which received 60 mg/kg STZ, but not in rats which received 20 mg/kg STZ. In the rats which developed diabetes mellitus after STZ, the plasma renin activity, renal renin content, plasma aldosterone, and urinary kallikrein activity were all significantly reduced. The urinary excretion of prostaglandin E2 (PGE2) was significantly increased at 1 week after STZ in diabetic rats, but it gradually returned to control values and showed a tendency to decrease at 4 weeks after the treatment compared to the rats of the control groups. The pressor responsiveness to norepinephrine in the conscious unrestrained state in the STZ-induced diabetic rats was not remarkably changed compared to that of control rats. These results indicate that blood pressure gradually increases with the progress of diabetes. It is suggested that the changes in urinary excretion of PGE2 and urinary kallikrein activity may be related to the regulation of blood pressure in STZ-induced diabetic rats. On the other hand, it is considered unlikely that the reninangiotensin system and vascular responsiveness play an important role in the occurrence of hypertension in diabetes mellitus.

Role of vasopressin V2 receptor in acute regulation of aquaporin-2.

Aquaporin-2 (AQP-2) has been shown to be a vasopressin-sensitive water channel in collecting duct (CD) cells of the kidney. To prove the role of the vasopressin V2 receptor (V2R) in the regulation of intracellular AQP-2 shuttling, we examined the acute effects of vasopressin and V2R antagonist on the distribution of AQP-2 in the cells. Normal Wistar rats were given continuous infusions of vasopressin, vasopressin V2R antagonist (OPC31260), or both. The kidneys were then processed for immunofluorescent studies with an affinity-purified specific antibody to AQP-2. One hour after the infusion of the V2R antagonist, AQP-2 staining was diffusely distributed in the CD cells from the cortex to the inner medulla. This tendency was not changed by the concomitant infusion with vasopressin. Vasopressin infusion without antagonist, however, induced intensified AQP-2 staining of the apical membrane in the CD cells. The ratio of the fluorescence intensity of the apical to subapical region was determined by confocal laser microscopy. In the inner medulla, this ratio was significantly increased in the vasopressin treatment group (2.26 +/- 0.76) as compared to the V2R antagonist group (1.03 +/- 0.34) and the combined treatment group (0.84 +/- 0.43). The increase in the ratio was also demonstrated in the cortex and the outer medulla in the vasopressin-treated group. In addition, Northern blotting studies clearly revealed that mRNA of AQP-2 in the vasopressin-treated group was increased when compared to the combined treatment animals. Our present results reveal that localization and gene expressions of AQP-2 are acutely regulated via vasopressin V2R.

Expression and Localization of the Water Channels in Human Autosomal Dominant Polycystic Kidney Disease

To characterize the cyst-lining cells in human autosomal dominant polycystic kidney disease (ADPKD), we performed immunohistological studies with specific antibodies against human aquaporin-2 (AQP-2, the vasopressin-regulated water channel) and aquaporin-3 (AQP-3), which are expressed only in collecting duct cells in the normal kidney. The polycystic kidney samples were obtained from 2 hemodialysis patient at uninephrectomy. Immunohistochemical studies revealed two types of staining of cyst-lining cells. Approximately 30% of all the cysts were simultaneously immunostained by both antibodies. Among these AQP-positive cysts, more than 90% of the cysts were intensely stained, with well-polarized localization of AQP-2 and AQP-3. In fewer than 10% of AQP-positive cysts, by contrast, immunostaining for AQP-2 and AQP-3 was faint and no clearly polarized localization of the channels was observed. We examined the immunostaining in further detail by electron microscopy. Staining specific for AQP-2 was mainly observed in the apical membrane of cyst-lining cells. Moreover, staining specific for AQP-3 was observed in all of the AQP-2-positive cysts. It appeared unlikely that the variations in immunostaining observed under the light microscope had been induced by total disruption of water-channel polarity. The present study suggests that about 30% of the cysts in our cases of ADPKD were derived from the collecting duct cells and that the cyst-lining cells were well differentiated in terms of AQP expression.

Renin and aldosterone in hypothyroidism: relation to excretion of sodium and potassium.

Changes in plasma renin activity (PRA) and plasma aldosterone (PA) were studied together with the urinary excretion of sodium and potassium in patients with hypothyroidism. The basal levels of PRA and PA were significantly less than those in normal subjects. However, there was no significant relationship between PRA and PA. The response of PRA after administration of 40 mg of frusemide in patients with hypothyroidism was significantly less than that in normal subjects, although the excretion of sodium was slightly higher than that in normal subjects. On the other hand, the excretion of potassium in patients with hypothyroidism was significantly lower than that in normal subjects. The responses of PA to various stimulations, such as ACTH, angiotensin II, potassium and frusemide, were equally suppressed. These results suggest that PRA and PA may be suppressed independently in hypothyroidism, probably due to dysfunction of juxtaglomerular cells and glomerulosa cells, respectively, and the possibility that suppression of PRA and PA in patients with hypothyroidism is related to exaggerated sodium excretion and a decrease in potassium excretion cannot be ruled out.

Effect of Aging on Single Nephron Renin Content in Rats

To investigate the relation between renin content in each juxtaglomerular apparatus and reduction of plasma renin activity (PRA) with aging, the PRA and microdissected superficial or juxtamedullary single nephron renin content (SNRC) were determined in 5 young (3-6 months) and 5 aged (13-18 months) rats fed on a normal salt diet. The mean value of the PRA in the aged group was significantly lower than that of the young group. A highly significant correlation was found between the RPA and mean values of the superficial SNRC. The mean values of both the superficial and deep SNRC in the aged rats were significantly lower than those of the corresponding zones in the young rats. It is suggested that decreased synthesis of renin in each juxtaglomerular apparatus is an important factor in the decreased PRA observed with aging.

A Case of Secondary Aldosteronism Induced by Pheochromocytoma

A case of secondary aldosteronism caused by a pheochromocytoma containing a large amount of norepinephrine is described. The increased level of angiotensin induced by norepinephrine seems to play an important role in the development of secondary aldosteronism.

Three cases of eosinophilic peritonitis during CAPD therapy.

CAPD (continuous ambulatory peritoneal dialysis) は腎不全の治療として血液透析や腎移植と共に重要な治療法の一つである. しかし最大の合併症として腹膜炎があり, これがCAPD継続の鍵を握っている. 最近この腹膜炎の中に無菌性のものがあることが知られている. 特に排液中の好酸球の割合が多く, 自然寛解をみる好酸球性腹膜炎が注目されてきている. 今回我々は3例の好酸球性腹膜炎を経験した. 3症例ともCAPD導入後2-3週間で発症し, 抗生剤を使用せずに1-3週間で自然治癒した. このうち2症例に肝機能障害が出現した. 好酸球性腹膜炎の発症機転はアレルギー反応と推察されているが, 我々の症例においては末梢血の好酸球の増加はなく, カテーテルの滅菌方法をEOGからオートクレーブに変更したところその後の症例においては1例も好酸球性腹膜炎の発症を認めていない. 好酸球性腹膜炎は自然治癒し腹膜機能も低下させないので, 腹膜炎発症時には白血球分画も測定し, 本疾患の診断をつけることが大切であり, 抗生剤治療は無意味である.

An approach to the profiling analysis of urinary prostaglandins by simple extraction methods

No useful profiling analyses of prostaglandins (PGs) in biological matrix have been reported. We present here a quantitative profiling analysis of urinary PGs determined by GC or GC-MS using a new simple extraction method. In both cases ODS silica column (Sep-Paks, Waters) was used for extraction of PGs from human urine. This column was pretreated with 20 mlethanol followed by 20 mlwater before use. The 20 mlsample was acidified with formic acid to pH 3.0 and passed through a ODS silica column using a glass syringe. The column was eluted successively with 20 mlethanol/water (15/85), 20 mlpetroleum ether and 10 mlmethyl formate. The recovery rate estimated using3H-PGs in methyl formate fraction was as follows. 6-keto-PGF1α: 68%, PGE2: 60%, PGF2α: 65%. The samples were derevatized to 6-keto-PGF1α-MOX-TMS-Bz, PGE2-MOX-TMS-Bz and PGF2α-TMS-Bz after extractive alkylation using THAH and benzyl bromide. GC peaks of the PG derivatives appeared at the same retention time as external standards. The quantitative determination of endogenous PGs were thus achieved by GC. d7-6-keto-PGF1α, d7-PGE2and d8-PGF2αwere added as internal standards to the human urine, which was treated with the same procedure as above. PGs in the methyl formate fraction were derivatized to 6-keto-PGF1αMOX-Me-TMS, PGE2-Me-TMS and PGF2α-Me-TMS. The sample was analysed by GC-MS (JEOL D-300) using selected ion monitoring.In conclusion, we have developed a new simple extraction method for PGs using a convenient ODS silica column chromatography in combination with the extractive alkylation. It seems to be very useful for the quantitative profiling analysis of urinary PGs.