Antibody to transforming growth factor-beta ameliorates tubular apoptosis in unilateral ureteral obstruction

BACKGROUND: Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-beta (TGF-beta), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-beta (1D11) in UUO. METHODS: Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-beta levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). RESULTS: Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-beta; 1D11 (10 microg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-beta as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-beta of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. CONCLUSION: The present study strongly suggests that an antibody to TGF-beta is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.     

Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats

BACKGROUND: Epidermal growth factor (EGF) markedly attenuates tubular apoptosis induced by unilateral ureteral obstruction (UUO) in the neonatal rat, and reduces apoptosis induced by mechanical stretch of cultured rat tubular cells. METHODS: To investigate the role of EGF in modulating apoptosis resulting from UUO, neonatal wild type and mutant mice lacking EGF (knockout), or with diminished EGF receptor activity (waved-2 mutant) were compared to control mice for tubular apoptosis and atrophy. Rat and mouse kidneys were compared for localization of the EGF receptor. Apoptosis was also measured in cultured mouse tubular cells subjected to stretch and exposed to EGF. RESULTS: UUO reduced endogenous renal EGF expression in wild-type mice. Unlike the rat, exogenous EGF did not decrease tubular apoptosis or atrophy in the obstructed kidney, and significantly increased stretch-induced apoptosis of cultured mouse tubular cells. Tubular apoptosis was 50% lower in the obstructed kidney of EGF knockout and waved-2 mice relative to wild type and heterozygous animals. Exogenous EGF increased tubular apoptosis and doubled atrophy in the obstructed kidney of waved-2 mice. Species differences in EGF receptor localization were detected in 3-day-old kidneys. CONCLUSION: EGF acts as a survival factor in the neonatal rat, but potentiates tubular cell death in the neonatal mouse. Species differences are maintained in cultured cells, suggesting that differences in EGF receptor signaling underlie these opposing effects.     

Smad3 deficiency attenuates renal fibrosis, inflammation,and apoptosis after unilateral ureteral obstruction

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO. METHODS: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice. RESULTS: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area. CONCLUSION: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.     

Chronic ureteral obstruction in the rat suppresses renal tubular Bcl-2 and stimulates apoptosis

Unilateral ureteral obstruction (UUO) results in widespread tubular apoptosis in obstructed kidneys of both adults and neonates. The oncoprotein bcl-2 inhibits many forms of apoptosis, whereas the related protein bax promotes apoptosis. To evaluate the interaction of bcl-2, bax, and apoptosis in the renal response to UUO, adult and neonatal rats were subjected to UUO or sham operation, and kidneys were harvested 14 days later. Apoptotic cells were identified by the Tunel technique, and the distribution of bcl-2 and bax was determined by immunochemistry. In both adults and neonates, tubular and interstitial apoptosis was present in the obstructed kidney, but not in intact kidneys. In both adults and neonates, there was diffuse tubular bcl-2 and bax staining of sham-operated and intact kidneys. While bcl-2 was increased in scattered nonapoptotic tubules of the obstructed kidney, there was minimal staining of dilated apoptotic tubules. These results are consistent with the premise that bcl-2 normally suppresses renal tubular apoptosis. The distribution of bax staining in tubules of the obstructed kidney overlapped that of bcl-2. We conclude that chronic UUO inhibits bcl-2 expression in selected tubules of the obstructed kidney which contributes to activation of apoptosis and progressive renal damage in either neonatal or adult kidneys. Dysregulation of apoptosis may be a response to renal injury similar to that underlying the development of cystic kidney disease or renal dysplasia.     

Selectins mediate macrophage infiltration in obstructive nephropathy in newborn mice

BACKGROUND: Urinary tract obstruction during development leads to tubular atrophy and causes interstitial fibrosis. Macrophage infiltration into the interstitium plays a central role in this process. Selectins, a family of three adhesion molecules, are involved in leukocyte recruitment to sites of inflammation and immune activity. We investigated the role of selectins in obstructive nephropathy in newborn mice. METHODS: Triple selectin-deficient mice (EPL-/-), L-selectin deficient mice (L-/-) and wild type mice (WT) were subjected to complete unilateral ureteral obstruction (UUO) or sham operation within the first 48 hours of life, and were sacrificed 5 and 12 days later. Kidneys were removed, and sections were stained for macrophage infiltration (mAb F4/80), apoptosis (TUNEL), tubular atrophy (periodic acid-Schiff) and interstitial fibrosis (Masson trichrome). RESULTS: Selectin deficient mice showed a marked reduction in macrophage infiltration into the obstructed kidney compared to WT at day 5 and day 12 after UUO. Tubular apoptosis was strongly reduced in EPL-/- at day 5 after UUO, and in EPL-/- and L-/- at day 12 after UUO when compared to WT. The number of apoptotic tubular cells was correlated with macrophage infiltration, suggesting that macrophages stimulate tubular apoptosis in obstructive nephropathy. In addition, tubular atrophy and interstitial fibrosis were significantly diminished in EPL-/- and L-/- compared to WT at day 12 after UUO. CONCLUSION: Following UUO, selectins mediate macrophage infiltration into the obstructed kidney, which in turn may induce tubular apoptosis, tubular atrophy and interstitial fibrosis.     

Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis.

BACKGROUND: Osteopontin is a macrophage adhesive protein that is expressed by renal tubules in tubulointerstitial disease. METHODS: To investigate the function of OPN, we induced tubulointerstitial disease in OPN null mutant (OPN-/-) and wild-type (OPN+/+) mice by unilateral ureteral ligation. Tissue was analyzed for macrophages (ED-1), types I and IV collagen deposition, TGF-beta expression, and for tubular and interstitial cell apoptosis. RESULTS: Obstructed kidneys from both OPN-/- and OPN+/+ mice developed hydronephrosis, tubular atrophy, interstitial inflammation and fibrosis. OPN was absent in OPN-/- kidneys but was increased in obstructed OPN+/+ kidneys. Macrophage influx, measured by computer-assisted quantitative immunostaining, was less in OPN-/- mice compared to OPN+/+ mice at day 4 (threefold, P < 0.02), day 7 (fivefold, P < 0.02), but not at day 14. Interstitial deposition of types I and IV collagen were also two- to fourfold less in obstructed OPN-/- kidneys (P < 0.02). There was also a reduction of TGF-beta mRNA expression in the interstitium at day 7 (by in situ hybridization) and a near significant 34% reduction in cortical TGF-beta activity (P = 0.06) compared to obstructed OPN+/+ kidneys at day 14. Obstructed kidneys from OPN-/- mice also had more interstitial and tubular apoptotic cells (TUNEL assay) compared to obstructed OPN+/+ mice at all time points. The ability of OPN to act as a cell survival factor was also documented by showing that the apoptosis of serum-starved NRK52E renal epithelial cells was markedly enhanced in the presence of neutralizing anti-OPN antibody. CONCLUSION: OPN mediates early interstitial macrophage influx and interstitial fibrosis in unilateral ureteral obstruction. OPN may also function as a survival factor for renal tubulointerstitial cells.     

Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss

BACKGROUND: To elucidate the sequence of renal responses leading to nephron loss in obstructive nephropathy, we examined the evolution of segmental nephron cellular changes consequent to chronic unilateral ureteral obstruction (UUO) in the neonatal mouse. METHODS: Neonatal mice were subjected to UUO or sham-operation, and kidneys were harvested 5, 12 or 19 days after surgery. Proximal tubules (PT), distal tubules (DT) and collecting ducts (CD) were identified with lectins. Histomorphometric quantitation was made for cellular necrosis, apoptosis, proliferation, tubular dilatation, tubular basement membrane (TBM) thickening, interstitial collagen, and glomerular maturation. The distribution of hypoxic tissue was determined using pimonidazole as a marker. Additional studies were performed by mechanically stretching monolayer cultures of mouse proximal tubular and collecting duct cells, and measuring apoptosis. RESULTS: Neonatal UUO induced an arrest of glomerular maturation throughout the period of study. Chronic UUO induced hypoxia, tubular necrosis, proliferation, and TBM thickening in the PT, but stimulated apoptosis in the DT and CD. Tubular dilation in the obstructed kidney was most severe in CD and least severe in PT. Tubular cell apoptosis closely paralleled tubular dilation (P < 0.05), and fibrosis surrounding individual tubules also correlated with tubular dilation (P < 0.001). Mechanical stretching of cultured mouse tubular cells induced apoptosis directly proportional to the magnitude of axial strain: apoptosis was consistently greater in CD than in PT cells (P < 0.05). CONCLUSIONS: Following UUO, the co-localization of hypoxia with cellular proliferation, necrosis, and TBM thickening of the PT is consistent with ischemic injury resulting from vasoconstriction. In contrast, a selective dilation of the distal portion of the nephron (DT and CD), which results from the greater tubular compliance there, leads to stretch-induced epithelial cell apoptosis, along with a progressive peritubular fibrosis. Nephron loss in the obstructed developing kidney likely results from complex, segment-specific cellular responses.     

Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction

Objectives. Acute unilateral ureteral obstruction (UUO) results in ipsilateral hydronephrosis characterized by a decrease in epidermal growth factor (EGF) mRNA expression and EGF protein levels in the distal renal tubules. UUO results in programmed cell death with increases in the characteristic markers of apoptosis. To suppress the apoptotic response during UUO, recombinant EGF was administered during renal obstruction and the ensuing molecular and histologic changes were studied.Methods. Mature Sprague-Dawley rats underwent left ureteral obstruction and the kidneys were harvested at 24, 48, and 72 hours. Markers of apoptosis included DNA laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated mRNA expression of SGP-2, and in situ hybridization for sulfated glycoprotein-2 (SGP-2) mRNA. Studies were repeated in rats following administration of 10, 20, and 40 μg of subcutaneous recombinant EGF on a daily basis after UUO.Results. Subcutaneous injection of EGF into unilaterally obstructed rats promotes renal tubular epithelial cell regeneration, as demonstrated by increased cortical mitotic activity. Systemic EGF supplementation in these unilaterally obstructed rats also resulted in a decrease in the intensity of the DNA laddering pattern associated with renal tubular apoptosis. An in situ labeling procedure to identify apoptotic nuclei in the ureterally obstructed kidneys revealed a 50% reduction in apoptosis after EGF administration. Northern blot analysis and in situ hybridization for SGP-2 mRNA or clusterin gene product also revealed a decreased expression in the obstructed and EGF-treated renal parenchyma.Conclusions. These data suggest that EGF, apart from its known role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally mature kidney.     

Telmisartan inhibits both oxidative stress and renal fibrosis after unilateral ureteral obstruction in acatalasemic mice.

BACKGROUND: Reactive oxygen species are involved in many of the angiotensin II signalling pathways. We have thus investigated whether the angiotensin II type 1 (AT1) receptor antagonist, telmisartan, can inhibit the accelerated renal fibrosis and excess oxidative stress, which occurs after unilateral ureteral obstruction (UUO) in acatalasemic mice. METHODS: The effect of daily intraperitoneal injection of telmisartan (0.1-0.3 mg/kg body weight) on the renal tubulointerstitial injury induced by UUO has been studied in homozygous acatalasemic mutant mice (C3H/AnLCs b Cs b) and wild-type mice (C3H/AnLCs a Cs a). We evaluated the systemic blood pressure of the mice on the seventh day. In addition, the tubulointerstitial expression of collagens type I and type IV, the p22-, p47- and p67-phox subunits of NADPH oxidase, 4-hydroxy-2-nonenal, and 4-hydroxy-2-hexenal lipid peroxidation products were assessed by immunohistochemistry. The level of apoptosis was determined by terminal deoxynucleotidyl transferase nick end-labelling analysis, while the mRNA level of the p22-, p47- and p67-phox subunits was quantified by real-time PCR. The renal content of each of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase was determined by specific assay. RESULTS: Obstructed kidneys from acatalasemic mice exhibited increased tubulointerstitial deposition in dilated tubules of collagens type I and IV, lipid peroxidation products, and the p22/p47/p67-phox subunits of NADPH oxidase. The level of the p22/p47/p67-phox subunit mRNA, and of apoptosis in tubular epithelial cells, was also increased compared with those from wild-type kidneys. Treatment with telmisartan attenuated all of the changes and prevented renal fibrosis in a dose-dependent manner; despite the low dose (0.1 mg/kg). The treatment did not lower the systemic blood pressure. The catalase activity remained low in acatalasemic obstructed kidneys without compensatory upregulation of glutathione peroxidase or superoxide dismutase activity; the level of neither anti-oxidant enzymes in obstructed kidneys was affected by telmisartan. CONCLUSIONS: The AT1 receptor antagonist telmisartan ameliorated renal fibrosis after UUO by inhibition of oxidative stress, even under acatalasemic conditions.     

Renal structural and functional repair in a mouse model of reversal of ureteral obstruction

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57bl/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.     

Osteopontin regulates renal apoptosis and interstitial fibrosis in neonatal chronic unilateral ureteral obstruction

Congenital obstructive nephropathy is a major cause of renal insufficiency in children. Osteopontin (OPN) is a phosphoprotein produced by the kidney that mediates cell adhesion and migration. We investigated the role of OPN in the renal response to unilateral ureteral obstruction (UUO) in neonatal mice. OPN null mutant (-/-) and wild-type (+/+) mice were subjected to sham operation or UUO within the first 2 days of life. At 7 and 21 days of age, fibroblasts (fibroblast-specific protein (FSP)-1), myofibroblasts (alpha-smooth muscle actin (SMA)), and macrophages (F4/80) were identified by immunohistochemical staining. Apoptotic cells were detected by terminal deoxy transferase uridine triphosphate nick end-labeling technique and interstitial collagen by Masson trichrome or picrosirius red stain. Compared to sham-operated or contralateral kidneys, obstructed kidneys showed increases in all parameters by 7 days, with further increases by 21 days. After 21 days UUO, there was an increase in tubular and interstitial apoptosis in OPN -/- mice as compared to +/+ animals (P<0.05). However, FSP-1- and alpha-SMA-positive cells and collagen in the obstructed kidney were decreased in OPN -/- compared to +/+ mice (P<0.05), whereas the interstitial macrophage population did not differ between groups. We conclude that OPN plays a significant role in the recruitment and activation of interstitial fibroblasts to myofibroblasts in the progression of interstitial fibrosis in the developing hydronephrotic kidney. However, OPN also suppresses apoptosis. Future approaches to limit the progression of obstructive nephropathy in the developing kidney will require targeting of specific renal compartments.     

Dietary arginine supplementation attenuates renal damage after relief of unilateral ureteral obstruction in rats

BACKGROUND: Progression of renal injury after relief of unilateral ureteral obstruction (UUO) has been demonstrated. Nitric oxide (NO) may be an effective intervention due to its vasodilatory, antifibrotic, and anti-apoptotic effects. Herein, we used dietary L-arginine (ARG) supplementation in a UUO relief model. METHODS: This study comprised group 1, control (no treatment). All other rats were subject to 3-day UUO, which was then relieved, and the rats maintained for 7 additional days. Group 2, no additional treatment; group 3, L-ARG; group 4, L-NAME, NO synthase inhibitor; group 5, ARG and L-NAME. Urinary NO(2/3) was quantified. GFR and ERPF were measured at day 10. Interstitial fibrosis and fibroblast expression, macrophage infiltration, tubular apoptosis, and proliferation, NOS expression, and the levels of tissue TGF-beta were evaluated. RESULTS: Urinary NO(2/3) was significantly increased by ARG treatment and decreased by L-NAME. GFR and ERPF measured 7 days following relief were not significantly different in the previously obstructed kidneys (POK) of groups 2 and 3. L-NAME significantly reduced GFR and ERPF in the POK. ARG significantly reduced apoptosis, macrophage infiltration, and fibroblast expression in the POK. L-NAME exacerbated the effects on apoptosis and fibroblasts. Fibrosis was minimal in groups 1 through 3, but was significantly increased by L-NAME. ARG did not affect renal NOS expression and tissue TGF-beta1 levels. CONCLUSION: Dietary ARG supplementation during UUO relief did not improve ERPF or GFR. However, renal damage, including fibrosis, apoptosis, and macrophage infiltration was significantly improved by ARG treatment. This suggests that increasing NO availability could be beneficial in the setting of UUO relief.     

Macrophage infiltration and renal damage are independent of matrix metalloproteinase 12 in the obstructed kidney

Aim: To determine whether matrix metalloproteinase‐12 (MMP‐12) plays a functional role in renal interstitial macrophage accumulation, interstitial fibrosis or tubular apoptosis in the unilateral ureteric obstruction (UUO) model. Background: MMP‐12 is an enzyme that can cleave a number of extracellular matrix proteins and plays a role in macrophage‐mediated injury in experimental models of emphysema and antibody‐dependent glomerular disease. Macrophages are thought to promote renal fibrosis and tubular damage in the obstructed kidney. Furthermore, upregulation of MMP‐12 expression by infiltrating macrophages in the obstructed kidney has been described, but the potential role of MMP‐12 in renal injury induced by this non‐immune insult is unknown. Methods: Groups of eight MMP‐12 gene deficient (MMP‐12^?/?) and wild type (WT) C57BL/6J mice were killed 3, 7 or 14 days after UUO. Results: Analysis of three different lineage markers found no difference in the degree of interstitial macrophage accumulation between MMP‐12^?/? and WT UUO groups at any time point. Examination of renal fibrosis by total collagen staining, α‐SMA + myofibroblast accumulation, and TGF‐β1, PAI‐1 and collagen IV mRNA levels showed no difference between MMP‐12^?/? and WT UUO groups. Finally, tubular damage (KIM‐1 levels) and tubular apoptosis (cleaved caspase‐3) in the obstructed kidney was not affected by MMP‐12 gene deletion. Conclusion: In contrast to lung injury and antibody‐dependent glomerular injury, MMP‐12 is not required for renal interstitial macrophage accumulation, interstitial fibrosis or tubular damage in the obstructed kidney.     

Effects of ICAM-1 antisense oligonucleotide on the tubulointerstitium in mice with unilateral ureteral obstruction

Effects of ICAM-1 antisense oligonucleotide on the renal tubulointerstitium in mice with unilateral ureteral obstruction. BACKGROUND: To extend our previous study of the therapy of the renal lesions of unilateral ureteral obstruction (UUO) in mice by an inhibitor of intercellular adhesion molecule-1 (ICAM-1), we investigated the blocking effects of ICAM-1 antisense oligonucleotides (ASONs) on the ICAM-1 expression in mouse kidney. METHODS: First, ICAM-1 ASON was transducted into mouse renal tubular epithelial cells to investigate the effects of ICAM-1 ASON in vitro. Second, fluorescein isothiocyanate (FITC)-labeled ICAM-1 ASON was injected intravenously to determine the distribution of the ASON in vivo. Third, the expression of ICAM-1 in kidney and the changes of renal morphology were observed to investigate the therapeutic effects of ICAM-1 ASON on the UUO mice in vivo. RESULTS: The expressions of ICAM-1 in the epithelial cells induced by interleukin-1beta were inhibited by ICAM-1 ASON at the dosages of 100 and 200 nmol/L. Twenty-four hours after an introvenous injection with FITC-labeled ICAM-1 ASON, the highest level of fluorescein was detected within the proximal tubules in mouse kidney. Results of immunohistology and Northern blot showed that the ICAM-1 expression was markedly reduced in the obstructed kidney after treatment with ICAM-1 ASON. The ASON also alleviated the infiltration of inflammatory cells and accumulation of the extracellular matrix in the tubulointerstitium of UUO mice without apparent side effects. CONCLUSION: Our data demonstrate that ICAM-1 ASON is taken up primarily by the proximal tubular cells of mouse kidney. ICAM-1 ASON can selectively inhibit the ICAM-1 expression of the renal tubular cells both in vitro and in vivo.     

The role of autophagy in unilateral ureteral obstruction rat model

Aim: Autophagy is a cellular process of degradation of damaged cytoplasmic components and regulates cell death or proliferation. Unilateral ureteral obstruction (UUO) is a model of progressive renal fibrosis in the obstructed kidney. And UUO is followed by compensatory cellular proliferation in the contralateral kidney. We investigate the role of autophagy in the obstructed kidney and contralateral kidney after UUO. Methods: To obtain the evidence and the patterns of autophagy during UUO, the rats were sacrificed 3, 7 and 14 days after UUO. To examine the efficacy of the autophagy inhibitors, 3‐methyladenine (3‐MA), the rats were treated daily with intraperitoneal injection of 3‐MA (30 mg/kg per day) for 7 days. Results: After UUO, autophagy was induced in the obstructed kidney in a time‐dependent manner. Inhibition of autophagy by 3‐MA enhanced tubular cell apoptosis and tubulointerstitial fibrosis in the obstructed kidney after UUO. In the contralateral kidney, autophagy was also induced and prolonged during UUO. Inhibition of autophagy by 3‐MA increased the protein expression of proliferating cell nuclear antigen significantly in the contralateral kidney after UUO. The Akt‐mammalian target of rapamycin (mTOR) signalling pathway was involved in the induction of autophagy after UUO in both kidneys. Conclusion: Our present results support that autophagy induced by UUO has a renoprotective role in the obstructed kidney and regulatory role of compensatory cellular proliferation in the contralateral kidney through Akt‐mTOR signalling pathway.     

GW274150, a potent and highly selective inhibitor of iNOS,reduces experimental renal ischemia/reperfusion injury

BACKGROUND: Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R. METHODS: Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured. RESULTS: GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation. CONCLUSIONS: These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.     

Apoptosis of circulating lymphocyte in rats with unilateral ureteral obstruction: role of angiotensin II

BACKGROUND: Unilateral ureteral obstruction (UUO) could induce increased renal angiotensin II (ANG II), which enhances apoptosis of renal tubular cells and renal tissue loss. Systemic ANG II is also increased in UUO. There are no data available about whether UUO can induce apoptosis of circulating lymphocytes or not. METHODS: UUO or sham-operated male Wistar rats (n = 8 in each group) were fed a drinking solution containing water, angiotensin II receptor type 1 antagonist (ARA; losartan, 500 mg/L) or angiotensin-converting enzyme inhibitor (ACEI; enalapril: 200 mg/L) for 1 day or 7 days. Blood samples were collected and circulating lymphocyte cells were separated. The apoptotic cells were detected by in situ terminal deoxynucleotidyl transferase (TdT assay)-mediated digoxigenin/antidigoxigenin conjugated fluorescein method and counted under a fluorescence microscope. The apoptotic index was calculated. RESULTS: UUO caused marked increases in the apoptotic index of circulating lymphocytes in UUO rats at both 1 day and 7 days when compared with the respective sham groups (P < 0.001). Neither ARA nor ACEI treatment had an effect on the apoptotic index values in the UUO rats at 1 day. In the UUO rats at 7 days, the apoptosis of circulating lymphocytes was markedly decreased from 29.2 +/- 2.7% to 11.9 +/- 2.7% (P < 0.01) in the ARA-treated rats and to 7.6 +/- 2.7% (P < 0.001) in the ACEI-treated rats. CONCLUSION: UUO, via stimulation of ANG II, could promptly enhance apoptosis of circulating lymphocytes. The apoptosis persisted throughout the 7 days of the study. Prolonged UUO would impair lymphocyte cell immunity and the host defense mechanism. Continuous treatment with either ARA or ACEI could abrogate ANG II-stimulated circulating lymphocyte apoptosis.