The prevalence of CKD has increased in recent decades, along with an increase in diabetes and hypertension, the two leading causes of CKD . Herein, we investigated the contribution of NF-κB and IL-6, IL-19, IL-34, and IL-37 in the pathogenicity of DN. Our data revealed that HbA1c% increased in all groups with diabetes compared with healthy controls. In addition, there was a marked increase in the serum levels of urea, uric acid, and creatinine, as well as a decrease in GFR levels, in both groups with kidney dysfunction. The increase in creatinine levels and the decrease in GFR levels were markedly observed in the DCKD group. Inflammation, which is closely associated with renal illness, is described as a complex network of interactions between renal parenchymal cells and local immune cells such as macrophages and dendritic cells, as well as recruitment of circulating monocytes, lymphocytes, and neutrophils .
Hyperglycemia is thought to reflect a state of elevated oxidative stress due to excessive production of ROS and a deficient antioxidant response . Our data showed that serum SOD activity levels were markedly decreased in patient groups compared with healthy controls. Enhanced ROS production in the glomerular microcirculation lowers nitric oxide bioavailability, leading to mesangial contraction dysfunction and arteriolar tone, as well as persistent oxidative stress, which causes endothelial disorder, leukocyte adhesion, and apoptosis of glomerular cells . In agreement with our findings, Liu et al.  demonstrated upregulation of renal SOD activity in diabetic mice. The antioxidant enzyme SOD is an important part of the cell’s defense against increased oxidative stress. It is therefore speculated that the decreased levels of SOD in patients with diabetes could lead to cytotoxic levels of superoxide overproduction, which may result in diabetic renal tissue damage . However, oxidative damage, inflammation, and cell death caused by hyperglycemia could be alleviated by SOD. Consequently, the reduction in serum SOD levels may contribute to the onset of DN.
The stimulation of the transcription factor NF-κB by oxidative stress caused by hyperglycemia may increase the levels of pro-inflammatory cytokines, which could promote DN [5, 6]. The current data revealed a noticeable increase in NF-κB expression levels in the groups with diabetes, CKD, and DCKD compared to the healthy control group. Moreover, NF-κB mRNA expression showed a positive correlation with HbA1c and creatinine and a negative correlation with GFR levels. Hyperglycemia leads to the production of ROS in mesangial cells, which upregulates NF-κB. Interestingly, NF-κB inhibitors protect against kidney injury and may be useful in inflammatory diseases . Notably, inhibiting the RAS and/or the NF-κB system may become an effective therapeutic asset to detain renal injury . Angiotensin II elevation, which is directly connected to hypertension, is a stimulus for NF-κB activation and the production of inflammatory responses. Additionally, NF-κB has been linked to the pathophysiology of kidney injury caused by hypertension . Importantly, the stimulation of NF-κB signaling regulates a variety of pro-inflammatory genes, including those that encode chemokines and cytokines like IL-6 .
IL-6 is a pro-inflammatory cytokine that strongly promotes the progression of insulin resistance and the pathophysiology of T2DM through the regulation of differentiation, proliferation, and cell death . In our study, serum IL-6 levels significantly increased in the groups with diabetes, DCKD, and CKD. In addition, IL-6 production showed a positive correlation with HbA1c and creatinine but a negative correlation with GFR levels. According to our findings, pro-inflammatory cytokines, such as IL-6, IL-1b, and TNF-α, have been linked with the severity of CKD. These are produced by adipose tissue (along with lymphocytes), which becomes dysfunctional during CKD . Notably, elevated IL-6 levels are not only a consequence of renal disease, but more importantly, it also acts as a trigger for the progression of renal disease and its related complications ; these results indicate that IL-6 contributes to the higher prevalence of CKD. Interestingly, it can be concluded that IL-6 impacts renal resident and infiltrating cells, affecting vascular permeability and promoting mesangial cell proliferation, glomerular basement membrane thickening, extracellular matrix synthesis, and neutrophil infiltration into the tubulointerstitium, resulting in DN progression . As a result, increased IL-6 levels in human sera are both an early and strong indicator of DN initiation.
It is worth mentioning that IL-6 trans-signaling induced by Ang II might activate STAT3 in podocytes, which would impact podocyte differentiation, cell cycle, and other physiopathologic processes . Importantly, increased IL-6 production by podocytes activated the JAK2/STAT3 pathway via enhanced phospho-JAK2 binding to gp130 and STAT3 phosphorylation. Likewise, hyperglycemia enhanced IL-6 production and signal transduction in podocytes, which was inhibited by an IL-6-neutralizing antibody or IL-6 siRNA. Therefore, the inhibition of IL-6 and its downstream mediators, including IL-6R and gp130, may reduce the onset of DN .
IL-19, a pro-inflammatory cytokine, promotes the T-helper 2 response . Indeed, Cuneo et al. revealed that IL-19 is activated by pro-inflammatory cytokines and is expressed in damaged vascular smooth muscle cells . IL-19 may be implicated in the development of vascular disease in diabetes. Serum IL-19 concentrations were positively correlated with Ang II and showed a significant association with insulin resistance and HbA1c%, suggesting that IL-19 is strongly linked with impaired glucose metabolism and vascular disorders in patients with T2DM . Regarding IL-19 expression, increased IL-19 expression in the groups with diabetes and both CKD and DCKD groups was observed. Furthermore, IL-19 mRNA expression showed a positive correlation with BMI, HbA1c, and creatinine but a negative correlation with GFR levels. Similarly, patients with DN had considerably greater serum levels of IL-19 compared with the controls  and revealed a positive correlation with HbA1c%, urea, creatinine, and CRP. Li et al.  proposed that chronic hyperglycemia may enhance the expression of IL-19 by activating endothelial cells, resulting in local inflammation and accelerating endothelial damage and atherosclerosis.
In mouse monocytes, IL-19 is elevated in an acute systemic inflammatory state and promotes the generation of IL-6, TNF-α, and apoptosis. In addition, IL-19 upregulates TGF-β1 and MCP-1 expression in renal cortical collecting duct cells (M-1) and also activates caspase-3 and caspase-9 to promote cell apoptosis through the p38 MAPK pathway in M-1 cells , which can lead to interstitial fibrosis caused by excess extracellular matrix deposition. In M-1 cells, IL-19 can activate ERK 1/2, JNK, and p38 MAPK and inhibit p38 MAPK signaling, which reduces IL-19-induced apoptosis; these results suggest that IL-19 causes apoptosis in renal epithelial cells via these three pathways . Moreover, Jennings et al.  revealed a substantial rise in IL-19 levels in the urine of patients with CKD, which is closely associated with calculated GFR levels, indicating that IL-19 was a possible novel translational marker of renal damage. Therefore, IL-19 or its receptor may represent novel therapeutic targets for addressing kidney impairment following DN.
The newly discovered pro-inflammatory cytokine, IL-34, has several functions in the modulation of inflammation and the immune response. Indeed, vascular diabetes complications may be predicted by serum IL-34 levels . Moreover, multiple chronic inflammatory conditions can be caused by IL-34, and it has a significant role in the development of insulin resistance in T2DM patients . In addition, IL-34 has been linked to impaired kidney function and severe anemia in patients with CKD . Our study revealed upregulation of IL-34 expression in the patients with DN groups. In addition, IL-34 mRNA expression revealed a positive correlation with BMI, HbA1c, and creatinine and a negative correlation with GFR levels. Importantly, IL-34 stimulates the protein tyrosine phosphatase ζ receptor; during acute kidney damage, both receptors and cytokines are upregulated. Moreover, IL-34 increased both intrarenal macrophage and bone marrow cell proliferation, increasing the levels of circulating neutrophils and monocytes and their recruitment to the kidney . It has been shown that TNF-α increased CSF-1 and IL-34 expression in intestinal epithelial cells via the NF-κB signaling pathway . In mice, redacting IL-34 improves nephritis via macrophage- and autoantibody-mediated pathways both inside and outside the kidney. IL-34 is a possible therapeutic target for tubular epithelial cell damage, and IL-34 inhibition may have reno-protective effects . Notably, IL-34 may be an effective biomarker and therapeutic target for treating inflammatory diseases, including DN.
IL-37 generates a complex with IL-18R and IL-1R8 that transduces anti-inflammatory signals by suppressing NF-κB and MAPK and activating the Mer-PTEN-DOK pathway. IL-37 exerts broad and complex anti-inflammatory and immunomodulatory effects, inhibits excess inflammation, and prevents tissue damage mediated by inflammation . Importantly, IL-37 is a possible therapeutic target for managing obesity-induced insulin resistance and T2DM . The biological effects of IL-37 can be attributed to its ability to inhibit NF-κB; the IL-6/STAT3 pathway; and TNF-α, IL-1β, and IL-6 synthesis . In addition, it has been shown that a high IL-37 concentration is correlated with increased systemic lupus erythematous activity and impaired renal manifestations .
IL-37 mRNA expression was found to be decreased in the patients with diabetes and both kidney dysfunction groups relative to the healthy control group. In addition, IL-37 mRNA expression revealed a negative correlation with BMI, HbA1c, and creatinine and a positive correlation with GFR levels. Notably, IL-37 can greatly reduce podocyte inflammation, oxidative stress, and apoptosis caused by hyperglycemia and can also block the STAT3-CypA signaling pathway . Moreover, IL-37 decreased the kidney expression of TNF-α, IL-6, and IL-1β and alleviated mononuclear cell infiltration and kidney damage in a mouse ischemia injury model. Thus, IL-37 may represent an effective approach for suppressing renal damage responses and promoting renal function after renal ischemic damage . In addition, understanding the role of IL-19, IL-34, and IL-37 in the pathogenicity of DN has been deemed crucial in creating novel anti-inflammatory agents to prevent or delay the onset of DN.
Hyperglycemia and insulin resistance are responsible for the micro- and macrovascular complications of diabetes. The metabolic insult of hyperglycemia and podocyte insulin resistance is a major source of podocyte damage, leading to albuminuria and proteinuric glomerulopathies in early DN . Importantly, IL-19, IL-34, and NF-κB expression levels and the levels of IL-6 in diabetic and DCKD patients were associated with insulin sensitivity and insulin resistance. The sustained rise in inflammatory cytokines, such as IL-19, IL-34, and NF-κB, as well as serum IL-6 levels, is involved in the progression of insulin resistance in diabetic and DCKD patients. In patients with diabetes, prolonged activation of pro-inflammatory and pro-fibrotic cell types causes excess extracellular matrix deposition . These elements are involved in the stimulation and proliferation of smooth muscle actin-positive myofibroblasts, which are essential for the excessive synthesis of extracellular matrix, ultimately leading to DN . Thus, IL-6, IL-19, IL-34, IL-37, and NF-κB may represent therapeutic targets for preventing or alleviating diabetic kidney disorders as well as their complications.
The current study has several limitations, including the small study population in the patient groups and the fact that the findings were separated depending on renal stages and disease duration. In addition, the lack of determination of IL-19, IL-34, and IL-37 at protein levels, immunohistochemistry staining slides for interleukin levels in the patients, and the addition of more inflammatory and anti-inflammatory cytokines could influence inflammation processes, pathogenesis, and DN development. Importantly, clinical investigations in larger cohorts are required to deeply understand the pathways that influence IL-19, IL-34, and IL-37 in the etiology and pathogenicity of DN.