The role of genetics and molecular aspects on HIV-associated kidney disease

Project title
Genetic and molecular characterization of HIV-associated kidney disease in black South Africans

Research question
We determined the prevalence of MYH9 and APOL1 high risk variants and examined their association with HIV- associated Nephropathy (HIVAN) compared to other forms of HIV-associated kidney diseases, HIV-positive controls and healthy population controls. We also profiled the N-terminal APOL1 mRNA expression in HIVAN compared to other kidney disease phenotypes and controls as well as determining and comparing the expression of the apolipoprotein L1 isoforms in human embryonic kidney (HEK) cells. Our results gave a positive direction towards the link between APOL1 high risk variants, apolipoprotein L1 isoforms and specific clinical entities such as HIVAN, HIVICK and possibly, other kidney diseases. Most importantly, our study highlighted the areas of focus in determining the mechanism of HIV-associated apolipoprotein L1-mediated kidney disease.

The burden of renal disease associated with HIV infection among black individuals is on the increase (Estrella et al., 2010; Post & Holt, 2009). According to the ESRD Patients in 2013: A Global Perspective report, an estimated 3.2 million people are on renal replacement therapy with an increased prevalence in individuals of African ancestry. This is mainly attributed to the fact that, other than environmental and socio-economic factors, these individuals also carry 1 or 2 risk alleles of the APOL1 gene which codes for an apolipoprotein L1. Mutants of this protein have been shown to have trypanolytic factors and therefore it protects against Trypanosomiasis but at the same time predisposes an HIV infected individual of African ancestry to CKD. Further studies are therefore needed in order to understand the genetic and molecular mechanism of apolipoprotein L1 and its role in inducing HIV-associated kidney disease.  The dramatic reduction in the incidence of opportunistic infections as a result of HAART has resulted in a shift of health concern toward chronic morbidities, including renal dysfunction, however, the burden of renal disease associated with HIV infection is increasing exponentially (D'Agati & Appel, 1997; de Silva et al., 2007; Wyatt & Klotman, 2007). The high prevalence of HIVAN and the impact on morbidity and mortality drives the need to come up with definitive indicators of disease progression and prognosis. There has been an increase in the number of patients progressing to ESRD, the majority of who are working-age adults, breadwinners and parents. Countries cannot afford to lose people in this age group especially in the developing world where it is not possible to offer renal replacement therapy to all those in need. Data from this study will enable us to determine the impact of genetic and molecular factors in the pathogenesis of HIVAN and other forms of HIV CKD and determine their potential role in terms of prediction or susceptibility to developing kidney disease in the presence of HIV infection. In addition, a better understanding of the genetic predisposition to kidney disease in a South African population will assist in alerting clinicians to kidney disease susceptibility in HIV-positive patients and the need to increase surveillance and possibly adjust treatment. At present, little is known about the biology of apolipoprotein L1 and its role in kidney disease although it is postulated that since it has the BH3 domain, it may use a similar mechanism described earlier in trypanolysis to cause kidney disease. This is the first study of its kind in Africa to functionally characterize apolipoprotein L1-related kidney disease.

Kidney biopsies from Charlotte Maxeke Johannesburg Academic Hospital were obtained from adults (aged ≥18 years) and selected according to histological diagnoses, clinical data were collected and patients were stratified according to age, gender and clinical presentation. Single nucleotide polymorphisms (SNPs) within and flanking MYH9 and APOL1 genes were selected by identifying all SNPs documented in 2011 from 1000 base pairs upstream and downstream of the two genes, and with a minor allele frequency of ≥0.05 in African populations based on the  International HapMap and 1000 Genomes Projects. SNPs with previous associations to kidney disease and literature SNPs were included, as was a panel of ancestry informative markers (AIMs). Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) kidney tissues and from whole blood. SNP genotyping was performed using the Illumina BeadXpress SNP genotyping assay and the TaqMan SNP genotyping assay. N-terminal apolipoprotein L1 mRNA expression was performed using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. Expression of N-terminal apolipoprotein L1 isoforms in HEK 293 cells was performed using PCR-based cloning and transfection techniques. Data analysis was performed using R program and Stata v11.1. Logic regression, Fisher’s exact test, Kruskal Wallis test and Student’s t test were employed in the data analysis.

A total of 228 samples [38 HIVAN, 41 HIV (+) CKD, 41 HIV (-) CKD, 54 HIV (+) Controls and 54 population controls] were successfully genotyped. Of 96 SNPs genotyped, 77 passed quality control. A total of nine MYH9 and APOL1 SNPs were significantly (P< 0.05) associated with HIVAN and not with other forms of CKD including focal segmental glomerulosclerosis (FSGS). However, the risk of MYH9 SNPs with P-values < 0.05 could not be determined. Single MYH9 E1 haplotype SNPs did not show any association, however, E1 haplotype block C-G-C showed a weaker significant factor with HIVAN (OR 3.45 P=0.008). The APOL1 risk alleles [G1 (0.560), G1 (0.500) and G2 (0.342)] had the highest prevalence among HIVAN individuals. G1GM haplotype was the most frequent in HIVAN (0.530).  19/38 (50.0%) of the HIVAN cases were compound heterozygous for G1 and G2 risk alleles; 11/38 were homozygous for either G1 or G2 risk alleles, 6/38 were heterozygous for a single risk allele and 2/38 were homozygous for non-risk allele. Thus, 30/38 (79.0%) of HIVAN but only 1/54 (2%) of population controls carried two risk alleles. Population allele frequencies were 7.3% for G1 and 11.1% for G2. In a recessive model, individuals carrying two APOL1 risk alleles had 89.0-fold higher odds 95% CI 17.68, 911.72; P=1.24x10-14 for developing HIVAN. APOL1 risk variants were not significantly associated with other forms of CKD [HIV (+) FSGS (OR=2.13 95% CI 0.03, 44.30; P=0.48) and HIVICK (OR=5.60 95% CI 0.4, 86; P=0.13) in the HIV (+) CKD group compared to HIV (+) controls] and [Primary FSGS (OR=6.30 95% CI 0.04, 248.70; P=0.26) in the HIV (-) CKD group compared to population controls]. Isoform B N-terminal apolipoprotein L1 mRNA levels were elevated in HIVAN and HIVICK while isoform C N-terminal apolipoprotein L1 levels were elevated in normal kidney. We showed that the three isoforms not only are they expressed in HEK 293 cells but also that they are expressed variably where isoforms B and C N-terminal apolipoprotein L1 that lack the secretory domain are seen to be more expressed within the HEK 293 cells than isoform A N-terminal apolipoprotein L1.

A combination of apolipoprotein L1 isoform B expression with C-terminal risk variants is a major contributor to the development of HIVAN and possibly other kidney diseases. There is a striking increase in the prevalence of APOL1 risk variants among HIVAN compared to other forms of kidney disease and we estimate that HIV-infected South African blacks with two APOL1 risk alleles not receiving anti-retroviral therapy (ART) have the greatest risk of developing HIVAN. Further studies are required to elucidate the mechanism of apolipoprotein L1-mediated kidney disease in order to develop effective therapeutic measures.

Research output
1. Alex N. Kasembeli*, Raquel Duarte, Michèle Ramsay, Pulane Mosiane, Caroline Dickens, Thérèse Dix-Peek, Sophie Limou, Efe Sezgin, George W. Nelson, Agnes B. Fogo, Stewart Goetsch, Jeffery B. Kopp, Cheryl A. Winkler, and Saraladevi Naicker (2015). APOL1 risk variants strongly associated with HIV-associated Nephropathy in Black South Africans. JASN 26 (3): 2611-2619
2. Alex N. Kasembeli*, Raquel Duarte, Michèle Ramsay and Saraladevi Naicker (2015). African origins and Chronic Kidney Disease susceptibility in the HIV era. WJN 4(2): 295-306

Prof. Michele Ramsay; Prof. Saraladevi Naicker; Dr. Raquel Duarte