年轻化内环境改善老年小鼠肾缺血再灌注损伤诱导的肾间质纤维化

doi:10.3877/cma.j.issn.2095-3216.2024.03.002

目的

探究年轻化内环境是否可以改善老年小鼠肾缺血再灌注损伤(IRI)诱导的肾间质纤维化。

方法

采用12周龄和22月龄雄性C57BL/6小鼠建立青年-青年、青年-老年和老年-老年联体共生模型3周后，应用受体小鼠、单体青年和老年小鼠构建单侧IRI(uIRI)模型。联体和单体模型小鼠分为青年-青年连体uIRI组(Y-IP uIRI组)、青年-老年连体uIRI组(O-HP uIRI组)、老年-老年连体uIRI组(O-IP uIRI组)、青年uIRI组(Y uIRI组)、老年uIRI组(O uIRI组)，共5组。分别于术后1 d、3 d和14 d采集血液和肾组织样本。检测小鼠血清肌酐和尿素氮等肾功能指标；天狼星红染色观察肾组织病理学变化；Western印迹检测α-平滑肌肌动蛋白、波形蛋白和I型胶原蛋白等纤维化标志物。

结果

Y-IP uIRI组小鼠的肾功能14 d恢复至基线水平，而O-IP uIRI组和O-HP uIRI组的的肾功能未恢复。然而，O-HP uIRI组的肾功能较O-IP uIRI组明显改善。14 d时O-IP uIRI组和O-HP uIRI组的肾间质纤维化较Y-IP uIRI组明显增加。而且，O-HP组较O-IP uIRI组小鼠的肾间质纤维化明显减轻。Western印迹检测肾间质纤维化标志物α-平滑肌肌动蛋白、波形蛋白和I型胶原蛋白的蛋白表达亦出现相似变化。

结论

年轻化内环境可以改善老年小鼠肾脏IRI诱导的肾间质纤维化，延缓其AKI-CKD进展。

关键词: 内环境, 老年, 缺血再灌注损伤, 肾间质纤维化

Objective

This experiment aimed to explore whether young internal environment could alleviate renal interstitial fibrosis induced by kidney ischemia-reperfusion injury (IRI) in elderly mice.

Methods

Male C57BL/6 mice aged 12 weeks and 22 months were used to establish youth-youth, youth-old, and old-old parabiosis models. Three weeks after establishment of the parabiosis models, the recipient mice, as well as young and old mice without parabiosis received induction of renal unilateral IRI (uIRI). The uIRI mice with and without parabiosis were divided into 5 groups, including youth-youth parabiosis uIRI group (Y-IP uIRI group), youth-old parabiosis uIRI group (O-HP uIRI group), old-old parabiosis uIRI group (O-IP uIRI group), youth uIRI group (Y uIRI group), and old uIRI group (O uIRI group). Blood and renal tissue samples were collected at 1 day, 3 days, and 14 days after surgery for tests of serum creatinine and blood urea nitrogen. The pathological changes of kidneys were observed with Sirius red staining. Fibrotic markers such as α-smooth muscle actin (α-SMA), vimentin, and type I collagen (COL-Ⅰ) were detected with Western blotting.

Results

The Y-IP uIRI group had its renal function to baseline level at day 14, while the O-IP uIRI group and the O-HP uIRI group did not have their renal function restore to baseline level. However, the renal function of O-HP uIRI group was significantly better than that of the O-IP uIRI group. At day 14, the renal fibrosis of both the O-IP uIRI group and O-HP uIRI group significantly increased compared with that of the Y-IP uIRI group. Moreover, the renal interstitial fibrosis of O-HP uIRI group significantly reduced compared with that of the O-IP uIRI group. Western blot results showed that renal fibrosis markers of α-SMA, vimentin, and Col-Ⅰ had similar changes.

Conclusion

Young internal environment could alleviate the renal interstitial fibrosis induced by IRI in the elderly mice so as to delay the progression of AKI-CKD.

Key words: Internal environment, Aged, Ischemia-reperfusion injury, Renal interstitial fibrosis

图1 动物模型构建与分组流程示意图

表1 各组小鼠缺血再灌注后肾功能的动态变化

肾功能指标	分组	基线水平	uIRI 1 d	uIRI 3 d	uIRI 14 d
血肌酐(μmol/L)	Y uIRI	19.88±2.22	119.70±11.13^a	47.18±5.45^a	21.95±1.85
	Y-IP uIRI	19.66±3.04	110.95±9.74^a	41.45±13.27^a	21.80±3.27
	O-HP uIRI	21.33±2.88	154.00±14.74^abc	75.53±9.39^abc	30.65±3.08^abc
	O-IP uIRI	20.88±2.76	201.15±12.75^ab	116.23±14.47^ab	47.00±3.62^ab
	O uIRI	24.00±5.96	228.35±8.59^a	139.45±6.54^a	55.59±5.71
尿素氮(mmol/L)
	Y uIRI	7.10±1.19	40.67±4.61^a	22.80±3.85^a	9.41±2.54
	Y-IP uIRI	7.03±1.10	41.29±5.54^a	23.45±5.40^a	8.85±1.94
	O-HP uIRI	8.11±0.82	55.12±7.34^abc	31.41±4.43^abc	20.71±3.81^abc
	O-IP uIRI	8.36±0.76	75.37±8.23^ab	51.84±5.07^ab	27.16±2.30^ab
	O uIRI	8.49±1.04	80.16±9.26^a	54.22±2.71^a	30.14±2.20

表1 各组小鼠缺血再灌注后肾功能的动态变化

肾功能指标	分组	基线水平	uIRI 1 d	uIRI 3 d	uIRI 14 d
血肌酐(μmol/L)	Y uIRI	19.88±2.22	119.70±11.13^a	47.18±5.45^a	21.95±1.85
	Y-IP uIRI	19.66±3.04	110.95±9.74^a	41.45±13.27^a	21.80±3.27
	O-HP uIRI	21.33±2.88	154.00±14.74^abc	75.53±9.39^abc	30.65±3.08^abc
	O-IP uIRI	20.88±2.76	201.15±12.75^ab	116.23±14.47^ab	47.00±3.62^ab
	O uIRI	24.00±5.96	228.35±8.59^a	139.45±6.54^a	55.59±5.71
尿素氮(mmol/L)
	Y uIRI	7.10±1.19	40.67±4.61^a	22.80±3.85^a	9.41±2.54
	Y-IP uIRI	7.03±1.10	41.29±5.54^a	23.45±5.40^a	8.85±1.94
	O-HP uIRI	8.11±0.82	55.12±7.34^abc	31.41±4.43^abc	20.71±3.81^abc
	O-IP uIRI	8.36±0.76	75.37±8.23^ab	51.84±5.07^ab	27.16±2.30^ab
	O uIRI	8.49±1.04	80.16±9.26^a	54.22±2.71^a	30.14±2.20

图2 缺血再灌注后14 d各组小鼠肾组织天狼星红染色(×400)病理改变与比较注：纤维化区域天狼猩染色呈红色，天狼星红染面积评分半定量分析：Y uIRI组(2.03±0.17)分，Y-IP uIRI组(2.00±0.18)分，O-HP uIRI组(2.30±0.14)分，O-IP uIRI组(3.23±0.15)分和O uIRI组(3.33±0.10)分；与Y-IP uIRI组比较，^aP<0.05；与O-HP uIRI组比较，^bP<0.05

图3 缺血再灌注后14 d各组肾组织的肾间质纤维化标志物表达水平的比较注：α-SMA：α-平滑肌肌动蛋白；Vimentin：波形蛋白；β-tubulin：β-微管蛋白(作为内参照)；CollagenⅠ(Col-Ⅰ)：Ⅰ型胶原蛋白；A：各组uIRI后14 d肾组织Western印迹检测结果；B、C、D：分别为α-SMA、波形蛋白及Ⅰ型胶原蛋白的蛋白表达定量分析比较；与Y-IP uIRI组比较，^aP<0.05；与O-HP uIRI组比较，^bP<0.05

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Young internal environment alleviated renal interstitial fibrosis induced by kidney ischemia-reperfusion injury in elderly mice

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Young internal environment alleviated renal interstitial fibrosis induced by kidney ischemia-reperfusion injury in elderly mice