Increased apoptosis in acute puromycin aminonucleoside nephrosis

Acute puromycin aminonucleoside nephrosis (PAN) in rats is characterized by heavy proteinuria associated with renal hypercellularity. The role of apoptosis in the resolution of renal hypercellularity was investigated in PAN. To study the participation of apoptosis in PAN, renal tissues were collected from nephrotic and control rats on weeks 1, 2 and 7 after a single puromycin aminonucleoside injection. Apoptosis was evaluated by light and electron microscopy. Apoptotic DNA fragmentation was detected by TUNEL staining. Renal tissues were also evaluated by the presence of leukocyte common antigen (LCA), ED1 (macrophages) and proliferating cell nuclear antigen (PCNA) with corresponding monoclonal antibodies. An increased number of apoptotic (TUNEL+) cells was observed in the glomerulus at week 1. Electron microscopy analysis showed glomerular apoptosis mainly in endothelial cells. In the interstitium and tubules, increased apoptosis was observed at weeks 1 and 2. Increased apoptosis was accompanied with increased LCA+, ED1+ and PCNA+ cells in the interstitium and with increased PCNA+ cells in tubules. There was a high significant correlation between the number of apoptotic cells and the number of interstitial LCA+, ED1+ and PCNA+ cells. Tubular PCNA expression was correlated with tubular apoptosis. We also observed significant correlation between glomerular, interstitial and tubular apoptosis with proteinuria during the nephrosis. Double staining analysis showed that about 13% of interstitial or tubular apoptotic cells were positive for PCNA. All these values returned to normal by week 7. These results indicate that apoptosis is involved in the repairing process of this disease model.     

细胞凋亡在脂质肾损害中的作用

目的 :研究细胞凋亡在脂质肾损害中的作用。方法 :高脂组大鼠饲以 4%胆固醇及 1%胆酸钠的鼠料 ,正常组大鼠饲以普通鼠料 ,分别于 4周及 8周时处死大鼠。采用TUNEL法检测肾组织中凋亡细胞 ,增殖性核抗原及层粘连蛋白在肾组织中的表达用免疫组化法检测 ,血脂及尿蛋白检测用生化法。结果 :与正常对照组相比 ,4周时高脂组增殖性核抗原表达增加 ,8周时高脂组凋亡细胞明显增多 (P <0 0 5 )。 8周时高脂组大鼠肾组织中层粘连蛋白质表达增加 ,2 4h尿蛋白定量明显增多 (P <0 0 5 ) ,肾小球、肾小管及其肾小管间质细胞凋亡指数与 2 4h尿蛋白定量呈正相关 (r =0 978,P <0 0 0 1;r =0 986 ,P <0 0 0 1;r =0 975 ,P <0 0 0 1)。结论 :细胞凋亡在脂质肾损害中起着重要作用 ,保持细胞凋亡与增殖的平衡有助于减轻肾损害     

Cytoskeletal protein expression and regenerative markers in schistosomal nephropathy.

BACKGROUND: Progression of renal diseases is related to the abnormal regulation of cellular and extracellular matrix turnover. Other factors in addition to schistosomal antigens may be relevant to the progression of schistosomal nephropathy (SN). The validity of markers of fibroblastic differentiation, alpha smooth muscle actin (alphaSMA), and vimentin, as well as the regenerative activity (PCNA/apoptosis index) in determination of progression of SN in comparison to other forms of non-schistosomal nephropathy (non-SN) is investigated. METHODS: Three groups were included; group I pure SN (n=16), group II a diverse group of non-schistosomal patients with comparable pathologic changes on renal biopsy (n=40) and a control group (n=5). Immunohistochemical staining of myofibroblasts (alphaSMA and vimentin) and proliferating cells (PCNA) and histomorphometric analysis was done. In situ end labelling (ISEL) of DNA was used to evaluate apoptosis. RESULTS: No differences in the patterns of distribution of positivity of the different studied markers were observed between the different nephropathy groups. Both alphaSMA and vimentin were detected in glomerular mesangial, tubular epithelial, interstitial inflammatory fibroblast-like cells and occasionally endothelial cells. PCNA and apoptotic cells were detected in tubular epithelial and interstitial cells with paucity of positive cells in the glomerulus. Significant positive correlations were detected in group I between glomerular sclerosis and interstitial markers including interstitial alphaSMA (r=0.609, P=0.001), interstitial vimentin (r=0.812, P=0.00) and interstitial apoptosis (r=0.733, P=0.001). On the other hand, glomerulosclerosis in group II showed significant positive correlations with predominantly the glomerular markers; glomerular alphaSMA (r=0.475, P=0.002), glomerular apoptosis (r=0.684, P=0.00) and glomerular PCNA (r=0.691, P=0.00). Interstitial fibrosis correlated significantly with interstitial markers in group I including interstitial alphaSMA (r=0.837, P=0.00) interstitial vimentin (r=0.929, P=0.00), interstitial apoptosis (r=0.807, P=0.00) and interstitial PCNA (r=0.617, P=0.01), while in group II it correlated with both interstitial and glomerular markers. In addition, the tubulo-interstitial ratio was significantly higher in group I in comparison with group II (P=0.024), with no difference between groups II and III. CONCLUSIONS: Although SN may start as glomerulopathy associated with increased mesangial cellularity, the interstitial rather than the glomerular markers of myofibroblastic differentiation and those of cell turnover are playing a crucial role in late stages of schistosomal, but not in non-schistosomal nephropathies.     

Rapid communication: renal apoptosis after shockwave application in rabbit model

PURPOSE: To identify any apoptotic effect of shockwave lithotripsy (SWL) on renal tubular and glomerular cells. MATERIALS AND METHODS: Thirty-five male New Zealand White rabbits were divided into five groups of seven rabbits each: I (control), II (sham), and III, IV, and GV (treated and sacrificed 1, 7, and 28 days after SWL, respectively). Intramuscular anesthetic agent (ketamine HCl; 20 mg/kg) and intravenous contrast medium (iohexol 300 mg of I/mL) were administered to animals in group II. The left kidneys of animals in groups III, IV, and V were exposed to 2000 shockwaves at 18 kV after administration of anesthesia and contrast medium. The animals were sacrificed on day 1, 7, or 28 after SWL, and the kidneys were removed. Apoptotic and proliferative indices of renal tubular and glomerular cells were determined by terminal deoxynucleotidyl transferase dUTP nick and label (TUNEL) and Ki-67 labeling methods, respectively, counting 1000 cells in each preparation. RESULTS: No apoptosis was detected in glomerular cells in any group. The mean apoptotic indices of the tubular cells in animals in groups I and II were 483.0 +/- 85 and 484.4 +/- 105, respectively with no significant difference between the groups. In groups III and IV, the mean apoptotic indices were 343.4 +/- 89 and 358.4 +/- 61, respectively. There were no statistically significant differences between groups III and IV and the control group. Similarly, there were no significant differences in the apoptotic indices in groups III and IV. However, the apoptotic index in group V was 821.4 +/- 57, significantly higher than in the control group. The proliferative indices of all SWL groups were lower than that of the control group. CONCLUSION: Shockwave lithotripsy has an apoptotic effect on renal tubular cells that can be detected 4 weeks after the procedures, but no apoptotic effect on glomerular cells. Treatment with SWL also attenuates the proliferation of both tubular and glomerular cells.     

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

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

Erythropoietin protects against ischaemic acute renal injury.

BACKGROUND: Erythropoietin (EPO) has recently been shown to exert important cytoprotective and anti-apoptotic effects in experimental brain injury and cisplatin-induced nephrotoxicity. The aim of the present study was to determine whether EPO administration is also renoprotective in both in vitro and in vivo models of ischaemic acute renal failure. METHODS: Primary cultures of human proximal tubule cells (PTCs) were exposed to either vehicle or EPO (6.25-400 IU/ml) in the presence of hypoxia (1% O(2)), normoxia (21% O(2)) or hypoxia followed by normoxia for up to 24 h. The end-points evaluated included cell apoptosis (morphology and in situ end labelling [ISEL], viability [lactate dehydrogenase (LDH release)], cell proliferation [proliferating cell nuclear antigen (PCNA)] and DNA synthesis (thymidine incorporation). The effects of EPO pre-treatment (5000 U/kg) on renal morphology and function were also studied in rat models of unilateral and bilateral ischaemia-reperfusion (IR) injury. RESULTS: In the in vitro model, hypoxia (1% O(2)) induced a significant degree of PTC apoptosis, which was substantially reduced by co-incubation with EPO at 24 h (vehicle 2.5+/-0.5% vs 25 IU/ml EPO 1.8+/-0.4% vs 200 IU/ml EPO 0.9+/-0.2%, n = 9, P<0.05). At high concentrations (400 IU/ml), EPO also stimulated thymidine incorporation in cells exposed to hypoxia with or without subsequent normoxia. LDH release was not significantly affected. In the unilateral IR model, EPO pre-treatment significantly attenuated outer medullary thick ascending limb (TAL) apoptosis (EPO 2.2+/-1.0% of cells vs vehicle 6.5+/-2.2%, P<0.05, n = 5) and potentiated mitosis (EPO 1.1+/-0.3% vs vehicle 0.5+/-0.3%, respectively, P<0.05) within 24 h. EPO-treated rats exhibited enhanced PCNA staining within the proximal straight tubule (6.9+/-0.7% vs vehicle 2.4+/-0.5% vs sham 0.3+/-0.2%, P<0.05), proximal convoluted tubule (2.3+/-0.6% vs vehicle 1.1+/-0.3% vs sham 1.2+/-0.3%, P<0.05) and TAL (4.7+/-0.9% vs vehicle 0.6+/-0.3% vs sham 0.3+/-0.2%, P<0.05). The frequency of tubular profiles with luminal cast material was also reduced (32.0+/-1.6 vs vehicle 37.0+/-1.3%, P = 0.05). EPO-treated rats subjected to bilateral IR injury exhibited similar histological improvements to the unilateral IR injury model, as well as significantly lower peak plasma creatinine concentrations than their vehicle-treated controls (0.04+/-0.01 vs 0.21+/-0.08 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls. CONCLUSIONS: The results suggest that, in addition to its well-known erythropoietic effects, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration and promotes renal functional recovery in hypoxic or ischaemic acute renal injury.     

Role of stem cell factor and mast cells in the progression of chronic glomerulonephritides.

BACKGROUND: Mast cells (MCs) have been implicated in the pathogenesis of atherosclerosis and tissue fibrosis. However, the role of MC in the development of renal fibrosis has not been fully elucidated. Stem cell factor (SCF; the ligand for MC c-kit receptor) is thought to attract and activate MCs. METHODS: The intensity of MC infiltration and SCF expression in renal biopsies from 56 patients with different forms of primary and secondary glomerulonephritis and five controls were investigated by immunohistochemistry, using a monoclonal anti-human MC tryptase antibody and a polyclonal antihuman SCF antibody. RESULTS: A large number of MCs were detected in the renal interstitium of the diseased kidneys. Immunostainable SCF was detected in tubular as well as interstitial cells. MC infiltration was significantly higher in glomerulonephritis (16.9 +/- 10.2 cells/field) compared with controls (2.8 +/- 2.1 cells/field, P = 0.03). Similarly, immunostainable SCF was 0.6 +/- 0.3% for controls and 3.3 +/- 2.1% in the glomerulonephritis group (P = 0.02). MC infiltration was highly correlated with SCF expression in diseased kidneys (r = 0.93, P = 0.0001). Double immunostain showed them to colocalize in some interstitial cells. Analysis of MC proliferation [proliferating cell nuclear antigen (PCNA) positivity] and apoptosis (in situ end labeling of DNA) showed these cells to be terminally differentiated. Both MCs and SCF were correlated with interstitial fibrosis (R = 0.71 for MC and R = 0.62 for SCF, P = 0.0001) and interstitial alpha-smooth muscle actin (R = 0.69 for MC and R = 0.60 for SCF P = 0.0001). Using regression analysis, the number of MC infiltration was found to be a very powerful determinant of interstitial fibrosis in the glomerulonephritis group (R2 = 91.4%). CONCLUSION: MCs as an infiltrating hematopoietic cell and its growth factor (SCF) seem to be up-regulated in glomerulonephritis, and may play a role in the development of renal fibrosis.     

Simulated ischemia induces renal tubular cell apoptosis through a nuclear factor-kappaB dependent mechanism

PURPOSE: Ischemia-reperfusion injury is a relatively common cause of renal tubular cell death and acute renal failure. While nuclear factor-kappaB has been implicated in the pathophysiology of renal ischemia-reperfusion injury, the effect of nuclear factor-kappaB inhibition on ischemia induced renal tubular cell death remains unknown. MATERIALS AND METHODS: Renal tubular cells (LLC-PK1) were exposed to simulated ischemia in the presence or absence of 10 microM. pyrrolidine dithiocarbamate (nuclear factor-kappaB inhibitor). Nuclear factor-kappaB activation (electrophoretic mobility shift assay and immunohistochemistry) and the effect of pyrrolidine dithiocarbamate on nuclear factor-kappaB activation (electrophoretic mobility shift assay) and ischemia induced apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling) were determined. RESULTS: Simulated ischemia induced nuclear factor-kappaB activation and renal tubular cell apoptosis versus controls (mean plus or minus standard error of mean 62 +/- 5.2 versus 0.4 +/- 0.3 apoptotic nuclei per high power field, p <0.05). In contrast, previous cellular exposure to pyrrolidine dithiocarbamate effectively inhibited nuclear factor-kappaB activation and prevented ischemia induced apoptosis (mean 14 +/- 6 apoptotic nuclei per high power field). CONCLUSIONS: Simulated ischemia induces nuclear factor-kappaB intranuclear translocation and activation in renal tubular cells. Furthermore, nuclear factor-kappaB mediates ischemia induced renal tubular cell apoptosis. Further elucidation of the complex role of nuclear factor-kappaB in inflammatory injury may lead to the development of targeted therapeutic strategies that ameliorate ischemic renal injury.     

Decrease of apoptosis rate in patients with renal transplantation treated with mycophenolate mofetil.

BACKGROUND/AIMS: Mycophenolate mofetil (MMF) is a powerful immunosuppressant that inhibits the proliferation of lymphocytes by blocking the enzyme inosine monophosphate dehydrogenase. MMF prevents acute graft rejection in organ transplants. The aim of this investigation is to study whether MMF has any influence on apoptosis and proliferation rates of cells other than lymphocytes. METHODS: We conducted a retrospective study of renal allograft biopsies taken during the 1st week after transplantation in 25 patients receiving triple therapy with prednisone, ciclosporin and azathioprine 75 mg/day and in 25 patients treated with MMF at a dose of 2 g/day instead of azathioprine, in order to investigate the differences in the proliferation and apoptosis rates of the glomerular, tubular, interstitial and endothelial cells of the kidney. Twelve normal kidneys were used as controls. Conventional histopathological techniques were applied as usual for pathological diagnosis. Proliferative activity was assessed by use of MIB-1 antibody. Sections of formalin-fixed, paraffin-embedded tissue blocks were stained for the presence of apoptotic cells by TUNEL assay. Evaluation of proliferative or apoptotic rates was made by counting the number of positive cells in 10 glomeruli and in 10 transversely cut tubuli in each biopsy. The positive cells in the interstitium were counted in ten high-power fields. Positive cells in the endothelium were scored semiquantitatively from 0 to 3: 0 = none, 1 = isolated cells, 2 = small groups of cells, 3 = most endothelial cells. Mann-Whitney U and chi-square tests were used for intergroup comparisons. RESULTS: All biopsies were normal or had borderline (Banff classification) acute rejection. MIB-1 rates were similar in both groups, without statistical differences (p > 0.05) between them. Significantly lower apoptotic rates were found in the group treated with MMF in tubular epithelium (23.41 +/- 8.86 vs. 57.4 +/- 13.42; p = 0.021), in glomerular (1.25 +/- 0.78 vs. 5.3 +/- 1.66; p = 0.027), and interstitial cells (1.58 +/- 0.6 vs. 5.8 +/- 1.54; p = 0.043). Apoptosis in endothelial cells (p > 0.05) was similar in both groups. CONCLUSION: We conclude that treatment with MMF of kidney transplant patients does not affect the proliferative rate of cells of the allograft, but decreases the number of apoptotic cells in tubular epithelium.     

Apoptosis versus necrosis during cold storage and rewarming of human renal proximal tubular cells

BACKGROUND: A recent clinical study demonstrated that in renal allografts preserved in the cold apoptosis occurred soon after reperfusion. The mode of cell death during cold storage is generally considered necrotic. Whether apoptosis occurs as a part of cold storage is uncertain. The objective was to determine in human renal tubular cells whether apoptosis is specific for rewarming or it also occurs during cold storage and whether it could be modified. METHODS AND RESULTS: Cold storage (4 degrees C) of primary human renal proximal tubular epithelial (RPTE) in University of Wisconsin (UW) solution up to 48 hr caused a time-dependent increase in cell death measured by lactic dehydrogenase (LDH) release and vital dye exclusion methods. Transmission electron microscopy (TEM) demonstrated that cell death in the cold was necrotic, involving considerable mitochondrial disruption, and was not apoptotic. The TUNEL assay that provides a specific, quantitative measure for apoptosis showed no increase in TUNEL-positivity during flow cytometry of cells stored in cold: 37 degrees C, 0.23+/-0.14%; 24 hr cold, 0.23+/-0.1%; 48 hr cold, 1.79+/-0.58%. Annexin-V staining, a sensitive method for detecting early apoptosis, similarly showed no increase in positively stained cells during cold storage. Addition of antioxidants 2-methyl aminochroman and deferoxamine to UW solution inhibited necrotic cell death and preserved mitochondrial structure. In contrast to cold storage alone, rewarming (37 degrees C for 24 hr) of cold stored cells, however, resulted in significant apoptosis (TUNEL positive: 48 hr cold: 2+/-0.6%, 48 hr cold and 24 hr rewarming: 54+/-17%), which was confirmed by the TEM based on typical apoptotic features. Addition of 2-MAC and DFO significantly inhibited rewarming-induced apoptotic cell death (plus 2-MAC: 3+/-1%, plus DFO: 3+/-2%). CONCLUSION: Our study in human tubular cells provides evidence that cold storage per se does not result in apoptosis, but is primarily necrotic. However, rewarming is associated with significant apoptosis in the presence of ongoing necrosis, speculatively due to the activation of the apoptotic enzymic process of sublethally injured cells. Inclusion of antioxidants in the storage solution confers protection against both cold storage and rewarming-induced necrosis and apoptosis.     

中性粒细胞细胞外网络与B淋巴细胞在狼疮肾炎患者肾组织的表达及其意义

目的 探讨中性粒细胞细胞外网络(NET)在狼疮肾炎(LN)患者肾组织中的表达与狼疮活动的关系.方法 采用免疫组织化学技术,分别检测NET(以瓜氨酸化的组蛋白H3为标志)在20例LN、8例微小病变(MCD)患者和3例健康对照的肾组织的表达,同时检测相应部位B淋巴细胞(CD19标志)的浸润.分析LN患者肾组织活动指数和慢性指数评分,以及它们与NET的关系.结果 LN患者肾小球中NET表达显著高于健康对照组和MCD组(1.056±0.985比0±0、0±0,均P＜0.01).在中、重度增生的系膜细胞、细胞新月体、有炎性细胞浸润的肾小管间质,NET表达显著上调;系膜细胞中、重度增生肾小球的NET表达显著高于其他类型肾小球,差异均有统计学意义(均P＜0.01).中、重度系膜细胞增生肾小球的平均NET阳性细胞数与LN病理活性指数呈正相关(r=0.620,P=0.004);与SLE-DAI评分呈正相关(r=0.492,P=0.027);与肾小管中NET阳性细胞数呈正相关(r=0.558,P=0.011).肾间质NET阳性细胞数与肾间质阳性B淋巴细胞数呈正相关(r=0.573,P=0.008);与LN病理慢性指数呈正相关(r=0.645,P=0.002).结论 NET在LN患者肾组织广泛表达,肾小球中浸润的NET可能参与了LN患者肾组织的活动性损伤.     

Activation of mitochondrial apoptotic pathways in human renal allografts after ischemiareperfusion injury

BACKGROUND: Ischemia-reperfusion injury in cadaveric (CAD) kidney allografts is associated with tubular cell injury, delayed graft function, and an increased incidence of acute and chronic rejection. We tested the hypothesis that activation of specific apoptotic pathways represents a mechanism for tubular cell death after CAD kidney transplantation. METHODS: Serial tissue sections from paraffin-embedded needle biopsy specimens obtained at approximately 1 hr of reperfusion after transplantation of 13 CAD and 12 living-related donor (LRD) renal allografts were examined by using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay to detect apoptosis and by immunohistochemistry for expression of key pro-apoptotic molecules (Bax, Bak, tumor necrosis factor receptor [TNFR]-1, Fas, and cytochrome c). RESULTS: Apoptosis was detected primarily in tubular cells, with a mean+/-standard deviation of 6.8+/-2.2 apoptotic cells per 100 cells examined in CAD renal allografts compared with 1.8+/-2.7 cells per 100 in LRD (P<0.001) renal allografts. There was a significant correlation between apoptosis rate and cold ischemia time in CAD (r=0.86, P<0.001) renal allografts. Bax was expressed in 100% of CAD versus 17% of LRD renal allografts (P<0.001), Bak in 92% of CAD versus 17% of LRD renal allografts (P<0.001), and TNFR-1 in 100% of CAD versus 58% of LRD renal allografts (P<0.05). Fas was expressed in only a small number of samples (23% of CAD and 17% of LRD renal allografts, P=not significant). Bax and Bak were expressed predominantly in apoptotic cells. Cytochrome c was detected as a mitochondrial pattern in LRD renal allografts, but in a diffuse cytosolic distribution in CAD renal allografts. CONCLUSIONS: Ischemia-reperfusion injury in CAD kidney transplants is associated with a duration-dependent increase in tubular cell apoptosis, mediated at least in part by activation of mitochondrial pathways.     

乙型肝炎病毒相关性肾炎患者肾组织Notch1受体表达与临床病理的相关性

目的 观察Notch1受体在乙型肝炎病毒相关性肾炎（HBV-GN）患者肾组织中的表达分布特点,探讨其与肾组织病理及临床表现的相关性。 方法 以2008年至2010年经肾活检确诊的HBV-GN患者48例为研究对象。用免疫组化方法观察HBV-GN肾组织中Notch1受体的分布特征;免疫荧光双标观察Notch1受体与HBsAg的分布关系;分析其与病理类型、肾小球病变、肾小管病变及临床指标的关系。 结果 Notch1受体主要分布在肾小管上皮细胞及间质区域,呈棕红色颗粒状,肾小球内也有少量表达。HBV-GN组患者肾组织Notch1受体阳性积分显著高于血清HBsAg阳性或阴性的原发性肾小球肾炎患者及正常肾组织对照。系膜增生性肾小球肾炎（MsPGN）组和膜增生性肾小球肾炎（MPGN）组Notch1受体阳性积分较高,但各病理类型组间Notch1积分差异无统计学意义。Notch1受体分布与HBsAg分布一致,其强度与肾间质纤维化（r = 0.473,P = 0.001）、小管萎缩（r = 0.690,P = 0.000）、炎细胞浸润（r = 0.616,P = 0.000）等肾小管间质病变呈正相关;患者肾功能与Notch1受体阳性积分呈负相关（r = -0.393,P = 0.006)。 结论 HBV-GN患者肾组织Notch1受体的表达增强,主要分布在肾小管上皮细胞及间质,与HBsAg分布一致;其表达水平与肾间质病变和肾功能变化相关。Notch1受体表达异常可能参与了HBV-GN的肾脏病理的进展。     

Cellular apoptosis and proliferation in experimental renal fibrosis

Background: The progression of chronic renal failure (CRF) is associated with the progressive deletion of renal cells along with the fibrosis of the kidney. We have studied the role of programmed cell death (apoptosis) in the progression of experimental CRF and renal scarring. Methods: The sub-total (5/6th) nephrectomy (SNx) model of CRF was studied in adult male Wistar rats, with renal tissue collected from experimental and control animals on days 7, 15, 30, 60, 90, and 120 post SNx (n-6 per group). These were examined for morphological signs of apoptosis by light and electron microscopy. Further, we stained the nuclear chromatin by the acridine orange fluorescent method and detected signs of DNA cleavage by endonucleases via the principal of TUNEL staining (ApopTag™). Rates of cellular proliferation were measured simultaneously by immunohistochemical staining for the proliferating cell nuclear antigen (PCNA). In addition, cell division was monitored by counting of morphologically mitotic motifs detectable by light microscopy. Results: Progressive renal insufficiency associated with glomerulosclerosis and tubulointerstitial fibrosis took place in the majority of SNx rats. In these animals, we noted a marked and progressive increase in the number of apoptotic glomerular, tubular as well as interstitial cells. The most significant apoptotic changes were seen in the tubules of remnant kidneys peaking at day 120 post-SNx. At this stage, the increase in apoptosis compared to controls was 10.33±2.67 (M±SEM) fold for glomerular cells (P⩽0.006), 26.20±4.56 fold for tubular cells (P<0.0001) and 4.66±0.81 fold for interstitial cells (P⩽0.01). Parallel changes in the number of PSNA positive renal cells were observed. Maximal PCNA staining was seen at day 120 when the increase with respect to controls was 14.00±4.93 fold (P⩽0.05) for glomerular cells, 60.01±12.20 fold (P⩽0.05) for tubular cells and 28.59±4.45 fold ((P⩽0.05) for interstitial cells. As expected the number of cells undergoing division and detectable by conventional light microscopy was lower at any time point to those expressing PCNA. We also observed a close correlation between the severity of tubular atrophy and tubulointerstitial fibrosis with the rate of tubular apoptosis (r=0.970, R²=0.941, P⩽0.001). Conclusions: We have shown a time-dependent increase in apoptosis and PCNA antigen positive staining in the sub-total nephrectomy model of chronic renal failure correlating with the progression of renal fibrosis. PCNA staining did not match analysis for mitosis and was considered to overestimate the number or proliferating cells in the tissue. With this reservation in mind and taking into account the relative time-frames in vivo of apoptosis and proliferation; apoptosis potentially outweighs proliferation by a factor of 2-8-fold, when examined over the same time period. Consequently, even small changes in the finite numbers of apoptotic cells become highly significant. Our results have shown the definite role of apoptosis within progression of renal damage and highlighted how it may contribute to the progression of tubular atrophy and play a role in the pathogenesis of tubulo-interstitial scarring.     

RGC-32在儿童IgA肾病肾组织中的表达及意义

目的 研究RGC-32（response gene to complement 32）在IgA肾病(IgAN) 儿童及正常肾组织中的表达及其意义。 方法 用免疫组织化学方法观察IgAN儿童及正常肾组织中RGC-32蛋白的表达与分布,并与α平滑肌肌动蛋白(α-SMA)、转化生长因子β1（TGF-β1）的表达、IgAN肾组织病理损伤程度及临床相关指标进行统计学分析。 结果 RGC-32蛋白在IgAN及正常肾组织的肾小管均明显表达,而在肾小球、肾小管间质及肾血管未见表达。RGC-32 在正常肾组织、IgAN轻度、中度及重度损伤组中的阳性表达指数分别为（18.29±6.22）%、（23.90±9.65）%、（31.23±9.86）%和（34.52±10.63）%。RGC-32在IgAN儿童肾组织的阳性表达指数与肾小球评分、肾小管间质评分均呈正相关（r = 0.385,0.347,P < 0.05）;与α-SMA、TGF-β1表达亦呈正相关（r = 0.594,0.521,P < 0.01）;而与Scr、尿NAG/Cr、尿Alb/Cr、尿 IgG/Cr、尿α1微球蛋白/Cr均无相关（r = 0.117,-0.115,-0.138,-0.176,-0.028,P均>0.05)。 结论 首次发现RGC-32蛋白在IgAN儿童和正常肾组织中表达于肾小管,而在肾小球、肾小管间质及肾血管未见表达。RGC-32可能参与了IgAN患儿的肾小管间质损伤,尤其是TGF-β1诱导的肾小管上皮细胞-间充质转分化（EMT）过程。     

Apoptosis in kidney and pancreas allograft biopsies

BACKGROUND: Apoptosis is a particular form of cell death involved in the elimination of somatic cells. In this study, the occurrence of apoptotic cells in kidney and pancreas allograft biopsies was analyzed and correlated with the number of infiltrating macrophages and lymphocytes and granzyme B expression. METHODS: Kidney and pancreas biopsies from patients submitted to simultaneous pancreas-kidney transplantation were classified into three groups: acute rejection, chronic rejection, and transplant cases without evidence of rejection. Formalin-fixed paraffin biopsies were used to identify apoptosis by the terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In normal kidney, only few apoptotic cells were observed. In contrast, in kidney-allograft biopsies, the TUNEL signal was detected in the nuclei of tubular epithelial cells and also in mononuclear cells scattered in the interstitium. In pancreas biopsies, numerous apoptotic cells were detected in acinar cells, in ducts, and occasionally in islets. The number of apoptotic cells in acute pancreas rejection was significantly higher compared with acute rejection of kidney grafts (50+/-14 vs. 21+/-4 cells/mm2; P<0.05). In kidney biopsies, there was a positive correlation between apoptosis and macrophages (r=0.51; P<0.005), and apoptosis versus T lymphocytes (r=0.45; P<0.05). In pancreas biopsies, the number of apoptotic cells correlated only with the number of macrophages (r=0.41; P<0.05). CONCLUSIONS: Apoptosis occurs in kidney and pancreas allograft biopsies, markedly in acute rejection in pancreas biopsies. Although apoptosis may reflect a mechanism of down-regulation of the allograft immune response by eliminating infiltrating cells, the elimination of graft cells may result in graft damage, particularly in pancreas transplantation.     

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.     

The role of interstitial infiltrates in IgA nephropathy: a study with monoclonal antibodies

The leucocyte subpopulations in the interstitium and the glomeruli in renal biopsies from 34 patients with IgA nephropathy were analysed using monoclonal antibodies and immunoperoxidase techniques. Monocyte/macrophages and T-cells constituted the predominant infiltrating cell type in the interstitium (278 +/- 24 and 269 +/- 37 cells/mm2 respectively). Few intraglomerular leucocytes were seen, the majority of them belonging to the monocyte/macrophage phenotype (1.1 +/- 0.1 cells/glomerular cross-section). CD4+ lymphocytes predominated among the interstitial and glomerular T-cell populations and the CD4:CD8 ratio was 2.1 +/- 1.1 and 2.4 +/- 1.5 respectively. Only small numbers of NK cells and B cells were found in the interstitium, and almost none in the glomeruli. In contrast, significantly increased numbers of DR-expressing interstitial cells were seen (487 +/- 29/mm2), whereas DR expression by the tubular cells was minimal (37 +/- 6/mm2). Numbers of total leukocytes and T-cells were well correlated with the degree of tubulointerstitial damage and there was a significant correlation between renal functional impairment at the time of biopsy and the numbers of interstitial T cells (P less than 0.05) and CD4+ T cells (P less than 0.01). In contrast, interstitial mononuclear cells did not correlate with subsequent progression of the disease over 2-3 years. However, a more rapid decline of renal function was associated with increased numbers of interstitial B cells. No association was found between intraglomerular cells and degree of renal impairment either at the time of biopsy or in the long term.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP结合元件A1与肾间质泡沫细胞形成、小管间质损害及血清胆固醇的相关性

目的 研究肾小球肾病患者肾间质区域ATP结合元件A1（ABCA1）蛋白的表达及其与泡沫细胞形成、肾小管间质损害、血清胆固醇的相关性。方法 选取诊断明确的Alport综合征患者5例、膜增生性肾小球肾炎（MPGN）患者28例、局灶节段性肾小球硬化（FSGS）患者35例、特发性膜性肾病（IMN）患者36例、IgA肾病（IgAN）患者34例为研究对象。采用免疫组织化学法检测患者肾间质区域（除肾小管）ABCA1蛋白的表达;用直线相关分析其与肾间质区域泡沫细胞相对面积、肾小管间质损害积分、血清胆固醇的相关性。 结果 在各类肾小球疾病患者的肾间质区域中,浸润的单核巨噬细胞、泡沫细胞及肾小管上皮细胞均表达ABCA1蛋白。泡沫细胞浸润者肾间质区域ABCA1蛋白的阳性表达量显著高于无泡沫细胞浸润者（P < 0.05）,但ABCA1的阳性表达量与泡沫细胞相对面积无相关。MPGN、FSGS、IMN患者肾间质区域ABCA1表达量与肾小管间质损害积分呈正相关（P < 0.05）。血清胆固醇与肾间质区域ABCA1蛋白的阳性表达量呈正相关（P < 0.05）。 结论 在各种病理类型的肾小球疾病患者中,血清胆固醇可促进肾间质ABCA1的表达;ABCA1对胞内脂质的转运具有双向作用,其与泡沫细胞形成缺乏肯定关联;ABCA1可能参与了肾小管间质的损害。