Diminished expression of aquaporin water channels in ureteral-obstructed kidney in rats

OBJECTIVE: We investigated whether regulation of aquaporin (AQP) water channels is altered in the ureteral-obstructed kidney. MATERIAL AND METHODS: Male Sprague-Dawley rats were unilaterally obstructed of their left proximal ureters for 48 h. The protein expression of AQP1-3 channels was determined in the kidney by Western blot analysis. The expression of AQP2 was also determined by immunohistochemistry. In order to specifically determine primary impairment of the pathway leading to an altered regulation of AQP channels stimulated by the arginine vasopressin (AVP)/cyclic adenosine monophosphate (cAMP) pathway, the catalytic activity and protein expression of different parts of the adenylyl cyclase complex were separately determined. RESULTS: In the previously obstructed kidney, urinary osmolality and free water reabsorption were greatly decreased. The expression of AQP2 proteins was decreased in the cortex, outer medulla and inner medulla. Immunohistochemistry also showed a marked decrease in AQP2 expression. The expression of AQP1 and AQP3 was decreased in the outer medulla and inner medulla. cAMP generation in response to AVP, sodium fluoride or forskolin was greatly decreased. The expression of Gsalpha and adenylyl cyclase VI proteins was decreased. The contralateral kidney showed minimal or no changes in these parameters. CONCLUSIONS: The reduced abundance of AQP water channels may at least partly account for the urinary concentration defect in the ureteral-obstructed kidney. The primary point of impairment of AQP channels regulated by the AVP/cAMP pathway may lie at the level of the catalytic unit of adenylyl cyclase.     

Developmental changes in cyclooxygenase mRNA expression in the kidney of rats

Prostaglandins, synthesized by cyclooxygenase (COX), regulate renal hemodynamics and also epithelial water and solute transport. To determine whether COX mRNA expression changes with age, we studied expression in renal medulla and in cortex in developing rats at various ages. We also examined age-related changes in COX mRNA expression induced by lipopolysaccharide (LPS). COX mRNA was quantitatively analyzed in a real-time polymerase chain reaction (PCR) with dual-labeled fluorogenic probes. COX-1 mRNA expression did not change with age in cortex or medulla. COX-2 mRNA expression was highest in 1-week-old rats and lowest in 4- and 8-week-old rats. Lipopolysaccharide treatment did not alter COX-1 mRNA expression in infantile or adult rats. In adults, LPS at 1, 5, and 10 mg/kg induced COX-2 mRNA expression in renal medulla; the higher doses, 5 and 10 mg/kg, induced COX-2 expression in cortex. In infantile rats, COX-2 mRNA, already high in the unmanipulated state, was further increased by only 1 mg/kg LPS in both renal cortex and medulla. Age-related changes in the expression of COX-2 mRNA might be responsible for changing physiologic characteristics of renal function during postnatal development in rats, and may be important in renal cortical development.     

Cellular localization of aquaporin 7 in the rat kidney

The cDNA for the seventh mammalian aquaporin (AQP7) was isolated from rat testis, and its expression was demonstrated at the tail of late spermatids [Ishibashi et al: J Biol Chem 1997;272:20782-20786]. AQP7 is also expressed in the kidney. The localization of AQP7 in the kidney is unknown. We examined the cellular localization of AQP7 in the kidney with Northern blot, reverse transcribed PCR, Western blot and immunohistochemistry in the rat kidney. In Northern blot, AQP7 was expressed in the cortex and the outer medulla but absent in the inner medulla of the kidney. Reverse transcribed PCR of rat nephron segments revealed the selective expression of AQP7 at the proximal straight tubules (PST). Western blot of the membrane fraction of outer medulla revealed a single band of approximately 33 kDa. Immunohistochemistry of the rat kidney showed the selective expression of AQP7 at the brush border membranes of PST (S3 segment). AQP7 is now shown to be localized selectively at the brush border membranes of PST in the rat kidney. The result suggests that AQP7 may function as a pathway for transcellular water transport in PST in concert with more widely expressed AQP1 in proximal tubules. Alternatively, as AQP7 transports urea as well as water, AQP7 may function as a passive urea secretory pathway in this segment and may play a role in the formation and/or maintenance of the medullary urea concentration gradient.     

阿霉素肾病大鼠肾组织中血管生成素样蛋白3基因表达

目的动态观察阿霉素肾病大鼠在发生蛋白尿的过程中肾组织血管生成素样蛋白3（ANGPTL3）mRNA表达水平的变化，探讨ANGPTL3在大鼠肾组织中表达与蛋白尿的关系。方法通过激光微分离技术分离阿霉素肾病大鼠肾小球和肾小管，采用实时荧光定量RT-PCR方法分析阿霉素注射后7、14、21和28d肾组织中ANGPTL3 mRNA表达水平的变化。结果（1）阿霉素注射后第14天开始出现大量蛋白尿（P〈0.01），血清白蛋白明显减少（P〈0．01），胆固醇（P〈0．01）和甘油三酯（P〈0．05）明显增高，以上各项指标均在第28天达高峰。（2）通过激光微分离系统成功地分离了大鼠肾组织中的肾小球和肾小管。（3）阿霉素注射后7及14d后，大鼠肾皮质中总ANGFTL3 mRNA表达水平与同一时间点正常对照组比较无明显变化；到第21天时为正常对照组的9．03倍（P〈0．01）；第28天时为正常对照组的3．95倍（P〈0．05），与第21天时相比已呈现下降趋势。（4）阿霉素注射后不同时间点肾小球中ANGPTL3 mRNA表达水平的变化趋势与肾组织总ANGPTL 3mRNA表达水平的变化一致，第7、14天时，肾小球中ANGPTL 3mRNA表达水平与同一时间点正常对照组比较无明显变化；到第21天时为同一时间点正常对照组的2．18倍（P〈0．05）；第28天时为正常对照组的1．90倍（P〈0．05）。（5）阿霉素注射后不同时间点肾小管中ANGPTL3 mRNA表达水平无明显改变，与同一时间点的正常对照组相比均无显著性差异。结论正常大鼠和阿霉索肾病大鼠的肾组织中均有ANGPTL3 mRNA表达。肾组织可以自身合成ANGPTL3；肾病的大鼠肾组织自身合成ANGPTL3增多，且这种改变是发生在肾小球。ANGPTL3可能通过影响肾小球的功能参与肾病蛋白尿的进展过程。     

Cisplatin decreases the abundance of aquaporin water channels in rat kidney

The present study examined whether the cisplatin induced urinary concentration defect can be related to an altered regulation of aquaporin (AQP) water channels in the kidney. Cisplatin (8 mg/kg) was injected intraperitoneally into male Sprague-Dawley rats. The control group was without cisplatin treatment. Four d later, the expression of AQP1, AQP2, and AQP3 proteins was determined in the kidney. To specify further the primary point of derangement in the pathway that activates the arginine vasopressin-mediated AQP channels, different components of adenylyl cyclase complex were examined separately. The cisplatin treatment caused a polyuric renal failure in association with decreases of free water reabsorption. The expression of AQP1 and AQP2 was decreased in the cortex, the outer medulla, and the inner medulla, whereas that of AQP3 was decreased in the outer medulla and the inner medulla. The expression of AQP2 proteins in the apical membrane-enriched fraction decreased in parallel with that in the subapical vesicle-enriched fraction, indicating a preserved targeting. Immunohistochemistry of the outer medulla also revealed that cisplatin decreased immunoreactivity for AQP1, AQP2, and AQP3. The arginine vasopressin-evoked generation of cyclic adenosine monophosphate was attenuated by cisplatin, being most prominent in the outer medulla. However, the cyclic adenosine monophosphate generation in response to forskolin was not affected, whereas that to sodium fluoride was diminished significantly. Cisplatin also decreased the expression of Gsalpha proteins in the outer medulla and the inner medulla. These results suggest that a reduced expression of AQP water channels accounts at least in part for the cisplatin-induced urinary concentration defect.     

高原低氧环境下大鼠肾脏水通道蛋白2的表达及意义

目的 研究高原低氧环境下大鼠肾脏内髓水通道蛋白2（AQP2）mRNA及蛋白的表达。 方法 将40只雄性SD大鼠随机分为24 h、48 h、72 h及1周组，同时设立对照组（西宁地区）。将4个实验组由西宁地区（2260 m）带至青海大学可可西里高原医学研究基地(4600 m)，在不同时间点处死取材。用放射免疫法检测血浆抗利尿激素(AHD)浓度，Western印迹、实时定量PCR、免疫荧光等方法检测AQP2蛋白及mRNA的表达。 结果 暴露于高原低氧环境后，大鼠AHD水平首先呈下降趋势，24 h为（142.46±10.57）；48 h时达到最低水平，仅为对照组的28.5%[（86.94±6.49） μg/L比（302.53±10.48） μg/L]；后逐渐上升，72 h组为（169.65±11.15） μg/L，与对照组差异均有统计学意义（均P < 0.01）；1周时为（306.46±11.14） μg/L达到对照组水平。AQP2 mRNA的表达与AHD水平的变化相一致，初入高原后呈下降趋势，48 h时达到最低水平，后逐渐上升，24 h、48 h、72 h组与对照组差异均有统计学意义（分别为0.04±0.005，0.03±0.002，0.04±0.003比0.09±0.008，均P < 0.01），1周时为（0.09±0.01），达到对照组水平。AQP2蛋白表达与mRNA的表达变化一致。免疫荧光结果显示，对照组及1周组大鼠肾脏荧光强度表达一致，其余各组较低，48 h组荧光强度最低。 结论 在高原低氧环境下大鼠肾脏内髓AQP2 mRNA和蛋白的表达在缺氧早期呈下降趋势，后逐渐上升到正常水平，其改变可能和机体对缺氧的程度和高原低氧的习服过程有关。     

Atrial natriuretic peptide in dehydrated Long-Evans rats and Brattleboro rats

Atrial natriuretic peptide (ANP) was measured by radioimmunoassay in atrial and plasma extracts from normal Long-Evans (LE) rats and Brattleboro-strain diabetes insipidus (DI) rats. LE rats, dehydrated for 72 hours, had an increased plasma osmolality and plasma vasopressin. They also demonstrated a higher atrial immunoreactive ANP (IR-ANP) content than hydrated animals (72 hr dehydration: 178.2 +/- 30.4 micrograms/g wet weight atria, mean +/- SE, control: 60.4 +/- 8.2; P less than 0.001). Plasma IR-ANP in dehydrated LE rats tended to be lower than hydrated LE but this was not statistically significant [72 hr dehydration: 61.9 +/- 5.9 pg/ml, control: 82.4 +/- 8.2]. IR-ANP concentration in atrial extracts from DI rats, without detectable plasma vasopressin levels but with increased plasma osmolality, was not different from that in hydrated LE rats (DI: 100.6 +/- 13.2 micrograms/g). There was also no significant difference between plasma IR-ANP in DI and hydrated LE rats (DI: 100.2 +/- 11.9 pg/ml). The atrial IR-ANP concentration in DI rats was decreased by infusion with either arginine-vasopressin (AVP) or 1-deamino-8-arginine vasopressin (DDAVP), and plasma IR-ANP was increased significantly by both infusions (AVP: 171.3 +/- 18.1 pg/ml, DDAVP: 179.5 +/- 24.6). Thus, changes in atrial and plasma IR-ANP concentration appeared to be associated with changes in water balance but not with plasma AVP levels, indicating that the changes in volume may be a more important factor controlling ANP release in vivo than vasopressin itself.     

水通道蛋白4在食管鳞癌中的表达

目的 观察水通道蛋白4(AQP4)在食管鳞癌组织中的表达并探讨其在食管癌发病中的作用.方法 取食管鳞癌组织、癌旁正常鳞状上皮组织各16例,应用免疫组织化学,逆转录.聚合酶链反应(RT-PCR)技术检测AQP4的表达及分布.结果 免疫组织化学显示,AQP4表达于正常食管黏膜鳞状上皮细胞,在食管鳞癌组中主要表达于肿瘤上皮细胞和癌巢中.RT-PCR法结果 显示,AQP4在癌旁正常组织和食管癌组织中的mRNA表达平均相对A值分别为0.45±0.12、0.70±0.23,差异有统计学意义(P＜0.05).结论 AQP4在正常食管黏膜鳞状上皮细胞以及食管鳞癌组中均有表达,而且在癌组织中表达增加;AQP4可能对人食管癌的发生、发展起促进作用.     

Diminished adenylate cyclase activity and aquaporin 2 expression in acute renal failure rats

BACKGROUND: The present study was aimed at investigating the changes of aquaporin 2 (AQP2) expression and its underlying mechanisms in ischemic acute renal failure (ARF). METHODS: ARF was induced by clamping the both renal arteries for 60 minutes in rats. Two or seven days later, AQP2 expression and trafficking were determined in the kidney by Western blot analysis and immunohistochemistry. The activity of adenylate cyclase was also measured. RESULTS: The urinary flow rates in ARF-2 and ARF-7 day were significantly increased in association with decreases of urine osmolality. While there was decreased expression of AQP2 in the cortex, outer medulla, and inner medulla in ARF, it was most pronounced in the outer medulla. The AQP2 expression was reduced in the apical membrane-enriched fraction as well the subapical vesicle-enriched fraction in ARF; however, the degree was greater in the former than in the latter. Immunohistochemical study also showed a markedly decreased expression of AQP2 in the collecting duct in ARF. cAMP generation in response to arginine vasopressin (AVP) in the kidney was attenuated in ARF, most prominently in the outer medulla. cAMP generation in the outer medulla in response to forskolin was not affected, but sodium fluoride was significantly blunted in ARF. CONCLUSIONS: The AVP-stimulated adenylate cyclase activity is impaired in ARF, secondary to a defect at the level of the G protein. The expression of AQP2 was reduced as a consequence, which may in part account for urinary concentration defect in ARF.     

Hyperosmolality in vivo upregulates aquaporin 2 water channel and Na-K-2Cl co-transporter in Brattleboro rats

There are considerable experimental results that indicate that arginine vasopressin (AVP)-independent factors are involved in urinary concentration. This study examined the role of hyperosmolality in vivo to modulate aquaporin 2 (AQP2) and Na-K-2Cl co-transporter (NKCC2), pivotal factors in urinary concentration, in AVP-deficient Brattleboro (BB) rats. Hyperglycemia with associated hyperosmolality occurred in diabetic BB rats (BBDM). Protein abundance of AQP2 increased and was reversed by insulin in the inner medulla (IM; control 100+/-5%; BBDM 146+/-8%; BBDM+Ins 122+/-9%; P<0.001) and inner stripe of outer medulla (ISOM; control 100+/-4%; BBDM 123+/-8%; BBDM+Ins 93+/-6%; P<0.05). These results were confirmed by immunohistochemistry studies. NKCC2 rose in the ISOM but was not reversed with insulin treatment. For investigation of the role of hyperosmolality in the absence of hyperglycemia on the regulation of the expression of renal AQP and NKCC2, studies were performed with hyperosmolality that was induced by 0.5% NaCl in drinking water in BB rats. Hyperosmolality that was induced by NaCl increased significantly the protein abundance of IM AQP2 (121+/-2 versus 100+/-5%; P<0.01), ISOM AQP2 (135+/-6 versus 100+/-5%; P<0.001), cortex plus outer stripe of outer medulla AQP2 (121+/-4 versus 100+/-1%; P<0.001), ISOM NKCC2 (133+/-1 versus 100+/-4%; P<0.05), and cortex plus outer stripe of outer medulla NKCC2 (142+/-16 versus 100+/-9%; P<0.05). In conclusion, hyperosmolality, secondary to either glucose or NaCl, upregulated renal AQP2 and NKCC2 in vivo in BB rats.     

尾加压素Ⅱ及其受体mRNA在糖尿病大鼠肾脏的表达

尾加压素Ⅱ（urotensinⅡ,UⅡ）是迄今体内最强的缩血管活性肽,其缩血管效应是内皮素1（ET-1）的10倍以上。研究证明人体一种孤立G蛋白耦联受体GPR14是UⅡ的特异性受体,UⅡ与其受体构成UⅡ-UⅡ受体系统,二者特异性结合可产生多种生物学效应。本研究观察UⅡ及其受体GPR14在糖尿病大鼠肾脏的表达规律,探讨它们在糖尿病肾病肾小管损伤发生中的可能作用。     

Topographic distribution of lymphatic vessels in the normal human prostate

BACKGROUND: The lymphatic endothelial hyaluronan receptor (LYVE-1) is a specific cell surface protein in lymphatic endothelium. The antiserum against human LYVE-1 was developed and was confirmed a powerful marker of lymphatic endothelium in human organs. With this novel marker we investigated the small network of intraprostate lymphatic vessels. METHODS: To identify intraprostatic lymphatic vessels, we performed an immunohistochemical staining method using LYVE-1 pAb and von Willebrand Factor (vWF), and assessed the difference in distribution of small lymphatic vessels between the components in the prostate. RESULTS: The density of lymphatic vessel was significantly high around ejaculatory ducts and in the fibromascular area between the globular area of peripheral zone and transitional zone. Predominat lymphatic vessels distributed in the fibromascular area in the anterior and posterior prostate of extrastromal area. CONCLUSIONS: Recognition of the distinctive features of the intraprostate lymphatic network, can help the investigation of lymphatic involvement in cancer of the prostate.     

Role of aquaporin water channels in kidney function studied using transgenic mice

Several aquaporin (AQP) water transporting proteins are expressed in mammalian kidney: AQP1 in plasma membranes of proximal tubule, thin descending limb of Henle, and descending vasa recta; AQP2 in collecting duct luminal membrane; AQP3 and AQP4 in collecting duct basolateral membrane; AQP6 in intercalated cells; and AQP7 in the S3 segment of proximal tubule. To define the role of aquaporins in renal physiology, we have generated and characterized transgenic null mice deficient in AQP1, AQP3, and AQP4, individually and in combinations, as well as AQP2 mutant mice, in which the T126M mutation causing human nephrogenic diabetes insipidus was introduced. AQP1-deficient mice are polyuric and unable to concentrate their urine in response to water deprivation or vasopressin administration. AQP1 deletion greatly reduces osmotic water permeability in proximal tubule, thin descending limb of Henle, and descending vasa recta, resulting in defective proximal tubule fluid absorption and medullary countercurrent exchange. Mice lacking AQP3 have low basolateral membrane water permeability in cortical collecting duct and excrete large quantities of dilute urine. Mice lacking AQP4 have low water permeability in inner medullary collecting duct, but manifest only a mild defect in maximum urinary concentrating ability. These data, taken together with phenotype analyses of brain, lung, and gastrointestinal organs, support the paradigm that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. The renal phenotype data in aquaporin knockout mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents. Received: March 19, 2001 / Accepted: March 22, 2001     

P-31 nuclear magnetic resonance spectral changes in obstructed or dehydrated kidney

Rat kidneys subjected to urinary obstruction or dehydration in vivo both develop marked increases in the area under a peak resonating in the phosphodiester region on their P-31 nuclear magnetic resonance spectra. The chemical species responsible for these changes were assessed using physiologic manipulations which altered the concentration of phosphate in the urine or increased the urine pH. Obstructed kidneys of rats fed a normal diet had a 140 +/- 50% increase in a peak resonating at 3.31 +/- 0.05 ppm after three hours of obstruction (P less than 0.01). Low phosphate diet which decreased urine phosphate concentration by 98% virtually eliminated this increase in peak area, where saline diuresis which decreased urine phosphate concentration by 50% markedly attenuated it. Acute phosphate loading which doubled urine phosphate concentration markedly accentuated the increase in peak area. Alkalinizing the urine with acetazolamide (changing urine pH from 6.2 +/- 0.2 to 8.0 +/- 0.1) shifted the resonance frequency of this increasing peak from 3.31 +/- 0.06 to 5.45 +/- 0.11 ppm (P less than 0.01). Rats fed a normal diet developed increases (57 +/- 15%, P less than 0.05) in a peak resonating at 2.84 +/- 0.03 ppm following 48 hours of dehydration. Rats fed a low phosphate diet had a comparable increase in the relative area of this peak (46 +/- 16%, P less than 0.05). Alkalinization of the urine did not affect the position or intensity of this peak under conditions of dehydration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats

BACKGROUND: Papillary cells adapt to their hyperosmotic environment by accumulating organic osmolytes and by enhanced synthesis of heat shock protein 70 (HSP70), which protect against high-solute concentrations. Because cyclooxygenase-2 (COX-2) is expressed abundantly in the renal papilla and is induced by dehydration, and because HSP70 expression is stimulated by specific prostaglandins, COX-2 inhibition may interfere with cellular osmoadaptation. METHODS: In vivo, rats received rofecoxib before water deprivation. Medullary expression of several tonicity-responsive genes was analyzed and apoptosis was monitored by transferase-mediated dUTP nick-end labeled (TUNEL) staining and determination of papillary caspase-3 activity. In vitro, inner medullary collecting duct 3 (IMCD3) cells were exposed to hypertonic medium containing a COX-2-specific inhibitor. Thereafter, expression of tonicity-responsive genes was analyzed and resistance to high-solute concentrations was examined. Further, the effect of Delta 12-PGJ2, a urinary prostaglandin, and of HSP70 overexpression on resistance against high urea concentration, was evaluated. RESULTS: Rofecoxib treatment significantly increased urine osmolality due to higher urea concentrations, but reduced papillary HSP70 abundance by 50%. TUNEL staining showed numerous apoptotic cells in the papilla, associated with increased caspase-3 activity. These in vivo results were confirmed by experiments on cultured IMCD3 cells, in which COX-2 inhibition impaired the tonicity-induced up-regulation of HSP70 expression and rendered the cells susceptible to high urea concentrations. Furthermore, Delta 12-PGJ2 increased both HSP70 expression and resistance against high urea, which was causally linked to higher HSP70 levels. CONCLUSION: These observations support the view that chronic COX-2 inhibition reduces medullary HSP70 expression, thus rendering papillary cells susceptible to damage by high urea concentrations, especially when accompanied by dehydration.     

补肾法对卵巢切除大鼠骨组织白细胞介素-6mRNA表达的影响

目的探讨补肾法治疗绝经后骨质疏松症的分子作用环节.方法以卵巢切除大鼠作为骨质疏松实验模型,通过斑点杂交技术,在mRNA水平分析模型组、密骨片组及假手术组白细胞介素-6(IL-6)表达.通过原位杂交技术,研究了IL-6 mRNA在骨组织细胞的分布.结果模型组大鼠IL-6 mRNA表达较假手术组明显升高(P＜0.01);而密骨片组治疗后IL-6 mRNA表达明显下降(P＜0.01).IL-6 mRNA主要在成骨细胞、骨衬里细胞和骨细胞胞浆内表达.结论 IL-6在骨质疏松骨丢失过程中起重要作用;补肾法抗骨质疏松疗效部分是通过降低IL-6产生而介导的.