Aliases: MOAT-B, MOATB
Gene name: ATP binding cassette subfamily C member 4 (ABCC4)
MRP4 is an ATP-dependent, unidirectional efflux transporter belonging to the C subfamily of the ABC protein superfamily. It is expressed in the kidney, blood-brain barrier (BBB), liver, and other tissues, and its localization to the basolateral or apical membranes is tissue-dependent. It plays an important role in alleviating the impact of cholestasis on hepatocytes by effluxing bile acids into the blood, and can be up-regulated in the liver in such instances. It may also be important in limiting CNS and hematopoietic cell exposure to xenobiotics, and as a transporter of cell signaling molecules. MRP4 has wide substrate specificity, including nucleoside analogs and antiviral drugs; however, reports of clinical DDIs due to MRP4 modulation are rare. MRP4 is highly polymorphic, and some of these polymorphisms may be clinically relevant. Current FDA and EMA guidances make no specific recommendations for MRP4. However, this transporter may be important where active renal secretion is suspected, or where changes in circulating levels of bile salts are observed.
MRP4 (recently reviewed in  is found in a variety of human tissues, with high levels of mRNA expression in the kidney and prostate, and lower levels in the liver, testis, ovary, lung, parotid gland, adrenal gland, and in various neurons and blood cells. MRP4 localizes to different membranes in different polarized cells. In the liver, choroid plexus, prostatic acinar cells, and the GIT, MRP4 is expressed at the basolateral membrane, while in the brain capillary endothelium and renal proximal tubule cells it is expressed at the apical membrane [2-6]. This means that, for instance, in the liver (where its abundance is relatively low) it effluxes its substrates into the bloodstream, whilst in the kidney (where it is one of the dominating ABC transporters ) it effluxes into the urine.
Function, physiology, and clinically significant polymorphisms
MRP4 with its 170-kDa molecular weight is the smallest among the MRP proteins. It comprises 12 putative membrane spanning helices and lacks the additional N-terminal helices found in MRP1, MRP2, MRP3, and MRP6 . MRP4 mediates the transport of endogenous substrates including signaling molecules (cAMP/cGMP, eicosanoids), bile acids, urate, and conjugated steroid hormones (e.g. pregnenolone sulfate and dehydroepiandrosterone sulfate ). Clinical drug substrates include antivirals (adefovir, tenofovir), antibiotics (cephalosporins, benzylpenicillin), paracetamol conjugates, diuretics (furosemide, hydrochlorothiazide), the antihypertensive olmesartan, and cytotoxic agents (methotrexate, 6-thioguanine, 6-mercaptopurine, topotecan) [3, 10-12]. Inhibitors of MRP4 include non-steroidal inflammatory drugs, phosphodiesterase inhibitors, cardiovascular drugs, and flavonoids, among others [10, 13]. MRP4 plays a role in the renal excretion of organic anions and drugs , in the modulation of signaling pathways , and in the restriction of drug penetration through the blood-brain, blood-cerebrospinal fluid, and blood-testis barriers [16-18]. Basolaterally expressed MRP4 in gastric and intestinal epithelia is thought to be involved in the oral absorption of cephalosporin antibiotics, dasatinib, and potentially other orally administered drugs [5, 19].
Glutathione as a conjugation partner or co-transported substrate plays multiple and partially undefined roles in the transport mechanism of MRPs, especially of MRP2. MRP4 has long been known to require reduced glutathione for the transport of bile acids , but the MRP4-mediated extrusion of other substrates such as halobenzoquinones may also depend on GSH, possibly via conjugate formation .
Given its ability to transport important intra- and intercellular mediators such as cyclic nucleotides and eicosanoids, the physiological repertoire of MRP4 is thought to cover platelet aggregation, cell migration and proliferation, angiogenesis, and cardiomyocyte contraction. For the same reason, it is also implicated in the progression of multiple cancer types  including acute myeloid leukemia , ovarian cancer , and clear cell renal carcinoma , and the pharmacological modulation of MRP4-cAMP binding is being actively explored .
Along with many other xenobiotic transporters and metabolizing enzymes, the expression of MRP4 is regulated by the xenobiotic-responsive transcription factors AhR, CAR, PXR, and PPARα [18, 26]. In acute myeloid leukemia cells, MRP4 expression is also upregulated by histamine .
The rare PEL-negative blood group has recently been associated with a large biallelic deletion in the ABCC4 gene, resulting in an MRP4-null phenotype . Cyclic nucleotide levels are normal in the red blood cells of PEL-negative individuals, suggesting compensation by other erythrocytic ABC transporters; however, platelet aggregation is impaired in the absence of functional MRP4. Albeit there is no evidence of diseases linked to single nucleotide polymorphisms in the ABCC4 gene, and an in vivo study showed no obvious abnormalities in Mrp4-null mice [29, 30], polymorphisms can alter the expression level and the transport rate of the protein, and may influence therapeutic outcomes and adverse events. Up to 2019, 12 variants with substantial clinical consequences had been identified; these SNPs affect both untranslated and coding regions of ABCC4 and result in altered transport of tenofovir, zidovudine, latanoprost, (Val)ganciclovir, methylated arsenic metabolites, bisphosphonates, cisplatin, furosemide, thioguanine drugs, methotrexate, imatinib, and cyclophosphamide .
There are no specific citations of clinically relevant DDIs ascribed to this transporter, and there is limited information on its role in the clinical ADME of drugs. However, there is evidence of its role in the renal elimination of tenofovir, as the renal clearance of tenofovir was 15% lower and its AUC was 32% higher in ABCC4 3463G carriers compared with wild types in one study . Since MRP4 also transports drugs which are used in HIV therapy, the efficacy of these drugs may be partially dependent on the expression of this transporter in T-cells [2, 8, 30, 32].
MRP4 transports bile acids in the presence of glutathione (GSH), and functions as a backup system for eliminating bile acids from hepatocytes. Although MRP4 expression in the liver is low, it can be induced by bile acids in cholestatic conditions [2, 14]; thus it plays an important compensatory role in protecting the liver from over-exposure to bile acids. The induction of MRP4 is FXR-independent and may instead be due to post-transcriptional regulation , as mRNA levels have been shown to remain unchanged .
MRP4 overexpression confers cellular resistance to nucleotide-base, nucleoside, and nucleotide analogues, as well as to certain tyrosine kinase inhibitors  and cytotoxic agents, therefore adversely affecting anticancer therapies. Overexpression of MRP4 confers doxorubicin resistance to osteosarcoma cells , and high MRP4 activity may be a determinant of resistance to arsenic-based chemotherapy regimens applied in leukaemia patients . High MRP4 expression was also associated with diminished response to methotrexate in childhood acute lymphoblastic leukemia . Therefore, selective MRP4 inhibitors are being developed to counter MRP4-mediated drug resistance, and some of them have markedly enhanced the sensitivity of MRP4-overexpressing HEK293 cells towards 6-mercaptopurine .
Since MRP4 transports arsenic metabolites such as dimethylarsenic acid, and polymorphic variants differ in their ability to do so, MRP4 SNPs potentially contribute to the risk of arsenic-induced toxicity and tumorigenesis . SNPs in ABCC4 also affect dose tolerance of 6-mercaptopurine in pediatric acute lymphoblastic leukemia .
MRP4 protects hematopoietic cells, both healthy and leukemic, against cytarabine, and counters the myelosuppressive effect of prolonged beta-lactam use . Conversely, the 3348A>G SNP in homozygous form predisposes to beta-lactam-induced neutropenia, and the 559G>T(G187W) nonsynonymous variant – which is fairly common in the Japanese population with a frequency of 12.5% – sensitizes cells to the active metabolite of irinotecan, SN-38 .
Currently, neither the FDA nor the EMA guidance specifically recommends the study of MRP4 interactions for NCEs. This is probably due to the lack of clear clinical citations of MRP4-mediated DDIs. However, this transporter may be relevant to NCEs where active renal secretion is suspected, or where changes in circulating levels of bile salts are observed.
|Location||Endogenous substrates||In vitro substrates used experimentally||Substrate drugs||Inhibitors|
|kidney, prostate, placenta, liver, BBB||taurocholic acid, cAMP, cGMP, urate, PregS, DHEAS, E2-17ßG, p-aminohippurate, PGE1 and PGE2||DHEAS, E2-17ßG||
antivirals: acyclovir, ritonavir, adefovir, tenofovir;
|NSAIDs: dipyridamole, sulindac, tolmetin, indomethacin, piroxicam, naproxen, celecoxib, flurbiprofen, ibuprofen, ketoprofen, diclofenac;
phosphodiesterase inhibitors: sildenafil, zaprinast, trequinsin;
cardiovascular drugs: verapamil, losartan, telmisartan, candesartan;
flavonoids: quercetin, silymarin;
probenecid, dilazep, dantrolene, ceefourin
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