GSTM1 Copy Number and Kidney Disease in People With HIV

Rachel K.Y. Hung, Kerry-Lee Rosenberg, Victor David, Elizabeth Binns-Roemer, John W. Booth, Rachel Hilton, Julie Fox, Fiona Burns, Andrew Ustianowski, Catherine Cosgrove, Lisa Hamzah, James E. Burns, Amanda Clarke, David Chadwick, David A. Price, Stephen Kegg, Lucy Campbell, Kate Bramham, Caroline A. Sabin, Frank A. Post, Cheryl A. Winkler, and on behalf of the GEN-AFRICA Study Group King’s College London, London, UK; Royal Free London Hospital NHS Foundation Trust, London, UK; Basic Research Laboratory, Frederick National Laboratory for Cancer Research and the National Cancer Institute, Frederick, USA; Barts Health NHS Trust, London, UK; Guy’s and St Thomas’ NHS Foundation Trust, London, UK; Pennine Acute Hospitals NHS Foundation Trust, Manchester, UK; St George’s Hospital NHS Foundation Trust, London, UK; University College London, London, UK; Central and North West London NHS Foundation Trust, London, UK; Brighton and Sussex University Hospital NHS Trust, Brighton, UK; Brighton and Sussex Medical School Department of Infectious Disease, Brighton, UK; South Tees Hospitals NHS Foundation Trust, Middlesbrough, UK; The Newcastle Upon Tyne Hospitals, Newcastle, UK; Lewisham and Greenwich NHS Trust, London, UK; and King’s College Hospital NHS Foundation Trust, London, UK

O xidative stress has been implicated in the pathogenesis and progression of chronic kidney disease (CKD). An imbalance between increased production of reactive oxygen species and reduced antioxidant defenses results in disruption to downstream cellular signaling and subsequent renal cell apoptosis and senescence, fibrosis, and vascular injury. 1 Genetic variants that improve the capacity to mitigate oxidative stress may therefore be protective against the development of CKD.
The glutathione-S-transferases play a role in the conjugation of prooxidant species with glutathione to facilitate the elimination of reactive oxygen species. GSTM1 is the gene encoding one such isoenzyme. This gene copy number has undergone gene deletion and expansion so chromosomes have no copies, 1 copy or, in rare cases, 2 copies of the gene. Two copies of the active allele are required for enzymatic activity (haploinsufficiency); those homozygous for the null allele, GSTM1(0), completely lack enzyme production. Individuals with the inactive GSTM1 genotypes (GSTM1 0/0 or 1/0) have been found to be at higher risk of common malignancies, atherosclerosis, coronary heart disease, and CKD progression. S1,S2 This study sought to investigate the relationship between GSTM1 genotype and prevalent CKD and the interaction between GSTM1 and APOL1 carrier status, 2-4 in a cohort of Black people with HIV in the United Kingdom. 5,6 Characteristics of the 2762 participants are summarized in Supplementary Table S1. Of these, 2075 (75.1%) had GSTM1 inactive genotypes whereas 687 (24.9%) carried 2 or 3 copies (active genotypes). The mean age of the participants was 48 years, and 57% were female. Most participants were established on antiretroviral treatment with suppressed HIV RNA levels; HIV parameters, hepatitis coinfection status, and prevalence of hypertension, diabetes, and cardiovascular disease did not differ by GSTM1 status. Kidney function (estimated glomerular filtration rate [eGFR]) and the prevalence of APOL1 risk variants and sickle cell trait were similar for the 2 GSTM1 groups (Figure 1a-c and Supplementary Figure S1).
In the overall study population, GSTM1 inactive genotypes were not associated with an increased risk of kidney disease (eGFR <60 or <90 ml/min per 1.73 m 2 or stage 5 CKD), whereas these genotypes were associated with reduced odds of albuminuria (Table 1). There was no significant interaction between GSTM1 genotype and APOL1 status for most kidney outcomes. When participants were stratified by APOL1 status (Supplementary Table S2), GSTM1 inactive genotypes in those with APOL1 low-risk genotypes were associated with reduced odds of eGFR <60 ml/min per 1.73 m 2 (odds ratio 0.65 [95% CI 0.49-0.87]) and albuminuria (odds ratio 0.77 [0.61-0.99]). In those with APOL1 high-risk genotypes, GSTM1 inactive genotypes were not associated with eGFR <60 and <90 ml/min per 1.73 m 2 or stage 5 CKD.
In contrast to some existing evidence in Black populations with impaired kidney function, 3,4 and consistent with recent data in people with HIV from the Eastern Congo, 7 we found no evidence for an increased risk of kidney disease in individuals with GSTM1 inactive genotypes. In addition, we found no evidence that GSTM1 inactive genotypes amplify the deleterious effect of the APOL1 high-risk genotypes.
Data from the African American Study of Kidney Disease and Hypertension revealed an association between GSTM1 inactive genotypes and accelerated progression of CKD in a cohort of 692 Black Americans with hypertensive kidney disease, with worse progression in APOL1 high-risk genotypes. 3 Our cohort is substantially larger than those included in the African American Study of Kidney analyses and differs in that only 32% (as compared with all participants in African American Study of Kidney) had a diagnosis of hypertension, and that most of our participants had normal kidney function. It is possible that GSTM1 loss is implicated in the pathogenesis of hypertensive renal disease but is less significant in other or HIV-associated pathologies. Alternatively, as oxidative stress is increased in CKD, 8 GSTM1 loss may have had a larger impact on kidney disease progression in the African American Study of Kidney study. It is possible that the potential protective effect of GSTM1 becomes important in declining eGFR and that this association was not captured in our cross-sectional study.  Figure 1. Distribution of eGFR in participants stratified by GSTM1 genotype, overall (a) and in those with APOL1 low-risk (b) and high-risk (c) genotypes. Evidence from the Atherosclerosis Risk in Communities Study revealed a 66% increased risk of kidney failure in both Black and White individuals with GSTM1 inactive genotypes, compared with those with active genotypes. 4 This study included 2254 Black participants with largely normal kidney function (mean eGFR 112 ml/min per 1.73 m 2 ). The increased risk persisted after adjustment for clinical risk factors, including diabetes and hypertension. No significant association was identified, however, between GSTM1 allele status and incident CKD. There is evidence to suggest that the protective, antioxidant effects of GSTM1 are of greater importance in a uremic environment (i.e., at lower GFR), and this may account for the disparity between risk of incident CKD and kidney failure in this cohort. 8 However, a large study by Zhang et al. 9 also failed to reveal an association between GSTM1 loss and kidney failure in either Black (n ¼ 796) or White participants (n ¼ 46,187).
Our study comprises the largest cohort of Black participants in which the association between GSTM1 status and CKD has been explored; the GSTM1 groups were indistinguishable in terms of HIV parameters and relevant comorbidities, such as hypertension and diabetes, and APOL1 renal risk status. This is also the largest study in which the association between GSTM1 status and kidney outcomes stratified by APOL1 genotype has been evaluated. Limitations include its cross-sectional study design, the positive HIV status of all participants which may preclude extrapolation to non-HIV populations, and the modest numbers of participants with the GSTM1 active genotypes and high-risk APOL1 genotypes, which may have rendered the study underpowered to detect an interaction between deleterious kidney outcomes and APOL1 carrier status. In summary, this cross-sectional study does not support some earlier observations that GSTM1 inactive genotype is a risk factor for kidney disease in Black individuals. Furthermore, GSTM1 inactive genotypes in this population do not seem to amplify the deleterious effects of the high-risk APOL1 genotype. Further studies in people with HIV are required to investigate the role of GSTM1 inactive genotypes in CKD progression among those with advanced kidney disease and proteinuria.

ACKNOWLEDGMENTS
The authors thank the study participants and all members of the GEN-AFRICA study group (Appendix). This study was supported by the Medical Research Council (United Kingdom) Confidence in Concept scheme (MC_PC_17164) and in part by the National Institutes of Health and the National Cancer Institute Intramural Research Program (CAW) and under contract HHSN26120080001E. The content of this publication does not necessarily reflect the view or policy of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the government.

AUTHOR CONTRIBUTIONS
The study was designed by CAW and FAP. JWB, RH, JF, FB, AU, CC, LH, JEB, AC, DC, DAP, SK, LC, and FAP were site (principal) investigators and coordinated recruitment and data collection at their sites. EBR performed the genotyping, and RKYH performed the analyses. RKYH, KLR, KB, CAW, and FAP interpreted the findings. KLR wrote the first draft of the manuscript with input from RKYH, KB, CAW, and FAP. All authors contributed to and approved the final version of the manuscript.

SUPPLEMENTARY MATERIAL
Supplementary File (PDF) Supplementary Methods. Supplementary References. Figure S1. Distribution of eGFR in participants stratified by GSTM1 copy number, overall (a) and in those with APOL1 low-risk (b) and high-risk (c) genotypes. Table S1. Characteristics of study participants stratified by GSTM1 status. Table S2. Characteristics of study participants stratified by APOL1 and GSTM1 status.