*4.1. Studies Addressing TNF-α and TNFRs in Animal Models*

Animal studies are the primary source of evidence for the role of TNF-α in the development of kidney diseases (Table 1). In the classical 5/6 nephrectomy CKD model, NF-kB is activated and other proinflammatory genes are upregulated [86]. The systemic administration of TNF-α in rat models of anti-glomerular basement membrane antibody-mediated nephritis worsened the severity of glomerular injury by increasing neutrophil influx, albuminuria, and the prevalence of glomerular capillary thrombi [83]. TNF-α blockade reduced proteinuria, inflammation status, and renal scaring in mice [87] and rat [88] models of glomerulonephritis. It was also shown that TNF-α blockade prevented the development of crescents in a rat model of crescentic glomerulonephritis [88] and reduced renal tubular cell apoptosis, caspase activity, and several markers of renal fibrosis, in a model of unilateral ureteral obstruction [89,90].

Studies addressing the deletion of TNFR1 and/or TNFR2 genes, in animal models, also illustrated the contributions of the TNFRs in the pathophysiology of kidney diseases. The deletion of TNFR1 was associated with an increase in GFR, in an angiotensin II-

induced model of hypertension [91]. Data from the same study showed that renal TNFR2 mRNA expression is increased in hypertensive TNFR1 knockout mice, along with increased urinary albumin excretion, compared to wild type mice and to TNFR1 knockout mice without induced hypertension. The authors suggested that TNFR2 has a leading role in the development of albuminuria [91]. Accordingly, TNFR2 knockout mice subjected to immune complex-mediated glomerulonephritis did not exhibit increased albuminuria and were protected from renal injury, despite preserving intact the immune system response [92]. In a model of unilateral ureteral obstruction, both TNFR1 and TNFR2 knockout mice showed a significantly reduced relative volume of the cortical interstitium, in the obstructed kidney, compared with the wild-type mice, as a result of the decreased deposition of pro-fibrotic proteins [93]. Additionally, the individual knockout of TNFR1 or TNFR2 resulted in decreased inflammation, demonstrated by the reduced activation of the NF-κB pathway. TNFR deletion was found to have comparable favorable effects in kidney disease development in several other animal studies [94–97].

TNFR participation in diabetic kidney disease has been the subject of specific research. Previous studies reported that the TNF-α inhibition protects against tubular injuries [97] and prevents renal hypertrophy [98] in diabetic rats. A diabetic mice model treated with a TNF-α inhibitor, Etanercept, showed improvements in albuminuria, decreased expression of inflammatory molecules, and decreased macrophage infiltration into the kidney [99]; renal levels of TNFR2, but not TNF-α or TNFR1, were decreased compared to non-treated mice [99]. The authors suggested that diabetic nephropathy is predominantly associated with the inflammatory action of TNF-α via the TNFR2 pathway. Other works also demonstrated that the administration of TNF antagonists inhibits salt retention, renal hypertrophy [98], and albuminuria [100], suggesting that TNF inhibition may slow the progression of diabetic nephropathy.

Transcriptomics further showed that both oxidative stress and inflammation play a role in the pathogenesis of CKD, and are correlated with cellular alterations that lead to systemic complications [101]. In ischemia–reperfusion mice models, proximal tubule cells at a late injury stage that mimic chronic progression confirmed a marked activation of the TNF, NF-κB, and AP-1 signaling pathways [102].


**Table 1.** Association of TNF-α and TNF receptors with renal dysfunction and disease in animal models.



dihydroxyadenine; ETN, Etanercept; GBM, glomerular basement membrane; LPS, lipopolysaccharide; NF-κB: factor nuclear kappa B; SLE, systemic lupus erythematosus; sTNFR1, soluble tumor necrosis factor receptor 1; STZ, Streptozotocin; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; TNF-α, tumor necrosis factor alpha; UUO, unilateral ureteral obstruction; WT, wild type.
