Disruption of cyclooxygenase-2 prevents downregulation of cortical AQP2 and AQP3 in response to bilateral ureteral obstruction in the mouse

Bilateral ureteral obstruction (BUO) in rats is associated with increased cyclooxygenase type 2 (COX-2) expression, and selective COX-2 inhibition prevents downregulation of aquaporins (AQPs) in response to BUO. It was hypothesized that a murine model would display similar changes in renal COX-2 and AQPs upon BUO and that targeted disruption of COX-2 protects against BUO-induced suppression of collecting duct AQPs. COX-2(-/-) and wild-type littermates (C57BL/6) were employed to determine COX-1, -2, AQP2, and AQP3 protein abundances and localization after BUO. In a separate series, sham and BUO wild-type mice were treated with a selective COX-2 inhibitor, parecoxib. The COX-2 protein level increased in wild-type mice in response to BUO and was not detectable in COX-2(-/-). COX-1 protein abundance was increased in sham-operated and BUO mice. Total AQP2 and -3 mRNA and protein levels decreased significantly after BUO in the cortex+outer medulla (C+OM) and inner medulla (IM). The decrease in C+OM AQP2 and -3 levels was attenuated/prevented in COX-2(-/-) mice, whereas there was no change in the IM. In parallel, inhibition of COX-2 by parecoxib rescued C+OM AQP3 and IM AQP2 protein level in wild-type mice subjected to BUO. In summary, 1) In C57BL/6 mice, ureteral obstruction increases renal COX-2 expression in interstitial cells and lowers AQP2/-3 abundance and 2) inhibition of COX-2 activity by targeted disruption or pharmacological blockade attenuates obstruction-induced AQP downregulation. In conclusion, COX-2-derived prostaglandins contribute to downregulation of transcellular water transporters in the collecting duct and likely to postobstruction diureses in the mouse.     

Renal prostaglandins in postobstructive diuresis. Comparative study of unilateral and bilateral obstruction in conscious dogs

An experimental model in conscious dogs was developed to investigate the role of prostaglandins (PG) in the obstructed kidney. Renal veins were separately catheterized. Urine flow was shunted to the skin by surgically implanted polyurethane loop ureterostomy so as to allow atraumatic manipulation with maintained continuous flow to the bladder between experiments. One week or more after surgery, renal function parameters as well as renal vein and urinary PGE2 and PGF2 alpha, and renal vein renin were studied during and after unilateral (UUO) and bilateral (BUO) ureteral obstruction. The release of ureteral obstruction produced a constant and marked elevation in urinary PGE2 and PGF2 alpha, two times higher after BUO than after UUO. A close correlation exists between PGE2 and sodium excretion in UUO and BUO. Increasing polyuria was observed only after chronic BUO. In BUO, renal vein renin concentration was augmented after 2 hours but was suppressed after 24 hours of BUO. Renal vein PG concentration was also elevated after chronic UUO and BUO but was in the normal range immediately prior to release of obstruction. The data obtained with the current experimental dog model indicate that the release of ureteral obstruction induces a striking increase in renal PGE2 and PGF2 alpha production which may mediate at least partly the phenomenon of postobstructive diuresis.     

Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.     

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.     

Effect of acute and chronic renal denervation on renal function after release of unilateral ureteral obstruction in the rat.

The role of the renal nerves in determining renal function after relief of 24-h unilateral ureteral obstruction (UUO) was studied using clearance techniques in anaesthetized rats. Acute renal denervation during the first 1--2 h after relief of UUO resulted in a significant increase in glomerular filtration rate (GFR), renal plasma flow (RPF), urine flow, and sodium and potassium excretion, changes which were not seen in the sham-denervated postobstructive kidney. Acute denervation of sham-operated normal kidneys caused a similar natriuresis and diuresis but with no change in GFR or RPF. Chronic renal denervation 4--5 days before UUO denervated postobstructive controls, while chronic denervation alone was associated with a significantly higher urine flow and sodium excretion rate from the denervated kidney. The effectiveness of renal denervation was confirmed by demonstrating marked depletion of tissue catecholamines in the denervated kidney. It was concluded that renal nerve activity plays a significant but not a major role in the functional changes present after relief of UUO. Chronic renal denervation did not protect against the functional effects of unilateral ureteral obstruction.     

Role of vasopressin in rats with bilateral ureteral obstruction

After unilateral release of bilateral ureteral obstruction (BUO), there is a significant increase in renal vasoconstriction that accounts for the marked decrease in glomerular filtration rate and effective renal plasma flow seen in this setting. We examined the potential role of antidiuretic hormone (ADH), a vasoconstrictor of the renal circulation, on renal hemodynamics in female Sprague-Dawley rats with BUO of 24-hr duration. Rats with BUO had significantly higher plasma values of ADH 65.1 +/- 12.2 vs. 12.1 +/- 4.1 pg/ml), sodium (145.4 +/- 0.91 vs 138.6 +/- 1.06 mEq/liter), and osmolality (375.6 +/- 2.0 vs 310.1 +/- 3.6 mOsm/kg) than sham-operated rats. Rats with BUO pretreated with enalapril, an angiotensin-converting enzyme inhibitor, before obstruction had somewhat higher, but not significantly different, plasma values for ADH (84.6 +/- 20.8 pg/ml) than rats with BUO not given enalapril. Rats with unilateral ureteral obstruction of 24-hr duration had plasma levels of ADH (8.2 +/- 1.3) not different from those in sham-operated rats. Rats with BUO pretreated with a specific antagonist of the V1-type receptor for ADH had significantly greater values for the glomerular filtration rate (2.31 +/- 0.24 vs 1.44 +/- 0.08 ml/min/kg body wt) and the effective renal plasma flow (8.95 +/- 0.71 vs 3.81 +/- 0.44 ml/min/kg body wt) and significantly lower values for mean arterial pressure (140.3 +/- 2.0 vs 159.1 +/- 5.5 mm Hg) than did BUO rats not given the antagonist. The results indicate that high levels of ADH play an important role in the decrease in the glomerular filtration rate and effective renal plasma flow observed in rats with BUO of 24 hr. The significant increase in ADH levels after BUO of 24-hr duration may be due to an increase in osmotic stimulation as a consequence of hypernatremia. Activation of the renin-angiotensin axis, known to occur after BUO or unilateral ureteral obstruction of 24-hr duration, does not appear to have a role in the increased circulating levels of ADH.     

依那普利在大鼠单侧输尿管梗阻再通模型中的抗纤维化作用

目的:探讨依那普利对大鼠单侧输尿管梗阻再通模型肾脏纤维化的影响.方法:18只SD大鼠随机分为两组:假手术组(6只)以及单侧输尿管梗阻模型组(12只).输尿管梗阻3天后,实施梗阻再通手术,再将大鼠随机分为模型组(6只)以及依那普利组(6只),术后,依那普利组给予依那普利灌胃10mg/kg/d,假手术组以及模型组给予等量0.5%CM-CNa溶液灌胃.用药2周后,取术侧肾组织做HE染色,并采用Raford评分系统对肾间质损伤程度进行评分;用Real-timePCR方法检测Ⅰ、Ⅲ型胶原以及CT-GFmRNA的表达;用Westemblot方法检测CTGF蛋白水平的表达.结果:模型组大鼠肾脏损伤程度,Ⅰ、Ⅲ型胶原mRNA表达水平,以及CTGFmRNA和蛋白表达水平均比假手术组明显上升(P<0.01).经依那普利治疗后,与模型组相比,以上指标均显著下降(P<0.01).结论:依那普利能有效阻止大鼠单侧输尿管梗阻再通后肾脏纤维化的进展.依那普利抗纤维化的作用机制可能与抑制CTGF的表达有关.     

羟苯磺酸钙通过抑制Ⅰ型胶原表达减轻肾间质纤维化

目的:研究羟苯磺酸钙对小鼠肾间质纤维化、Ⅰ型胶原表达的影响。方法:将C57小鼠随机分为假手术组(Sham组,n=4)、肾间质纤维化模型组(UUO组,n=5)及羟苯磺酸钙治疗组(CDT组,n=4);采用单侧输尿管梗阻制备肾间质纤维化模型,CDT组给予羟苯磺酸钙灌胃、Sham组和UUO组给予双蒸水灌胃;采用HE染色、Masson染色、免疫组化、实时定量PCR以及蛋白免疫印迹观察单侧输尿管梗阻术后14 d小鼠术侧肾脏的肾间质纤维化程度和Ⅰ型胶原表达情况。结果:与Sham组比较,UUO组小鼠术后14 d术侧肾脏肾发生显著肾间质纤维化,Ⅰ型胶原表达显著增强(Ⅰ型胶原基因相对表达量:Sham组:1.00000,UUO组:114.92289,P0.0001)。与UUO组比较,CDT组小鼠术后14 d术侧肾间质纤维化程度显著减轻,Ⅰ型胶原表达显著减弱(Ⅰ型胶原基因相对表达量:UUO组:114.92289,CDT组:45.33516,P0.005)。结论:羟苯磺酸钙通过抑制小鼠肾间质Ⅰ型胶原表达从而减轻单侧输尿管结扎小鼠肾间质纤维化。     

Angiotensin AT(1) receptor inhibition-induced apoptosis by RhoA GTPase activation and pERK1/2 signaling pathways in neonatal obstructive nephropathy

Intrarenal renin-Angiotensin system (RAS) activity is increased during early development and is further enhanced by unilateral ureteral obstruction (UUO). We studied the involvement of mitogen-activated protein (MAP) kinase members and the RhoA GTPase signaling pathways on the regulation of renal cell response after AT1 Angiotensin II receptor inhibition in obstruction. Neonatal rats subjected to sham operation or complete UUO within the first 48 hours of life received saline vehicle, Losartan (AT1 inhibitor), or PD-123319 (AT2 inhibitor) during the first 14 days of life. Cortex tubular epithelial cell apoptotic response was shown by TUNEL and confirmed by electron microscopy associated with mitochondrial signaling pathway through the increased proapoptotic ratio Bax/BcL-2, and consequently increased caspase 3 expression and activity in obstructed kidney before and after Type 1 (AT1) receptor blockade. Non injury of contralateral kidney was shown. The convergence of two independent signal pathways, the RhoA GTPase and pERK and concurrent inhibition of JNK MAP kinase, were required for the apoptotic response in 14 day kidney obstructed tubular cells either with or without Losartan treatment. Absence of increased AT2 protein expression after AT1 receptor inhibition on day 14 of obstruction was shown. Selective AngiotensinAT2-receptor inhibition with PD-123319 had no protective effect on the renal response to complete 14 day UUO. We suggest a role of both RhoA GTPase activation and the opposing actions of the ERK and JNK-MAP kinase signaling pathways as events involved in tubular cell apoptosis regulation in neonatal UUO. The selective AT1-receptor inhibition had no effect on the renal cellular response in the kidney subjected to UUO for 14 days.     

Transplantation of endothelial progenitor cells alleviates renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction

AimsThe present study investigated whether transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) in renal capillary network improves renal interstitial fibrosis in unilateral ureteral obstruction (UUO) model in mice.Main methodsEx vivo generated, characterized, and cultivated mice BM-EPCs were identified by their vasculogenic properties in vitro. BM-EPCs were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) before transplantation. The animal models of UUO were used. Histological changes in renal tubular interstitium were observed with HE and Masson staining. The protein levels of vascular endothelial growth factor(VEGF), hypoxia inducible factor-1α (HIF-1α) and connective tissue growth factor (CTGF) were analyzed by western blotting and immunohistochemistry. Transforming growth factor-β1 (TGF-β1) was detected by immunohistochemistry. Peritubular capillary (PTC) density was determined by CD31 immunostaining.Key findingsTransplanted BM-EPCs were successfully incorporated into the capillary network in the obstructed kidney in vivo. UUO induced a significant decrease in VEGF levels and PTC density in the kidney tissue, which was accompanied by a significant increase in HIF-1α, CTGF and TGF-β1. Transplantation of BM-EPCs increased PTC density, VEGF expression and alleviated the development of renal interstitial fibrosis in UUO mice. No significant pathological changes were found in control mice.SignificanceThe reduction of PTC density and up-regulation of HIF-1α are the important mechanisms of interstitial fibrosis in UUO mice. BM-EPCs transplantation may increase the number of capillary density and alleviate the development of renal fibrosis in obstructive nephropathy in mice.     

Renal elimination of organic anions in rats with bilateral ureteral obstruction

Urinary tract obstruction is an important cause of acute renal failure. Several abnormalities in renal tubular function may occur in obstructive nephropathy. The tubular secretion of organic anions is an important function of the kidney that eliminates potentially toxic organic anions from the body, however, the mechanisms involved in organic anions renal elimination in rats with bilateral ureteral obstruction (BUO) have not been elucidated. In this study, it was evaluated the renal handling of p-aminohippurate (PAH) in adult male Wistar rats with BUO. A diminished renal clearance of PAH was observed in BUO rats as consequence of a diminution in the secreted load of this organic anion. The increase in the abundance of organic anions transporter 1 (OAT1) and the absence of modification in cortical renal blood flow, measured with fluorescence microspheres, do not explain the altered secretion of PAH. The diminished Na,K-ATPase activity in cortex from obstructed kidneys might condition OAT1 function. Additionally, it is also possible to conclude that in the presence of BUO, PAH clearance is not a good estimate of renal plasma flow.     

V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells

The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.     

Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine.

Using a cDNA probe for rat renal 24-hydroxylase, expression of its mRNA was compared in the rat kidney and intestine. Vitamin D-deficient rats received a single injection of 1 alpha,25-dihydroxyvitamin D3. Expression of 24-hydroxylase mRNA was first detected in the kidney at 3-h post-injection and increased thereafter. Similarly, 24-hydroxylase mRNA was expressed in the intestine after 1 alpha,25-dihydroxyvitamin D3 injection. However, the dose level of 1 alpha,25-dihydroxyvitamin D3 required to induce the intestinal 24-hydroxylase mRNA expression was only 1/100 the amount required to induce renal 24-hydroxylase mRNA. Induction of intestinal 24-hydroxylase mRNA expression by 1 alpha,25-dihydroxyvitamin D3 was far more rapid than that of renal 24-hydroxylase mRNA. Thyroparathyroidectomy shortened the time required to induce expression of renal, but not intestinal, 24-hydroxylase mRNA. Administration of either parathyroid hormone or cAMP to vitamin D-deficient rats greatly reduced the expression of 24-hydroxylase mRNA in the kidney but not in the intestine. When rats were fed a vitamin D-repleted diet containing 0.7% (adequate) or 0.03% (low) calcium for 2 weeks, intestinal expression of 24-hydroxylase mRNA could be induced only in the low calcium group. In contrast, renal mRNA expression was preferentially stimulated in the adequate calcium group. These results clearly demonstrate that the expression of 24-hydroxylase mRNA is down-regulated by parathyroid hormone in the kidney but not in the intestine.     

Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis

The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.     

Vitronectin accumulates in the interstitium but minimally impacts fibrogenesis in experimental chronic kidney disease

Vitronectin (Vtn) is a glycoprotein found in normal serum and pathological extracellular matrix. Given its known interactions with plasminogen activator inhibitor-1 (PAI-1) and Vtn cellular receptors, especially αvβ3 integrin and the urokinase receptor (uPAR), this study was designed to investigate its role in renal fibrogenesis in the mouse model of unilateral ureteral obstruction (UUO). Kidney Vtn mRNA levels were increased ×1.8-5.1 and Vtn protein levels ×1.9-3 on days 7, 14, and 21 after UUO compared with sham kidney levels. Groups of age-matched C57BL/6 wild-type (Vtn+/+) and Vtn-/- mice (n = 10-11/group) were killed 7, 14, or 21 days after UUO. Absence of Vtn resulted in the following significant differences, but only on day 14: fewer αSMA+ interstitial myofibroblasts (×0.53), lower procollagen III mRNA levels (×0.41), lower PAI-1 protein (×0.23), higher uPA activity (×1.1), and lower αv protein (×0.32). The number of CD68+ macrophages did not differ between the genotypes. Despite these transient differences on day 14, the absence of Vtn had no effect on fibrosis severity based on both picrosirius red-positive interstitial area and total kidney collagen measured by the hydroxyproline assay. These findings suggest that despite significant interstitial Vtn deposition in the UUO model of chronic kidney disease, its fibrogenic role is either nonessential or redundant. These data are remarkable given Vtn's strong affinity for the potent fibrogenic molecule PAI-1.