open access

Vol 73, No 5 (2022)
Original paper
Submitted: 2021-11-16
Accepted: 2022-03-25
Published online: 2022-09-20
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The KLF4–p62 axis prevents vascular endothelial cell injury via the mTOR/S6K pathway and autophagy in diabetic kidney disease

Xinxing Wang1, Wei Su2, Mingze Ma3, Lichao Zhu4, Ruxin Gao1, Chuan Qin1, Shuai Shao1, Dexuan Gao5, Junlin Gao2, Zhenhai Zhang6
·
Pubmed: 36621906
·
Endokrynol Pol 2022;73(5):837-845.
Affiliations
  1. Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
  2. Liver Gall Bladder and Pancreatic Surgery Ward, Qinghai Red Cross Hospital, Xining, Qinghai, China
  3. Department of Infectious Diseases, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
  4. Department of Paediatric Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
  5. Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
  6. Department of Hepatobiliary Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong, China

open access

Vol 73, No 5 (2022)
Original Paper
Submitted: 2021-11-16
Accepted: 2022-03-25
Published online: 2022-09-20

Abstract

Introduction: Diabetic kidney disease (DKD) is a complication of systemic diabetic microangiopathy, which has a high risk of developing into end-stage renal disease and death. This study explored the mechanism underlying autophagy in DKD vascular endothelial cell injury.

Material and methods: DKD and vascular endothelial cell injury models were established using Sprague Dawley rats and human umbilical vein endothelial cells (HUVECs). HUVECs overexpressing Kruppel-like factor 4 (KLF4) were constructed by transient transfection of plasmids. Biochemical determination of urinary protein and blood urea nitrogen (BUN), superoxide dismutase (SOD), and creatinine (Scr) levels was performed. Renal pathology was observed by periodic acid–Schiff (PAS) staining. Cell Counting Kit-8 (CCK8), terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and immunocytochemistry (ICC) were used to analyse the growth and apoptosis of HUVECs. Microtubule-associated protein light chain 3 (LC3) expression was observed by immunofluorescence (IF). The reactive oxygen species (ROS) levels were measured using flow cytometry. Monocyte chemoattractant protein-1 (MCP-1), KLF4, and tumour necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA). The expression of KLF4, p62 protein, and LC3 was analysed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). S6 kinase (S6K), p70 ribosomal S6 kinase (p-S6K), Beclin1, ATG5, LC3, p62, Caspase-3, mammalian target of rapamycine (mTOR), and phsophorylated mTOR (p-mTOR) expressions were detected by western blotting.

Results: PAS-positive substances (polysaccharide and glycogen) and S6K protein levels increased, and LC3 protein expression decreased in DKD rats. The levels of urinary protein, BUN, and Scr increased, and KLF4 decreased in DKD rats. High glucose (HG) levels decreased the proliferation and increased the apoptosis rate of HUVECs. The expression of ROS, TNF-α, MCP-1, and p62 increased, while the expression of SOD, KLF4, Beclin1, ATG5, and LC3 decreased in HG-induced HUVECs. KLF4 overexpression significantly increased Beclin1, ATG5, and LC3 protein expression and decreased p62 protein expression compared to the oe-NC group in HG-induced HUVECs. KLF4 overexpression inhibits the expression of Caspase-3, p-mTOR, and p-S6K in HG-induced HUVECs.

Conclusions: KLF4–p62 axis improved vascular endothelial cell injury by regulating inflammation and the mTOR/S6K pathway in DKD.

Abstract

Introduction: Diabetic kidney disease (DKD) is a complication of systemic diabetic microangiopathy, which has a high risk of developing into end-stage renal disease and death. This study explored the mechanism underlying autophagy in DKD vascular endothelial cell injury.

Material and methods: DKD and vascular endothelial cell injury models were established using Sprague Dawley rats and human umbilical vein endothelial cells (HUVECs). HUVECs overexpressing Kruppel-like factor 4 (KLF4) were constructed by transient transfection of plasmids. Biochemical determination of urinary protein and blood urea nitrogen (BUN), superoxide dismutase (SOD), and creatinine (Scr) levels was performed. Renal pathology was observed by periodic acid–Schiff (PAS) staining. Cell Counting Kit-8 (CCK8), terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and immunocytochemistry (ICC) were used to analyse the growth and apoptosis of HUVECs. Microtubule-associated protein light chain 3 (LC3) expression was observed by immunofluorescence (IF). The reactive oxygen species (ROS) levels were measured using flow cytometry. Monocyte chemoattractant protein-1 (MCP-1), KLF4, and tumour necrosis factor alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA). The expression of KLF4, p62 protein, and LC3 was analysed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). S6 kinase (S6K), p70 ribosomal S6 kinase (p-S6K), Beclin1, ATG5, LC3, p62, Caspase-3, mammalian target of rapamycine (mTOR), and phsophorylated mTOR (p-mTOR) expressions were detected by western blotting.

Results: PAS-positive substances (polysaccharide and glycogen) and S6K protein levels increased, and LC3 protein expression decreased in DKD rats. The levels of urinary protein, BUN, and Scr increased, and KLF4 decreased in DKD rats. High glucose (HG) levels decreased the proliferation and increased the apoptosis rate of HUVECs. The expression of ROS, TNF-α, MCP-1, and p62 increased, while the expression of SOD, KLF4, Beclin1, ATG5, and LC3 decreased in HG-induced HUVECs. KLF4 overexpression significantly increased Beclin1, ATG5, and LC3 protein expression and decreased p62 protein expression compared to the oe-NC group in HG-induced HUVECs. KLF4 overexpression inhibits the expression of Caspase-3, p-mTOR, and p-S6K in HG-induced HUVECs.

Conclusions: KLF4–p62 axis improved vascular endothelial cell injury by regulating inflammation and the mTOR/S6K pathway in DKD.

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Keywords

Kruppel-like factor 4; human umbilical vein endothelial cells; diabetic kidney disease; mTOR/S6K pathway; autophagy

About this article
Title

The KLF4–p62 axis prevents vascular endothelial cell injury via the mTOR/S6K pathway and autophagy in diabetic kidney disease

Journal

Endokrynologia Polska

Issue

Vol 73, No 5 (2022)

Article type

Original paper

Pages

837-845

Published online

2022-09-20

Page views

4497

Article views/downloads

1154

DOI

10.5603/EP.a2022.0072

Pubmed

36621906

Bibliographic record

Endokrynol Pol 2022;73(5):837-845.

Keywords

Kruppel-like factor 4
human umbilical vein endothelial cells
diabetic kidney disease
mTOR/S6K pathway
autophagy

Authors

Xinxing Wang
Wei Su
Mingze Ma
Lichao Zhu
Ruxin Gao
Chuan Qin
Shuai Shao
Dexuan Gao
Junlin Gao
Zhenhai Zhang

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