【作者】 杨海春；

【导师】 林善锬； Agnes Fogo； 顾勇； 马骥；

【作者基本信息】 复旦大学 ， 内科肾脏病学， 2005， 博士

【摘要】 第一部分PPARγ激动剂保护足细胞损伤所致FSGS的机制研究背景大鼠和人的肾小球硬化时，我们观察到过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptorsγ，PPARγ)在足细胞上表达。在肾大部切除所导致的FSGS(Facal segmental glomerulosclerosis)模型中，给予PPARγ激动剂可以改善非糖尿病性肾小球硬化，说明在硬化时上调足细胞上的PPARγ可能是拮抗肾脏病变的有益反应。另外体外实验也证明PPARγ激动剂可以保护嘌呤霉素(PAN)诱导的足细胞坏死和凋亡。因此，本研究的目的是观察体内激活PPARγ是否能够改善足细胞损伤所导致的肾小球硬化。方法成年SD(Sprague-Dawley)大鼠诱导PAN肾病(一种足细胞损伤的FSGS模型)：0周时行单侧肾切除，2周时给予腹腔注射PAN(剂量为100mg／kg体重)，随后在第6、7、8周各给一次PAN(剂量为40mg／kg体重)。动物随机分为三组：Cont组(对照组)，给予正常饮食；P0组(前治疗组)，在做单侧肾切除时即给予吡咯列酮；P6组(后治疗组)，从第六周给予吡咯列酮。在第12周取肾组织做组织形态学和分子生物学检测。结果在12周，三组的蛋白尿和血压没有明显差异。前治疗组与对照组的肾功能(GFR)下降基本相同，而后治疗组的GFR明显增高，同时小球的硬化指数也明显降低(P＜0.05，与对照组相比)。给予PAN后，PPARγ可以在节段硬化小球的足细胞和系膜细胞上表达，部分在壁层上皮细胞表达。6周后给予吡咯列酮治疗，可以减少系膜区的PPARγ染色，但足细胞上的染色明显增强，伴有小球硬化明显减轻。给予吡咯列酮治疗的两组都可以明显减轻小球内WT-1阳性细胞的丢失(P＜0.05，与对照组相比)，并有下降小球内PCNA阳性细胞／凋亡细胞的趋势。两个治疗组中，小球内巨噬细胞的浸润均明显减少，皮质PAI-1mRNA的表达降低(P＜0.05，与对照组相比)。与此相反，吡咯列酮治疗不改变。肾脏内TGF-β1和TIMP-1的表达。此外，治疗组皮质血管生成素样蛋白4(Aglp4)的mRNA水平仅为对照组的20％左右，同时小球内VEGF阳性细胞数及CD31染色增加(P＜0.05，与对照组相比)。结论给予PPARγ激动剂(吡咯列酮)可以保护足细胞损伤所导致的FSGS。这种保护作用可能与PPARγ减少足细胞的凋亡、降低巨噬细胞的浸润和PAI—1的表达、上调VEGF并下调Aglp4维护小球内皮细胞功能有关。同时前治疗组与后治疗组在功能和形态学上存在差异，只有在病变启动后给予PPARγ激动剂才有PPARγ相关的保护作用。我们的结果提示PPARγ激动剂可能是治疗足细胞损伤相关性FSGS的一种新方法。第二部分残余肾小鼠模型中整合素β6缺失不改变间质纤维化但加重小球硬化的机制研究背景整合素αvβ6可以激活局部的TGFβ(transforming growth factor-β)。在肾脏β6主要表达于小管上皮细胞和球旁器。我们以前已观察到β6缺失的小鼠(β6knock-out小鼠)可以保护单侧输尿管结扎所导致的间质纤维化。本研究运用残余肾模型，一种原发性小球硬化并有继发性间质纤维化的模型，观查小管上β6的缺失对小球硬化的的影响。方法成年野生型(WT组，129背景)和β6敲除(β6-／-组)小鼠行5／6肾切除。在0周和第12周取肾脏，观察形态学改变，并通过免疫组化和Western Blot的方法检测P-Smad2、PAI-1、肾素(renin)、nNOS(neuronal nitric oxide synthase)和COX-2(cyclooxygenase-2)。结果在12周，WT和β6-／-组均有类似的尿蛋白排泄增高(P＜0.05，与手术前相比)。β6-／-组较WT组的血压显著降低。尽管皮质间质容量分数类似，β6-／-组有更重的小球损伤(P＜0.05，与WT组相比)。SI(小球硬化指数)与间质纤维化的比值显示β6-／-组小球／小管病变较WT组更加不对称。同时，β6-／-组小球内PAI-1表达上调。在正常时，β6缺失并不改变P-Smad2、nNOS、renin和COX-2的蛋白水平。肾大部切除后12周，与WT组相比，β6-／-小鼠的P-Smad2降低5.7倍、nNOS降低2.4倍，而renin的表达升高1.8倍(P＜0.05)。COX-2的表达两组没有差别。免疫组化显示：0周时，nNOS和renin主要表达于球旁器，而12周时，β6-／-组renin的广度和强度增加、nNOS则下降(与WT组相比)；两组在5／6肾切除后，致密斑和髓质间质细胞处的COX-2表达均升高。结论整合素β6缺失会加重残余肾小鼠的小球硬化，而间质纤维化相对WT小鼠改变不明显，同时球旁器renin表达上调、nNOS表达下调。我们推测球旁器β6的缺失，导致管—球反馈的失调及局部RAS的改变，可能是小球硬化加重的原因。更多还原

【Abstract】 PART 1 Treatment with Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) Agonist Is Protective in Podocyte Injury-Associated FSGSBackground We have previously observed increased expression of PPARγ in podocytes in both rat and human sclerotic conditions in vivo. PPARγ agonist ameliorated non-diabetic glomerulosclerosis (FSGS) in the hypertensive subtotal nephrectomy model in the rat, suggesting that up-regulated podocyte PPARγ in sclerosis could be a beneficial counterregulatory response. Our recent in vitro data show that PPARγ activation protects against puromycin aminonucleoside (PAN)-induced apoptosis and necrosis. The aim of the present study was to investigate whether activation of PPARγ can also attenuate podocyte injury-associated glomerulosclerosis in vivo.Methods PAN nephropathy, a podocyte-injury model of FSGS, was induced in adult male Sprague-Dawley rats. Following uninephrectomy at week 0, i.p. PAN was given at weeks 2 (100mg/kg Bwt), 6, 7 and 8 (40mg/kg Bwt). The animals then either received no further treatment, Control group (Cont); or pioglitazone (10 mg/kg/d, Pio) starting at week 0, P0 group; or pioglitazone starting at week 6, P6 group. Morphological and molecular biological analyses were performed at week 12. Results At week 12, there were similar urinary protein excretion and SBP during three groups. GFR was decreased in CONT and P0 groups by week 12; Rats with posttreatment with Pio had increased GFR, and by morphological analyses, this group showed parallel amelioration of the development of glomerulosclerosis compared with control. PPARγ expression in PAN-treated rats at 12 weeks was present in podocytes and in mesangial cells in segmentally sclerotic glomeruli, with occasional parietal epithelial cell staining. In PAN+Pio rats, mesangial staining was lessened, but podocyte staining was strongly accentuated, correlating with less severe overall sclerosis. Treatment with Pio partially restored podocyte WT-1 staining and showed a numerical trend to decrease the ratio of PCNA-positive to apoptoic cells in glomeruli. Pre- or post-treatment with Pio significantly reduced infiltrating glomerular macrophages and PAI-1 mRNA expression in cortex. In contrast, Pio did not change renal mRNA levels for TGF-β1 and TIMP-1. Pio treatment decreased renal cortical Aglp4 expression to almost 20% at Cont group and increased VEGF positive glomerular cells.Conclusions We conclude that treatment with the PPARγ agonist pioglitazone has protective effects on progression of podocyte injury-associated glomerulosclerosis. These beneficial effects appear to be related to the regulation of podocyte apoptosis, decreased macrophage infiltration and PAI-1 expression, and protection of glomerular capillary loss by increasing VEGF expression and decreasing Aglp4. Our results further indicate that there are key functional and morphological differences in altimate responses to PPARγ agonist given before, or after, podocyte injury, with PPARγ protection only occurring in the latter. The beneficial affects of delayed time point of interaction is clinically relevout, as therapies are initiated in humans following the initial insult. Our results thus suggest that PPARγ agonists can provide a novel approach with therapeutic potential in podocyte injury-associated FSGS.PART2Absence of integrin αvβ6 does not change interstitial fibrosis but worsens glomeruloslerosis in remnant kidney miceBackground The heterodimeric integrin αvβ6 activates transforming growth factor-β (TGFβ) locally. Integrin αvβ6 is expressed in the tubular epithelium and the juxtaglomerular apparatus (JGA) in kidney. We have previously observed that mice deficient in β6 (β6-/-) were protected against interstitial fibrosis induced by unilateral ureteral obstruction. The aim of the present study was to investigate whether the effect of β6 deficiency on development of glomerulosclerosis was parallel to that on interstitial fibrosis, by study of the remnant kidney model of glomerulosclerosis and associated, secondary interstitial fibrosis.Methods Adult male wild type (WT 129) and β6-/- mice on 129 background underwent 5/6 nephrectomy (Nx). Kidneys were harvested for morphologic, P-Smad2, renin, neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) analyses by western and immunohistochemistry at week 0 and 12. Result At week 12, both WT and β6-/- had significantly and similarly increased proteinuria when compared with baseline (P<0.05). β6-/- showed lower systolic blood pressure compared with WT. Although the cortical interstitial volume fraction was similar, β6-/- group had more severe glomerular injury than WT (P<0.05). Ratios of SI vs interstitial fibrosis thus showed disproportionally milder interstitial lesions vs glomerular lesions in β6-/- vs WT. At baseline, β6 -/- did not show altered expression by western blot of P-Smad2, nNOS, renin or COX-2. At week 12 after Nx, β6 -/- micehad decreased P-Smad2 by 5.7-fold and nNOS by 2.4-fold, while renin expression increased 1.8 fold vs WT Nx (P<0.05). There was no difference in COX-2 expression. By immunohistochemistry, nNOS and renin were observed mainly in JGA at week 0 with increased renin extent and intensity and decreased nNOS intensity in β6 -/- vs WT at week 12. Increased COX-2 was detected in macula densa/cTALH and medullary interstitial cells after 5/6 nephrectomy in both WT and β6 -/-.Conclusion We conclude that absence of integrin β6 worsens glomerulosclerosis after 5/6 nephrectomy, with disproportionally milder tubulointerstitial fibrosis at least in part by increasing renin and decreasing nNOS expression in the JGA. We speculate that dysregulated tubuloglomerular feedback due to absent JGA β6 expression in the remnant kidney might be involved in this augmented sclerosis.更多还原