Changes in regional renal perfusion following ischemia/reperfusion injury to the rat kidney

Summary Post-ischemic renal failure is associated with a zone of vascular hyperaemia in the outer medulla of the kidney. The effect of this lesion on regional renal perfusion is, however, unclear. Acute unilateral renal ischemia was applied to four groups of ten adult male Wistar rats for a period of 60 min, followed by revascularisation for 0, 15, 30 or 60 min. The aorta was then clamped and Microfil was injected at a standard pressure to fill the renal vasculature. Gross and histological examinations of the renal parenchyma and vasculature were then performed. Regional renal Microfil perfusion was quantified by examination of unstained histological sections, giving rise to a vascular perfusion index (VPI) for each vascular region of the kidney. The VPIs were similar in control and ischemic kidneys that were not subjected to reflow (group 1). In contrast, the VPI was markedly decreased in the inner stripe and inner medulla in animals in which revascularisation had occurred (groups 2–4), and the vasculature in these regions was histologically shown to be packed with red blood cells. Post-ischemic renal failure is associated with hyperperfusion of the medulla resulting from blockage of the vasculature that occurs during revascularisation.     

常温肝缺血再灌注损伤的实验研究

目的：探讨肝缺血再灌注损伤的作用机制。方法：采用大鼠部分肝缺血再灌注模型，将健康雄性SD大鼠24只随机分为三组：A组（手术对照），B组（肝缺血90min），C组（肝缺血90min再灌注120min）。观察每一动物肝组织病理切片；分别检测血浆谷草转氨酶（AST）、谷丙转氨酶（ALT）、乳酸脱氢酶（LDH）、肿瘤坏死因子（TNF－α）、白介素1β（IL-1β）浓度；测定肝组织中髓过氧化物酶（MPO）含量。结果：肝缺血再灌注后，光镜下大鼠肝组织有明显的肝血窦和中央静脉瘀血，内皮细胞及肝细胞普遍水肿变性；C组肝细胞坏死较B组明显；血浆中肝功能酶学指标显著升高，（B、C组与A组比及C组比B组P均＜0．01）；肝组织中MPO活性升高，以再灌注120min组为著（C组比A组P＜0．01）；与血浆中TNF－α、IL-1β的变化趋势相同（TNF－α：C组比A组P＜0．05，IL－1β：B、C组比A组P均＜0．01）。结论：肝脏微循环障碍是肝缺血再灌注损伤的病理基础；TNF－α、IL-1β介导中性粒细胞参与的肝缺血再灌注损伤过程。     

CC趋化因子受体2/单核细胞趋化蛋白1与肾脏疾病的研究进展

CC趋化因子受体2(CC chemokine receptor,CCR2)是单核细胞趋化蛋白-1(mono-cyte chemoattractant protein,MCP-1)的主要受体,可与MCP-1发生特异性结合将信号转入细胞内,趋化并激活单核/巨噬细胞,促进炎症的发生发展.研究表明,CCR2/MCP-1参与了多...     

Bone marrow stem cells-derived microvesicles protect against renal injury in the mouse remnant kidney model

Aims: Several studies have demonstrated administration of mesenchymal stem cells (MSC) could reverse kidney injury by paracrine mechanisms rather than by MSC transdifferentiation. Recently, a few researchers found microvesicles (MV) derived from MSC might be a paracrine mechanism for cell‐to‐cell communication. The aim of this study was to investigate the repair effects of MV in a 5/6 subtotal nephrectomy (Nx) mice model. Methods: The animals were randomly divided into four groups: Control, Nx, Nx + MSC and Nx + MV group. MSC were injected (1 × 10⁶/mouse) through caudal vein in Nx + MSC group at the second day after the surgery and MV were injected (30 µg/mouse) through caudal vein in Nx + MV group on alternate days. Mice were killed on day 7 after the first time of administration. Blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA) and proteinuria were evaluated. Histopathology of kidney was analysed. Results: In Nx mice, the levels of Scr, UA and proteinuria were significantly decreased with administration of MV and MSC (P < 0.05). The remnant kidneys of MV and MSC‐treated Nx mice showed less fibrosis, interstitial lymphocyte infiltrates and less or absent tubular atrophy compared with the untreated Nx group. The Histological Score of Kidney in untreated mice was 3.13 ± 0.74, while in the MSC‐treated group it was 1.67 ± 0.47 and in the MV‐treated group it was 1.80 ± 0.44, nearly preserving normal morphology of the kidney (P < 0.01). Conclusion: This study showed MV protects against renal injury induced by 5/6 Nx, which could mimic the role of MSC in kidney repair. The research showed a newly potential therapeutic approach to kidney diseases.     

Amelioration of cisplatin-induced acute renal injury by renal progenitor-like cells derived from the adult rat kidney

The replacement of a necrotic tubular epithelium with functional tubular epithelial cells is required for recovery from acute renal failure (ARF). A rat renal progenitor-like (rKS56) cell line was recently established derived from the S3 segment of renal proximal tubules. The therapeutic efficacy of rKS56 cells was examined in a rat model of cisplatin-induced ARF. rKS56-lacZ cells expressing 3-galactosidase were injected into SD rats either at the subcapsule of the left kidney (rKS-SC) or via the left renal artery (rKS-IA) 2 days after the injection of cisplatin. Bluo-gal(+) rKS56-lacZ cells were observed in the subcapsule in the rKS-SC group on day 5, and were further increased in number on day 9, accompanied by partial distribution in the corticomedullary junction, but not in the rKS-IA group. A portion of Bluo-gal(+) cells coexpressed Ki-67, aquaporin-1, hepatocyte growth factor (HGF), and c-Met. rKS-SC treatment significantly improved the tubular injury scores, ameliorated tubular cell apoptosis, and induced cell proliferation. The renal function also significantly improved in the rKS-SC group on day 5. These results demonstrate that locally implanted rKS56 cells could differentiate into tubular epithelial cells, thereby accelerating the recovery from tubular injury, most likely by producing tubular trophic factors. These results suggest the therapeutic potential of this novel approach for patients with end-stage renal failure.     

Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat

BACKGROUND: Activation of the cysteine protease calpain has been implicated in renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of calpain inhibitor-1 (Cal I-1) in an in vivo model of renal I/R injury. METHODS: Male Wistar rats were administered Cal I-1 (10 mg/kg, IP) 30 minutes before undergoing bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Plasma concentrations of urea, creatinine, Na(+), gamma-glutamyl transferase (gamma GT), aspartate aminotransferase (AST) and urinary Na(+), glutathione S-transferase (GST), and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal dysfunction and I/R injury. Creatinine clearance (C(Cr)) and fractional excretion of Na(+) (FE(Na)) were used as indicators of glomerular and tubular function, respectively. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of neutrophil infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RESULTS: Cal I-1 significantly reduced I/R-mediated increases in urea, creatinine, gamma GT, AST, NAG, and FE(Na) and significantly improved C(Cr). Cal I-1 also significantly reduced kidney MPO activity and MDA levels. Cal I-1 also reduced histologic evidence of I/R-mediated renal damage and caused a substantial reduction in the expression of iNOS and COX-2, both of which involve activation of nuclear factor-kappa B (NF-kappa B). CONCLUSIONS:: These results suggest that Cal I-1 reduces the renal dysfunction and injury associated with I/R of the kidney. We suggest that the mechanism could involve the inhibition of I/R-mediated activation of NF-kappa B.     

血红素氧合酶-1对大鼠肾缺血再灌注损伤的保护作用

目的　探讨钴卟啉 (CoPP)诱导的血红素氧合酶 (HO) 1高表达对大鼠肾缺血再灌注损伤 (IRI)的保护作用。方法　建立大鼠肾缺血再灌注损伤模型 ,随机将动物分为假手术组 ,对照组和实验组。动态检测血尿素氮 (BUN)、肌酐 (Cr)、超氧化物岐化酶 (SOD)、丙二醛 (MDA)的含量以及进行肾组织光镜形态学观察和酶联免疫吸附试验 (ELISA)和Westernblot分析HO 1。结果　对照组BUN、Cr升高 ,SOD下降 ,MDA升高 ,HO 1中度提高 (14 4.5± 13 .6) ,肾组织结构紊乱。实验组除HO 1含量大幅提高外 (62 9.4± 78.9) ,尚能显著逆转上述改变 ,两组间差异具有统计学意义 (P <0 .0 5 )。结论　CoPP预处理诱导HO 1在肾缺血之前高表达可通过清除氧自由基 (OFRs)而减轻大鼠肾IRI。     

Urinary kidney injury molecule-1 levels in renal stone patients

Objective

To study kidney injury molecule-1 (KIM-1) biomarker levels, indicating renal tubular damage, in patients with kidney stones and in those who underwent minimally invasive method stone treatment.

Patients and methods

Sixty patients with renal stones between 10 and 20 mm were included into the present study. Patients who were divided into three cohorts underwent micropercutaneous nephrolithotomy (microperc), retrograde intrarenal stone surgery (RIRS), and percutaneous nephrolithotomy (PNL). Urine samples were obtained from all participants before, 4 h and 14 days after the procedure. In all the samples obtained, urinary KIM-1 and creatinine (Cr) levels were measured and KIM-1/Cr ratios (ng/mg creatinine) were calculated.

Results

Preoperative urine KIM-1/Cr ratio was higher than postoperative 14th day. The bigger the renal stone size, the higher was the ratio (correlation coefficient 0.353, p = 0.006). According to preferred treatment procedure, there was a statistically significant decrease in preoperative and postoperative 4th hour and 14th day urine KIM-1/Cr rates in the RIRS and PNL, yet none in the microperc group (p = 0.010, p = 0.001, p = 0.212, respectively).

Conclusion

In renal stone patients, the elevated urine KIM-1/Cr ratio levels increase further according to stone size. KIM-1/Cr ratio is a promising marker might be helpful in monitoring the damage created by stone disease.

     

Calcium and hypoxic injury in the renal medulla of the perfused rat kidney

To study the interaction between calcium and the medullary hypoxic lesions found in isolated perfused rat kidneys, the acute effects of high extracellular calcium upon renal function and morphology were evaluated in kidneys perfused with cell-free medium at a total calcium concentration of 8 to 9 mg/dl (controls), 13 to 14 and 19 to 20 mg/dl (high Ca++). High Ca++ increased hypoxic damage to medullary thick ascending limbs from 58.2 +/- 4.0% of tubules in controls to 80.2 +/- 4.0% (P less than 0.005) in the deepest area of the outer medulla. Morphological changes in the cortex were minimal. The increase in damage to medullary thick limbs induced by high Ca++ was prevented by the calcium channel blocker verapamil. Addition of the calcium ionophore A23187 to controls reproduced the effects of high Ca++ with an increase in the proportion of damaged thick limbs to 92.1 +/- 4.1% (P less than 0.001 vs. controls). Addition of equimolar amounts of magnesium chloride did not reproduce the effect of high calcium perfusions. When transport activity was reduced with ouabain, high calcium perfusions were no longer associated with structural damage. In kidneys perfused with a medium enriched with amino acids, the proportion of tubules with severe, irreversible damage increased from 12 +/- 3 to 43 +/- 10% (P less than 0.01) after high calcium perfusion, and to 75 +/- 12% (P less than 0.001) after perfusion with the calcium ionophore. High extracellular and intracellular calcium appear to act in concert with hypoxia to increase the susceptibility of the renal medulla to injury by mechanisms potentially operative in hypercalcemic and ischemic nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit anti‐rat thymocyte immunoglobulin preserves renal function during ischemia/reperfusion injury in rat kidney transplantation

Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction in humans. Increases in cold and warm ischemia times lead to a higher risk of early post‐transplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long‐term kidney graft loss. The protective effect of rabbit anti‐rat thymocyte immunoglobulin (rATG) was evaluated in a rat model of I/R injury following syngeneic kidney transplantation. Serum creatinine concentration was evaluated at 16 h and 24 h post‐transplant. Animals were sacrificed 24 h post‐transplant for evaluation of histology, infiltrating leukocytes, nitrotyrosine staining, and apoptosis. rATG was effective in preventing renal function impairment, tissue damage and tubular apoptosis associated with I/R only when was given 2 h before transplantation but not at the time of reperfusion. Pretransplant rATG treatment of recipient animals effectively reduced the amount of macrophages, CD4⁺, CD8⁺ T cells and LFA‐1⁺ cells infiltrating renal graft subjected to cold ischemia as well as granzyme‐B expression within ischemic kidney. On the other hand, granulocyte infiltration and oxidative stress were not modified by rATG. If these results will be translated into the clinical setting, pretransplant administration of Thymoglobuline^® could offer the additional advantage over peri‐transplant administration of limiting I/R‐mediated kidney graft damage.     

Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells

Endothelial progenitor cells are known to reverse acute kidney injury by paracrine mechanisms. We previously found that microvesicles released from these progenitor cells activate an angiogenic program in endothelial cells by horizontal mRNA transfer. Here, we tested whether these microvesicles prevent acute kidney injury in a rat model of ischemia-reperfusion injury. The RNA content of microvesicles was enriched in microRNAs (miRNAs) that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from acute kidney injury by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. Microvesicles also protected against progression of chronic kidney damage by inhibiting capillary rarefaction, glomerulosclerosis, and tubulointerstitial fibrosis. The renoprotective effect of microvesicles was lost after treatment with RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells, or depletion of pro-angiogenic miR-126 and miR-296 by transfection with specific miR-antagomirs. Thus, microvesicles derived from endothelial progenitor cells protect the kidney from ischemic acute injury by delivering their RNA content, the miRNA cargo of which contributes to reprogramming hypoxic resident renal cells to a regenerative program.     

Local macrophage and myofibroblast proliferation in progressive renal injury in the rat remnant kidney

Background. We have recently shown that blockade of angiotensin II activity inhibits local macrophage and myofibroblast proliferation in progressive non-immune renal injury in the rat remnant kidney. However, it is not known whether this local proliferation contributes to macrophages and myofibroblast accumulation and the development of renal injury. Therefore, we examined this issue in a detailed time-course study of the rat remnant kidney. Methods. Groups of five rats were killed 4, 8, 12 or 16 weeks after 5/6 subtotal nephrectomy (STNx) or a sham operation. Macrophage and myofibroblast proliferation was assessed by two-colour immunostaining for ED1⁺ macrophages or &agr;-smooth muscle actin (&agr;-SMA)-positive myofibroblasts with the proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine. Results. All parameters of renal function and histology remained normal in the sham operated controls, and no macrophage or myofibroblast accumulation was evident. In contrast, prominent macrophage accumulation developed in both the glomerulus and tubulointerstitium in STNx animals, peaking at week 12. Many ED1⁺ macrophages showed PCNA expression, accounting for 19-34% of the total macrophage population. There was a highly significant correlation between proliferating macrophages and total macrophage accumulation in the glomerulus (r = 0.82, P <0.0001) and tubulointerstitium (r = 0.70, P <0.001). Macrophage proliferation was largely restricted to focal areas of renal damage, such as glomerular segmental lesions and severe tubulointerstitial damage. Also, the subpopulation of proliferating macrophages gave a highly significant correlation with loss of renal function, proteinuria, and glomerular and tubulointerstitial lesions. In addition, many &agr;-SMA myofibroblasts were evident within expanded mesangial areas and the tubulointerstitium following STNx. Interestingly, active lesions contained many large &agr;-SMA⁺ cells double-stained for PCNA, accounting for 24-29% of total myofibroblasts. There was a highly significant correlation between the number of proliferating myofibroblasts and total myofibroblast accumulation during the evolution of this disease, and both populations correlated with progressive renal injury. Conclusions. This study has shown that local proliferation is an important mechanism in both macrophage and myofibroblast accumulation during the development of renal injury in the rat remnant kidney. In addition, local macrophage proliferation is postulated as a mechanism for amplifying kidney damage in non-immune renal injury.     

PI3K－Akt 信号通路在内皮祖细胞移植减轻大鼠缺血再灌注肾损伤中的作用

目的探讨磷脂酰肌醇－3－激酶－丝氨酸／苏氨酸激酶（ PI3K－Akt）信号通路在内皮祖细胞（ EPC）移植减轻大鼠缺血再灌注肾损伤中的作用。方法抽取大鼠外周血,采用密度梯度离心的方法分离、培养内皮组细胞。22只雄性SD大鼠随机分为正常对照组、缺血再灌注（ I／R）组、EPCs移植组3组,分别于术后第1 d收获所有大鼠肾脏标本和血标本。流式细胞仪及细胞免疫荧光鉴定 EPC 表面标志（ CD34／VEGFR－2）;测定血标本尿素氮和肌酐值;行western bolt检测各组大鼠p－AKT蛋白的表达情况。结果与正常对照组比较,其余各组血肌酐及尿素氮均升高,I／R组、EPC组肾脏p－Akt表达上调（ P ＜0．05）;与I／R组比较,EPC组的肌酐及尿素氮降低,p－Akt表达上调（ P ＜0．05）。结论 EPC移植可能通过激活PI3K－Akt信号通路减轻大鼠缺血再灌注肾损伤。     

Minocycline attenuates testicular damages in a rat model of ischaemia/reperfusion (I/R) injury

Testicular torsion is a serious urological disease leading to testicular damage. This study aimed to assess the effect of minocycline on testicular ischaemia/reperfusion (I/R) injury caused by testicular torsion/detorsion. Male adult Wistar rats (n = 32) were assigned into four groups of sham, I/R, I/R + minocycline and minocycline. I/R injury was induced by two sets of surgical operations, including the rotation of the left testis (720°, counterclockwise), followed by detorsion after 4 hr. The administration of minocycline was carried out 30 min before detorsion and then continued for 8 weeks. At the end of the 8th week, rats were killed and sampling was done. Johnson's score, the height of seminiferous tubule epithelium, the mean seminiferous tubule diameter, as well as biochemical parameters, SOD, GPx and CAT, were significantly enhanced in the I/R + minocycline group compared with the I/R group. The administration of minocycline led to a marked decrease in expression levels of Caspase-3, Bax, IL-1β and TNF-α genes, and a remarkable increase in expression levels of Bcl-2, 3β-HSD and 17β-HSD3 genes compared with the I/R group. Administration of minocycline could also reduce the rate of germ cell apoptosis (TUNEL staining). Hence, minocycline was useful in the management of testicular torsion/detorsion.     

Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155

BackgroundTo prove the internal connection, we deciphered the effect of cinnamaldehyde on kidney senescence through establishing animal and cell models.MethodsIn vivo, a rat senescence model was constructed using D-galactose (D-gal), and the modeled rats were further treated with cinnamaldehyde. In vitro, rat renal tubular epithelial cells (NRK-52E) were transfected with miR-155 mimic or inhibitor and then treated with cinnamaldehyde, D-gal or PI3K inhibitor (LY294002). The serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) of the rats were measured by an automatic biochemical analyzer. Pathological changes of kidney were determined by hematoxylin-eosin staining. The senescence and viability of NRK-52E cells were assessed by SA-β-gal staining and CCK-8 assay, respectively. The levels of miR-155, p-PI3K/PI3K, p-Akt/Akt, LC3B (LC3-II and LC3-I) and Beclin1 were detected by qRT-PCR, immunohistochemistry, or western blot.ResultsD-gal elevated the levels of BUN, Scr and miR-155 in the kidney, induced the renal pathological damage, inhibited the cell viability, increased the numbers of SA-β-gal-, LC3B- and Beclin1-positive cells and upregulated the levels of LC3-II/LC3-I and Beclin1 both in the kidney and cells. Cinnamaldehyde reversed D-gal-induced effects on the kidney and cells, and moreover, the cinnamaldehyde-induced anti-D-gal effects on cells could be suppressed by miR-155 mimic but promoted by miR-155 inhibitor. LY294002 potentiated D-gal-induced effects, and reversed cinnamaldehyde- and miR-155 inhibitor-caused impacts on the PI3K/Akt pathway and LC3-II/LC3-I level in D-gal-induced cells.ConclusionCinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155.     

Kidney injury molecule-1 as a biomarker of acute kidney injury in renal transplant recipients

This Practice Point commentary discusses a study by Zhang and colleagues that investigated the usefulness of kidney injury molecule-1 (KIM-1) for the early diagnosis of tubular injury on renal biopsy. The study found that KIM-1 protein expression was present in 100% of biopsies that showed acute tubular injury, and in 28% of protocol kidney biopsies that showed no morphological pathology. KIM-1 levels showed positive correlation with serum creatinine and blood urea nitrogen levels, and inverse correlation with estimated glomerular filtration rate. We discuss the findings of Zhang et al., and describe other potential biomarkers for detecting early tubular injury that are currently under investigation. KIM-1, potentially in combination with other biomarkers, might be useful for diagnosing early acute tubular injury on renal biopsy, and might also be of prognostic significance.     

血红素氧合酶1基因转移对大鼠肾缺血再灌注损伤的防护作用

目的探讨血红素氧合酶1(HO-1)基因转移对大鼠自体移植肾缺血再灌注损伤的保护作用。方法构建HO-1腺病毒表达载体,经肾动脉灌注转染26只大鼠(实验组)移植肾,4℃保存24h后行自体移植,移植后5d切除对侧肾脏;以25只大鼠为对照。于移植后3h、3d,应用逆转录聚合酶链反应(RT-PCR)及免疫组织化学方法检测移植肾HO-1基因及蛋白的表达;应用酶联免疫吸附试验(ELISA)法测定肾组织匀浆中HO-1蛋白的含量(以吸光度值表示)。结果移植后3h及3d,实验组移植肾HO-1mRNA的表达强度分别为0·65±0·11及0·86±0·17,而对照组分别为0·09±0·01及0·15±0·02,两组相比差异具有统计学意义(t=14·38,11·73,P均<0·05);实验组移植肾HO-1蛋白含量分别为(297±61)及(468±51)ng/g,而对照组分别为(98±30)及(155±31)ng/g,两组相比差异具有统计学意义(t=8·27,14·83,P均<0·05)。与对照组相比实验组移植肾病理改变明显减轻(P<0·05),血肌酐水平明显降低(t=8·41,P<0·05)。结论腺病毒载体可成功介导HO-1基因对大鼠肾脏的转移,对自体移植肾缺血再灌注损伤具有保护作用。