Integrated Transcriptomic and Secretome Proteomic Analysis of Hyperglycemia-Stimulated Endothelial Cells and Screening of Target Compounds

Summary

This protocol establishes an integrated multi-omics pipeline (transcriptome and proteome) combined with network pharmacological screening to identify molecular drivers and therapeutic targets for endothelial dysfunction in diabetic complications.

Abstract

Endothelial dysfunction is a key driver of diabetic kidney disease (DKD), but its systemic molecular mechanisms remain incompletely decoded. We hypothesized that integrated multi-omics analysis could map hyperglycemia-induced endothelial damage and identify reusable therapeutics. A reusable computational pipeline was applied to integrate transcriptomic/secretome profiles from hyperglycemic endothelial cells and diabetic kidneys. This identified 534 commonly upregulated genes/proteins. Functional enrichment revealed activation of extracellular matrix remodeling, intercellular communication, and inflammation pathways. Cross-database validation refined 278 high-confidence mediators, and protein-protein interaction network analysis pinpointed ten hub genes. Using network pharmacology, we screened an approved drug library, identifying several candidate compounds (e.g., bruceantin, idelalisib) that potentially target this network. Furthermore, transcription factor regulation and exemplary molecular docking simulations (e.g., idelalisib with CTCF/BRD4) provided mechanistic hypotheses for experimental validation. In conclusion, this study establishes a reusable multi-omics framework that delineates endothelial pathogenic mechanisms in DKD and nominates repurposable drug candidates, offering a strategic approach for mechanistic and therapeutic discovery.

Introduction

Diabetes mellitus, as a global health challenge, affected 529 million people worldwide in 2021, with projections indicating it will impact 1.31 billion people by 2050¹. Beyond glycemic control, long-term complications are the primary source of morbidity, mortality, and reduced quality of life. These include microvascular complications (e.g., diabetic kidney disease (DKD), retinopathy, neuropathy) and macrovascular complications (e.g., accelerated atherosclerosis). Critically, DKD affects 30%-40% of diabetic patients, representing the leading cause of end-stage renal disease (ESRD) worldwide². The profound burden of these....

Protocol

The animal experiments were conducted with the approval of the Committee on Animal Research and Ethics of the Hebei Yiling Medical Research Institute (approval number: N2024082). Check the Table of Materials for the experimental materials and instruments used in this study. Personnel must strictly wear appropriate personal protective equipment (PPE) when handling hazardous reagents such as TRIzol (skin/eye irritant, contains phenol) and protease inhibitors (potential respiratory/skin sensitizers). Segregate biological/liquid waste in autoclavable bags, and hazardous chemical waste in designated containers for institutional hazardous disposal. Decontam....

Results

Demonstration of the pathogenic impact of the hyperglycemic endothelial secretome on renal tubules

This study commenced with the validation of the pathogenic effect of the hyperglycemic endothelial secretome. Conditioned medium from hyperglycemic human glomerular endothelial cells (HG-CM) significantly impaired the viability of human proximal tubule epithelial cells (HK-2) compared to the control groups.

Discussion

This study establishes hyperglycemia-induced endothelial secretome dysfunction as a critical driver of diabetic kidney injury through integrated multi-omics and computational approaches. Demonstration that secretome-mediated cytotoxicity toward renal tubules coincides with systematic dysregulation of 534 endothelial-derived factors converging on matrix remodeling, oxidative stress, and inflammation-core pathways in DKD pathogenesis. Identification of 278 high-confidence mediators, including mechanistically validated hubs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CCL2 ELISA kit	Thermo Fisher Scientific	#88-7399-88
CCR2 inhibitor	Merck Millipore	#227016
Cell Viability Assay Kit	Seven Biotechnology	CCK-8, SC119-01
DNA Sequencer	Illumina	NovaSeq 6000
High-Performance Liquid Chromatography System	Thermo Fisher Scientific	UltiMate 3000 Binary RSLC
HRGECs Culture medium	Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.	1001
Human Renal Glomerular Endothelial Cells (HRGECs)	Shanghai Zhongqiao Xinzhou Biotechnology	PRI-H-00038
Human Renal Tubular Epithelial Cell Complete Medium	Procell	CM-H193
Human Renal Tubular Epithelial Cells (HK2)	Procell	CP-H193
Male db/db and db/m mice (4-week-old)	Hangzhou Ziyuan Biotech Co., Ltd.	SCXK 20190004
Mass Spectrometer	Thermo Fisher Scientific	Astral
Microplate Absorbance Reader	Molecular Devices	SpectraMax iD5
Protease Inhibitor Cocktail (EDTA-free)	Roche	cOmplete, #5056489001
RNA Extraction Reagent	Thermo Fisher Scientific	TRIzol, #15596026
RNA Quality Analyzer	Agilent Technologies	5300 Fragment Analyzer, M5311AA
RNA Quantification Instrument	Thermo Fisher Scientific	Qubit 3.0, Q33216
ROS detection kit	Thermo Fisher Scientific	#EEA019
Ultrafiltration Centrifugal Devices (3 kDa MWCO)	Merck Millipore	Amicon Ultra-4