Iron is a mineral that the body obtains from the environment in order to maintain its physiological activity, and the majority of it is found in the form of hemoglobin in red blood cells . Iron homeostasis imbalances can cause significant disorders by disrupting mitochondrial activity, as well as DNA repair and synthesis . Metabolic changes and altered skeletal muscle function can be caused by iron insufficiency . Iron excess can cause oxidative stress and iron deposition in parenchymal organs , which can harm the organism . Traditionally, inorganic iron sulphate has been added to the feed to promote normal growth in animals and to prevent clinical deficiencies caused by low iron utilization . The NCR advises a basic iron intake of 30 mg/Kg of feed dry matter for sheep, with a maximum tolerance of 500 mg/Kg of feed dry matter.
The kidneys are an essential endocrine organ that play an important role in maintaining fluid balance and normal organ function . Diets supplemented with various iron dosages were found to have a close linear association with iron contents in the kidneys in studies. The kidneys complete the transport and reabsorption of iron through divalent metal ion transporters and transferrin receptors [10, 11], and unused iron excretion in urine . As a result, the kidney plays a crucial role in maintaining iron homeostasis and preventing excessive iron. Excessive iron intake can persist in kidney tubular cells , affecting the innate immune response and even leading to cell death . Inflammation induces the creation of iron regulatory proteins, which govern iron homeostasis , and disorder of inflammatory factors may lead to dysregulation of tissue repair and fibrosis . These findings imply that in vivo iron levels are linked to inflammation and fibrosis. While it is well established that excessive iron causes liver inflammation and fibrosis , and the certainty of this relationship is enhanced by the reduction of serum iron levels in the follow-up of inflammation-induced experimental infections , the association between excessive iron and kidney inflammation and fibrosis in sheep is unclear and need to investigate further. Our study provides a theoretical foundation for further research into the harmful effects of excessive iron and the rational use of iron in sheep feeding management.
Inflammation is a defense mechanism in the body that is triggered by irritation or infection , inflammatory mediators play a role in cellular interactions and signal transduction. Hepcidin is a key factor in iron homeostasis management, which can suppress the expression of IL-1β, IL-6, NF-κB and TNF-α , and it is a major product released in response to IL-6 . Excessive iron stimulation resulted in increased expression and release of IL-6 in the liver, according to previous studies . IL-1β stimulates hepcidin expression via the same mechanism that activates IL-6 mediated hepcidin transcription . Chelation reduces intracellular iron, which inhibits NF-κB induction of TNF-α and other cytokines. NF-κB is an important regulator of the inflammatory response, and the NF-κB pathway can be induced by TNF-α . Iron can phosphorylate the inhibitor protein κB (IKB)  and dissociate it from NF-κB, resulting in NF-κB activation and inflammatory disease , which can be inhibited by the use of an iron chelating ligand (deferasirox) . IL-2 is a pro-inflammatory factor that impacts cell proliferation and differentiation and is essential for immune cell proliferation, activation, and dynamic homeostasis. IFN-γ and TNF-α are important regulators of macrophage iron status and immunological function, causing cytokine-mediated effects that contribute to the inflammatory response .
Fibrosis is a pathological process in which inflammation causes necrosis of the parenchymal cells of certain organs and abnormal tissues, but it is unclear whether inflammation play a role in the development of kidney fibrotic disease in sheep . However, previous studies have demonstrated that IL-1β stimulates macrophages and neutrophils , promotes cellular conversion to the epithelial mesenchyme, expresses collagen, and induces fibrosis , and induces tubulointerstitial renal tubular disease by promoting proximal tubular damage and fibrosis . TNF-α is the primary cytokine responsible for pro-inflammatory cell recruitment and cellular inflammasome activation after macrophage activation. IL-1β and TNF-α induce NF-κB translocation from the nucleus to active NF-κB, which is involved in pro-inflammatory and pro-fibrotic production . TNF-α, IL-1β, and IL-6 activate the NF-κB pathway, which causes fibroblast activation and kidney fibrosis . TGF-β1 is activated and released by macrophages, promotes intrinsic cell activation, which results in the production of large amounts of collagen and extracellular matrix , it is a crucial mediator in the recruitment of peripheral cells and the development of renal fibrosis in chronic kidney disease , and treatment of mouse renal tubular epithelial cells causes kidney fibrosis . As a result, inflammatory factor disorders are virtually usually the cause of kidney fibrosis.
In these studies, in vivo experiments were used to assess the development of the inflammatory response in the sheep kidney to verify the extent of inflammatory factor disruption. The inflammatory response was triggered by increasing the amount of iron in the diet, which resulted in increased secretion of IL-2, IL-6, TGF-β1, and NF-κB and decreased secretion of IL-1β, IFN-γ, and TNF-α. Excessive iron leads to disturbance of the balance of inflammatory factors, causing tissue fibrosis to worsen.