DKD is a significant cause of chronic kidney disease (CKD) and end-stage renal disease (ESKD). DKD pathogenesis includes renal hemodynamic changes, tubular atrophy, glomerular hypertension, oxidative stress, ischemia and hypoxia, and the upregulation of the renin-aldosterone system [13]. Many studies have focused on renal tubular lesions in diabetic nephropathy, including atrophy and interstitial fibrosis [14]. The renal tubule is the leading reabsorption site; renal tubule cells account for approximately 90% of the cortex. Many factors, such as excessive reabsorption and enhanced gluconeogenesis, in the early stage of diabetic neuropathy (glomerular hyperfiltration stage) lead to hypoxia and mitochondrial dysfunction in proximal tubular cells. This induces and exacerbates renal tubule lesions [15, 16]. Exosomes can participate in the abnormal signal transduction of local vascular cells. Moreover, the content of exosomes in urine is not easily affected by some peak proteins, which better reflects the progression of diabetic nephropathy [17]. Recently, exosomes have become a new focus of DKD research as diagnostic markers [18].
Proteomic analysis revealed 34 target proteins in the exosome satisfying the thresholds of a |log2(FC)|>1 and a p value < 0.05. Renal tubular transcriptomics revealed 985 genes in the transcriptome, meeting the thresholds of a |log2(FC)|>1 and a p-value < 0.05. The two intersect to obtain 22 intersectional targets (COL4A1, C3, TGM2, TFPI2, ANXA2, STAT1, CFH, NRP1, MCM6, FBN1, RPA1, BGN, PLAT, VWF, EIF3M, ARCN1, VLDLR, SEMA5A, SPOCK1, HTRA1, DHX15, and PCOLCE2). GO and KEGG enrichment analyses of these 22 intersecting targets revealed that they were mainly involved in renal and urogenital system development in BP. The CCs were mainly associated with the extracellular matrix, which contains collagen, basement membrane, and vacuolar lumen, and the MFs were mainly associated with extracellular matrix structural components and enzyme inhibitor activity. KEGG pathway enrichment analysis revealed enrichment of genes related to the complement and coagulation cascades and the PI3K-Akt signaling pathway. The complement system is a main component of human innate immunity. The complement and coagulation cascades are activated and initiated through three different pathways: the classical, alternative, and lectin pathways [19, 20]. Innate immune activation could have a potential role in DKD pathogenesis and progression[21]. PI3K is an intracellular phosphatidylinositol kinase, and Akt is a protein kinase called protein kinase B (PKB) due to its high homology with protein kinase A (PKA) and protein kinase C (PKC) [22]. Renal tubulointerstitial fibrosis is induced by high glucose after activation of the PI3K-Akt signaling pathway [23]. Renal fibrosis is the main pathological marker of late DKD. Thus, regulating the PI3K-Akt signaling pathway can effectively prevent DKD progression.
The RF, SVM, and GLM models constructed in R were used to identify nine key targets. Then, three genes with AUC > 0.9 were further screened by constructing ROC curves (CFH, COL4A1, and ANXA2) as biomarkers for diagnosing DKD. Differential expression analysis and clinical correlation verification of the three biomarkers revealed that CFH, COL4A1, and ANXA2 were differentially expressed and clinically correlated. Complement factor H (CFH) is a sialic acid-containing glycoprotein that causes inflammation and complement system disorders through the NF-κB pathway [24]. Inflammation and oxidative stress are often considered key DKD mechanisms [25]. In existing studies, [26] CFH was significantly associated with fasting insulin and prediabetes in an age-and-sex model. Therefore, CFH can be detected throughout the disease course, such as in prediabetic nephropathy, and can be used as an early diagnostic DKD marker. COL4A1 (collagen type IV α 1 chain) is a vital basement membrane component closely related to the severity of renal fibrosis [27, 28]. ANXA2, a member of the annexin family, is a calcium-dependent phospholipid-binding protein that has multiple biological functions, such as cell proliferation, apoptosis, and migration, and interferes with high glucose-induced apoptosis and inflammation [29, 30]. However, no studies have described the specific mechanism of DKD, especially the role of CFH, COL4A1, and ANXA2 in diabetic renal tubulopathy. Investigating the roles of CFH, COL4A1, and ANXA2 in DKD will help to decipher DKD pathogenesis and help develop new therapeutic strategies for DKD.