IgAN, the most common glomerulonephritis worldwide, has a high risk of progression to ESRD . The progression of IgAN is not completely related to glomerular lesions, for example, in some patients with controlled glomerular lesions, the renal function continues to decline, which implies that tubulointerstitial injury may play a role. The Oxford MEST (interstitial fibrosis/tubular atrophy, segmental sclerosis, hypercellularity, endocapillary, and mesangial) histologic score in IgAN suggested that T, S, and M lesions are related to the prognosis of the disease [2, 17]. Although guidelines recommend histological risk factors and clinical features so that disease prediction and therapy selection can be applied properly, specific genes related to glomerular and renal tubular damage are still poorly understood. The mechanisms of crosstalk between glomeruli and tubulointerstitium in the pathogenesis of IgAN are lacking. Therefore, we performed integrated bioinformatics analysis to identify key genes and explore the correlation between glomeruli and tubulointerstitium in the pathogenesis of IgAN.
In the present research, 583 DEGs were detected in glomeruli, comprising 213 down-regulated genes and 370 up-regulated genes. Meanwhile, 272 DEGs were detected in tubulointerstitium, including 150 up-regulated genes and 122 down-regulated genes. Among these DEGs, 119 genes were overlapping in both groups (Fig. 2). The overlapping DEGs suggested that both glomerular and tubulointerstitial lesions may be caused by the same genes and pathways, which could explain that two lesions always occur concurrently in IgAN nephropathy.
Based on WGCNA, eight and four co-expression modules were detected in glomerular and tubulointerstitial samples (Fig. 3), respectively. The positively related modules were “blue” “yellow” “green-yellow” and “magenta” in glomerular samples and “grey60” module in tubulointerstitial samples. While the negatively related modules were “black”, “tan”, “turquoise” and “purple” modules in glomerular samples and “dark-green” and “black” modules in tubulointerstitial samples.
Through gene functional enrichment analysis of the positively related gene modules, we found that both glomeruli and tubulointerstitium were involved in the adaptive and innate immune systems. Since bacterial or viral infection may trigger the occurrence of IgAN and recurrent infections may worsen the disease. Previous studies have shown that streptococcus may aggravate inflammatory damage in IgAN via the chemotaxis of Th22 cells . The activation of Toll-like receptors (TLRs) might promote the production of IgA and elevate IgA glycosylation. Moreover, TLR 9 and TLR 4 were correlated with IgAN severity . The yellow module in glomeruli was related to vasculogenesis, which suggested that vascular endothelial growth factor (VEGF) and other related inflammatory factors may lead to inflammation and proliferation of the mesangial cells and further cause glomerulosclerosis . Plate-derived growth factor (PDGF) not only directly stimulates the proliferation of mesangial cells but also can lead to renal fibrosis . The grey60 module in tubulointerstitial samples positively correlated with IgAN was predominately enriched in the complement activation pathway. It has been confirmed that in human and rodent experiments, IgA (mainly poly IgA involved) can activate complement alternative pathway (AP) . The modules negatively related to IgAN indicated that the biological process of ion transmembrane transport has a protective effect on IgAN.
Concerning the pathogenesis of IgAN, the “multi-hit” hypothesis, including production of galactose-deficient IgA1 (Hit 1), anti-glycan response (Hit 2), formation of IgA1-containing immune complexes (Hit 3), and glomerular deposition (Hit 4), has been widely supported by many studies . Tubulointerstitial damage in IgAN is considered to be a secondary event of glomerular injury developing later in IgAN. However, a recent single-cell RNA sequencing (scRNA-seq) study revealed that proximal tubular cells are injured already at the early stage of IgAN . This may be partially mediated by albuminuria and glomerulotubular cross-talk. Meanwhile, the tubulointerstitial injury may also lead to increased glomerular damage. Tubulointerstitial fibrosis can activate the macula densa and result in abnormal tubuloglomerular feedback, with continuing arteriolar vasodilation, and consequently result in glomerulosclerosis . Therefore, we propose that this adverse cross-talk of glomeruli and tubulointerstitium will enhance IgAN progression in all stages.
In our study, the key genes screened from the crosstalk network were mainly associated with adaptive and innate immunity, such as ITGAM, ITGB2, TYROBP, CSF1R, HCK, and LAPTM5. As mentioned above, the “first hit” is that individuals with a genetic susceptibility develop abnormal immune responses to common and environmental antigens, which leads to an increase of galactose-deficient IgA1 in circulation. A growing body of evidence suggests that abnormality of intestinal mucosal immunity plays an important role in IgAN . Integrin alpha M (ITGAM) encodes the integrin alpha M chain (CD11b) and belongs to the integrin family. CD11 is an important leukocyte differentiation antigen, which is widely expressed in a variety of immune cell subsets, such as dendritic cells, neutrophils, NK cells, and B cells. CD11 integrin participates in innate immunity, adaptive immunity, and inflammatory response, and plays an important role in regulating immune tolerance [26, 27]. ITGAM is proven to participate in the modulation of intestinal IgA-producing plasma cells in mice, which indicates the function of the intestinal immune in the pathogenic mechanism of IgAN . Integrin subunit beta 2 (ITGB2) also belongs to the integrin family and is implicated in binding between endothelial cells and inflammatory cells, inflammatory cells chemotaxis . According to a previous study, there is a negative correlation between ITGB2 and eGFR in patients with chronic kidney disease (CKD). However, the mechanisms of ITGB2 mediating glomerular and tubulointerstitial injuries are not clear, which needs further study. Transmembrane immune signaling adaptor (TYROBP) encodes a transmembrane signaling polypeptide that encompasses an immunoreceptor tyrosine-based activating motif in its cytoplasmic domain. TYROBP binds non-covalently to NK cell activity receptors and activates signal transduction. It has been previously reported that TYROBP is highly correlated with proteinuria in systemic lupus erythematosus (SLE) , but its role in IgAN is unclear. Cytochrome b-245 beta chain (CYBB) has been postulated as a major constituent of the phagocyte microbicidal oxidase system . Fibronectin 1 (FN1) is a well-known protein that has great binding activity and is also the primary constituent of the extracellular matrix. The aggregation and chemotaxis of FN1 and collagen serve as critical building blocks for the proliferation of endothelial cells, mesangial cells, and fibroblasts. The expression of FN1 in the glomeruli indicates the presence of active mesangial cell growth and the progression of the lesion . Cathepsin S (CTSS) participates in antigen presentation and cytokine secretion. It has been demonstrated that CTSS inhibits apoptosis and promotes cell proliferation through PI3K/Akt or MAPK pathway . A recent study on proteomics of the tubulointerstitium in IgAN shows that the abundance of cathepsin G (CTSG) was related to disease progression . Both CTSG and CTSS belong to the cathepsins super-family, which are lysosomal enzymes that participate in important physiological processes, such as tissue remodeling, senescence and adaptive and innate immunity. Hemopoietic cell kinase (HCK) plays an important part in regulating innate immune response, phagocytosis, cell survival and proliferation, cell adhesion, and migration . HCK can activate TGF-β–mediated pro-fibrotic pathway, as well as other proliferation contributing factors, which are implicated in renal tubular cell damage and fibrosis and even the modulation of the immune system . It has been demonstrated that lysosomal protein transmembrane 5 (LAPTM5) performs an instrumental function in the lysosomal disintegration of B cell and T cell antigen receptors (BCR/TCR) by transporting endosomes to lysosomes.
Based on the analysis presented above, we hypothesize that inflammatory mediators (cytokines and chemokines) released by ligand-receptor interactions between glomeruli and tubulointerstitium, together with albuminuria, may participate in glomerulotubular crosstalk.
In our study, another dataset was used to verify the key genes, and LN was served as the control group. The result revealed that key genes were shared partially between IgAN and LN, while CYBB, CTSS and TYROBP were specific for IgAN. Both IgAN and LN are immunity-related renal diseases and share many similarities. For example, both have proteinuria and hematuria as common clinical manifestations, and renal IgA deposition as pathological manifestations. Not only that, co-occurrences of IgAN and LN were found in some patients . The results of our study are basically consistent with the previous studies [16, 37].
As mentioned above, some of the key genes have been shown to perform a vital part in the pathogenic process of IgAN, such as ITGAM , ALB , FN1  and CTSS . However, there is little known about CYBB, TYROBP, ITGB2, CSF1R, HCK, and LAPTM5, which need further research to reveal their mechanisms.
The limitation of this study. Because of the scarcity of clinical data in the GEO database, it is hard for us to link gene modules to specific clinical characteristics. In addition, the data of our study were obtained by bioinformatic analysis of microarray datasets; consequently, more in vivo and in vitro tests are required to validate the findings.