Screening models of nephrotoxicity and their molecular mechanism

Kidney is among the most vital organs in the anatomy of human body which filters 200 litres of fluid every 24 hours and has many more function which include regulating acid-base balance, maintaining electrolytes balance, eliminating waste and toxins products from the human body. Nephrotoxicity refers as the deterioration in the kidney function due to toxic effect of drugs and chemical and is characterized by alterations in glomerular hemodyanmics, renal tubular failure and thrombotic microangiopathy. Drug-induced nephrotoxicity is becoming more well identified as a principal reasons of kidney illness, such as acute kidney injury (AKI) and chronic kidney disease (CKD) (CKD). Nephrotoxicity covers a broad range, displaying damage to various nephron segments as a result of distinct pharmacological actions. There are various agents which exerts nephrotoxic effects through pathogenic mechanisms like non-steroidal agents (Indomerhacin, Paracetamol, Mefenamic acid), anticancer agents (Carmustine, Cisplatin, Methotrexate), antibiotics (Gentamicin, Tobramycin, Amikacin), antiviral (Acyclovir, Tenofovir, Cidofovir), antifungal (Amphotericin B, Vancomycin, Nystatin)and immunosuppresant drugs(Cyclosporine, Tacrolimus). Alteration in glomerular hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy are all possibilities for drug-induced nephrotoxicity. In this review, we have explained different experimental models for nephrotoxicity that might assist in developing novel drugs to cure nephrotoxicity.


Graphical Abstract
Nephrotoxic effect of various drugs on kidney

Introduction
The kidney is a significant organ in the body that regulates the balance between acidity and alkalinity, electrolyte balance, blood filtration, as well as waste product excretion.Acute kidney damage is frequently caused by drugs and chronic kidney injury.When kidney function is destroyed by exogenous and endogenous toxicants, nephrotoxicity occurs.Nephrotoxicity occurs when kidney-specific detoxification and excretion do not work effectively.It is noted that hemodynamic changes in the glomerulus followed by irritation, nephritis caused by crystals, and thrombotic thrombocytic purpura results from nephrotoxicity [1].Medication-induced nephrotoxicity has a variety of mechanisms that vary by pharmacological and drug class, however, they are generally classified based on the histological component of the kidney that is harmed.[2].Renal impairment caused by nephrotoxic medicines is known as drug-induced nephrotoxicity.In several clinical circumstances, such as underlying renal dysfunction, cardiovascular disease, diabetes, and sepsis, it is a common concern.It was observed that chemicals like Amphotericin B, Carbon tetrachloride, Paracetamol, Acyclovir, Tacrolimus, Mefenamic acid, Aspirin, Gentamicin, Colistin, Tenofovir, Cyclosporin, Deltamethrin, and metals like Lithium, Chromium, Cisplatin, Mercury cause mild to moderate nephrotoxicity.To avoid drug-induced nephrotoxicity and progression to end-stage renal disease, early detection of deteriorating drug effects, as well as the patient's clinical history, basic renal function, drug-related risk factors, and nephrotoxic drug combinations, is critical.[3].Analgesics like Paracetamol, Aspirin, mefenamic acid induce nephrotoxicity by causing Chronic Interstitial Nephritis, altered Intraglomerular Hemodynamics.Antimicrobials like Amphotericin B, Acyclovir, Gentamicin, Colistin promote Crystal Nephropathy, Acute Interstitial Nephritis, Tubular cell toxicity which ultimately results in Nephrotoxicity.Tacrolimus and Deltamethrin are immunosuppressive which induces nephrotoxicity by provoking interstitial nephritis, glomerulonephritis.Calcineurin Inhibitors like Tacrolimus and Cyclosporin induce nephrotoxicity by causing Thrombotic microangiopathy, altered intraglomerular hemodynamics, chronic interstitial nephritis [4].In order to find a viable pharmaceutical target for nephrotoxicity, many experimental models are used to produce nephrotoxicity.However, there is currently insufficient literature for a nephrotoxicity animal model.This review focuses on the chemicals and metals-induced nephrotoxicity-based animal model which will be further helpful in understanding the basic pathophysiology behind the drug-induced kidney damage or renal failure [5]

Experimental models for nephrotoxicity
In practically all biomedical research, animal models are a key tool for studying mechanisms.They involve the entire animal's complexity, making in vivo system monitoring extremely difficult.As the compounds are exposed in a sequential manner through absorption from the initial exposed location, metabolism, distribution, and elimination, an in vivo system accurately replicates the exposure profile and cellular function.However, the mechanism should be essentially the same as in human reactions, and the unfavorable effect must be clinically significant.Small animals such as rats, mice, rabbits, and guinea pigs, as well as large animals such as pigs, cattle, sheep, and primates, can be used to research nephrotoxic effects, distribution, and clearance.They may be utilized to deduce the basic mechanism of xenobiotic actions, which will help researchers better understand how they affect human health.The experimental model, on the other hand, provides a road map for the identification of new molecular, noble signalling pathways for the benefit of humanity.[5] Figure 1 Various Models of Nephrotoxicity

Paracetamol induced nephrotoxicity
Paracetamol (PCM), the most extensively utilized as antipyretic and analgesic drug in the world and it is safe when used within the clinically recommended dose.

Aspirin induced nephrotoxicity
Aspirin is a class of medications used for analgesic and anti-inflammatory and is responsible for kidney damage in higher doses.[13]A major mechanism in the pathophysiology of Aspirin nephrotoxicity is oxidative damage.It has been reported that aspirin causes nephrotoxicity by lowering antioxidant production.[14]  Single dose of 600 mg/kg orally Female Wistar Albino rats.17

Mefenamic acid introduced nephrotoxicity
Mefenamic acid non-steroidal anti-inflammatory medication (NSAID) act against inflammation, pain and pyrexia.Along with its potential medicinal applications, mefenamic acid is applied in clinical practice.[18]Mefenamic acid is utilized for inflammatory pain that includes post traumas and dental pain.Regardless of the wide usage, they cause are asserted with severe toxicity such as severe gastrointestinal tract disorders, hepatotoxicity and nephrotoxicity.Prostaglandin I2 and PGE2, which are released by both glomerular and medullary interstitial cells, are reported to be responsible in the mechanism.Vascular resistance will be alleviated, renal vascular beds will dilate, and organ perfusion will be restored.Blood flow will be redirected in juxtamedullary area starting from the renal cortex.NSAIDs impede prostaglandin production, causing acute ischemic renal insufficiency by reducing blood flow to the nephrons.[19] Table 3

Amphotericin b induced nephrotoxicity
Amphotericin B (AMB) is the gold standard for treatment if mycosis.[22]Despite its broad range of activity and widespread clinical use, drug-induced nephrotoxicity is prevalent.During treatment using amphotericin B, up to 80% of patients will see an episode of renal impairment.[23]Tubular damage followed by acute renal failure is a well-known problem associated with AMB.[24]Following AMB administration, there was an uptick in the lipid peroxidation products MDA,NO, and SOD activity, as well as a lower in the antioxidant enzyme catalase.It has been documented that increased generation of reactive oxygen species play a vital role in pathogenesis of AMB induced nephrotoxicity.Additionally, AmB induces tubular dysfunction by forming pores in membranes.[25]

Acyclovir (acv) induced nephrotoxicity
Acyclovir is an important antiviral agent.Although the drug is well tolerated but severe nephrotoxicity has been observed with this drug.[30] Crystalluria has long been thought to be the aetiology of acyclovir-induced nephrotoxicity.Clinical evidence of nephrotoxicity in the absence of crystal formation, on the other hand, suggested that acyclovir could elicit direct injury to renal tubular cells.[31] ACV has been associated with a multitude of nephrotoxicity symptoms, including crystal nephropathy, acute interstitial nephritis, acute tubular necrosis, and obstructive nephropathy, according to studies.Direct assault on renal tubular cells and oxidative stress have been proposed as methods by which ACV induces nephrotoxicity.ACV also had a deleterious effect on kidney redox state, reducing antioxidants (SOD, CAT, GSH, and GPx) and elevating MDA levels.The activation of oxidative stress as a result of ROS production by ACV may have impaired kidney antioxidant concentrations.The released ROS may have depleted kidney antioxidants and promoted the oxidation of renal lipids (polyunsaturated fatty acids), resulting in an increase in MDA levels.It's possible that oxidative damage is a key indicator of ACV-induced renal impairment.[32]

Gentamicin induced nephrotoxicity
Gentamicin, aminoglycoside antibiotic, has widely been utilized as bactericidal agent that act against severe bacterial infection by gram negative bacterial infections.However long term us , it can also produce nephrotoxicity.[37] Gentamicin has been shown to cause kidney damage in 10-20% of patients after seven days of therapy, in addition to its therapeutically helpful effects.Despite the discovery of new antibiotics, gentamicin is still used because of its effectiveness against lactam-resistant bacteria, low levels of Enterobacteriaceae resistance, and inexpensive cost.[38] Some of the mechanisms of Gentamicin-induced nephrotoxicity include free radical generation, increased lipid peroxidation, and decreased endogenous antioxidant activity resulting in decreased glomerular filtration rate and renal dysfunction.Gentamicin exacerbated tubule damage by necrosing tubular epithelial cells, specifically in the proximal segment, and affecting the function of critical cellular components involved in water and solute transport.[39] Table 6 Various Dosage of Gentamicin induced Nephrotoxicity

Tenofovir induced nephrotoxicity
The nucleoside reverse transcriptase inhibitor tenofovir is used to treat hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections.Approximately 15% of individuals when treated with tenofovir for a period of 2-9 years experienced tenofovir-induced nephrotoxicity.Fanconi syndrome, gradual deterioration in renal function, and nephrogenic diabetic insipidus (NDI) due to distal tubular dysfunction have been documented as the most common renal consequences of TDF.[52] It causes proximal tubulopathy, which is thought to be caused in part by the obstruction of mitochondrial DNA polymerase, which reduces mitochondrial DNA (mtDNA) replication.Furthermore, mitochondrial damage may result in apoptosis.

Chromium induced nephrotoxicity
In nature, chromium (Cr) exists in a variety of forms, the most stable of which are Cr (III) and Cr (VI).Chromium is a strong oxidizing agent, and serious or persistent chromium exposure (through inhalation, skin contact, or drinking water) causes toxicity in cells, cancer, mutagenesis, or genotoxicity in the body's important organs such as the lungs, liver, and kidneys.[76]Because heavy metals are excreted mostly through the kidneys, excess levels of hexavalent chromium [Cr (VI)] are accumulated there which causes nephrotoxicity.[77]Chromium has the ability to influence cellular functioning by blocking antioxidant enzymes and binding to antioxidant components such as Glutathione (GSH), contributing to oxidative stress.In, proximal convoluted tubules, chromium compounds are selectively deposited where they produce acute tubular necrosis in large dosages after parenteral administration.[78] Figure 7 Motion-related protein 1 (DRP1) regulates mitochondrial fission, whereas mitofusin 1 (MFN1) and mitofusin 2 control mitochondrial fusion (MFN2).Peroxisome proliferation-activated receptor-g coactivator-1a (PGC-1a) controls DRP1 and MFN2 expression.PGC-1a is activated by the silent information regulator two ortholog 1 (Sirt1) through deacetylation.Defects in mitochondrial fusion and fission cause oxidative stress and apoptosis, among other things.The level of pro-apoptotic protein (Bax) increased in a dose-dependent manner, while the level of antiapoptotic protein like B cells lymphoma 2 and B cells lymphoma 2-Extra large(Bcl-2 and Bcl-xl) decreased in a dosedependent manner.Increased level of CYT-c and caspase-3 causes overproduction of cleaved Poly adenosine diphosphate ribose polymerase (PARP) which leads to apoptosis and kidney injury

Mercury induced nephrotoxicity
Mercury (Hg) is a poisonous metal that can be found in its elemental (metallic), inorganic, and biological forms.
Occupational and environmental contexts can expose humans to various types of mercury.The most common method of human exposure, however, is through the consumption of MeHg-contaminated food, particularly fish.MeHg is quickly absorbed by the gastrointestinal tract after consumption, after which mercuric ions enter the systemic circulation and are supplied to target organs.[83].When animals or people are exposed to Hg°, the body quickly converts it to Hg+ and after oxidation it causes nephrotoxicity by promoting the development of reactive oxygen species (ROS).ROS causes alteration in mitochondria by blocking the permeable transition pore.[84].Because mercuric ions can cause kidney injury, which may not be working at full capacity, may be more susceptible to the effects of Hg than healthy kidneys.

Lithium induced nephrotoxicity
Lithium is considered as the first therapeutic line for treatment of bipolar affective disorders and manic-depressive illness [89].Lithium has been used in healthcare for almost 150 years.Garrod and Hammond advocated lithium salts as a therapeutic for gout and uric acid nephrolithiasis in the eighteenth century.However, the medicine was taken off the market by the Food and Drug Administration (FDA) in the same year when several patients suffered a heart attack or hypertension as a result of lithium intoxication.However, it has the possibility to trigger renal damage, such as decreased urine concentrating ability and natriuresis, renal tubular acidosis, tubulointerstitial nephritis progressing to chronic kidney disease, and hypercalcemia.[90] Renal toxicity can be categorized as the undesirable side effect of lithium therapy [91]Lithium detrimental effects are caused by processes that have yet to be elucidated.However, oxidative stress has been identified as one of the primary contributors of Li's harmful effects.ROS, such as superoxide and nitric oxide (NO), in particular, may play a role in the pathogenesis of Li-induced renal impairment.Lithium builds up in the collecting tubule, which is where its ability to change renal water excretion is most likely to be found.The ability of the cortical section of the collecting tubule in the kidneys to create cyclic adenosine monophosphate in response to antidiuretic hormone stimulation has been demonstrated decisively.[92]

Cisplatin induced nephrotoxicity
Cisplatin is a cancer chemotherapeutic drug that is extensively used and extremely effective.Nephrotoxicity is one of the most serious adverse effects of cisplatin.Increased activation of caspase3/7 and PARP(poly-ADA-ribose polymerase) cleavage was observed after comparing with NT3 cells in cisplatin treatment.

Conclusion
This study reveals that overdose of drugs like PCM, Cisplatin, Chromium, Aspirin, Tacrolimus, Lithium, Gentamicin, Amphotericin B etc. induces nephrotoxicity.Nephrotoxicity is caused by drugs through processes including their intrinsic toxicity, as well as their transport and management by the kidneys.The incidence of nephrotoxicity is expected to be reduced by avoiding modifiable risk factors and overseeing therapeutic medication monitoring for high-risk groups.If the reversibility of renal damage has been proven, continuous dosage of the experimental medicine may be explored, but with more regular safety evaluations to see if renal function would stabilize or worsen.From the above study it can be concluded that early detection of high doses of various drugs reduces the risks by discontinuing the drug when worsening of renal function observed.Early detection of a medication-induced nephrotoxicity would be the most important factor in lowering the expenses of new drug safety testing.For the growth of pharmaceuticals, clinical trials should be executed, taking into deliberation of various drugs administration schemes as high or low doses used in preclinical studies.Above data shows that avoiding of that dose at which drug causes toxicity for better protection of kidney.Thus, the overview of this study will help in easy identification of suitable model of nephrotoxicity for further research and provides the road map for the future study on nephrotoxicity.

43 9.Figure 3
Figure 3 Colistin induces nephrotoxicity by upregulating cytochrome C (Cyt C) which increases the level of caspase 9 and caspase 3 leads to apoptosis and necrosis.Further, overproduction of ROS elevate P53 level results in mitochondrial dysfunctioning

Figure 4
Figure 4 Exposure to DLM increases oxidative stress by suppressing the production of the Nrf-2 gene and by enhancing p38 MAPK which activates inflammatory and apoptotic pathways leads to nephrotoxicity.[49]

[ 53 ]Figure 5 Table 9 Figure 6
Figure 5 Oxidative stress including ROS are responsible for the activation of Nuclear Factor kappa B (NFkB) resulting in cellular damage.The targeted genes of NF-KB like cycloxygenase-2( COX-2), Tumor necrosis factor -α(TNF-α) and Inducible nitric oxide synthase (iNOS) promotes apoptosis, endothelial damage and renal failure

[ 85 ]Figure 8
Figure 8 Activated NF-kB stimulates oxidative stress which further increases pro-inflammatory responses.Decreased antioxidants enhances the pro-apoptotic bodies and P53, increased cyt-c , caspase 9 and 3 results in apoptosis which further cause nephrotoxicity

[ 97 ]Figure 9
Figure 9 Cisplatin accumulation triggers increased production of TNF-α and NF-kB which further stimulates inflammation.Increased oxidative stress and Bax along with Cyt-c leads to apoptosis.Interleukin-1, Monocyte chemoattractant protein 1(MCP-1) and C-X-C Motif Chemokine Ligand 1(CXCL-1) cause renal tubular injury.Extrinsic apoptosis is caused by Tumor necrosis factor receptor ½(TNFR ½) signaling caspase-B and Caspase-3.Upregulation of MAPK due to increased P38 level resulting in apoptosis

Table 1
Various Dosage of PCM induced Nephrotoxicity

Table 2
Various Dosage of Aspirin induced Nephrotoxicity

Toxic dose of Mefenamic acid Route of Administration Reference Animal Species
Various Dosage of Mefenamic acid induced Nephrotoxicity

Table 4
Various Dosage of AMB induced Nephrotoxicity

Table 5
Various Dosage of Acyclovir induced Nephrotoxicity

Table 7
Various Dosage of Colistin induced Nephrotoxicity

Toxic dose of colistin Route of administration Species References
[48](deltamethrin) is a broad-spectrum pyrethroid insecticide.DLM accumulation can harm the kidneys by a variety of ways, including oxidative stress, apoptosis, and inflammation.[48].

Table 8
Various Dosage of Delametharin induced Nephrotoxicity

Table 10
[63]ous Dosage of Cyclosporine induced NephrotoxicityTacrolimus is a calcineurin inhibitor possessing immunosuppressive properties which can be used widely in renal transplantation.Long term use of tacrolimus induces nephrotoxicity.[61]Tacrolimuscausesirreversiblekidneydamage, notably hyalinization and thickening of the arteriolar walls, vasoconstriction and ischemia, tubulointerstitial fibrosis, apoptosis, and atrophy.[62]Themechanismsunderlying nephrotoxicity induced by tacrolimus are still ambiguous.Tacrolimus propensity to generate reactive oxygen species via activation of the nicotinamide adenine dinucleotide phosphate oxidase pathway has been linked to its nephrotoxicity in previous investigations.In nephrotoxic agent-induced nephrotoxicity, apoptosis is crucial.In apoptosis, the caspase family plays a vital function.Caspase-3, in particular, is a key effector enzyme in apoptosis-related kidney damage.Tacrolimus can create reactive oxygen species (ROS) and hinder antioxidant defences in the proximal tubules.[63]

Table 11
Various Dosage of Tacrolimus induced Nephrotoxicity

. Carbon tetrachloride induced nephrotoxicity Carbon
[70]achloride (CCL4) a widely known haloalkane, belong to the class of chemicals utilized as anthelmintics and grain fumigants.It is also used as intermediates which can result in nephrotoxicity by synthesis of chlorofluorocarbons.[68]Fatty liver disease, cirrhosis in the liver, centrilobular necrosis and acute tubular necrosis in the kidney ensue after CCl4 exposure.[69]Accordingtoseveral studies, elevation of extracellular matrix triggers renal fibrosis.Further many studies have shown that CCL4 when processed in proximal tubule cells of kidneys can produce oxidative radicals.It has been observed that CCL4 therapy increases oxidative stress resulting fibrogenesis and renal inflammation.Exposure to CCL4 both in vitro and invivo can result in kidney inflammation.By inhibiting the Nrf2 pathway, CCl4 induces oxidative stress, inflammation, and fibrosis in the kidneys.[70]

Table 12
Various Dosage of CCL4 induced Nephrotoxicity

Table 13
Various Dosage of Chromium induced Nephrotoxicity

Table 14
Various Dosage of Mercury induced Nephrotoxicity

Table 15
Various Dosage of Lithium induced Nephrotoxicity

Table 16
Various Dosage of Cisplatin induced Nephrotoxicity