Endostatin expression in the murine model of ischaemia/reperfusion-induced acute renal failure

BACKGROUND: Renal ischaemia-hypoxia is a leading cause of acute renal failure, a clinical condition associated with rapid loss of renal function and high rates of mortality. Renal proximal tubular cells are the most severely injured during renal ischaemia, caused by the breakdown of the extracellular matrix of the tubular basement membrane. Endostatin is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage and it is well-known as being an inhibitor of angiogenesis. In vitro, endostatin inhibits endothelial cell proliferation and migration, as well as tubule formation. In vivo, it has a potent inhibitory effect on tumour growth. In this study, we analysed endostatin gene expression in C57BL/6 mouse kidneys subjected to ischaemia/reperfusion. METHODS: Ischaemic renal failure was induced via 45 min of bilateral occlusion of the renal artery and vein, followed by 12 h or 24 h of reperfusion. Whole-kidney homogenate and total RNA were extracted for examination by western blot analysis and quantitative polymerase chain reaction. The immunohistological examination revealed increased endostatin expression in injured kidney, mainly in the proximal tubule and collecting ducts. RESULTS: Endostatin/collagen XVIII mRNA and protein expression increased during ischaemia and within 12 h of reperfusion. In the western blot assay, we identified increased expression of the 30 kDa endostatin-related fragment and of matrix metalloproteinase-9. CD31 was significantly expressed during reperfusion (P < 0.05). Immunohistological examination revealed glomerular and tubulointerstitial expression of endostatin. CONCLUSION: These data suggest the local synthesis of a 30 kDa endostatin-related fragment following acute renal failure and suggest its role in the modulation of renal capillary density.     

Relative roles of endothelin-1 and angiotensin II in experimental post-ischaemic acute renal failure.

BACKGROUND: The relative roles of endothelin (ET)-1 and angiotensin (ANG) II in post-ischaemic acute renal failure (ARF) have not been fully established so far. With the aim of contributing to this goal, we assessed in this study the effect of ANG II and ET-1 blockade on the course of post-ischaemic-ARF. METHODS: Anaesthetized Wistar rats received i.v. either bosentan (a dual ET receptor antagonist; 10 mg/kg body weight) or losartan [ANG II type 1 (AT(1)) receptor antagonist; 5 or 10 mg/kg body weight] or both, 20 min before, during and 20 min after ischaemia. Rats in the control group received the vehicle via the same route. Survival and renal function were monitored up to 8 days after the ischaemic challenge, while haemodynamic parameters were measured 24 h after ARF. RESULTS: Our results demonstrate that bosentan treatment has a more beneficial effect on experimental ARF than losartan. The survival rate was remarkably higher in bosentan-treated rats than in both rat groups treated with losartan. In the ARF group treated with bosentan, renal blood flow (RBF) was increased by 129% in comparison with the untreated ARF group, whereas in the losartan-treated ARF groups, RBF was only approximately 35 or 38% higher than in control ARF rats. The glomerular filtration rate was markedly higher in bosentan-treated rats than in all other ARF groups on the first and second day after ischaemia. Tubular cell injury was less severe in bosentan-treated rats than in the control ARF rats, but in losartan-treated groups it was similar to that in the ARF group. Concurrent blockade of both ET and AT(1) receptors did not improve ARF because this treatment induced a marked decrease in blood pressure. CONCLUSIONS: These results suggest that ET-1 blockade is more efficient in improving the early course of post-ischaemic renal injury than ANG II inhibition, and that blockade of ET-1 might be effective in prophylaxis of ischaemic ARF.     

Macrophages contribute to the development of renal fibrosis following ischaemia/reperfusion-induced acute kidney injury.

BACKGROUND: Ischaemia/reperfusion is a major cause of acute kidney injury and can result in poor long-term graft function. Although most of the patients with acute kidney injury recover their renal function, significant portion of patients suffer from progressive deterioration of renal function. A persistent inflammatory response might be associated with long-term changes following acute ischaemia/reperfusion. Macrophages are known to infiltrate into tubulointersitium in animal models of chronic kidney disease. However, the role of macrophages in long-term changes after ischaemia/reperfusion remains unknown. We aimed to investigate the role of macrophages on the development of tubulointerstitial fibrosis and functional impairment following acute ischaemia/reperfusion injury by depleting macrophages with liposome clodronate. METHODS: Male Sprague-Dawley rats underwent right nephrectomy and clamping of left renal vascular pedicle or sham operation. Liposome clodronate or phosphate buffered saline was administered for 8 weeks. Biochemical and histological renal damage and gene expression of various cytokines were assessed at 4 and 8 weeks after ischaemia/reperfusion. RESULTS: Ischaemic/reperfusion injury resulted in persistent inflammation and tubulointerstital fibrosis with decreased creatinine clearance and increased urinary albumin excretion at 4 and 8 weeks. Macrophage depletion attenuated those changes. This beneficial effect was accompanied with a decrease in gene expression of inflammatory and profibrotic cytokines. CONCLUSIONS: These results suggest that macrophages play an important role in mediating persistent inflammation and fibrosis after ischaemia/reperfusion leading to a development of chronic kidney disease. Strategies targeting macrophage infiltration or activation can be useful in the prevention of development of chronic kidney disease following ischaemic injury.     

Protective effect of adenosine A2A receptor activation in small-for-size liver transplantation

The aim of the present study was to investigate the potential role of adenosine A(2A) receptor (A(2A)R) activation in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed by using 40% (range: 36-46%) liver grafts. Recipients were given either saline (control group) or CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride, a selective A(2A)R agonist), or CGS 21680+ ZM 241385 (a selective A(2A)R antagonist) immediately after reperfusion for 3 h. Compared with control group, CGS 21680 used at both low dose (0.05 microg/kg/min) and high dose (0.5 microg/kg/min) increased the survival rate from 16.7% (2/12) to 83.3% (10/12) and 66.7% (8/12), respectively. These effects correlated with improved liver function and preserved hepatic architecture. CGS 21680 effectively decreased neutrophil infiltration, suppressed pro-inflammatory (TNF-alpha, IL-1beta and IL-6) expression, promoted expression of antiapoptotic molecules, and inhibited apoptosis. The effects of CGS 21680 were prevented when ZM 241385 was co-administrated. In conclusion, the present study showed that A(2A)R activation alleviated portal hypertension, suppressed inflammatory response, reduced apoptosis, and potentiated the survival of small-for-size liver grafts. Our findings provide the rationale for a novel therapeutic approach using A(2A)R activation to maximize the availability of small-for-size liver grafts.     

Expression of bone type 1 PTH receptor in rats with chronic renal failure

Some researchers have speculated that a decrease in bone type 1 PTH receptor (PTH1R) may be among the causes of “skeletal resistance” in chronic renal failure (CRF). Indeed, the down-regulation of PTH1R mRNA has been identified in uremic bones. However, few studies have identified the patterns of PTH1R protein expression. In this article we compare the bone expression of PTH1R protein and mRNA under control and CRF conditions. Sprague–Dawley rats underwent 5/6 nephrectomies (Nx) or sham operations (control), and were killed 16 weeks later. Blood urea nitrogen (BUN), serum Cr, P, and parathyroid hormone (PTH) were higher in the Nx group than in the controls, while serum Ca and 1,25(OH)₂D₃ were lower in the Nx group. Immunohistochemical images of lumbar bone samples were analyzed by an image processing system. PTH1R was essentially identified in all osteoblasts. The expression of osteoblast PTH1R protein was quantified based on the gray value of PTH1R staining. The mean gray scale of osteoblasts was 25% lower in Nx rats than in control rats (P < 0.01), whereas osteoblast cell counts and cell sizes were not significantly different between the two groups. Thus, down-regulation of PTH1R protein expression under the CRF condition appeared likely. Total RNA extracted from the bone samples was reverse transcribed for real-time polymerase chain reaction (PCR). PTH1R mRNA expression was 33% lower in the Nx group than in the control group in the quantitative PCR analysis (P < 0.05). Our findings suggested that osteoblast PTH1R expression is down-regulated at both the protein and mRNA levels in the steady state of CRF.     

Ozone oxidative preconditioning is mediated by A1 adenosine receptors in a rat model of liver ischemia/ reperfusion

The liver is damaged by sustained ischemia in liver transplantation, and the reperfusion after ischemia results in further functional impairment. Ozone oxidative preconditioning (OzoneOP) protected the liver against ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the role of A₁ adenosine receptor on the protective actions conferred by OzoneOP in hepatic I/R. By using a specific agonist and antagonist of the A₁ subtype receptor (2-chloro N6 cyclopentyladenosine, CCPA and 8-cyclopentyl-1,3-dipropylxanthine, DPCPX respectively), we studied the role of A₁ receptor in the protective effects of OzoneOP on the liver damage, nitiric oxide (NO) generation, adenosine deaminase activity and preservation of the cellular redox balance. Immunohistochemical analysis of nuclear factor-kappa B (NF-κB), tumor necrosis factor alpha (TNF-α) and heat shock protein-70 (HSP-70) was performed. OzoneOP prevented and/or ameliorated ischemic damage. CCPA showed a similar effect to OzoneOP + I/R group. A₁AR antagonist DPCPX blocked the protective effect of OzoneOP. OzoneOP largely reduced the intensity of the p65 expression, diminished TNF-α production, and promoted a reduction in HSP-70 immunoreactivity. In summary, OzoneOP exerted protective effects against liver I/R injury through activation of A₁ adenosine receptors (A₁AR). Adenosine and ^.NO produced by OzoneOP may play a role in the pathways of cellular signalling which promote preservation of the cellular redox balance, mitochondrial function, glutathione pools as well as the regulation of NF-κB and HSP-70.     

50例急性肾衰竭患者尿沉渣镜检与肾活检病理对比分析

目的探讨尿沉渣谱是否能反映急性肾衰竭患者肾脏病理损伤。方法50例急性肾衰竭患者以肾活检病理为评估金标准。取肾活检日晨尿，由专人双盲用相差显微镜检测。根据肾脏病理结果将患者分为3组：以肾小球增殖性病变为主、非增殖性病变为主和小管间质病变为主。尿沉渣所见包括红细胞、有核细胞和各种管型，也分为3类沉渣谱：Ⅰ类以变形红细胞和红细胞管型为主的多种细胞、多种管型，伴蛋白尿；Ⅱ类呈少细胞、细颗粒或透明管型中镶嵌有核细胞，伴大量尿蛋白；Ⅲ类少细胞、透明管型为主或其中嵌入几个有核细胞，蛋白量少。比较不同病理改变的尿沉渣特点。结果肾小球增殖性病变为主33例中30例（91％）为Ⅰ类尿沉渣谱；肾小球非增殖性病变为主9例中8例为Ⅱ类尿沉渣谱；小管间质病变为主8例中5例为Ⅲ类尿沉渣谱。结论尿沉渣分析能在一定程度上反映肾损伤的部位和严重性，这项无创、经济、方便的检查方法值得临床重视与应用。     

Protective effects of sumatriptan on ischaemia/reperfusion injury following torsion/detorsion in ipsilateral and contralateral testes of rat

This study was planned to evaluate the effects of sumatriptan, 5‐HT1B/1D receptors agonist, on ischaemia/reperfusion injury in bilateral testes after unilateral testicular torsion/detorsion in rats. Male Wistar rats (n = 42) were allocated into a sham‐operated group, a control group and treatment groups which were injected sumatriptan (0.1, 0.3 and 1 mg/kg), GR‐127935 (0.01 mg/kg)—5‐HT1B/1D receptors antagonist—and sumatriptan (0.1 mg/kg) + GR‐127935 (0.01 mg/kg). Torsion was induced for 1 hr by rotating right testis 720⁰ in the clockwise direction, and after 7 days of detorsion, bilateral orchiectomy was conducted. While the level of TNF‐α rose in testicular tissue after inducing torsion/detorsion, sumatriptan injection notably lowered TNF‐α level in ipsilateral (torted) and contralateral (nontorted) testes (p < 0.001). Moreover, after inducing testicular torsion/detorsion, SOD activity was decreased, whereas administration of sumatriptan significantly increased SOD activity in bilateral testes (p < 0.001). After induction of torsion/detorsion, macroscopic and histological analyses also showed severe damages which were improved by sumatriptan injection. Interestingly, co‐administration of sumatriptan with GR‐127935 reversed the beneficial impacts of sumatriptan on macroscopic appearance, microscopic pattern and biochemical markers. It is concluded that sumatriptan presumably via stimulation of 5‐HT_1B/1D receptors decreased inflammation, oxidative stress and deteriorations induced by ischaemia/reperfusion injury following testicular torsion/detorsion.     

Pretreatment with granulocyte colony-stimulating factor attenuated renal ischaemia and reperfusion injury via activation of PI3/Akt signal pathway

Aim: Granulocyte colony-stimulating factor (G-CSF) has been shown to exert protective effects in various tissues and experimental models of ischaemia-induced injury. However, the mechanism of renoprotective action in ischaemia/reperfusion (I/R) renal injury of G-CSF was unknown. Methods: Male C57BL/6J mice, subjected to renal ischaemia for 45 min, 48 h and 7 days reperfusion, were administered either saline, wortmannin, G-CSF, and G-CSF plus wortmannin 3 days prior to I/R. Saline-treated group served as the control. At 48 h and 7 days of reperfusion, the mice were killed. Results: Significantly, renal dysfunction and morphological injury were identified at 48 h and 7 days after I/R. Wortmannin pretreatment worsened the renal injury significantly. However, G-CSF pretreatment significantly attenuated renal injury, reduced the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive ratio of renal tubular epithelial cells and inflammation cytokine expression in the kidney. Moreover, G-CSF pretreatment inhibited the expression of Bax and increased the expression of bcl-2 and p-Akt in the kidney. Wortmannin blunted the beneficial effects of G-CSF. Conclusion: The cytoprotective action of G-CSF against I/R injury seems to be associated with its anti-apoptotic action mediated by upregulation of p-Akt signal pathway.     

Ultrastructural and immunocytochemical study of P1, protamine localization in human testis

Summary. Two monoclonal antibodies (MAbs) directed against human protamine P ₁ were realized. Anti- P ₁ specificity was assessed by western-blot and confirmed by elisa. Monoclonal antibody 97-3 was selected. Protamine P ₁, was specifically demonstrated in human testis by immunoelectron microscopy, using 97-3 MAb and an indirect post-embedding immunogold technique. Our results clearly demonstrated the precise time of appearance of P ₁ protamine in the nuclei of human spermatids. P ₁, first appeared in the nucleus of step 5 spermatids and its concentration was increased in steps 6–8 spermatids, cytoplasm was not labelled.     

Polymorphonuclear leucocyte rheology and cytosolic Ca2+ content after activation in chronic renal failure

SUMMARY: We evaluated polymorphonuclear leucocyte (PMN) flow properties in patients with clinically stable chronic renal failure (CRF) and in control subjects at baseline and after activation with 4-phorbol 12-myristate 13-acetate (PMA) and N -formyl-methionyl-leucyl-phenylalanine (fMLP). Initial relative flow rate (IRFR) and clogging particles (CPs) were obtained using the St. George's Filtrometer, and PMN membrane fluidity was assessed by marking PMNs with 1-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). PMN cytosolic Ca²⁺ concentration was determined by marking PMNs with Fura 2-AM. At baseline, CRF patients showed a significant increase only in PMN cytosolic Ca²⁺ content. After activation with PMA and fMLP, a decrease in IRFR and an increase in CP were observed in both control subjects and CRF patients, although the variation in IRFR present in the group of CRF patients was greater than in the control group. After activation with PMA and fMLP, we found a decrease in PMN membrane fluidity only in CRF patients, but no variation in PMN cytosolic Ca²⁺ concentration in either group was observed. These results provide evidence for PMN dysfunction in chronic renal failure.     

Up-regulation of cell cycle regulatory genes after renal ischemia/reperfusion: differential expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes depending on reperfusion time

The aim of this study was to evaluate the influence of renal ischemia, cold preservation and reperfusion on the degree of renal kidney senescence. An experimental model of ex vivo renal hemoperfusion was used. Expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes (CDKIGs) was studied immunohistochemically in kidney biopsy samples at baseline and different time points after reperfusion. All three markers were up-regulated in kidney tissue after the reperfusion; however, their activation in different renal cells varied according to the reperfusion time. Expression of p16 was significantly increased in tubular cells at 180 min of reperfusion when compared with the baseline. Activation of p27 was detected in glomerular cells at 15 min and was significantly higher at 60, 120 and 180 min of reperfusion. The marker started increasing in tubular cells at 15 min and was elevated at every time point afterwards. p21 was significantly over-expressed in all renal cells after the reperfusion. It has been shown by the results of the current study that renal ischemia/reperfusion is associated with over-expression of CDKIGs indicating on substantial DNA damage and/or accelerated tissue senescence. For the first time it has been shown that tissue expression of CDKIGs is positively related with the reperfusion time.     

Sphingosine-1-phosphate reduces hepatic ischaemia/reperfusion-induced acute kidney injury through attenuation of endothelial injury in mice

Aim: Hepatic ischaemia/reperfusion injury (IRI) frequently complicates acute kidney injury (AKI) during the perioperative period. This study was to determine whether hepatic IRI causes AKI and the effect of the sphingosine‐1‐phosphate (S1P) on AKI. Methods: S1P and vehicle were given to mice before ischaemia and mice were subjected to hepatic IRI. Plasma creatinine (PCr), alanine transaminase (ALT), urinary neutrophil gelatinase‐associated lipocalin (NGAL) and renal histological changes were determined. As a marker of endothelial injury, vascular permeability was measured. The effect of VPC 23019, a S1P₁ receptor antagonist, was also assessed. Results: Hepatic IRI resulted in liver injury (increased ALT) and systemic inflammation. Kidneys showed elevated inflammatory cytokines, leucocyte infiltration, increased vascular permeability, tubular cell apoptosis and increased urinary NGAL, although PCr did not increase. Pretreatment with S1P resulted in an attenuation of systemic inflammation and kidney injury without any effect on plasma ALT or peripheral lymphocytes. The protective effect of S1P was partially reversed by VPC 23019, suggesting the important contribution of the S1P/S1P₁ pathway to protect against hepatic IRI‐induced AKI. Conclusion: The study data demonstrate the important contribution of systemic inflammation and endothelial injury to AKI following hepatic IRI. Modulation of the S1P/S1P₁ receptor pathway might have some therapeutic potential in hepatic IRI‐induced kidney injury.     

Sitagliptin protects male albino rats with testicular ischaemia/reperfusion damage: Modulation of VCAM-1 and VEGF-A

Twisting of the spermatic cord is considered a popular problem in the urological field, which may lead to testicular necrosis and male infertility. Sitagliptin, a glucose-lowering agent, proved to have a vindicatory function in myocardial and renal ischaemia/reperfusion (I/R), but its role in testicular I/R has not yet been studied. The current work investigates its capability to recover the testicular I/R injury with shedding more light on the mechanism of its action. Four groups were used: sham, sham pretreated with sitagliptin, I/R and sitagliptin/I/R-pretreated groups. The outcomes proved that I/R significantly decreased the serum testosterone, with a major increase in oxidative, inflammatory and nitrosative stress, along with a reduction in testicular vascular endothelial growth factor-A level with marked germinal cell apoptosis. However, pretreatment with sitagliptin significantly reversed the profound testicular I/R damaging effects, on the basis of its antioxidant, anti-inflammatory and anti-apoptotic activities with the ability of recuperation of the testicular vascularity.     

Theophylline protects against glycerol-induced acute renal failure: Regulation of heme oxygenase-1 and glutathione in rat kidney

Background To investigate the mechanism of glycerol-induced renal injury, we examined kidney levels of the cellular antioxidant glutathione and heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, after glycerol injection. Methods Male Wistar rats were injected with glycerol to induce acute renal failure. Serum creatinine levels were used as a marker of renal function at 24 hours after glycerol injection. Theophylline and buthionine sulfoximine or vehicle were administered to the rats after glycerol injection, and we determined renal glutathione levels (by biochemical assay) and the levels of HO-1 protein and messenger ribonucleic acid (mRNA; using immunoblot analysis [kidney only]) in various rat organs at 24 hours after glycerol injection. Results Glutathione levels abruptly declined after glycerol injection, reached a minimum at 4 hours, then returned to about two thirds of control levels at 24 hours. HO-1 protein was detected at 4 hours and reached a maximum at 24 hours. The induction of HO-1 protein was observed only in the kidney. HO-1 mRNA was faintly expressed at 2 hours, increased until at least 8 hours, and was not detected 24 hours after the treatment. When theophylline was administered to glycerol-injected rats, renal function improved and glutathione levels increased. In addition, the levels of HO-1 protein decreased, compared with those of glycerol-treated rats not given theophylline. Conclusions These results suggest that theophylline may act by modulating the HO-1 directly or indirectly.     

End-stage renal failure in New Zealand Maori: an analysis of circulating transforming growth factor-β1

SUMMARY: There is a high incidence of end-stage renal disease in New Zealand Maori. Reasons for this have not been established. Transforming growth factor-β, (TGF-β₁) is a profibrogenic cytokine, which stimulates the secretion of extracellular matrix components, and has been implicated in the pathogenesis of kidney failure. the aim of this study was to examine TGF-β₁ in the serum of haemodialysis patients at our institution, in order to determine whether there was an upregulation of TGF-β₁ in Maori. A TGF-Prspecific sandwich enzyme-linked immunosorbant assay was used to measure active TGF-β from the sera of 74 haemodialysis patients, and 19 healthy Maori without renal disease, diabetes or hypertension. In addition, clinical and laboratory markers were examined in the haemodialysis patients studied. There was no association between TGF-β₁ and ethnicity in the groups studied. Transforming growth factor-β₁ protein appeared to be inversely related to age. but was not associated with parameters of survival on dialysis such as serum albumin or measures of dialysis adequacy. Although there was a significantly higher incidence of type II diabetes mellitus in the Maori ( P < 0.001) in comparison to European patients, the glycaemic control was comparable between the groups, as were all other laboratory and clinical parameters studied. This is the first study to examine the fibrogenic growth factor TGF-β₁ in New Zealand Maori. Thus, an endogenous increase in TGF-β₁ in Maori does not appear to be implicated in the increased incidence of end-stage renal disease in this population.