N6-甲基腺嘌呤甲基化修饰下调Klotho介导脓毒症急性肾损伤肾小管上皮细胞衰老的机制

doi:10.3969/j.issn.1006-5725.2026.11.019

Abstract

Abstract:

Objective To elucidate the molecular mechanism whereby N6-methyladenosine （m6A） RNA methylation regulates Klotho expression and drives renal tubular epithelial cell （RTEC） senescence in septic acute kidney injury （SAKI）. Methods An in vitro SAKI cellular model was established using lipopolysaccharide （LPS）-stimulated human proximal tubular epithelial cells （HK-2）. Cell viability， senescence-associated β-galactosidase （SA-β-gal） activity， and global and gene-specific m6A levels were quantified. Functional gain- and loss-of-function experiments were performed via transient transfection of small interfering RNAs （siRNAs） targeting METTL3 or YTHDF2， and plasmid-mediated overexpression of wild-type Klotho or a Klotho mutant lacking functional m6A consensus sites. Results In the SAKI model， RTECs exhibited significantly reduced viability （P < 0.05）， increased SA-β-gal positivity （P < 0.05）， elevated global m6A abundance， and site-specific hypermethylation of Klotho mRNA （P < 0.05）. Concurrently， METTL3 and YTHDF2 protein expression were upregulated （P < 0.05）， whereas Klotho mRNA and protein levels were markedly suppressed （P < 0.05）. METTL3 or YTHDF2 overexpression enhanced m6A deposition on Klotho mRNA （P < 0.05）， accelerated its YTHDF2-dependent decay （P < 0.05）， and consequently diminished Klotho expression （P < 0.05） and exacerbated cellular senescence （P < 0.05）. Conversely， siRNA-mediated knockdown of METTL3 or YTHDF2， or expression of the m6A-site-deficient Klotho mutant， restored Klotho expression and attenuated senescence phenotypes. Conclusion Dysregulated m6A methylation-mediated by METTL3/YTHDF2 axis-promotes RTEC senescence in SAKI through targeted suppression of Klotho. These findings identify the m6A–Klotho regulatory axis as a mechanistically defined and therapeutically relevant pathway in sepsis-induced renal aging.

Key words: N6-methyladenosine, Klotho, cell senescence, septic kidney injury

CLC Number:

R692.5

Zhu LI,Shiqi NIE,Jianglu TAN,Xiaoyue LI,Lili TANG,Zhi KOU. N6-methyladenosine methylation regulates klotho expression in septic acute kidney injury-underlying mechanism of renal tubular cell senescence[J]. The Journal of Practical Medicine, 2026, 42(11): 2037-2048.

Figures/Tables 19

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References 26

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基因		序列
p53	F	5'-AGTTCCTGCATGGGCGGC-3'
p53	R	5'-TCTGGGAAGGGACAGAAGGA-3'
p21	F	5'-CCAGCGGAACAGTTTACCTT-3'
p21	R	5'-GAAGATCAGCCGGCGTTTG-3'
p16INK4A	F	5'-CTCGTGCTGATGCTACTGAGG-3'
p16INK4A	R	5'-GGTCGGCGCAGTTGGGCTCC-3'
Ki67	F	5'-AGCCAGCGTTCCGAAGTT-3'
Ki67	R	5'-TCCAGCGTGTCCGTCTTCT-3'
METTL3	F	5'-AGCTGACAGCAGCTACAGTC-3'
METTL3	R	5'-TCTGCTGCTGCTGCTGCTGT-3'
YTHDF2	F	5'-GCTGCTGCTGCTGCTGCTGC-3'
YTHDF2	R	5'-AGCAGCAGCAGCAGCAGCAG-3'
Klotho	F	5'-CGCTGCTGCTGCTGCTGCTG-3'
Klotho	R	5'-TCTGCTGCTGCTGCTGCTGA-3'
GAPDH	F	5'-GGAGCGAGATCCCTCCAAAAT-3'
GAPDH	R	5'-GGCTGTTGTCATACTTCTCATGG-3'

检测指标	Control组	SAKI组	t值	P值
CCK-8（OD）	1.24 ± 0.13	0.66 ± 0.06	6.950	0.002
CCK-8/%	100.00 ± 10.47	53.25 ± 5. 09	6.949	0.002
P53	1.02 ± 0.42	5.35 ± 0.71	-9.097	0.001
P21	1.04 ± 0.22	4.91 ± 0.52	-11.978	< 0.001
p16INK4A	1.03 ± 0.27	4.17 ± 0.43	-10.666	< 0.001
Ki67	5.29 ± 0.64	1.00 ± 0.27	10.693	< 0.001
IL-1β	61.44 ± 19.27	245.01 ± 17.01	-12.368	< 0.001
IL-8	108.69 ± 44.54	797.13 ± 43.16	-19.226	< 0.001
IL-6	128.97 ± 43.96	381.39 ± 24.24	-8.708	0.001
TNF-α	122.70 ± 17.70	241.62 ± 21.17	-7.465	0.002
TGF-β	205.65 ± 49.60	1 167.51 ± 93.79	-15.702	< 0.001

目的基因	SAKI组	SAKI + METTL3组	SAKI + YTHDF2组	F值	P值
METTL3	2.23 ± 0.26	5.15 ± 0.40^??	2.32 ± 0.33	73.518	< 0.001
YTHDF2	3.35 ± 0.65	3.62 ± 0.46	8.33 ± 0.45^??	84.082	< 0.001
Klotho	2.63 ± 0.40	1.55 ± 0.29^?	1.16 ± 0.34^?	14.515	0.005

检测指标	SAKI组	SAKI + METTL3组	SAKI + YTHDF2组	F值	P值
CCK-8（OD）	0.99 ± 0.06	0.22 ± 0.04	0.14 ± 0.02	371.198	< 0.001
CCK-8/%	100.24 ± 5.80	23.21 ± 4.25	15.94 ± 2.14	348.526	< 0.001
p53	2.58 ± 0.28^?#	4.09 ± 0.98^*	6.37 ± 0.53^#	24.926	0.001
p21	2.99 ± 0.88^?#	4.80 ± 0.75^*	7.30 ± 0.61^#	24.607	0.001
p16INK4A	2.96 ± 0.30^?#	5.76 ± 0.67^*	5.67 ± 1.09^#	13.192	0.006
Ki67	2.88 ± 0.95^?#	0.95 ± 0.26^*	1.03 ± 0.18^#	10.660	0.011
IL-1β	267.23 ± 48.47^?#	572.94 ± 41.89^*	639.00 ± 40.66^#	61.488	< 0.001
IL-8	803.16 ± 53.72^?#	1 660.72 ± 129.89^*	2 133.22 ± 162.90^#	88.385	< 0.001
IL-6	403.22 ± 56.31^?#	818.41 ± 69.64^*	760.82 ± 75.80^#	33.078	0.001
TNF-α	272.32 ± 26.02^?#	435.33 ± 38.76^*	485.67 ± 80.05^#	13.035	0.007
TGF-β	1 155.65 ± 99.81^?#	1 795.33 ± 144.89^*	1 895.67 ± 173.70^#	23.727	0.001

目的基因	SAKI组	SAKI + si-METTL3组	SAKI + si-YTHDF2组	F值	P值
METTL3	2.23 ± 0.26	1.05 ± 0.26	2.58 ± 0.23	30.794	0.001
YTHDF2	3.35 ± 0.65	3.68 ± 0.77	0.69 ± 0.18	23.301	0.001
Klotho	2.63 ± 0.40	7.67 ± 0.58	9.70 ± 0.71	119.204	< 0.001

N6-methyladenosine methylation regulates klotho expression in septic acute kidney injury-underlying mechanism of renal tubular cell senescence

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检测指标	SAKI组	SAKI + si-METTL3组	SAKI + si-YTHDF2组	F值	P值
CCK-8（OD）	0.99 ± 0.06	1.42 ± 0.06	1.73 ± 0.11	65.699	< 0.001
CCK-8/%	100.24 ± 5.80	143.65 ± 5.38	174.84 ± 11.21	67.061	< 0.001
p53	2.58 ± 0.28^?#	1.72 ± 0.16^*	1.58 ± 0.19^#	18.445	0.003
p21	2.99 ± 0.88^?#	1.76 ± 0.38^*	1.59 ± 0.25^#	5.430	0.045
p16INK4A	2.96 ± 0.30^?#	1.63 ± 0.22^*	0.92 ± 0.34^#	37.275	< 0.001
Ki67	2.68 ± 0.64^?#	5.78 ± 0.29^*	5.12 ± 0.75^#	22.543	0.002
IL- 1β	287.23 ± 31.04^?#	168.06 ± 29.56^*	163.97 ± 20.51^#	19.537	0.002
IL-8	803.16 ± 53.72^?#	441.75 ± 34.31^*	458.12 ± 68.45^#	42.85	< 0.001
IL-6	403.22 ± 56.31^?#	179.09 ± 30.50^*	207.25 ± 23.97^#	28.692	0.001
TNF-α	272.32 ± 26.02^?#	183.62 ± 16.63^*	171.38 ± 20.10^#	20.113	0.002
TGF-β	1 155.65 ± 99.81^?#	636.96 ± 55.18	484.71 ± 77.29^#	58.697	< 0.001

目标基因	组1	组2	t值	P值
Klotho	SAKI组: 2.63 ± 0.40	SAKI + Klotho mRNA mut组: 8.24 ± 0.65	-12.786	<0.001
METTL3	SAKI + METTL3组: 5.15 ± 0.40	SAKI + METTL3 + Klotho mRNA mut组: 3.96 ± 0.45	3.427	0.027
YTHDF2	SAKI + YTHDF2组: 8.33 ± 0.46	SAKI + YTHDF2 + Klotho mRNA mut组: 7.02 ± 0.53	3.242	0.032

检测指标	SAKI组	SAKI + Klotho mRNA mut组	t值	P值
CCK-8（OD）	0.99 ± 0.06	1.64 ± 0.06	-13.231	< 0.001
CCK-8/%	100.24±5.80	165.9 ± 6.54	-13.006	< 0.001
p53	2.58 ± 0.28	1.72 ± 0.16	8.963	0.001
p21	2.99 ± 0.88	1.76 ± 0.38	3.394	0.027
p16INK4A	2.96 ± 0.30	1.63 ± 0.22	9.926	0.001
Ki67	2.68 ± 0.64	5.78 ± 0.29	-8.98	0.001
IL-1β	267.23 ± 48.47	101.08 ± 12.37	5.752	0.005
IL-8	803.16 ± 53.72	333.82 ± 34.04	12.782	< 0.001
IL-6	403.22 ± 56.31	138.48 ± 18.79	7.725	0.002
TNF-α	272.32 ± 26.02	128.63 ± 16.31	8.104	0.001
TGF-β	1 155.65 ± 99.81	389.92 ± 75.67	10.589	< 0.001

检测指标	SAKI + METTL3组	SAKI + METTL3 + Klotho mRNA mut组	t值	P值
CCK-8（OD）	0.22 ± 0.04	0.66 ± 0.05	-11.643	< 0.001
CCK-8/%	23.21 ± 4.25	67.30 ± 5.02	-11.601	< 0.001
p53	4.09 ± 0.98	1.25 ± 0.12	4.979	0.008
p21	4.80 ± 0.75	1.54 ± 0.30	7.000	0.002
p16INK4A	5.76 ± 0.67	1.03 ± 0.18	11.809	< 0.001
Ki67	0.95 ± 0.26	7.16 ± 0.94	-11.015	< 0.001
IL-1β	572.94 ± 41.89	220.89 ± 25.18	12.475	< 0.001
IL-8	1 660.72 ± 129.89	550.04 ± 130.77	10.437	< 0.001
IL-6	818.41 ± 69.64	233.01 ± 23.95	13.768	< 0.001
TNF-α	435.33 ± 38.76	160.90 ± 13.38	11.592	< 0.001
TGF-β	1 795.33 ± 144.89	830.90 ± 59.10	10.675	< 0.001

检测指标	SAKI + YTHDF2组	SAKI + YTHDF2 + Klotho mRNA mut组	t值	P值
CCK-8（OD）	0.14 ± 0.02	0.62 ± 0.04	-19.089	< 0.001
CCK-8/%	15.94 ± 2.14	63.71 ± 3.60	-19.772	< 0.001
p53	6.37 ± 0.53	1.36 ± 0.30	14.329	< 0.001
p21	7.30 ± 0.61	1.56 ± 0.61	11.480	< 0.001
p16INK4A	5.67 ± 1.09	1.55 ± 0.21	6.447	0.003
Ki67	1.03 ± 0.18	6.41 ± 0.58	-15.396	< 0.001
IL-1β	639.00 ± 40.66	99.25 ± 24.46	19.700	< 0.001
IL-8	2 133.22 ± 162.90	365.78 ± 53.99	17.838	< 0.001
IL-6	760.82 ± 75.80	187.68 ± 28.64	12.251	< 0.001
TNF-α	485.67 ± 80.05	144.59 ± 16.57	7.227	0.002
TGF-β	1 895.67 ± 173.7	581.26 ± 50.24	12.591	< 0.001

目标基因	SAKI组	SAKI + Klotho mRNA mut组	t值	P值
METTL3	1.55 ± 0.29	2.57 ± 0.60	-2.882	0.045
YTHDF2	1.16 ± 0.34	2.26 ± 0.15	-5.104	0.007