TFV was first approved by the FDA in 2001 and is currently a widely used antiretroviral agent [28
]. However, KTD has been reported with long-term TFV use [4
], with reported cases growing in number from 2 in 2002 [29
] to >15 in the last 2 years, including several large scale studies [4
]. In a retrospective cross-sectional analysis of 1647 patients (964 on TFV-containing regimens, 683 on TFV-sparing regimens), TFV exposure was associated with a greater risk of developing proximal tubular dysfunction [4
]. Importantly, adverse renal effects of TFV were also shown to persist.
Inhibition of renal efflux transporters may result in increased intracellular TFV, which in turn may cause renal toxicity. However, apart from a well-established role for ABCC4 [12
] and a tentative role for ABCC2 [14
], no other transporters have been identified for TFV renal efflux. The recent discovery and characterization of ABCC10 [18
], its functional similarity with ABCC4 [31
], and its high expression in kidney [21
] provided a rationale to explore its specificity for TFV and possible role in KTD.
-expressing embryonic kidney cell lines, C17 and C18, were used to establish that TFV is a substrate for ABCC10; the same in vitro models have elsewhere been used to characterize the transport of other substrates [18
]. Furthermore, cepharanthine resulted in an increased intracellular accumulation of TFV, illustrating the importance of ABCC10 for TFV in renally derived cells. To assess the importance of ABCC10 at more physiologically relevant expression levels, siRNA experiments were conducted in CD4+
cells and MDMs. CD4+
cells and MDMs represent primary cell types that are readily available in large numbers and express transporters at physiologically relevant densities. They also allow assessment of the role of transporters in an HIV replication–competent cell system. Intracellular accumulation of radiolabeled TFV was significantly increased in siRNA-treated immune cells compared with untreated controls. Hence, the experimental evidence obtained in this study confirms a role for ABCC10 in the efflux transport of TFV.
A case-control association study was then undertaken to investigate common variants in ABCC10
in HIV-positive patients on TFV-containing regimens. This is the first genetic association analysis reported for ABCC10
. Two SNPs and their haplotype were significantly associated with KTD: rs9349256 located in intron 4 and rs2125739, a nonsynonymous SNP, in exon 12, resulting in an amino acid change (p.Ile920Thr). A recent study showed carriers of the ABCC4
3463G variant (associated elsewhere with KTD [15
]) to have 35% higher intracellular TFV [32
]. The functional effects of polymorphisms identified in ABCC10
in this study are not known; a bioinformatic approach using FastSNP software (http://fastsnp.ibms.sinica.edu.tw/
) found rs2125739 to be located in a putative splice site. Splice site polymorphisms have been shown to affect pre-mRNA splicing and may cause the splicing apparatus to use nearby cryptic splice sites or skip exons, leading to an altered protein [33
]. Based on HapMap data (www.hapmap.org
), the minor allele frequency for rs2125739 in various populations are as follows: Northern and Western European ancestry (26.7%); Sub-Saharan African (34.2%); Han Chinese (3.4%), and Japanese (8.0%). No functional effect was predicted for rs9349256, suggesting that the association may be due to linkage disequilibrium with another functional marker. A marginally significant increase in the frequency of the haplotype defined by the 2 associated alleles was also observed in KTD compared with controls, further strengthening a role for ABCC10
in TFV-associated KTD.
TFV-associated KTD is multifactorial [10
] with risk factors including polymorphisms along with nongenetic factors, such as age [3
] and body weight [3
]. The previous study using the same cohort found an association between KTD and ABCC2
rs717620 and a haplotype containing it [16
]. Therefore, the joint contribution of ABCC10
haplotypes was investigated. The combination haplotype, GGC-CGTC, was significantly more prevalent in patients with KTD than in controls. The ABCC10
combined haplotype was more strongly associated with KTD (OR, 3.0) than the individual haplotypes (OR, 2.1 and 1.96 for ABCC10
, respectively). Interestingly, in a multivariate analysis, the carriage of ABCC10
combined haplotype showed a trend toward independent significant association (P
= .06) with KTD.
Median TFV plasma levels were significantly higher in a subset of patients with KTD than in controls, but they did not show any association with ABCC10 SNPs or ABCC10-ABCC2 combined haplotype. Smaller sample size could be one reason for the lack of association observed here; the relationship therefore needs to be explored in larger cohort studies. However, it is important to note that it is unclear whether the increase in TFV plasma levels observed in KTD is the cause or the consequence of the alterations in the tubular cells. Moreover, the intracellular kidney concentrations for TFV could be more important than the plasma TFV levels in the pathogenesis of KTD; it remains to be explored whether ABCC10 polymorphisms are associated with intracellular TFV levels.
rs9349256 was associated with urine phosphorus wasting and β2-microglobulinuria. The ABCC10
combination haplotype was also more prevalent in patients with abnormal urine phosphorus excretion. This is interesting, because reports have suggested that fractional excretion of urine phosphate [5
] and urinary β2-microglobulin [34
] are more sensitive markers of tubular dysfunction than conventional markers. Conventional markers, such as serum creatinine and urine albumin, are primarily indicators of glomerular disease [8
]. Urinary β2-microglobulin is freely filtered at the glomerulus and is avidly taken up and catabolized by the proximal renal tubules. Therefore, high levels of this protein indicate various pathologic conditions involving the proximal renal tubule. Urine phosphorus wasting is among the 3 criteria defining Fanconi syndrome. TFV is the drug most associated with this syndrome [35
], which leads to bone demineralization and osteomalacia [36
These results confirm that ABCC10 is involved in the efflux transport of TFV. Renal elimination is the dominant mode of TFV clearance, and these findings add to our understanding of the molecular pharmacology of TFV. ABCC10 mRNA has been found to be highly expressed in various tissues including kidney, brain and colon, suggesting an important role for this protein in the transport of drugs and other endogenous molecules. However, it is unclear whether ABCC10 is expressed on the luminal or basolateral side of the tubular cell. We have also identified a role for ABCC10 genetic variants in the pathogenesis of KTD using a limited sample size; replication of these results in an independent disease cohort is now required to validate these findings. Nevertheless, the findings of this study improves our understanding of genetic susceptibility factors involved in KTD pathogenesis and therefore may help in the development of avoidance strategies (stratification of medicines and better drug design) and intervention strategies (cotherapies to prevent manifestations of KTD).