A large number of studies have confirmed that BMI, hypertension, proteinuria and renal function effect on the prognosticate IgA nephropathy, however, the genetic mechanism of these clinical characteristics caused progression of IgA nephropathy are not clear, and the relationship and association between factors (like age sex) and IgA nephropathy are still needed more study and evidence on biological processes at the molecular level. Noteworthy, disease always involved thousands of gene expression changes with a huge and complex gene regulated network, which means are search for a single gene is superficial and unreliable and it is hard to explain the mechanism of a disease. WGCNA is an advanced, successful and comprehensive algorithm for co-expression analysis, and it was not only used to construct gene co-expression network and screen gene modules, but also a powerful tool to identify hub genes associated external information, especially clinical characteristics, and help researchers to understand the mechanism of the disease and providing a theoretical basis for the diagnosis and treatment of the disease.
In the present study, we collected 32 human samples from the European Renal cDNA Bank and used RMA to preprocession and utilize the limma to obtain DEGs in renal interstitium and glomeruli. For glomeruli, there were altogether 1470 differentially expressed unique genes (fold change>1.2, FDR<0.05). These DEGs were used to construct the gene co-expression and identify the hub genes associated with clinical characteristics by WGCNA: brown module has the highest GS related to IgA nephropathy occurrence; blue, yellow and green modules were correlation with BMI with highest GS of yellow module; turquoise module was correlated to GFR, Scr and proteinuria with highest GS. Howerer, since the genes assoiated with age or sex are quite few and the genes assoiated with Bp allocated among many modules, no modules specifically assoiated with age, sex or Bp. Furthermore, we identify 10 hub genes ( HK1, HEY1, MCAM, GPR4, SPRY4, NETO2,DCBLD2, MSX1,GPR124, LPPR2) associated with age, 8 hub genes (CTH, EAF2, LAMTOR5, ZNF331, PRKX , CD99, FECH, DDX3X) associated with sex, 48 hub genes associated with Bp, 223 hub genes associated with BMI, 82 hub genes associated with proteinuria and 136 hub genes associated GFR.
Then, the results of GO, KEGG pathways and PPI network analysis validated the results of WGCNA:223 hub genes associated with BMI in glomerulienriched in small molecule catabolic process, organic acid catabolic process and fatty acid degradation pathway; PPI network analysis indicated that 223 hub genes associated with BMI belong to abiologically cluster. Above all, these 223 hub genes not only interrelated with IgA nephropathy but also specificity associated with BMI, which provides directions for future research of relationship between BMI and IgA nephropathy.
Meanwhile, 48 hub genes associated with Bp enrichd in ERK1 and ERK2 cascade, cellular response to organic substance, central carbon metabolism in cancer and Rap1 signaling pathway; 136 hub genes associated GFR in glomeruli enriched in immune response, cellular response to chemical stimulus and PI3K-Akt signaling pathway, indicated that IgA stimulus and immune responsewere dominant in impaction of glomerular filtration. It is known that glycosylated IgA has a transferrin receptor (CD71) on the surface of mesangial cells  and abnormal glycosylated IgA immune complexes are specifically recognized and deposited in the mesangium causing proinflammatory cytokines and angiotensinⅡreleased , and tumor necrosis factor alpha (TNF-ɑ) derived from IgA nephropathy patients podocytes cells autocrine synthesis caused TNF receptor 1 (TNFR1), TNF receptor 2 (TNFR2) and IL - 6 were up - regulated. Elevated expression of TNFR1 leads to podocyte apoptosis and up-regulation of TNFR2 expression leading to chronic inflammation ; Phosphatidylinositol-3 kinase (PI3K)-serine/threonine kinase (Akt) is an important pathway in intracellular involve in cell metabolism, apoptosis, proliferation and differentiation [32, 33]. A study by Cox et al. reported that PI3K-Akt signaling pathway was hyperactive in IgA nephropathy patients and played animportant role in IgA nephropathy . Additionally, based on the results of WGCNA, we believed that PI3K-Akt signaling pathway specificity impact the renal function in IgA nephropathy.
For proteinuria,82 hub genes in glomeruli enriched in extracellular matrix organization, extracellular structure organizationand PI3K-Akt signaling pathway, meaning that these genes may played an important role in changing the extracellular matrix and leading to proteinuria. Traditionally, the disruption of glomerular filtration barrier (GFB), a 3-layer structure consisted of endothelial cells, glomerular base membrane (GBM) and podocyte, is the main reason leading to proteinuria. There is plenty of evidence supporting GFB molecular sieve effect and solute with different size are obstructed in varying degrees with the water and small molecules solute free permeability and macromolecular selective permeability, while GFB also is charge barrier preventing anionic molecules such as albumin passing through the GBM [35, 36]. It is now believed that GFB also consists by two additional layers, endothelial surface layer (ESL) and sub podocyte space (SPS). ESL and SPS have solute molecular screening characteristics of glomerular filtration and has a significant impact in renal function [37, 38].
In tubulointerstitium, there were 480 DEGs between IgA nephropathy and healthy control. Among 480 DEGs, 6 hub genes (HES1, ACAD10, GEM, RHOB, CREM, FILIP1L) associated with age,15 hub genes (MFAP1, RPL21 , ZMYM4, CTNNA1, EIF5, CCT6A, DNAJC10, MIR7110, TRAPPC11, CALCR, TTC37, EPHA7, TUFT1, NUP62, ELL2) associated with sex,35 hub genes associated with Bp enriched in positive regulation of apoptotic process, cellular response to nutrient levels and regeneration. Moreover, 87 hub genes associated with GFR in tubulointerstitium enriched in negative regulation of macromolecule metabolic process, negative regulation of metabolic process and RNA transport, and 33 hub genes associated with proteinuria enriched in regulation of apoptotic process, regulation of programmed cell death and FoxO signaling pathway. Proteinuria is closely associated with poor cardiovascular outcomes and progression of end-stage renal disease in patients with chronic kidney disease [39, 40].Our results shown both Bp and proteinuria are related to apoptotic process in tubulointerstitium, indicating hypertension casued damage or apoptosis of cell in tubulointerstitium also leading to Proteinuria.Additionally, in vitro, mesangial cell in IgA nephropathy patients mainly produces humoral factors TNF,TGF - β and angiotensin Ⅱ, which passed through glomerular filtration barrier or transported by blood to the renal tubules Interstitial, causing localized inflammation cascade amplification and damaging renal tubular epithelial cell . It's remarkable that cysteine-rich angiogenic inducer 61 (Cyr61) , a heparin binding activity of secreted protein as matrix related signaling molecules involved in cell proliferation, differentiation, transformation and apoptosis, is one of the hub gene associated with GFR in tubulointerstitium. Moreover, our previous studies have shown protection of renal tubular epithelial cells from apoptosis by Cyr61 expression under hypoxia [43, 44]. Therefore, our results of WGCNA and Gene ontology enrichment analysis were consistent with previous studies results, at least in part, Cyr61 as a matrix molecule related to apoptosis although the underlying mechanisms of proteinuria in IgA nephropathy still need to be explored.