Progression after release of obstructive nephropathy

Progressive glomerular and tubulointerstitial fibrosis develop in 1-year-old rats even after relief (R) of unilateral ureteral obstruction (UUO) at 5 days of age. The present study investigated whether a progressive renal injury model of UUO could be achieved after reversal of UUO (RUUO) in adult instead of neonatal rats. The potential for α-tocopherol modulation of mRNA for the fibrogenic cytokine, transforming growth factor-β1 (TGFβ1), apoptosis (TUNEL assay), and the presence of the stress protein, heat shock protein-70 (HSP-70), was also studied in this post-obstructive model. Male Sprague-Dawley rats weighing 125–150 g were randomly assigned to groups of 4 animals each for durations of 7 or 14 days of α-tocopherol supplementation after RUUO. The groups included: (i) sham, regular chow; (ii) RUUO, regular chow; (iii) RUUO, contralateral nephrectomy (NX); and (iv) RUUO, NX plus α-tocopherol supplementation. We found a significant increase in the ratio of kidney weight/body weight in the RUUO+NX group at 14 days compared with the sham and RUUO groups. This rise in the RUUO+NX group was significantly reduced after 14 days of α-tocopherol administration. The elevated level of kidney TGFβ1 mRNA in the RUUO+NX group was only partially reduced at 7 days. But at 14 days this became significantly reduced with the continued α-tocopherol treatment. The HSP-70 staining and the apoptosis of the kidney showed results parallel to those of TGFβ mRNA at 14 days. To separate the effects of hypertrophy after unilateral NX from the RUUO studies, we carried out a second experiment in control animals subjected to NX, with and without α-tocopherol supplementation. Fourteen days after NX, the apoptosis and TGFβ1 mRNA showed no significant differences from the control animals. Our data suggest that a model of progressive renal injury in RUUO can be established in adult rats. After contralateral NX, the progressive injury is evidenced by the increase in the ratio of kidney weight/total body weight, the apoptotic counts, as well as fibrogenic cytokine TGFβ1 mRNA in the post- obstructed kidney. Finally, our data also support the concept that α-tocopherol is renal protective, as judged by TGFβ1 mRNA, apoptosis, and HSP-70 staining, even in the progressive disease process of the post-obstructed model. Received: 17 February 2000 / Revised: 21 September 2000 / Accepted: 21 September 2000     

Protein Kinase C-ζ is Critical in Pancreatitis-Induced Apoptosis of Kupffer Cells

Protein kinase C-zeta (PKC-ζ) regulates cell death via NF-κB; therefore, we tested the hypothesis that PKC-ζ plays a critical role in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in rats by cerulein injection 24 h later, livers were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, and apoptosis was assessed with Caspase-3 and DNA fragmentation. Kupffer cells from unoperated rats were infected with a PKC-ζ domain-negative adenovirus (AdPKCζ-DN) to inhibit PKC-ζ, or transfected with pCMVPKC-ζ to overexpress PKC-ζ, and then stimulated with pancreatic elastase; cellular extracts were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation. Cerulein-induced pancreatitis upregulated PKC-ζ protein and activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3 and increased DNA fragmentation in rat livers (all p < 0.001 vs control). AdPKCζ-DN abolished elastase-induced upregulation of PKC-ζ activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3 and DNA fragmentation (all p < 0.001 vs infection control), whereas overexpression of PKC-ζ augmented elastase-induced upregulation of IKKβ, IKKγ, Fas/FasL, Caspase-3 and DNA fragmentation (p < 0.001 vs control). PKC-ζ plays a critical role in pancreatitis-induced Kupffer cell apoptosis via NF-κB and Fas/FasL. The ability of Kupffer cells to autoregulate their stress response by upregulating their death receptor/ligand and key proapoptotic cell signaling systems warrants further investigation. Presented as a poster during the Annual Meeting of the SSAT, Los Angeles, May 2006.     

Application of gene therapy to kidney diseases

There is little doubt that molecular biological intervention therapy has come of age, and tremendous excitement has emerged from its potential. A gene transfer technique, the HVJ liposome method, is now applicable to analysis of the molecular aspects of the pathophysiology of renal diseases and is a tool for gene therapy. We demonstrated that overexpressed transforming growth factor-β (TGF-β) in the normal rat glomeruli by gene transfer of TGF-β cDNA leads to glomerulosclerosis. Inhibition of TGF-β action by direct introduction of antisense oligodeoxynucleotides (ODNs) into glomeruli could suppress glomerular matrix accumulation in experimental glomerulonephritis. In addition, continuous delivery of decorin or chimeric soluble TGF-β receptor from gene-transferred skeletal muscle also inhibits extracellular matrix expansion in experimental glomerulonephritis. We examined whether in vivo gene transfer of antisense ODNs for TGF-β into interstitial fibroblasts can suppress the progression of interstitial fibrosis in unilateral ureteral obstruction model rats. Introduction of TGF-β1 antisense ODNs significantly reduced levels of TGF-β1 and type I collagen mRNA expression in obstructed kidneys and consequently suppress the progression of interstitial fibrosis following ureteral obstruction. Taken together with clinical observations that upregulation of TGF-β plays an important role in the formation of various kidney fibrosis, manipulation of the overexpression of TGF-β may provide a novel therapeutic intervention to ameliorate the progression of renal disease. Received: June 7, 1999 / Accepted: June 21, 1999     

Protein Kinase C-Zeta (PKC-ζ) Regulates Kupffer Cell Apoptosis During Experimental Sepsis

Background Kupffer cells play an important role in sepsis-mediated liver injury. We tested the hypothesis that PKC-ζ plays a critical role in Kupffer cell apoptosis during sepsis. Methods Sepsis was induced in rats by cecal ligation and puncture (CLP); 12 h later, livers were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation. Kupffer cells from control rats were infected with AdPKC-ζ DN to inhibit PKC-ζ, or transfected with pCMVPKC-ζ to overexpress PKC-ζ, and then treated with lipopolysaccharide (LPS). Cellular extracts were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation. Results During sepsis, PKC-ζ localized in cells positive for the macrophage marker (F4/80). CLP upregulated PKC-ζ protein and activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and increased DNA fragmentation in rat livers (all p < 0.001). AdPKC-ζ DN attenuated the LPS-induced upregulation of PKC-ζ activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation in Kupffer cells (all p < 0.001), whereas overexpression of PKC-ζ augmented LPS-induced upregulation of IKKβ, IKKγ, NF-κB, Caspase-3, and DNA fragmentation (p < 0.001). Conclusion PKC-ζ plays an important role in sepsis-induced apoptosis of Kupffer cells via activation of NF-κB and Fas/FasL. Manipulating the response of Kupffer cells to cellular stress may have important therapeutic implications. Presented as a poster during Digestive Disorders Week, Washington, DC, May 2007.     

Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1

BACKGROUND: The administration of insulin-like growth factor-1 (IGF-1) has been shown to ameliorate the renal injury resulting from ischemic acute renal failure. As there are a number of similarities between acute renal failure and obstructive nephropathy, we examined the effects of IGF-1 on the renal cellular response to unilateral ureteral obstruction (UUO) in the neonatal rat. METHODS: Forty-five rats were subjected to UUO or sham operation within the first 48 hours of life and received IGF-1 (2 mg/kg/day) or saline for the following three or seven days, after which kidneys were removed for study by morphometry and immunohistochemistry. To determine the effects of UUO on endogenous expression of IGF-1 and its receptor, six additional rats were subjected to UUO or sham operation, and mRNA was measured by solution hybridization. RESULTS: There was no effect of seven days of UUO on the renal expression of endogenous IGF-1 or its receptor. Moreover, seven days of exogenous IGF-1 did not improve the suppression of nephrogenesis, the delay in glomerular maturation, or the reduction in tubular proliferation induced by ipsilateral UUO. However, in the obstructed kidney, IGF-1 reduced tubular expression of vimentin, apoptosis, and tubular atrophy by 38 to 50% (P < 0.05). In addition, IGF-1 also decreased renal interstitial collagen deposition in the obstructed kidney by 44% (P < 0.05). Following three days of UUO, the administration of IGF-1 also reduced tubular apoptosis (P < 0.05), but did not alter tubular proliferation. CONCLUSIONS: IGF-1 has a profound salutary effect on the tubular and interstitial response to UUO in early development, without affecting glomerular injury or development. These results suggest that IGF-1 may have therapeutic potential in the management of congenital obstructive nephropathy.     

Oxidation of lipoprotein in injury and repair of obstructive nephropathy

Background. To test the hypothesis that oxidized low density lipoprotein (oLDL) is involved in the renal injury of obstructive nephropathy, male Sprague-Dawley rats weighing 125–150 g were used. Methods. Three days after arrival, the rats were randomly assigned to undergo: (1) sham operation, (2) left unilateral ureteral obstruction (UUO), or (3) reversal of the unilateral ureteral obstruction (R-UUO). Seven days after the reversal operation or 10 days after the sham or UUO procedure, all animals were killed by exsanguination under anesthesia, with blood taken from the abdominal aorta. LDL was prepared by gradient ultracentrifugation and used immedi-ately after isolation. Rat mesangial cells were utilized with an LDL concentration of 100 μg/ml/protein in the media. After 72 h, cell survival was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method. Cell survival was determined by comparing the optical density between the control wells and the experimental wells. In order to investigate the mechanisms of injury and repair of obstructive nephropathy, data for kidney apoptosis, malondialdehyde (MDA), and transforming growth factor β1 (TGFβ1) mRNA were obtained in the sham-operated, UUO, and R-UUO groups. Results. Our results showed that LDL malondialdehyde during UUO was increased 87% over baseline values (P < 0.005). With R-UUO, the oxidized LDL dropped 23% from the peak values during UUO (P < 0.005), but was still different from that of the baseline values (P < 0.025). Rat mesangial cell survival, after 72 h exposure to oLDL, inversely correlated to oLDL cytotoxicity and showed a 14% drop during UUO compared with sham-operated animals (P < 0.01). Cell survival increased 11% after R-UUO (P < 0.02) and was not different from control values (P = NS). The apoptotic counts by the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) technique showed significant increases during UUO and a noticeable reduction after R-UUO. Conclusion. Our data support the proposition that UUO stimulates oxidation of LDL. The cytotoxicity of oLDL plays a significant role in the injury of UUO. A decrease in cytotoxicity was associated with the repair in R-UUO. Our observations that apoptosis follows this same pattern, point to the importance of apoptosis in the injury and repair of obstructive nephropathy. Future studies to interrupt these processes of injury may lead to novel treatment modalities in reversing the injury and hastening the repair of obstructive nephropathy. Received: January 26, 1999 / Accepted: April 23, 1999     

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.     

Molecular and cellular pathophysiology of obstructive nephropathy

Congenital obstructive nephropathy remains one of the most-important causes of renal insufficiency in children. This review focuses on the unique interactions that result from urinary tract obstruction during the period of renal development in the neonatal rodent. Following unilateral ureteral obstruction (UUO), growth of the obstructed kidney is impaired and compensatory growth by the intact opposite kidney is related directly to the duration of obstruction. Development of the renal vasculature is delayed by UUO, and the activity of the intrarenal renin-angiotensin system is enhanced throughout the period of obstruction. Glomerular maturation is also delayed by UUO, and nephrogenesis is permanently impaired. The effects of UUO on the developing tubule are also profound, with a suppression of proliferation, stimulation of apoptosis, and the maintenance of an immature phenotype by tubular epithelial cells. Expression of tubular epidermal growth factor is suppressed and transforming growth factor-β1 and clusterin are increased. Maturation of interstitial fibroblasts is delayed, with progression of tubular atrophy and interstitial fibrosis resulting in part from continued activation of the renin-angiotensin system and oxygen radicals. Future efforts to prevent the consequences of congenital urinary tract obstruction must account for the dual effects of obstruction: interference with normal renal development and progression of irreversible tubulointerstitial injury. Received: 9 September 1998 / Revised: 9 November 1998 / Accepted: 13 November 1998     

Altered expression of immune modulator and structural genes in neonatal unilateral ureteral obstruction

BACKGROUND: Congenital obstructive nephropathy is a condition characterized by hydronephrosis, tubular dilatation, apoptosis, and atrophy, as well as interstitial cellular infiltration and progressive interstitial fibrosis. The renal consequences of chronic unilateral ureteral obstruction (UUO) in the neonatal rat are similar to those of clinical congenital obstructive nephropathy. METHODS: To define alterations in renal gene expression induced by chronic neonatal UUO, Sprague-Dawley rats were subjected to UUO or sham operation within the first 2 days of life, and kidneys were harvested after 12 days. RESULTS: Microarray analysis revealed that the mRNA expression of multiple immune modulators, including krox24, interferon-gamma regulating factor-1 (IRF-1), monocyte chemoattractant protein-1 (MCP-1), interleukin-1beta (IL-1beta), CCAAT/enhancer binding protein (C/EBP), p21, c-fos, c-jun, and pJunB, was significantly increased in obstructed compared to sham-operated kidneys (all P < 0.05). Western blot analysis revealed significant changes in immune modulator protein abundance in the obstructed versus sham-operated kidney for krox24 (P = 0.0004), IRF-1 (P = 0.005), MCP-1 (P = 0.01), and JunD (P = 0.0008). Alternatively, the abundance of all of the immune modulator proteins was similar in sham-operated and obstructed kidneys in rats subjected to acute (4 days) neonatal UUO. Microarray analysis studies also reveal that structural genes that comprise the cytoskeleton and cell matrix are significantly up-regulated by chronic neonatal UUO, including calponin, desmin, dynamin, and lumican (all P < 0.05). CONCLUSION: Multiple genes are aberrantly expressed in the kidney of rats subjected to chronic neonatal UUO. Elucidation of these genes involved in neonatal UUO may lead to new insight about congenital obstructive nephropathy.     

Transient urethral obstruction predisposes to ascending pyelonephritis and tubulo-interstitial disease: studies in rats

Chronic tubulo-interstitial disease, an important cause of end-stage renal disease, often results from the combined effects of a disturbed urinary outflow tract and urinary tract infection. Acute unilateral ureteral obstruction in rats rapidly induces foci of medullary necrosis, confined to the region of the papilla and fornices. This injury may provide a nidus for bacterial invasion and may invoke reactive and regenerative changes, ultimately leading to chronic pyelonephritis and tubulo-interstitial nephropathy. To explore this possibility, adult rats underwent renal morphological evaluation 2–7 days following transient 24-h unilateral ureteral obstruction. In some experiments the bladder was inoculated with bacteria (10⁸–10⁹ cfu/ml Escherichia coli in 0.5 ml) after release of ureteral obstruction, with subsequent cultures obtained from the pelvis of both kidneys and from the urinary bladder. Morphologic evaluation of perfusion-fixed kidneys, 2–7 days after the release of 24-h ureteral obstruction disclosed papillary necrosis, urothelial proliferation, marked inner-stripe interstitial expansion, and fibrosis and proximal tubular (S₃) dilatation. The lateral (perihilar region) was predominantly affected, with lesions spreading from the fornices. There was some progression of interstitial fibrosis during the postobstructive time course or following more prolonged ureteral obstruction. By contrast, infection hardly contributed to the tubulointerstitial changes. In rats subjected to infection, cultures were positive in all 15 postobstructive kidneys, as opposed to five contralateral kidneys (P < 0.0001). Viable counts from the postobstructive kidney were also higher than those from the contralateral side (79,000 ± 12,000 vs 2900 ± 1600 cfu/ml, mean ± SEM, P < 0.0001), and were comparable to those obtained from the bladder (77,000 ± 13,000 cfu/ml). We conclude that transient ureteral obstruction predisposes to ascending pyelonephritis and to tubulointerstitial disease. This vulnerability may relate to altered urodynamics and medullary tissue destruction. Received: 28 December 1999 / Accepted: 28 September 2000     

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.     

Expression of fibrosis-associated molecules in IgA nephropathy treated with cyclosporine

Cyclosporine (CsA) treatment in immunoglobulin A nephropathy (IgAN) is controversial and has not been widely studied. The aim of this study was to investigate the effects of CsA on renal histology and the expression of interstitial fibrosis-associated molecules in childhood IgAN. The subjects were 18 children (age 4.2–13.9 years; male:female 13:5) who had been treated with CsA for 8 or 12 months and who had renal biopsies before and after treatment. Renal biopsies were assessed by routine histology and immunohistochemistry against osteopontin (OPN), transforming growth factor-β (TGF-β), CD68, and CD34. The degree of proteinuria and mesangial IgA deposits decreased or disappeared after treatment in all cases, and the percentage of patients with diffuse mesangial proliferation decreased from 44.4 to 22.2%. However, interstitial fibrosis developed or was aggravated in nine patients (50%) after treatment and was associated with an increased degree of interstitial inflammation in five patients. Tubular OPN expression (45.3 ± 23.4 vs. 37.6 ± 19.3%) and the degree of CD68-positive macrophage infiltration (136.1 ± 88.2 vs. 132 ± 86.0/mm²) were not increased after CsA treatment, but TGF-β expression was significantly increased (6.4 ± 4.2 vs. 13.3 ± 9.9%; p = 0.025). Microvascular density was increased and peritubular capillaries were of small caliber in inflamed areas. We conclude that increased levels of TGF-β and the development of interstitial fibrosis limit the long-term use of CsA in IgAN patients. Osteopontin and macrophages may be indirectly involved in renal fibrosis by prolonging interstitial inflammation rather than by directly increasing TGF-β expression.     

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.     

Ureteral obstruction in the neonatal rat: Renal nerves modulate hemodynamic effects

In the neonate, chronic unilateral ureteral obstruction (UUO) reduces renal blood flow (RBF) of the ipsilateral kidney and increases RBF of the opposite kidney. To determine whether renal nerves mediate or modulate these responses complete left UUO in the neonatal rat was used as a model of severe obstructive uropathy, and was compared with sham-operated controls. At 24–28 days of age, animals underwent left or right mechanical renal denervation or left sham renal denervation. One week after denervation, animals were anesthetized and blood pressure and heart reate were measured. Cardiac output and RBF were determined by the radioactive microsphere technique. UUO increased blood pressure and heart rate, and decreased RBF in the obstructed kidney, regardless of denervation. While left UUO increased RBF to the intact opposite kidney in rats with left renal denervation, this was attenuated by right renal denervation. Thus, in the neonatal rat, UUO modulates systemic renal hemodynamics, possibly through activation of the renin-angiotensin system. While renal nerves do not mediate the vasoconstriction of the obstructed kidney, renal nerves modulate vascular tone of the kidney contralateral to UUO.     

α-Tocopherol modulates lipoprotein cytotoxicity in obstructive nephropathy

Oxidative stress in unilateral ureteral obstruction (UUO) contributes to the development of glomerular and tubulointerstitial lesions. The present study investigated whether oxidized low-density lipoprotein (oLDL) contributes to the pathogenesis of kidney injury in UUO, and whether α-tocopherol modulates such cytotoxicity and promotes repair. Male Sprague-Dawley rats weighing 100–125 g were assigned to three groups of 6 animals each: (1) sham, regular chow; (2) UUO, regular chow; and (3) UUO, α-tocopherol supplementation. We found a significant increase in the level of oxidative stress in the UUO group as measured by malondialdehyde (MDA) content in both plasma and kidneys. The LDL isolated from this group was cytotoxic to rat mesangial cells. The level of oxidation and cytotoxicity was significantly reduced when animals were treated with α-tocopherol. Plasma cholesterol concentration, kidney MDA, and transforming growth factor β1 mRNA expression were all significantly increased in the UUO animals, and partially reduced in α-tocopherol-treated animals. Our data suggest that oxidative modification of LDL is associated with the renal injury in UUO. Taken together, our data support the concept that α-tocopherol can modulate LDL oxidation and its cytotoxic effects on rat mesangial cells in vitro. Received: 23 July 1999 / Revised: 1 November 1999 / Accepted: 1 November 1999     

Expression of α5β1 and α6β1 integrins in IgA nephropathy (IgAN) with mild and severe proteinuria. An immunohistochemical study

Immunoperoxidase staining was carried out using monoclonal antibodies against integrin α5β1 and integrin α6β1 on renal biopsy specimens from patients with IgA nephropathy with mild proteinuria (n = 15) and with severe proteinuria (n = 10). Our study revealed increase in glomerular immunoexpression of α5β1 in renal biopsies in IgA nephropathy with severe proteinuria. There were no statistical differences between interstitial α5β1 immunostaining, as well astubular α6β1 immunoexpression in renal tissue between patients with mild and severe proteinuria. The intensity of interstitial α5β1 integrin immunoexpression was positively correlated with the degree of interstitial fibrosis in both studied groups, meanwhile the intensity of tubular α6β1 integrin immunoexpression was not related to the degree of renal interstitial fibrosis in patients with mild and severe proteinuria. Our results suggest that elevated immunoexpression of α5β1 integrin on endothelial glomerular cells in the renal tissue in patients with severe proteinuria may point to the role of this integrin in the mechanism of glomerular injury in these cases of IgA nephropathy. The positive association between the interstitial expression of α5β1 integrin and the relative interstitial cortical volume in renal biopsies in patients with mild and severe proteinuria suggests that α5β1 integrin may play a role in the pathogenesis of chronic progressive renal disease in both studied group. Lack of positive correlation between tubular α6β1 integrin immunoexpression and the relative interstitial cortical volumen may indicate that this molecule play no role in the patomechanism of interstitial fibrosis in IgA nephropathy with mild and severe proteinuria. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapamycin attenuates unilateral ureteral obstruction-induced renal fibrosis

Unilateral ureteral obstruction (UUO) is a well-characterized hydronephrosis model exhibiting interstitial inflammatory-cell infiltration and tubular dilatation followed by tubulointerstitial fibrosis of the obstructed kidney. Our recent report indicates that rapamycin is effective for 50% of transplant recipients with chronic allograft nephropathy. In this study, we investigate the effect of rapamycin on UUO-induced renal fibrosis. UUO or sham-operated rats were randomly assigned to rapamycin or vehicle and were killed on days 7 and 14 after UUO or sham operation. Rapamycin decreased cross-sectional and gross-morphology changes in the obstructed kidney significantly. Rapamycin markedly blunted the increase in weight of the obstructed kidney, obstructed kidney length, and the obstructed/non-obstructed kidney weight ratio (by 74.6, 42.8, and 61.6% on day 14, respectively, all P<0.01). The scores for tubular dilatation, interstitial volume, interstitial collagen deposition, and alpha-smooth muscle actin (alpha-SMA) after UUO were significantly reduced by rapamycin. Rapamycin also decreased the number of infiltrative anti-ED1-positive cells and the gene expression of transforming growth factor (TGF)-beta1 (84.8 and 80.2% on day 7) after UUO (both P<0.01). By double immunostaining and Western analysis, rapamycin blocked the TGF-beta1-induced loss of E-cadherin expression and de novo increase of the expression of alpha-SMA in a dose-dependent manner. In conclusion, rapamycin significantly attenuated tubulointerstitial damage in a UUO-induced rat model of renal fibrosis, suggesting that rapamycin may have the potential to delay the progression of tubulointerstitial renal fibrosis.     

N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对肾间质纤维化大鼠MCP-1及NF-κB表达的影响

目的：观察N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸（AcSDKP）对单侧输尿管梗阻（UUO）所致大鼠肾间质纤维化的的保护作用并初探其机制。方法：雄性Wistar大鼠18只,随机分为假手术组、UUO组、治疗组（AcSDKP＋UUO）,每组各6只。于造模第14天处死大鼠,取其梗阻侧肾脏组织行HE、Masson染色,光镜下观察肾组织病理改变,评价肾小管变性程度及肾间质纤维化指数,免疫组织化学检测各组肾脏组织MCP-1、ED-1及NF-κB蛋白表达,RT-PCR检测MCP-1 mRNA的表达,Western blot检测NF-κB的蛋白质表达。结果：与假手术组相比,UUO组大鼠肾脏肾小管变性及肾间质纤维化程度严重,AcSDKP治疗后可明显改善UUO组大鼠肾小管变性和间质纤维化（P〈0.05）;免疫组化染色显示：MCP-1、ED-1及NF-κB的蛋白表达UUO组和治疗组明显多于假手术组,但治疗组较UUO组明显减少（P〈0.05）;AcSDKP治疗组MCP-1 mRNA及NF-κB蛋白质的表达均显著弱于UUO组,二者相比差异有统计学意义（P〈0.05）。结论：AcSDKP通过抑制NF-κB激活并进一步减少下游炎症细胞因子的表达以达到治疗肾间质纤维化的作用。     

Role of nitric oxide in renal tubular apoptosis of unilateral ureteral obstruction

BACKGROUND: The obstructed kidney in unilateral ureteral obstruction (UUO) is characterized by renal atrophy and tissue loss, which is mediated by renal tubular apoptosis. We sought to determine whether NO is involved in renal tubular apoptosis in vitro and in vivo. METHODS: Rat renal tubular epithelial cells (NRK-52E) were subjected to mechanical stretch, and apoptosis and cell size were analyzed by flow cytometry. Furthermore, we studied UUO in mice lacking the gene for inducible nitric oxide synthase (iNOS-/-) and their wild-type littermates. Tubular apoptosis and proliferation were detected by immunostaining. NOS activity and NOS expression were assessed by a citrulline assay and Western blot, respectively. RESULTS: Stretching-induced apoptosis in NRK-52E, which was reduced when NO was increased; conversely, stretch-induced apoptosis was increased when a NOS inhibitor was added to the cells. Stretched cells are larger and more apoptotic than unstretched cells. In UUO, the obstructed kidney of iNOS-/- mice exhibited more apoptotic renal tubules than the wild-type mice through 14 days of UUO. The obstructed kidney of iNOS-/- mice at day 3 showed more proliferative tubules compared with wild type. The obstructed kidney of wild-type mice exhibited higher total NOS activity until day 7 after UUO compared with iNOS-/- mice. However, the obstructed kidney of day 14 wild-type mice exhibited significantly lower iNOS activity and protein compared with the day 0 kidney. CONCLUSION: These results suggest that mechanical stretch is related to renal tubular apoptosis and that NO plays a protective role in this system in UUO.