Iron-Overload Conditions: Manifestations to the Kidney Organs – A Review

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INTRODUCTION
Iron overload is when too much iron accumulates in the body.Increased iron absorption in the small intestinal mucosa and frequent red blood cell transfusions can lead to iron overload in patients with chronic anemia such as β-thalassemia major.Before recently, long-term iron chelation therapy with iron chelating medications was the standard for managing iron overload, particularly in patients with transfusion-dependent thalassemia 1 .Hemosiderosis is an iron overload condition with increased iron deposition in tissues without clinical signs 2 .More than 3% of people worldwide carry the thalassemia gene, with Southeast Asia having the highest incidence -up to 40% -of this single gene inherited disease.Thalassemia is an inherited disorder of globin chain synthesis.Various mutations and deletions in the α-and β-globin gene clusters reduce globin chain synthesis to varying degrees and produce thalassemia traits or clinical features of thalassemia.Chronic anemia brought on by inefficient erythropoiesis, intra and extramedullary hemolysis, and iron overload dominates the clinical picture of thalassemia 3 .Iron is essential for transporting oxygen and various metabolic processes.Its ability to take and donate electrons, switching between its ferrous (Fe 2+ ) and ferric (Fe 3+ ) forms, enables it to participate in this process 4 .Hereditary anemia caused by thalassemia is typically treated with routine blood transfusions, which may raise the body's overall iron levels.As soon as proteins that control iron are functional (e.g., ferritin, transferrin) 5 .Based on the severity of the clinical phenotype, β-thalassemia has historically been categorized into three primary subgroups: major, intermedia, and minor.Red blood cell transfusion requirements have been used as the basis for a more straightforward and clinically applicable classification system over the past ten years.Thalassemia patients are classified as transfusion-dependent (TD) or non-transfusion-dependent (NTD).While NTD patients frequently have anemia, they do not require blood transfusions for daily function or survival; TD thalassemia patients must receive regular blood transfusions throughout their lifetimes.Patients with NTD thalassemia may occasionally need transfusions, notably during pregnancy, surgery, and infections, but they typically maintain hemoglobin (Hb) concentrations between 7 and 10 g/dL 6 .Reactive oxygen species (ROS) and free radicals, which harm cellular and subcellular structures and result in metabolic dysfunction, are produced by metals like iron and some low molecular weight substances containing iron.The presence of disturbed iron metabolism can also produce free radicals 3 .Iron deposits in essential organs like the kidneys, liver, and heart may occur from circulating nontransferrin-bound iron (NTBI), brought on by plasma transferrin saturation and iron overload 7 .Iron chelating agents must be supplied continually and for the rest of one's life to remove excess iron from the body and stop it from building up in tissues to combat the accumulation of too much iron (iron overload) 8 .Renal illness, like in other aging populations, potentially may become more prevalent with the arrival of efficient chelating medicines that can decrease the iron burden and its effects and extend patients' survival 9 .Iron overload is brought on by a buildup of iron that starts in the blood plasma and eventually gets deposited in tissues and organs, causing organ damage, especially in patients receiving routine blood transfusions.Kidneys, as one of the vital organs involved in excretion, might be affected by their function if damaged.There has yet to be much discussion on how iron overload affects kidney damage, how it manifests, or how it relates to renal function damage markers.How iron can harm the kidney will be discussed in further detail.

THE ROLE AND FUNCTION OF THE KIDNEYS
The kidneys play a critical role in maintaining homeostasis by controlling the amounts of various plasma elements, including electrolytes and water, and by eliminating all metabolic wastes other than CO2 exhaled by the lungs.Plasma will be repeatedly filtered through the kidneys, retaining the constituents of value to the body and removing unwanted or excessive materials into the urine.Controlling the salt and water balance allows the kidneys to regulate the volume and osmolarity of the solute concentration of the internal fluid environment 10 .Besides playing a crucial regulatory role in preserving fluid and electrolyte balance, the kidneys are vital for the body's excretion of potentially hazardous metabolic waste products and foreign substances.These wastes must be eliminated in solution since they cannot be eliminated as solids.As a result, the kidneys must produce at least 500 mL of waste-containing urine per day.Figure 1 shows the locations of the three fundamental procedures that contribute to the creation of urine: glomerular filtration, tubular reabsorption, and tubular secretion 11 .The kidneys are the primary excretory organs for eliminating metabolic waste products and are crucial for preserving the body's normal electrolyte and fluid balance.Additionally, the kidneys perform two endocrine tasks: they secrete erythropoietin, which encourages the creation of red blood cells, and renin, which controls blood pressure 12 .

IRON HOMEOSTASIS
Plasma iron is quantitatively much less than erythrocytic iron and has a total mass of around 5 mg, and erythrocytic iron, which makes up roughly half of the body's iron stores, is the two main types of blood iron that circulate.Erythrocytic iron is the constituent of heme incorporated into the four globin molecules to create the hemoglobin macromolecule.Furthermore, plasma iron must be attached to transferrin since it exists as a metal and cannot flow freely in a liquid.A transferrin molecule can only bind a maximum of two iron atoms 13 .The primary sources of iron are duodenal enterocytes and recycled macrophages.Absorbed iron is transported across the apical membrane of enterocytes after reduction.Imported iron can be used for cell-intrinsic metabolism, stored bound to the protein ferritin, or exported to plasma bound to the protein transferrin 14 .Dietary iron is present in the oxidized form Fe 3+ and must be reduced to Fe 2+ before absorption occurs in the intestine.This reduction is thought to be mediated by ferrireductase in the apical membrane of intestinal cells 15 .Cytochrome b (Dcytb) in the duodenal enterocyte membrane reduces Fe 3+ to Fe 2+ , then Fe 2+ enters the cell through divalent metal transporter 1 (DMT1) on the membrane.The protein ferritin, which has enormous cavities to store hundreds of iron atoms and is coupled to extracellular iron, resists the dissociation of iron and results in oxidative cell damage 16 .Under typical circumstances, transferrin-bound iron makes up most of the iron in the bloodstream.Iron is kept in an inert form by transferrin, which may carry up to two iron molecules 15 .Intracellular iron is released into the circulation via the only known iron exporter, ferroportin, commonly referred to as FPN1, and encoded by the SLC40A1 gene 16,17 .After Fe 2+ comes out into the circulation, it will be oxidized to Fe 3+ by ferroxidases such as hephaestin (HEPH) or its homolog: ceruloplasmin (CP) and will then be bound to transferrin protein/transferrin bound iron (TBI) and transported by the bloodstream 16 .Specific uptake mechanisms in the body transfer circulating iron to erythrocytes and other cells.The main mechanism is transferrin-bound iron uptake by TFR1 via receptor-mediated endocytosis into the clathrin-coated (Figure 2).Iron will be released in an acidic environment from the endosome, then Fe 3+ is reduced to Fe 2+ by the STEAP3 ferrireductase, and Fe 2+ is exported out of the endosome by DMT1, while TFR1 will be recycled to the cell surface 15 .Hepcidin, an iron hormone released by the liver, is the mechanism by which systemic iron is regulated under physiologically normal circumstances.Hepcidin binds to ferroportin (a transmembrane iron-exporting protein) on macrophages and iron-storing hepatocytes, degrading ferroportin and inhibiting iron entry into the circulation.Moreover, hepcidin binds to ferroportin on enterocytes, reducing the expression of DMT-1 protein that facilitates non-heme iron uptake on the apical surface of enterocytes and decreasing intestinal iron absorption 18 .Mutations that cause a lack of hepcidin or resistance to it cause excessive amounts of iron to be absorbed from the duodenum, released uncontrollably into the circulation by macrophages, and deposited excessively in several organs 19 .Iron is necessary for healthy physiology, but too much of it can be dangerous because it speeds up the Fenton reaction, which creates reactive oxygen species (ROS) that can destroy cells and tissues.Maintaining body iron homeostasis is critical because there is no natural method to eliminate excess iron from the body 15 .Iron overload results in plasma transferrin saturation and hence the emergence of circulating nontransferrin-bound iron (NTBI), which causes iron deposition in vital organs such as the kidneys, liver, and heart 7, 19 .

IRON OVERLOAD CONDITIONS
β-thalassemia is a hereditary disorder of Hb production characterized by impaired Hb synthesis, leading to shorter red blood cell survival through hemolysis and premature death of red blood cell precursors in the bone marrow.This condition results in chronic severe anemia and bone marrow expansion.Hepatosplenomegaly, bone deformities brought on by bone marrow growth, and heart failure brought on by severe anemia can all result from thalassemia if left untreated 20 .Red blood cell transfusions are essential for the survival of patients with β-thalassemia.Pediatric thalassemia patients require erythrocyte transfusions more frequently than 8-12 times per year in order to ensure their survival as well as healthy growth and development 21 .Blood transfusions help treat chronic anemia, avoid bone deformities, stimulate healthy growth, and improve patients' quality of life.For adult men and women, Hb levels exceeding 120 and 130 g/L, respectively, are considered normal values 20 .Due to repeated transfusions, the body cannot store enough iron to reach acceptable levels of excess iron in the plasma.By producing hydroxyl free radicals, which lead to oxidative stress, excessive iron has the potential to penetrate cells in the liver, heart, endocrine glands, and other organs and gradually destroy surrounding tissue.By binding plasma iron to transferrin, which becomes highly saturated in individuals with iron overload, the body can only manage iron to a certain extent.This causes the production of a significant amount of iron not linked to transferrin, including labile plasma iron, and speeds up the creation of free radicals in some organs and tissues.Apart from transferrin, the human body itself does not have any physiological mechanisms to remove excess iron 22 .

PATHOPHYSIOLOGY OF KIDNEY DAMAGE DUE TO IRON OVERLOAD
Urine formation is regulated by a highly sophisticated mechanism involving the glomerulus, proximal convoluted tubule, loop of Henle, and distal convoluted tubule.The wound's severity and location can significantly impact the outcomes of any incident that damages the tubular epithelial cells.According to experimental research, mice with an excess of iron can develop mild proteinuria, considerable glomerulosclerosis, tubular atrophy, interstitial fibrosis, and visible iron deposits in the proximal tubules, glomeruli, and interstitium 23 .Chronic renal hyperfiltration causes increased proteinuria and, in the long term, can lead to a progressive decrease in glomerular filtration rate (GFR) and renal fibrosis.The reasons for the nephrolithiasis seen in TDT patients have been hypothesized to be hemolysis, hypercalciuria, and hyperuricosuria.In 2 to 10% of TDT patients, hematuria has been noted, and it may be linked to an increased risk of nephrolithiasis 24 .Almost every living thing on Earth depends on iron, the second most abundant metal in the world.Humans require iron as a cofactor for hemoproteins (catalase, cytochrome, hemoglobin, or myoglobin) and other non-heme proteins involved in cell division, proliferation, DNA synthesis, and drug metabolism.On the other hand, through the Fenton reaction, iron can also create dangerous oxygen-free radicals, specifically hydroxyl radicals.Normal cellular metabolism produces a slight excess of free radicals, which are then neutralized by natural cellular antioxidant systems.However, in conditions of excess iron, such as hemochromatosis, the level of non-transferrin bound iron (NTBI), i.e., free iron circulating in the plasma, will increase in number.The availability of free iron will form the basis of iron toxicity because it accelerates the Fenton reaction to produce an increase in the amount of ROS that cannot be suppressed so that saturation of the antioxidant system can occur 19 .A condition of imbalance between excessive oxidants, also known as free radicals, and the antioxidant system's insufficient ability to degrade these radicals as a kind of defense against them is known as oxidative stress.Reactive oxygen and reactive nitrogen species (RNS) are two examples of oxidants created and eliminated by various antioxidant defense systems during physiological conditions 25 .Reactive oxygen species play a significant role in cell signaling involved in cell proliferation, differentiation, apoptosis, and immune defense in various cells, including kidney cells, under normal settings.Numerous inflammatory cells are attracted by the creation of ROS, which also promotes the production of inflammatory cytokines, growth factors, and transcription factors.Reactive oxygen species and RNS levels within cells are enhanced during oxidative stress, frequently seen in several kidney illnesses.Reactive oxygen species are typically considered hazardous substances, resulting in oxidative damage since they interact with lipids, proteins, and DNA.Both the renal cortex and the medulla can experience ROS generation or altered ROS production, which can have a variety of repercussions, from altered renal blood flow to sodium/fluid retention to inflammation and cellular fibrosis, as well as the onset of proteinuria 26 .In the course of renal failure, it has been reported that high levels of free radicals are associated with disease pathogenesis and progression 27 .Albumin can be found in the urine of patients with glomerular or tubular renal injury, but higher urinary albumin levels are usually found in glomerular injury 28 .Albuminuria is a recognized indicator of renal impairment that develops early in many types of chronic kidney disease (CKD) and plays a significant role in the course of the illness by causing mesangial and tubular toxicity as well as activating systemic inflammatory pathways 29 .The occurrence of barrier dysfunction in glomerular filtration in the presence of podocyte injury is a sign of the development of proteinuria and subsequent glomerulosclerosis.Evidence from experimental and clinical studies suggests podocytes are particularly vulnerable to oxidative harm.It appears that glomerular injury is directly mediated by an increase in ROS in an experimental model of the disease that develops into focal segmental glomerulosclerosis (FSGS) 30 .

Ferritin and transferrin saturation
A significant protein that stores iron, ferritin is also involved in several physiological and pathological processes and is crucial for maintaining iron homeostasis.Its primary function is as a ferroxidase in iron absorption, converting Fe (II) to Fe (III).Iron is hazardous to cellular systems because it can generate reactive species that can obliterate DNA and proteins.Iron pools within cells are supported and captured by ferritin.Ferritin is mainly utilized in clinical medicine as a serum marker of total body iron reserves.Serum ferritin is crucial for diagnosing and treating both iron deficiency and excess 31 .The ratio of serum iron to total iron binding capacity, or transferrin saturation (TSAT), is another significant biochemical marker for determining the body's overall iron status.In CKD, transferrin saturation can track a child's response to iron therapy and ESAs (erythropoiesis-stimulating drugs).Iron disutilization for erythropoiesis is thought to occur in patients with high ferritin and TSAT levels.Even if the body appears to have adequate iron stores, iron disutilization for erythropoiesis occurs when insufficient iron is incorporated into erythroid precursors.Patients with infectious diseases, persistent inflammation, chronic heart disease, chronic kidney disease, and malignant diseases are affected by this condition 32 .According to the Kidney Disease Outcomes Quality Initiative (K/DOQI), serum ferritin and transferrin saturation were the primary markers in evaluating iron management in patients with anemia and CKD.According to the K/DOQI guidelines, patients with serum ferritin levels greater than 800 ng/mL may experience iron overload, albeit this condition is also very variable.In patients undergoing dialysis, serum ferritin levels reach 2000 ng/mL, which is a picture of the condition of patients with hemochromatosis, where the deposition of iron in the tissue clinically begins to occur 33 .

Urea
The liver produces and distributes urea, the byproduct of protein and amino acid catabolism, through internal and extracellular fluids, which are then filtered in the glomerulus.Urea examination is beneficial in establishing the diagnosis of acute renal failure.The clinical index most often determined to estimate renal function depends on the serum urea concentration.An increase in Blood Urea Nitrogen (BUN) is linked to the presence of kidney failure and obstruction of the urinary tract by kidney stones.Urea clearance is a measure of abnormalities in the glomerular filtration rate.When the BUN value exceeds 100 mg/dL, it may be a sign of significant kidney damage 34 .

Creatinine
Creatinine is a product of the breakdown of creatinine phosphate in muscles, and depending on the amount of muscle mass, the body typically produces it at a reasonably steady rate.Typically, creatinine is used to gauge renal function.Men's normal creatinine readings are 110-150 mL/min, whereas women's normal values are 100-130 mL/min.The progression of renal disease is tracked using measurements of creatinine clearance 35 .According to the National Kidney Disease Education Program of the National Institutes of Health, the estimated GFR should be determined using serum creatinine readings as an early indicator of kidney disease 36 .When the serum creatinine level is higher than the top limit of the normal range, the diagnosis will result in renal failure.The glomerulus and tubules eventually excrete less creatinine in patients with chronic renal failure and uremic diseases 34 .Based on the evidence from Ige et al. 36 showed that in experimental Wistar rats treated with ferrous sulfate treatment as much as 15 and 30 mg/kg BW every day for 21 days, the serum analysis showed an increase in urea and creatinine levels in both treatment groups significantly compared to the control group.

Renal tissue antioxidant
There have been reports of renal impairment occurring alongside oxidative tissue damage when there is inflammation 37 .Chronic kidney disease's pathophysiological foundation includes OxS, apoptosis, and inflammation.These characteristics are consistently present in both humans and animals.They act similarly in rat models of chronic renal failure, making them essential disease mediators.High plasma concentrations of inflammatory mediators (C-reactive protein, tumor necrosis factor-α (TNF-α), and other cytokines) and many stress markers are present in patients and laboratory animals with CKD 38 .Early reactions to kidney injury include activating inflammatory pathways inside the kidneys and recruiting inflammatory cells to the area of injury.Injury may be followed by healing mechanisms or chronic inflammation that progresses over time and eventually results in fibrosis 39 .The first line of defense against free radicals or ROS/RNS produced by enzymatic reactions or auto-oxidation mechanisms in mammalian systems is antioxidants.The measurement of total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) in plasma or body tissues provides the cumulative action of all the antioxidants and oxidants present in the body 40 .An imbalance between the production of free radicals and the antioxidant system results in oxidative stress (OxS), which harms biomolecules.These antioxidant systems are vital in preserving the cell's oxide/ reduction equilibrium 41 .Polyunsaturated fatty acids in membrane phospholipids are the primary target substrates for free oxygen radical activity, and their alteration leads to the disruption of cell structure and function.The end product of these reactions is malondialdehyde (MDA).It is excreted in urine, blood, and other fluids and, therefore, serves as a marker of lipid peroxidation and the presence of oxidative stress, respectively.Reactive oxygen species must be deactivated and removed to the activation of antioxidant defense mechanisms, such as the following enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) 42 .

Proinflammatory mediators
Tumor necrosis factor-α, interleukin 1 (IL-1), IL-6, IL-8, IL-12, monocyte chemoattractant protein 1, tissue inhibitor of metalloproteinase 1, and interferon (IF) are a few examples of chemokines and cytokines that have been found to have higher levels of proinflammatory mediators.These mediators are crucial in the inflammatory process that harms organs 37 .Interleukin 6 influences the dynamics of the extracellular matrix, general renal hypertrophy, thickening of the basement membrane in the glomeruli, podocyte hypertrophy, and cell cycle arrest, all linked to albuminuria.Interleukin 6 also contributes to neutrophil infiltration in the interstitial tubules 43 .Compared to other proinflammatory mediators in the inflammatory response during acute renal impairment, it has been shown that the major proinflammatory mediator, IL-6, is a better marker in renal impairment patients 39 .Both glomerular and tubular cells displayed elevated TNF mRNA expression in the early stages of diabetes.Tumor necrosis factor-causes various effects, including the production and differentiation of inflammatory cells, renal cell cytotoxicity, apoptosis activation, glomerular hemodynamics modifications, increased vascular endothelial permeability, and increased oxidative stress.Blood cells and numerous intrinsic renal cells, including glomerular, endothelial, tubular, and mesangial cells, can produce inflammatory cytokines in the kidney.As nephropathy worsens, these chemicals' concentrations rise, and there is no connection between these inflammatory markers and urinary albumin excretion 43 .Proinflammatory cytokines, including IL-1, IL-6, and TNF-α, produce changes in renal structural hemodynamics, cellular necrosis, and alterations in the kidney's glomerular endothelial permeability 44 .Based on research conducted by Ige et al. 36 , which showed that in experimental animals, Wistar rats treated with ferrous sulfate treatment of 15 and 30 mg/kg BW every day for 21 days showed that in serum analysis, there was an increase in TNF-α and IL-6 levels in both groups were significantly compared with the control animal group.Finally, people with thalassemia who require blood transfusions may develop an iron overload disease that may result in organ damage.The condition of excess iron occurs due to the accumulation of iron in various organs.The accumulation that forms in the vital organs of the kidneys can cause oxidative stress, which can harm cells and organs, with the kidney being one of the most susceptible organs.Damage can occur in any area of the kidney, beginning with the glomerulus and progressing to the proximal tubule, the loop of Henle, and the distal tubule.As the kidney's primary function, it will impact every step of the excretion process.Being a crucial organ that also serves as an excretory organ, it is only reasonable that iron overload would substantially impact it if its function decreased and worsened.This happens because physiologically, excess iron cannot be expelled in its whole; it must be in the form of a chelate.

CONCLUSION
Ultimately, iron overload can disrupt iron homeostasis, increase NTBI, and increase free radical generation, all of which contribute to and induce kidney organ dysfunction.Appropriate treatment is needed to prevent iron accumulation in the organs: iron chelation drugs.Iron chelation improves the quality of life of individuals who receive routine blood transfusions.Indonesia, one of the countries with abundant botanical species, has become an opportunity for drug development research on iron chelation therapies based on natural ingredients.Natural product-based treatments are currently being developed as complementary therapies to the primary treatment to help patients maximize the treatment outcomes.

Figure 1 .
Figure 1.Kidney nephron, created by https://www.biorender.com/.The fundamental mechanism in the renal system results in urine generation.Glomerular filtration: nondiscriminant filtration of protein-free plasma from the glomerulus into Bowman's capsule.Tubular reabsorption: selective movement of filtered substances from the tubular into the peritubular capillaries.Tubular secretion: selective movement of nonfiltered substances from the peritubular capillaries into the tubular lumen 10 .

Figure 2 .
Figure 2. Iron homeostasis, created by https://www.biorender.com/.Non-heme iron is mainly absorbed by intestinal enterocytes expressing the ferric reductase DCYTB, which converts Fe 3+ to Fe 2+ and facilitates its transport through the apical side of the enterocyte cell membrane by DMT1 subsequently stored in cells as bound Ferritin protein.Iron enters the circulation by being exported through FPN on the basolateral membrane of enterocytes.Next, the formed TBI complex 18 .