Protein restriction ameliorates renal tubulointerstitial nephritis and reduces renal transforming growth factor-beta expression in unilateral ureteral obstruction.

In contrast to the substantial evidence for attenuation of the glomerular lesions by a low-protein (LP) diet, it remains to be determined whether and how such a diet lessens the progression of tubulointerstitial lesions, which show the strongest correlation with renal function. Chronic unilateral ureteral obstruction (UUO) results in interstitial fibrosis of the affected kidney. We investigated the therapeutic effects of an LP diet on the progression of interstitial fibrosis in UUO mice. Sixty ICR mice underwent UUO or sham operation; half of these mice were fed a normal-protein (NP) and the other half a LP diet. They were sacrificed at 3, 7 and 14 days postoperatively. The degree of tubular lesion, the distribution of transforming growth factor-beta (TGF-beta), alpha-smooth muscle actin and fibronectin and the activated TGF-beta 1 level were determined. The LP diet significantly reduced the progression of tubular injury, depositions of fibronectin, tubulointerstitial myofibroblast formation, the interstitial expression of TGF-beta-positive cells (at 14 days; NP = 6.91 +/- 3.35 vs. LP = 1.67 +/- 0.41; p < 0.005), and renal active TGF-beta 1 concentration (at 14 days; NP = 5.72 +/- 2.03 vs. LP = 2.96 +/- 0.72; p < 0.01). We conclude that protein restriction may aid the attenuation of progression of tubulointerstitial fibrosis through the reduction in tubulointerstitial expression of TGF-beta.     

Tranilast ameliorates renal tubular damage in unilateral ureteral obstruction

PURPOSE: We determined whether tranilast, the anti-allergic agent N-(3, 4-dimethoxyciannamoyl)-anthranilic acid, would diminish renal transforming growth factor-beta (TGF-beta) levels in unilateral ureteral obstruction and concomitantly affect renal tubular apoptosis and proliferation in that condition. MATERIALS AND METHODS: Tranilast (150 mg./kg.) was administered to rats 1 day before unilateral ureteral obstruction and each day thereafter. Kidneys were harvested day 14 after unilateral ureteral obstruction. Tissue TGF-beta was measured by bioassay using mink lung epithelial cells. Renal tubular proliferation and apoptosis were detected by immunostaining proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay, respectively. Fibrosis was assessed by measuring collagen deposition with trichrome stained slides. RESULTS: TGF-beta bioassay showed that obstructed kidneys in controls contained significantly higher mean TGF-beta plus or minus standard deviation than unobstructed kidneys in controls (73.7 +/- 13.6 versus 14.1 +/- 5.5 pg./mg. tissue) and tranilast significantly decreased tissue TGF-beta in obstructed kidneys (15.9 +/- 4.8 pg./mg. tissue). The terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay demonstrated that obstructed kidneys in controls had significantly more mean tubular apoptosis than the unobstructed counterparts (36.6 +/- 6.7 versus 5.8 +/- 5.5 nuclei per high power field) and tranilast significantly decreased mean renal tubular apoptosis in obstructed kidneys (16.2 +/- 1.7 nuclei per high power field). In addition, immunostaining proliferating cell nuclear antigen showed that obstructed kidneys in controls had significantly more mean renal tubular proliferation than unobstructed kidneys (20.7 +/- 3.4 versus 6.2 +/- 2.1 per high power field) and tranilast significantly increased proliferating renal tubules in obstructed and unobstructed kidneys (26.5 +/- 8.3 and 14.5 +/- 3.4 per high power field, respectively). Control obstructed kidneys exhibited significantly more fibrosis, which was also blunted by tranilast. CONCLUSIONS: Tranilast significantly decreases tissue TGF-beta, resulting in a reduction in tubular apoptosis and an increase in tubular proliferation. This finding suggests that tranilast is a promising agent for preventing renal tubular damage in unilateral ureteral obstruction.     

Elevated plasma concentrations of transforming growth factor-beta 1 in patients with unilateral ureteral obstruction

We measured plasma concentrations of TGF-beta 1 in patients with obstructive ureteral calculi and compared them with the plasma concentrations of healthy volunteers. The present study was a prospective study containing a homogenous group of patients with unilateral ureteral obstruction (UUO). The study consisted of patients with ureteral stones less than 7 mm in diameter that caused mild to moderate obstruction. All patients were referred by the emergency department of our hospital and examined between April 2003 and April 2004. The presence and characteristics of both stone and obstruction were determined by plain abdominal x-ray and gray-scale ultrasonography (US). Blood samples were collected from both patients and control individuals on admission and 1 week after conservative follow-up. The plasma TGF-beta 1 concentration was determined using a quantitative sandwich enzyme immunoassay specific for TGF-beta 1. There were 35 patients with 20 women and 15 men (average age 26.8±5.9 years), and 15 volunteers in the control group, with nine women and six men (average age 24.2±4.5 years). Average stone size was 5.6 mm±1.2 mm (range 3.5–7) for the patient group. US showed the presence of mild hydronephrosis in 24 and moderate hydronephrosis in 11 patients. Plasma concentrations of TGF-beta 1 in patients with ureteral obstruction (1,117±5.8 ng/ml, range 36–2,442 ng/ml) were significantly higher than those in the healthy control group (32±4 ng/ml) on admission (P<0.001). There was a significant increase in TGF-beta 1 plasma concentrations in the patient group (33,525±6.8 ng/ml, range 1,107–73,288 ng/ml) after 1 week follow-up (P<0.001). Ureteral obstruction increases plasma TGF-beta 1 concentrations in patients with ureteral stones as in UUO models in animal studies. A concomitant treatment with an anti-fibrotic agent may reduce the incidence of renal injury during obstruction.     

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.     

Transforming growth factor-beta 1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction

BACKGROUND: Interstitial expression of transforming growth factor-beta1 (TGF-beta1) is important in tubulointerstitial fibrosis, a common process in most progressive renal diseases. However, no effective therapy for progressive interstitial fibrosis is known. Recently, we developed an artificial viral envelope (AVE)-type hemagglutinating virus of Japan (HVJ) liposome-mediated retrograde ureteral gene transfer method, which allowed us to introduce the genetic material selectively into renal interstitial fibroblasts. METHOD: We introduced antisense or scrambled oligodeoxynucleotides (ODNs) for TGF-beta 1 into interstitial fibroblasts in rats with unilateral ureteral obstruction, a model of interstitial fibrosis, to block interstitial fibrosis by retrograde ureteral injection of AVE-type HVJ liposomes. RESULTS: TGF-beta 1 and type I collagen mRNA increased markedly in the interstitium of untreated obstructed kidneys, and those were not affected by scrambled ODN transfection. Northern analysis and in situ hybridization revealed that the levels of TGF-beta 1 and type I collagen mRNA were dramatically decreased in antisense ODN-transfected obstructed kidneys. Consequently, the interstitial fibrotic area of the obstructed kidneys treated with antisense ODN was significantly less than that of the obstructed kidneys untreated or treated with scrambled ODN. CONCLUSION: The introduction of TGF-beta 1 antisense ODN into interstitial fibroblasts may be a potential therapeutic maneuver for interstitial fibrosis.     

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.     

单侧输尿管梗阻对对侧肾小管上皮细胞凋亡影响的研究

目的探讨单侧输尿管梗阻对对侧肾小管上皮细胞凋亡的影响。方法新西兰大白兔48只,随机建立右侧输尿管完全梗阻4周和8周组;部分梗阻4周和8周组;假手术4周和8周组动物模型,每组8只。分别于梗阻4周、8周时取对侧肾脏,称重后利用TUNEL法检测对侧肾小管上皮细胞凋亡,并行HE染色。结果梗阻4周、8周均引起对侧肾实质代偿性增生,与梗阻程度及时间正相关（P〈0.05）。梗阻4周组HE染色见少数肾小管扩张,腔内存在坏死脱落细胞,以完全梗阻明显,但8周较4周相应各组无明显变化。TUNEL法检测完全梗阻、部分梗阻及假手术4周组对侧近曲小管上皮细胞凋亡指数分别为（6.02±1.21）%、（3.87±1.70）%、（2.32±1.39）%;远曲小管上皮细胞凋亡指数分别为（12.08±2.33）%、（7.22±3.35）%、（3.38±2.34）%;8周组对侧近曲小管上皮细胞凋亡指数分别为（7.00±2.19）%、（4.47±2.11）%、（2.29±0.85）%;远曲小管上皮细胞凋亡指数分别为（13.02±3.16）%、（8.11±2.99）%、（3.63±1.96）%。相应各组组间比较均有统计学意义（P〈0.05）,但8周与4周相应各组比较均无统计学意义（P〉0.05）。结论单侧输尿管梗阻早期可引起对侧肾小管上皮细胞损害,但随梗阻时间的延长并未进一步加重。     

COMP-angiopoietin-1 ameliorates renal fibrosis in a unilateral ureteral obstruction model

Injury to the renal microvasculature may be a major factor in the progression of renal disease; therefore, protection of endothelial cells (EC) in renal vasculature may have a therapeutic role in renal fibrosis. Recently, a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, was developed. The contribution of COMP-Ang1 in renal interstitial fibrosis, however, remains to be clarified. This study investigated the effects of COMP-Ang1 on peritubular capillary EC in the renal cortex and the renal fibrogenic process that is triggered by unilateral ureteral obstruction. COMP-Ang1 preserved renal platelet-EC adhesion molecule-1-and Tie2-positive EC. Morphologic examination indicated less tubular injury and tubulointerstitial fibrosis in mice that received COMP-Ang1 than vehicle-treated mice. Interstitial type I collagen and myofibroblast accumulation were significantly suppressed by COMP-Ang1 treatment. COMP-Ang1 increased Tie2 and Akt phosphorylation in ureteral obstructed kidneys. Renal surface microvasculature and renal blood flow were higher after treatment with COMP-Ang1 than with vehicle. COMP-Ang1 treatment decreased monocyte/macrophage infiltration, tissue levels of TGF-beta1, and Smad 2/3 phosphorylation and increased Smad 7 in the obstructed kidney. These results demonstrate that COMP-Ang1 treatment can decrease the progression of renal fibrosis in unilateral ureteral obstruction. COMP-Ang1 may be an endothelium-specific therapeutic modality in fibrotic renal disease.     

Effects of suppressing intrarenal angiotensinogen on renal transforming growth factor-beta1 expression in acute ureteral obstruction

BACKGROUND: Angiotensin II (Ang II) mediates the up-regulation of fibrogenic factors such as transforming growth factor-beta1 (TGF-beta1) in chronic renal diseases. In addition, it has been proposed that the intrarenal renin-angiotensin system (RAS) is as important as the systemic RAS in kidney disease progression. METHODS: We suppressed angiotensinogen (AGT) gene expression in the kidney by transferring recombinant adenoviral vectors carrying a transgene expressing AGT antisense mRNA, and determined the effect of the local inhibition of the RAS on TGF-beta1 synthesis in the kidneys of rats with unilateral ureteral obstruction (UUO). Immediately after UUO, recombinant adenovirus vectors were injected intraparenchymally into the cortex of obstructed kidneys. RESULTS: beta-galactosidase (beta-gal)-stained kidney sections revealed the efficient transduction of the recombinant adenoviral vectors into tubular epithelial cells. Kidney cortex injected with AGT antisense showed significantly lower native AGT mRNA and protein expressions than control UUO kidneys at 24 hours and 5 days post-UUO. TGF-beta1 was significantly up-regulated in the renal cortex 24 hours and 5 days post-UUO, whereas AGT antisense-injected UUO rats showed significantly reduced TGF-beta1 expression compared to control UUO rats. Both fibronectin and collagen type I expressions were increased 24 hours and 5 days post-UUO, and these augmentations were considerably reduced by AGT antisense RNA treatment. CONCLUSION: This study demonstrates that the suppression of intrarenal RAS prevents the formation of renal cortical TGF-beta1, and of related fibrogenic factors, in early UUO.     

Renal tubular apoptosis after complete ureteral obstruction in the presence of hyperoxaluria

Hyperoxaluria is a well-known cause of renal stone disease and in vitro studies have shown that oxalate crystals have a stimulatory effect on apoptosis of renal tubular epithelial cells. Total and partial ureteral obstruction also have an accelerating effect on apoptosis of renal tubular epithelial cells. The aim of the present study was to investigate the apoptotic effect of unilateral ureteral obstruction in the presence of hyperoxaluria on the rat kidney. Twenty-eight male Wistar rats were divided into four groups, with seven rats in each. The groups were named G1 (control), G2 (hyperoxaluric), G3 (obstructive) and G4 (hyperoxaluric + obstructive). G2 and G4 rats were given 1% ethylene glycol (a precursor for oxalates) in their drinking water. G1 and G2 rats underwent sham operation, while left proximal ureteral ligation with a 5-zero silk suture was performed on G3 and G4 animals. The rats were sacrificed 2 weeks after the operation; left nephrectomy was then performed. We searched for the apoptotic cells by direct immuno-peroxidase detection of digoxigenin-labeled genomic DNA. The mean ± SD values of the apoptotic cell count was 0.86 ± 0.90 in G1 and 4.33 ± 3.81 in G2. The values for G3 and G4 were 30.17 ± 16.85 and 302.67 ± 184.45, respectively. We found a statistically significant difference between all groups (P < 0.001). When compared with the control group (G1), the mean apoptotic cell count was fivefold that of G2 and 35- and 351-fold those of G3 and G4, respectively. Our study demonstrated that hyperoxaluria with complete ureteral obstruction induces an excessive level of apoptosis, which is responsible for renal damage, and that ureteral obstruction is a more important factor for apoptosis than hyperoxaluria. Considering these data, we also believe that research studies for medical preventive measures must be considered for patients with ureteral obstruction and/or hyperoxaluria. Received: 14 January 2000 / Accepted: 12 May 2000     

Partial unilateral ureteral obstruction in rats

This review comprises an overview of the current knowledge on experimental partial unilateral ureteral obstruction (PUUO) and a summary of our latest original experimental PUUO studies in rats. Neonatal PUUO is the type of obstruction that is most often encountered in pediatric clinical practice. However, the pathogenesis of PUUO is still incompletely understood. Most of our knowledge on PUUO has been derived from experimental studies in a variety of animal models. Although progress has been made, the natural history of congenital hydronephrosis is still incompletely described. The effects on kidney functions of long-term urinary tract obstruction, especially PUUO, have been less intensively studied. Recently, we created models with mild and severe PUUO in young rats by embedding the upper one fourth or the upper two thirds of the left ureter into the psoas muscle, respectively. Thereafter, the technique was used to create mild and severe PUUO in newborn rats and magnetic resonance imaging studies showed that both mild and severe obstruction caused a time-dependent decrease in renal blood flow. Compensatory increase in total kidney volume and renal vein blood flow in contralateral non-obstructed kidneys was not detectable when functional deterioration in the partially obstructed kidneys was present. Finally, we investigated the dynamic changes in renal relative signal intensity (RSI) of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) using magnetic resonance imaging in rats with partial, complete unilateral ureteral obstruction and sham-operated controls. The results showed that changes in Gd-DTPA RSI are compatible with the known physiological and anatomical changes in kidneys in response to ureteral obstruction and useful for distinguishing an obstructed from a non-obstructed collecting system and also for differentiating a partially obstructed from a completely obstructed collecting system.     

Gene expression in response to acute unilateral ureteral obstruction

Acute unilateral ureteral obstruction results in differential growth characteristics of both the ipsilateral and contralateral kidney. The obstructed kidney undergoes cellular atrophy following an initial phase of interstitial proliferation while the contralateral kidney hypertrophies. To evaluate the molecular events occurring in both kidneys after obstruction, we examined the expression of growth related (c-fos, c-myc, cH-ras, HSP 70), cell maintenance (beta-actin), and cellular damage (TRPM-2) genes at the mRNA level. In the contralateral kidney an early and transitory induction of c-fos and c-myc expression occurred while a bimodal induction was noted in the obstructed kidney. The patterns of cH-ras, HSP 70 and actin expression also differed in both kidneys. Induction of TRPM-2 was noted only in the obstructed kidney. Rapid gene activation is evident in both the contralateral and obstructed kidney following unilateral ureteral obstruction. The patterns of expression are distinct and may reflect the cellular response to stress (cell death and stromal proliferation) in the obstructed kidney versus a response to a systemic stimulus resulting in cellular hypertrophy in the contralateral kidney.     

Ginsenoside-Rg1 inhibits endoplasmic reticulum stress-induced apoptosis after unilateral ureteral obstruction in rats

Abstract

Aim: Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) are implicated in many fibrotic diseases, including renal fibrosis. Whether Ginsenoside-Rg1 (G-Rg1) could attenuate renal fibrosis via suppression of ER stress and UPR has not been reported. The aim of this study was to explore the effect of G-Rg1 on ER stress and UPR-induced apoptosis in kidneys with unilateral ureteral obstruction (UUO) rat model. Methods: Twenty-four male Sprague–Dawley rats were randomly divided into control group, model group and G-Rg1 treatment group. G-Rg1 was administered to rats by intraperitoneal injection. Renal interstitial fibrosis in the model group was developed by UUO in rats. Renal function was estimated by the levels of serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathological damage was evaluated by hematoxylin and eosin (HE) and Masson’s trichrome staining. The ER stress was assessed with glucose-regulated protein (GRP) 78 expression, and the proapoptotic response was detected with CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 expressions by Western Blot. The number of apoptotic cells was determined by Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) analysis. Results: UUO for 14 days aggravated renal function, renal damage and renal interstitial fibrosis, activated ER stress response (induction of GRP78 protein), enhanced the proapoptotic response (increase in CHOP and caspase-12 proteins) and increased the number of apoptotic cells (shown by the TUNEL assay). Treatment with G-Rg1 significantly ameliorates the renal pathological lesions and decreases expressions of ER stress-associated proteins and the level of apoptotic cells in kidneys. Conclusion: G-Rg1 suppresses renal cell apoptotic and fibrotic process partly through inhibition of ERS- and UPR-related apoptotic pathway in the kidneys after UUO.     

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.