Preclinical trials are essential for evaluating drug safety, efficacy, toxicity, and pharmacokinetics. It is necessary to understand the difference between humans and animals at the levels of drug transporters. Along with quantitative real time-polymerase chain reaction (qPCR) and Western blot, the highly sensitive liquid chromatography tandem-mass spectrometry (LC-MS/MS) has become one of the most effective methods in the quantitative detection of drug transporters (Fallon et al., 2016; Nakamura et al., 2016; Basit et al., 2019). Table 2 has shown the species differences of renal drug transporters found in past decades. Basit et al. (2019) showed that the abundances of OAT1, OCT2, and MATE1 in human renal tissue were higher than the other eight drug transporters. The levels of OAT1, OAT3, OATP4C1, OCT2, MDR1, MATE1, OAT4, MRP2, and MRP4 in monkey were from 1.6-fold to 3.7-fold higher than that in human. OATP4C1, MATE1, MATE2-K, OAT4C1, and MRP2 in rat and mouse, as well as OATP4C1, OCT2, MATE1, MATE2-K, OAT4C1, MRP2, and MRP4 in dog, were not detected as no conserved peptide available (Basit et al., 2019). Fallon et al. (2016) have revealed three renal efflux transporters in human, monkey, dog, and rat. The concentrations of MDR1 and MRP2 were similar in human and monkey, but higher than that in rat. However, the BCRP level was the highest in rat (4.5 pmol mg⁻¹ protein), which is 50-fold than that in human (0.09 pmol mg⁻¹ protein). In another study, 11 transporters were detected in the pooled microsomal fraction of human kidney (Nakamura et al., 2016). All transporters, except for OATP4C1, have sensitive responses when detection. The content of BCRP (3.6–4.5 pmol mg⁻¹) was similar as the other reports (Fallon et al., 2016; Nakamura et al., 2016; Al-Majdoub et al., 2020). Many exogenous substances show species differences in excretion. Taking per- and polyfluoroalkyl substances (PFAS) as an example, a longer serum half-life in human (t_1/2), compared to that in animal species, is potentially due to the difference in excretion which was mediated by renal tubular OATs and OATPs (Ducatman et al., 2021). Therefore, the differences on cross-species kidney transporters should be carefully considered in drug preclinical-to-clinical stage.

As an important factor, sex-gender should attract a full attention in the context of drug development and clinical use. Between 1995 and 2000, 11 out of 300 new drug applications recorded by the Food and Drug Administration (FDA) showed >40% sex-gender differences in pharmacokinetics (Anderson, 2005). Taking methotrexate as an example, after 1,277 osteosarcoma patients were treated with a high dose of methotrexate, the incidence of delayed excretion in female patients was about 1.75%, which was significantly higher than that in male patients (0.37%) (Zhang et al., 2016). It has been reported that OAT1, OAT3, OATP4C1, OCT2, MDR1, MATE1, OAT4, MRP2, and MRP4 in the kidney show no sex-gender differences in healthy monkeys. However, significant sex-gender differences of OAT1, OCT2, MDR1, BCRP, and MRP4 are observed in rats and/or mice with different trends (Buist and Klaassen, 2004; Basit et al., 2019). For example, the abundance of MDR1 was 1.6-fold greater in male rats than that in the females, whereas its expression was twice as high in female mice compared with that in the males. It has been shown that OATP4C1, BCRP, and MATE2-K levels exhibited sex-based differences between healthy men and women (Basit et al., 2019). Due to sex-gender differences in transporter expression, full consideration should be given to pharmacokinetic, safety, efficacy, and toxicity studies of drug to allow better interpretation of clinical data. For example, 2 h after oral administration of metformin at a dose of 500 mg/kg, the cumulative urinary excretion and renal tissue-to-plasma concentration ratio in female rats (26,689 ± 1,266 µg and 2.96 ± 0.47 ml/g, respectively) were markedly lower than that in male rats (32,949 ± 1,384 µg and 4.20 ± 0.31 ml/g, respectively), which are potentially explained by gender-related differences on renal OCT2 expression, as the metformin is the substrate of OCT2 (Ma et al., 2016). Pregnancy is an especial physiological status for women. Lu et al. have proved no significant differences among OAT1/3, OCT2, MRP2, and MATE1 mRNA expressions in the kidneys between pregnant and non-pregnant rats (Lu et al., 2020).

Age affects the transcription and translation of drug transporters. Joseph et al. (2015) have determined 30 drug transporters in two groups of human kidneys (age <50 years and ≥50 years). Among them, the mRNA expression of the OCT2 in the <50 years group was greater than that in the ≥50 years group (Joseph et al., 2015). The nephrotoxicity of cisplatin exhibits an age dependence in human. Wen et al. (2015) had analyzed 182 cisplatin-treated patients to illuminate the influence of nongenetic factors on cisplatin-induced nephrotoxicity, and age (≥50 years) is closely associated with the decline of renal function with an odds ratio (OR) of 11.771. This might be due to the fact that the MATE1, which excretes cisplatin into urine, was significantly decreased with the increasing age (≥50 years), resulting in the specific accumulation of cisplatin in renal cells. Although it is difficult to investigate the changes of drug transporters in the human kidney throughout life, an age-related study on OAT1, OAT3, OATP4C1, OCT2, MDR1, BCRP, MATE1, MATE2-K, MRP2, and MRP4 mRNA expressions was performed in male rat kidneys with qPCR and Western blot methods (Xu et al., 2017). In this preclinical study, all the detected drug transporters showed different increasing trends. As shown in Figure 2, the mRNA expressions of OAT1, OATP4C1, OCT2, BCRP, MATE1, and MATE2-K reached the peaks at the 180th day and subsequently declined. Compared with the mRNA expressions at the -2nd day and/or the 1st day, the expressions at the peak were 35-fold, 35-fold, 470-fold, 18-fold, 18-fold, and 37-fold greater for six drug transporters, respectively. The mRNA expressions of OAT3 and MRP2 reached the peak at the 850th day. For OAT3, the mRNA expression at the 180th day was 22-fold greater than that at the 1st day, while it was 3-fold greater at the 850th day than that at the -2nd day for MRP2. Interestingly, the mRNA expression of MRP2 was reduced almost 50% at the 14th and the 21st day compared with the -2nd day. The mRNA expression of MRP4 reached peak at the 28th day, and it was 6-fold higher in comparison with that at the -2nd day. Ren et al. (2015) used both young rats (aged 3 months) and old rats (aged 12 months) to illuminate age-related excretion of metformin. The values of t_1/2, accumulation in urine, and total clearance (CL_tot) (1.717 ± 0.30 h, 6,088.402 ± 931.55 μg, and 93.414 ± 11.47%, respectively) in young rats were significantly different from those indexes in old rats (2.002 ± 0.51 h, 4,287.087 ± 458.08 μg, and 57.161 ± 18.59%, respectively). A significant age-related decrease of OCT2 is probably responsible for renal excretion of metformin (Ren et al., 2015). Although the lives of experimental animals are much shorter than that of human, we still clarity that the drug transporter undergoes a dynamic change throughout the life. Thus, drug transporters should receive close attentions at fetal, neonatal, mature, and old ages during drug development and clinical use (Andreollo et al., 2012; Sengupta, 2013; Gleeson et al., 2021; Vinarov et al., 2021).

Kidney diseases, such as chronic kidney disease (CKD), acute kidney injury (AKI), and renal failure, can change the excretion rate of endogenous and/or exogenous substances, mainly due to a decrease in the glomerular filtration rate and the changes in drug transporters and metabolic enzyme activities. Levels of renal drug transporters are crucial for drug excretion, as drugs were mainly excreted through these transporters. As shown in Table 3, the mRNA and/or protein levels of drug transporters in renal-related disease models were summarized.

Uric acid nephropathy, mainly caused by hyperuricemia, is a common kidney disease associated with hypertension, proteinuria, oedema, etc. (Perry et al., 1976). In animal experiments, adenine and potassium oxonate are often employed to construct uric acid nephropathy models for drug screening, development, and drug interaction research (Liu et al., 2017; Wu et al., 2017; Wang et al., 2020b). To date, OAT1 and OAT3 are the two most studied drug transporters in uric acid nephropathy, as these proteins are responsible effects for uric acid excretion. Reduced OAT1/3 mRNA and protein levels are consistently noted in uric acid nephropathy (Liu et al., 2015; Liu et al., 2017; Wang et al., 2020b; Zhang et al., 2020). The same trend was observed in the uptake transporter OCT2 and efflux transporter BCRP (Wu et al., 2017; Wang et al., 2019a). Nephrotoxic substances, such as cisplatin, gentamicin, and heavy metals, are causative agents of AKI in clinic. In the cisplatin-induced nephrotoxicity model, the mRNA and protein levels of renal OAT1/3 were decreased in both rats and mice, which could prevent the absorption of cisplatin from blood into renal tubular epithelial cell (Wang et al., 2018a; Deng et al., 2020b). MATE1 is an efflux transporter that mediates the excretion of cisplatin from renal tubules into urine. Previous study has proved that the survival time of Mate1^−/− mice was significantly shorter than that of the control group. After cisplatin administration, the blood concentration and accumulation in the kidney of Mate1^−/− mice were higher than that in the control mice. Using combination of cisplatin and the MATEs inhibitor pyrimethamine was found to be more nephrotoxic than using cisplatin alone. These results suggest that MATE1 mediates the renal excretion of cisplatin and participates in its nephrotoxicity (Kusuhara et al., 2011; Song et al., 2016). However, OCT2 mRNA and protein levels exhibited the species differences, which were decreased in mice but were increased in rats (Aleksunes et al., 2008; Soetikno et al., 2019). It is worth noting that mRNA and protein levels may also show different trends during the course of disease. For instance, oral administration of NH₄Cl caused downregulation of OAT1 mRNA and protein levels on the 2nd day, but the levels returned to its original levels on the 7^th day (Gottier Nwafor et al., 2020). In the model of heavy metal-induced nephrotoxicity, the uptake transporters OAT1/3, OATP4C1, and OCT2 showed decreasing trends, whereas the efflux transporters MDR1, MATE2-K, and MRP2/4 showed increasing trends (Liu et al., 2016; Ljubojević et al., 2016; Zhang et al., 2017a), which decreased absorption, enhanced urinary clearance of heavy metals, and prevented accumulation in the kidney. Ischemia-reperfusion injury (IRI) in the kidney is another most common clinical cause of AKI, which often occurs during the clinical course of shock, acute renal artery occlusion, or renal transplantation. At present, the nonclinical IRI model is usually constructed by sealing the bilateral renal pedicle with a mini-artery clip (Shiva et al., 2020). Interestingly, the drug transporters OAT1/3, OCT2, MDR1, MATE1, and MRP2/4 in this model showed reducing trends. Schneider et al. (2007) reported that the OAT1/3 levels are associated with recovery time after IRI in kidney. Compared with the sham group, OAT1/3 mRNA and protein levels were completely restored to the preoperative level after 7 days postischemic reperfusion. Given that renal OAT1/3 play significant roles in the excretion of many anionic drugs, IRI induced AKI changes the pharmacokinetics of these drugs, including increases in plasma levels and t_1/2. An animal model with 5/6 nephrectomy is often used to simulate chronic renal failure in humans. Previous studies have reported the downregulation of uptake transporters OAT1/3 and OCT2, as well as the downregulation of efflux transporters MDR1, BCRP, and MRP2 in animal kidneys during the disease statuses (Laouari et al., 2001; Nagura et al., 2016; Kong et al., 2017). Nephrotic syndrome (NS) is a group of clinical symptoms characterized by increased glomerular basement membrane permeability, high proteinuria, hypoproteinemia, high oedema, and hyperlipidemia (Hull and Goldsmith, 2008). During the 12-week experiment, OAT1 mRNA and protein levels consistently exhibited trends of reduction. OCT2 protein level showed a downward trend within 9 weeks, but increased in the 12th week. The same trends were observed in MRP2 and MRP4 (Dong et al., 2020). The unilateral ureteral obstruction (UUO) animal model is often used to simulate clinical obstructive nephropathy. A study reported reduced OAT1/3 protein levels in the UUO model, indicating reduced renal excretion rates and pharmacokinetic changes of some drugs (Brandoni and Torres, 2015). Downregulation of OCT2 protein level was observed in the kidney transplanted rats. Atenolol, pindolol, and ofloxacin are the substrates of OCT2, and their appropriate doses are difficult to achieve in kidney transplanted patients (Ciarimboli et al., 2013).

In summary, in renal-related diseases, the expressions of OAT1/3 transporters are invariably reduced. The OAT1/3 are responsible for the uptake of endogenous and/or exogenous substances from the blood into renal tubular epithelial cells. The reduction in the renal-related diseases is like a double-edged sword. On the one hand, it prevents some toxic substances (e.g., indole sulfate) from entering renal tissue during the kidney injury, subsequently reduces renal damage, and thus plays a role in protecting the kidney. On the other hand, it also reduced drug excretion, resulting in increased drug concentration in plasma, which leads to toxic side effects. Clinical pharmacokinetics of morinidazole has proved that sulfate conjugate of morinidazole M7 was a substrate of OAT1 and OAT3, while the glucuronide conjugates of M8-1 and M8-2 were substrates of OAT3. After intravenous infusion of 500 mg morinidazole, the area under the curve (AUC) values for M7, M8-1, and M8-2 were 15.1-, 20.4-, and 17.4-fold, respectively, higher in patients with severe renal impairment than those in healthy subjects (Zhong et al., 2014; Kong et al., 2017). The expressions of efflux transporters are different in different renal diseases. It is noteworthy that the increased MRP2/4 in nephrotoxic pathology will transfer the toxic substances from renal tubular epithelial cells into the urine to exert the detoxification effect. However, the increased MRP2/4 also promote the transfer of some therapeutic drugs. Therefore, both changes should be carefully considered in the treatment of the renal-related diseases.

The Inner Canon of Huangdi, an ancient Chinese medical scripture that is the basis for traditional Chinese medicine, states that “the liver and kidney are homologous.” This means that, although the structure and function of the liver and kidney are different, they exhibit a close relationship based on physiological and pathological features. For example, renal injury is often also associated with liver-related diseases, such as hepatorenal syndrome (Raj et al., 2020; Simonetto et al., 2020). In Table 4, we have summarized the characteristics of nonalcoholic fatty liver disease (NAFLD), ischemia reperfusion (IR), and bile duct obstruction pathology on renal drug transporters, providing information for optimizing the drug prescriptions. NAFLD is a global disease (with an approximately 25% of global incidence rate) induced by many factors except alcohol and other specific factors, characterized by excessive deposition of fat in hepatocytes (Younossi et al., 2016). Nonclinical NAFLD models have shown that OAT1 mRNA expression was decreased in mice, but no significant difference was found in rats. Elevated OAT3, MDR1, and MPR2/4 mRNA expression levels were observed in both mouse and rat models, but BCRP protein level was decreased (Canet et al., 2015). Clinical treatments for NAFLD typically include metformin, vitamin E, and statins to exert anti-IR, antioxidant, and lipid-lowering effects. Clarke et al. (2015) revealed that metformin exposure is increased in nonalcoholic steatohepatitis mice model (60% of metformin dose excreted in urine) than that in control group (82% of metformin dose excreted in urine), which might be caused by the decreases of OCT2 and MATE1 in the kidneys of disease mice. In patients, 25% of NASH may lead to an impaired kidney function that highlights a need to adjust drug dosage (Targher et al., 2010). A recent research showed that the novel targeting therapeutic agents mainly target peroxisome proliferator-activated receptors (PPARs), farnesoid X receptor (FXR), and glucagon-like-1 (GLP-1) (Zhou et al., 2020b). As one of the subtypes of PPARs, PPARα is widely expressed in the liver, adipose tissue, heart, skeletal muscle, and kidney. Given that PPARα has a regulatory effect on the drug transporters MATE1 and OCT2 (Freitas-Lima et al., 2020), attention should be given to drug disposition in vivo during the clinical use of this novel drug simultaneously with other drugs. IRI of the liver is an inevitable complication during liver resection and liver transplantation. The animal model confirmed that hepatic IRI could increase the kidney MRP2/4 levels (Tanaka et al., 2008). Interestingly, the increases of proteins showed obvious delays, compared to those of mRNAs. The increased renal MRP2 accompanied with decreased hepatic MRP2 may protect against oxidative stress and inflammation after hepatic IRI. Extrahepatic cholestasis is a pathophysiological process caused by the obstruction of bile secretion and excretion. This disease status could induce the expressions of the efflux transporter MRP2/4 in the kidney which will promote the excretion of bile acids and some therapeutic drugs (Slitt et al., 2007). OAT1/3 levels are different in various animal models, which may be related to the way and/or time of modeling (Brandoni et al., 2006; Liu et al., 2012). In general, with liver and bile duct-related diseases, the expressions of MRP2/4 are increased, which accelerates the excretion of endogenous/exogenous toxic substances in the kidney to play a protective role in the body.

As shown in Table 5, metabolic diseases can also cause changes in drug transporters in the kidney. Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder, and the condition is primarily caused by excessive production and reduced excretion of uric acid. A cross-sectional study in China reported that, with the improvement of people’s quality of life, the prevalence of hyperuricemia has increased from approximately 26.1% in 2010 to approximately 34.4% in 2019 among men in Wuhan city (Wan et al., 2021). High uric acid level is associated with hypertension, CKD, obesity, metabolic syndrome, etc. Therefore, a combined medication is a common treatment for hyperuricemia. Thus, the levels of renal transporters should attract clinician attentions. In animal models, the OAT1/3, OCT2, and BCRP expressions are decreased under the pathological condition, whereas MDR1 and MRP2/4 expressions are not affected by hyperuricemia (Nishizawa et al., 2019; Le et al., 2020). A growing number of studies have reported that OAT1/3, OCT2, and BCRP are involved in urate efflux from epithelial cells to urine (Taniguchi et al., 2021), and these transporters have been extensively studied in the hyperuricemia. Cephalexin is a substrate of OAT1 and is excreted via MATE1. Compared with that in control group, the AUC_inf of cephalexin was 2.66-fold and the CL_R was 0.36-fold in hyperuricemic rats, which were responsible for variations of drug transporters (Nishizawa et al., 2019). Clinically, it is suggested that, in the treatment of the hyperuricemia, it is necessary to pay special attentions to the dosage of the drugs that are primarily excreted by OAT1/3. Streptozotocin is the most commonly used agent to construct animal models of diabetes, due to its selective destruction of islet β cells in animals (Goyal et al., 2016). In the diabetes model, the decreased OAT1/3 and OCT2 expressions and increased BCRP and MRP2/4 expressions were observed in the kidney (Xu et al., 2015a; Zhang et al., 2017b; Wang et al., 2018b). The excretion of antidiabetic drugs could be affected by these changed transporters. For example, metformin is excreted through OCT2, and sitagliptin is transported by OAT3 (Chu et al., 2007; Koepsell, 2013). Therefore, the clinical uses of these drugs during different disease statuses should take their pharmacokinetic changes into consideration. In general, most renal drug transporters are not affected by a long-term administration of a high-fat diet, but decreased expressions of the uptake protein OAT1/3 was observed in the kidney (Lu et al., 2019), which can lead to an accumulation of endogenous toxic substances that may contribute to obesity-related diseases, such as hyperlipidemia, NAFLD, CKD, and diabetes.

Rheumatoid arthritis (RA) is an autoimmune disease mainly characterized by synovitis and with a prevalence of 0.24% in the world. This disease could decrease the protein level of OAT1, which may lead to an increased t_1/2 for some drugs (Wang et al., 2020a). In the high-altitude condition, the efflux drug transporters of MDR1 and MRP2 were increased, and clinicians should take it into consideration when drug prescription (Luo et al., 2017). It is noteworthy that different viral infections bring different changes of the renal transporter levels. The poly I:C infected pregnant rat showed lowered levels of OAT1/3, OATP4C1, BCRP, and MRP2 (Karimian Pour and Piquette-Miller, 2020), but the HIV-1 transgenic rat showed reduced OCT2 and MATE1 levels (Karimian Pour and Piquette-Miller, 2018). A hepatitis B virus infection could also damage kidneys, and it could downregulate some kidney transporters (Zuo et al., 2020). This difference should call attentions to pharmacokinetic performance shift of the used drug when different virus-infection in clinic. In an acute lymphoblastic leukemia mice model, the MRP 2/4 levels and the clearance of MRP-mediated drugs were significantly reduced, but the MDR1, OAT3, and OCT2 levels were increased, corresponding to the increased clearances of MDR1-mediated digoxin, OAT3-mediated furosemide, and OCT2-mediated metformin (Zhou et al., 2020a). A study reported that female obese Zucker spontaneously hypertensive fatty rats showed a strongly decreased expression of OAT2 in the kidneys, and OAT2-mediated furosemide renal excretion could be impaired in these rats (Babelova et al., 2015). There may be other diseases affecting the levels of renal transporters and needing further explorations.