Identification of key cuproptosis-related genes and their targets in patients with IgAN

Immunoglobulin A nephropathy (IgAN) was initially described in 1968 by a French pathologist, Dr. Jean Berger, and his colleague, Dr. Nicole Hinglais (an electron microscopist), as a kidney disease with glomerular “intercapillary deposits of IgA-IgG” [1]. IgAN is characterized by dominant IgA glomerular deposits in renal biopsy that are usually accompanied by local cellular proliferation and matrix expansion [2]. It is also one of most common types of primary glomerulonephritis worldwide and remains a leading cause of chronic kidney disease and renal failure [3, 4]. An epidemiological survey of the global prevalence of IgAN in 2018 showed that the incidence of IgAN in Asian countries was significantly higher than that in Europe, the Americas and Africa. Among Asian countries, and the incidence rates in China and Japan were significantly higher than those in South Korea, India and other countries [5]. The clinical markers of the severity of kidney disease, including proteinuria, hypertension and impaired renal function, are nonspecific and only present when severe kidney injury occurs. Therefore, studies exploring the new possible pathogenic mechanisms of IgAN and identifying potential therapeutic targets are very important.

Copper (Cu) is an essential element required for the maintenance of the precise activities of eukaryotes, similar to iron (Fe) and zinc (Zn). Cu plays an important role in our body; on the one hand, Cu is a vital component of many mitochondrial enzymes, such as cytochrome c oxidase (COX) and superoxide dismutase (SOD1) [6, 7]. On the other hand, mitochondria are central for Cu metabolism [8]. The term “cuproptosis” was proposed by Tsvetkov et al [9]. They defined it as a Cu-elesclomol-triggered, ferredoxin-dependent form of cell death distinct from other modalities of cell death, such as apoptosis, ferroptosis, necroptosis, and pyroptosis [9, 10]. Recently, cuproptosis has been recognized as a possibly new mechanism in some diseases [9]. However, researchers have not determined whether cuproptosis is involved in the development of IgAN.

At present no studies have examined whether cuproptosis is involved in the pathogenesis of IgAN. In this study, we screened the pyroptosis-related genes (CRGs) that may be involved in IgAN according to the data in three GEO databases, established models and screened some possible therapeutic drugs.

In this study, we screened 11 CRGs that may be involved in the occurrence and development of IgAN by analysing three GEO databases. Then, we constructed a logistic regression model for these genes and drew a nomogram and a ROC curve; the results indicated that these genes may be involved in the occurrence of IgAN. We selected the gene with a significant difference in expression (LIPT1) to draw the ROC curve, with an AUC area of 0.729, suggesting that it is a good predictor of the risk of IgAN. Some studies have found that LIPT1 may lead to the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway by affecting mitochondrial function and the TCA cycle, activating the immune response, and promoting the occurrence of IgAN [11, 12]. In addition, we performed enrichment analyses of these genes, and the results showed that these genes were mainly involved in a variety of metabolic pathways in vivo. Finally, using these genes, we screened relevant drugs that may be used to treat IgAN in the future. Compared with previous studies on patients with IgAN, we first analysed the differences in CRG expression in patients with IgAN and healthy people, established a model and screened potential therapeutic drugs. We screened 293 possible drugs. Interestingly, D-penicillamine was used to treat a patient with IgA nephropathy with Wilson’s disease. After three months of treatment, the patient’s urinary protein level was significantly improved [13].

The main causes of IgAN may be the deposition of glomerular IgA immune complexes (ICS) or IgA immune aggregates and the activation of innate immunity, followed by the activation of T cells and the resulting inflammatory response [14, 15]. However, the exact pathogenesis of IgAN remains unclear, and the specific treatment of renal disease is still insufficient. The internationally recognized theoretical basis of IgAN is the “four hit” hypothesis [1]: ① the serum level of galactose-deficient IgA1 (GD-IgA1) in the circulation is significantly increased; ② GD-IgA1 is recognized by an IgG antibody in vivo to induce the production of autoantibodies; ③ antigen–antibody complexes are formed and widely deposited in mesangial cells; and ④ mesangial cell proliferation, glomerular inflammation and injury occur [16]. These immune complexes may be deposit in the glomerular mesangial area and then activate mesangial cells, which damage kidney tubular cells and podocytes (apoptosis and foot effacement) by secreting many detrimental bioactive mediators, such as IL-6, tumour necrosis factor (TNF), aldosterone, transforming growth factor-β (TGFβ), and angiotensin II (Ang II) [17]. However, this hypothesis also has some limitations. For example, it does not explain why GD-IgA1 also exists in the circulation of healthy individuals, and IgA deposition has also been observed in the glomerular mesangium [18]. Therefore, a better understanding of the pathogenesis of IgAN may help to identify new targets and better treat patients with IgAN.

The aetiology of IgAN consists of many factors, among which immunity plays an important role in the pathogenesis of IgAN. T cell subsets are commonly measured in the clinic to reflect the state of immune function. The immunoregulatory function of T cells is mainly completed by CD4 + T cells and CD8 + T cells. Some studies have documented an increase in the number of CD8 + T cells in patients with IgAN [19], consistent with our results. The cytotoxic effect of CD8 + T cells may accelerate damage to the glomerulus. The synthesis and secretion of low glycosylated IgA1 are regulated by Th cells. Many studies have found that an imbalance in the Th1/Th2 cell ratio leads to an increase in the number of Th2 cells, which might lead to the synthesis of low glycosylated IgA1 and a sustained immune response in a variety of pathways [20, 21]. In our study, we also observed significant differences in the numbers of Th1 cells and Th2 cells in glomerular samples between patients with IgAN and healthy people. However, we did not analyse the changes in Th1/Th2. Our study also found that B cells may also be involved in the occurrence of IgAN. Some studies suggest that B cells may promote the secretion of GD-IgA1 through Toll-like receptors (TLRs) and a proliferation-inducing ligand (APRIL) [22]. Consistent with our findings, some studies have found that natural killer (NK) cells and dendritic cells (DCs) may participate in the development of IgAN [19], and more mechanisms require further exploration. In adults and children with IgAN, a larger number of CD68 + cells (markers of macrophages) were correlated with worse outcomes [23, 24]. We also observed a significant increase in the number of macrophages in patients with IgAN compared with healthy people. However, we did not detect a difference in the number of glomerular Treg cells between patients with IgAN and healthy people, suggesting that Treg cells may not play a significant role in IgAN glomerulopathy. Our results also show that a variety of immune-related functions are altered in patients with IgAN. In the present study, we found that LIPT1 may be involved in IgAN. Recently, LIPT1 was reported to affect fatty acid synthesis by regulating metabolism. For example, increased expression of LIPT1 may promote fatty acid synthesis and reduce fatty acid oxidation [12]. In addition, some researchers have shown that P-aIgA1 (aggregated IgA1 from patients with IgAN) promotes the proliferation of human kidney mesangial cells, while the administration of short-chain fatty acid inhibitors reverses the increased proliferation of mesangial cells and extracellular matrix synthesis [25]. However, few relevant studies are available, and further evidence is needed to determine how LIPT1 participates in IgAN.

In recent years, progress has been achieved in determining how essential trace metals exert toxic effects on mammalian cells. These different metals kill cells by activating a process that does not require an apoptotic pathway. For example, excess zinc triggers nonapoptotic cell death by inhibiting the synthesis of adenosine triphosphate (ATP) [26, 27]. Iron catalyses the formation of toxic membrane lipid peroxides. Iron sagging is a unique form of nonapoptotic cell death [28]. Copper and other trace metals are essential for life, but research has revealed that copper also causes cell death [29]. Copper-binding agents have been used to treat cancer because they bind copper and cause cancer cell death. A drug forming a bond with copper called elesclomol has been used in clinical trials to treat cancer, and although the success rate is limited [30, 31], it provides a new idea for the treatment of cancer in the future. However, the mechanism of cuproptosis is not yet clear. Evidence from yeast and mammalian cells suggests that copper toxicity is related to changes in mitochondrial function [10]. Our GO enrichment analysis also showed that these CRGs were enriched in the mitochondrial matrix, suggesting that mitochondria play an important role in cuproptosis. According to a previous study, cuproptosis is activated by mitochondrial stress, which is characterized by the assembly of lipoylated mitochondrial enzymes and the reduction of Fe-S cluster proteins rather than mitochondrial reactive oxygen species (ROS) [32]. A recent study found that copper directly binds to fatty acylated components in the tricarboxylic acid (TCA) cycle, leading to the accumulation of fatty acylated proteins and the subsequent loss of sulfur cluster proteins, leading to protein toxic stress and eventually cell death [9]. study indicates that the TCA cycle plays an important role in cuproptosis, consistent with the results of our enrichment analysis. Copper mediates a variety of kidney diseases [33‐36], and the use of copper bonds can treat renal injury caused by cisplatin [37]. These studies show that copper may participate in the occurrence and development of some kidney diseases through a variety of pathways. Therefore, research on cuproptosis-mediated kidney diseases may become a new hotspot in future kidney disease research.

Our research has several advantages, as described below. First, we systematically analysed the expression of CRGs in patients with IgAN and constructed a model based on these databases to predict the risk of IgAN. Second, we suggested that a correlation may exist between cuproptosis and immune changes in the process of IgAN occurrence and development, which provides a new direction for the treatment and research of IgAN. However, the study also has some limitations. First, the sample size in the database is not sufficient, which may lead to errors. In addition, we did not conduct in vivo and in vitro experiments to verify these results, and further research is needed to reveal the potential mechanism of cuproptosis in IgAN.

In conclusion, this study systematically analysed the immune changes related to cuproptosis in patients with IgAN. Our study shows that these CRGs may play a crucial role in the occurrence and development of IgAN. Our results provide a new direction for IgAN research and treatment in the future.