Anna Vinnikova, MD APOL1

I have no relevant financial relationships with commercial interests

But I have a passionate interest in the following problem:

National decline in Nephrology Fellowship applications

Jhaveri et al., Why Not Nephrology? AJKD 2013

wp.vcu.edu/avinniko

Why are African Americans susceptible to kidney disease?

Data from USRDS and US Census www.presciousbodyfluids.com

>30% AA with incident ESRD have a relative w ESRD Clustering of disparate forms of CKD within single AA families: DM, FSGS, HIVAN, H-GS, SLE, SSD Freedman and Murea, Curr Hypertens Rep 2012

Hypertensive nephrosclerosis in African Americans: Global Glomerulosclerosis Solidified GS Obsolescent GS Marcantoni et al., KI 2002

Hypertensive nephrosclerosis in African Americans: Global Glomerulosclerosis Segmental and global GS together Marcantoni et al., KI 2002

Hypertensive nephrosclerosis in African Americans: Global Glomerulosclerosis Disappearing Glomeruli Marcantoni et al., KI 2002

Does treatment of hypertension change the course of hypertensive nephrosclerosis?

AASK trial, JAMA 2002

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ESRD or 50% GFR loss AASK cohort, NEJM, 2010

ESRD or 50% GFR loss no proteinuria \_( )_/ AASK cohort, NEJM, 2010

ʘ ʘ ESRD or 50% GFR loss proteinuria no proteinuria \_( )_/ AASK cohort, NEJM, 2010

Large population of African Americans develop CKD attributed to hypertension

However, control of hypertension does not change the outcomes

What is the real cause of this disease?

Let s turn to population genetics

Linkage Equilibrium

Linkage Disequilibrium

Population Admixture Reshiram Zekrom

Population Admixture

Disease cases Kyurem

Are any gene alleles at linkage disequilibrium? Kyurem

Mapping by Admixture Linkage Disequilibrium (MALD) Reshiram markers { Kyurem

Friedman and Pollak, JCI, 2011

1000 Genomes Project

Single Nucleotide Polymorphisms (SNPs)

Genome-wide association studies (GWAS)

Let s combine MALD+GWAS studies

to look for genetic variants in patients with kidney disease which are at linkage disequilibrium for African ancestry

Genome-wide admixture scan in 1,372 ESRD cases and 806 controls found an association between excess African ancestry and non-diabetic ESRD (lod score = 5.70) but not diabetic ESRD (lod = 0.47) on chromosome 22q12. Kao et al, Nat Genet, 2008

* Genome-wide admixture scan in 1,372 ESRD cases and 806 controls found an association between excess African ancestry and non-diabetic ESRD (lod score = 5.70) but not diabetic ESRD (lod = 0.47) on chromosome 22q12. Kao et al, Nat Genet, 2008

* Genome-wide admixture scan in 1,372 ESRD cases and 806 controls and found an association between excess African ancestry and nondiabetic ESRD (lod score = 5.70) but not diabetic ESRD (lod = 0.47) on chromosome 22q12. Kao et al, Nat Genet, 2008

Kao et al, Nat Genet, 2008

Kopp et al, Nat Genet, 2008 *

Genovese et al., Science, 2010

Friedman and Pollak, JCI, 2011

Etty Kruzel-Davila et al. Nephrol. Dial. Transplant. 2015 Structure of APOL1 APOL1 G0 tails19950.deviantart.com

Etty Kruzel-Davila et al. Nephrol. Dial. Transplant. 2015;ndt.gfu391 Structure of APOL1 APOL1 G1 APOL1 G0 tails19950.deviantart.com

Etty Kruzel-Davila et al. Nephrol. Dial. Transplant. 2015;ndt.gfu391 Structure of APOL1 APOL1 G1 APOL1 G2 tails19950.deviantart.com

Trypanolytic? 2004 Dennis Kunkel Microscopy, Inc

African Trypanosomiasis T. brucei brucei T. brucei rhodeseinse T. brucei gambiense

Warren Photographic

Corbis

Frevert U, Movila A, Nikolskaia O, Raper J, Mackey Z, Abdulla M, McKerrow J, Grab D

APO L1 as Trypanolytic Factor

HDL3 APOA1 APOL1

Friedman and Pollak, JCI, 2011

Pays et al. Nature Reviews Microbiology 4, 477 486 (June 2006) doi:10.1038/nrmicro1428

Friedman and Pollak, JCI, 2011

Schematic structure of APOL1

APOL1 WT SRA

Friedman and Pollak, JCI, 2011

APOL1 G1 APOL1 G2 SRA

T b. brucei infects many mammals, but is harmless to humans due to TLF containing APOL1

T b. rhodesiense and T b. gambiense evolved a defense against TLF and are able to infect humans

A single copy of APOL1 G1 or G2 restores TLF and makes carrier immune to sleeping sickness ~50% of Africans carry one of these risk variants

However, APOL1 G1 or G2 homozygotes and G1G2 heterozygotes are at 10-fold increased risk of kidney disease 12% of African Americans carry these risk genotypes

Balanced polymorphism!

APOL1 nephropathy spectrum

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APOL1 Genetic Variants in FSGS and HIVAN Kopp et al., JASN 2011 APOL1 genotypes and haplotypes obtained for 1378 AA and EA w FSGS or HIVAN

APOL1 Genetic Variants in FSGS and HIVAN Kopp et al., JASN 2011 Carrying APOL1 risk alleles confers: OR 17 for FSGS OR 29 for HIVAN Earlier onset Faster progression Similar sensitivity to steroids

APOL1 Genetic Variants in FSGS and HIVAN Kopp et al., JASN 2011 The effect of carrying two APOL1 risk alleles explains 18% of FSGS and 35% of HIVAN; Eliminating this effect would reduce FSGS and HIVAN by 67%

Background: We hypothesize that the apolipoprotein L1 gene (APOL1) nephropathy risk variants G1/G2, common in AA and rare in EA contribute to ethnic disparities in risk. Methods: APOL1 G1 and G2 nephropathy variants were genotyped in 855 AA with LN-ESKD (cases) and 534 AA with SLE lacking nephropathy (controls) and tested for association under a recessive genetic model via logistic regression. Results: Mean±SD duration of SLE to LN-ESKD was 7.3±0.3 years in cases. The G1/G2 risk variants were strongly associated with SLE-ESKD, with 25% of cases and 12% of controls possessing two nephropathy risk variants (odds ratio [OR]=2.57, recessive model p=1.49x10-9 ), and after age, gender and ancestry adjustment (OR=2.72, p=6.23x10-6 ). ). The age, gender, and admixture adjusted population attributable risk for the G1/G2 polymorphisms is 0.26, compared to 0.003 in EA. Time from SLE diagnosis to ESKD was ~2 years earlier for individuals with APOL1 risk genotypes (p=0.01). Conclusions: APOL1 G1/G2 variants strongly impact the risk of LN-ESKD in AA and progression to ESKD. The high frequency of these variants in AA with near absence in EA explains an important proportion of the increased risk of LN-ESKD in AA.

Background: We hypothesize that the apolipoprotein L1 gene (APOL1) nephropathy risk variants G1/G2, common in AA and rare in EA contribute to ethnic disparities in risk. Methods: APOL1 G1 and G2 Methods: APOL1 G1 and G2 nephropathy variants were genotyped in 855 AA with LN-ESKD (cases) and 534 AA with SLE lacking nephropathy (controls) and tested for association under a recessive genetic model via logistic regression. nephropathy variants were genotyped in 855 AA with LN-ESKD (cases) and 534 Results: Mean±SD duration of SLE to LN-ESKD was 7.3±0.3 years in cases. The G1/G2 risk variants were strongly associated with SLE-ESKD, with 25% of cases and AA 12% with of controls SLE possessing lacking two nephropathy risk variants (odds ratio [OR]=2.57, recessive model p=1.49x10-9 ), and after age, gender and ancestry adjustment (controls) (OR=2.72, and p=6.23x10 tested -6 ). ). The for age, gender, association and admixture adjusted population attributable risk for the G1/G2 polymorphisms is 0.26, compared to 0.003 in EA. Time from SLE diagnosis to ESKD was ~2 years earlier for individuals with APOL1 risk genotypes (p=0.01). Conclusions: APOL1 G1/G2 variants strongly impact the risk of LN-ESKD in AA and progression to ESKD. The high frequency of these variants in AA with near absence in EA explains an important proportion of the increased risk of LN-ESKD in AA.

Results Table 3. Test for association between APOL1 compound risk* and systemic lupus erythematosus with ESKD Model N cases** N controls** OR (95% CI) P-value Unadjusted 855 534 2.57 (1.89-3.50) 1.49x10-9 Adjusted for admixture 699 407 2.80 (1.95-4.02) 2.21x10-8 Adjusted for age, gender, admixture 494 283 2.72 (1.76-4.19) 6.23x10-6 * Compound risk is coded as a binary variable where the risk genotypes are homozygous for the G1 allele, homozygous for the G2 alleles or compound heterozygous for G1/G2 (i.e., two copies of any risk allele). ** Frequency of G1/G2 compound heterozygotes is 0.25 in systemic lupus erythematosus ESKD cases and 0.12 in systemic lupus erythematosus non-nephritis controls.

Results Table 4. Tests of whether the APOL1 G1/G2 risk allele is associated with age at ESKD, age at SLE onset, and duration of SLE until ESKD No admixture adjustment Admixture adjusted 0/1 risk alleles 2 risk alleles P-value 0/1 risk alleles 2 risk alleles P-value Age at ESKD (Years) 33.9±12.1 (33) 33.5±10.1 (33) 0.9152 33.8±12.0 (32.5) 33.4±10.0 (33) 0.8603 Age at SLE (Years) Case Control Duration SLE to ESKD (Years) 30.9±12.9 (29) 27.0±11.1 (25) 39.4±12.4 (41) 30.0±11.2 (29) 28.1±10.2 (27) 40.0±11.0 (39) 0.1278 0.1372 0.6925 31.3±13.0 (30) 27.0±11.1 (25) 39.4±12.4 (41) 30.4±11.4 (29) 28.3±10.3 (27) 40.0±11.0 (39) 0.0840 0.1554 0.3268 7.89±7.30 (6) 5.81±6.64 (4) 0.0003 7.87±7.33 (6) 5.81±6.06 (4) 0.0111 Data expressed as N (%) or mean ± SD (median)

These studies suggest a second-hit model : APOL1 risk variants act as progressor genes for many renal diseases

Is there a rationale for APOL1 genotyping in renal transplantation?

Effect of Recipient APOL1 status Lee at al., Am J Transplant, 2012

Effect of Donor APOL1 status Reeves-Daniel et al., Am J Transpl, 2011

What do we know about APOL1 in the kidney?

APOL1 carried by HDL particles Which ApoL1?

Which ApoL1? APOL1 carried by HDL particles Free circulating APOL1?

Which ApoL1? APOL1 carried by HDL particles Free circulating APOL1? Intracellular APOL1

Localization of APO L1 in normal kidney Madhavan et al, JASN 2011

Localization of APO L1 in diseased kidney Predisposition to podocytopathy and vasculopathy? Madhavan et al, JASN 2011

APO L1 in normal kidney of a European American 2015 by American Society of Nephrology Lijun Ma et al. JASN 2015;26:339-348

APO L1 in normal kidney of a European American APOL1 Podocyte marker Nuclei 2015 by American Society of Nephrology Lijun Ma et al. JASN 2015;26:339-348

Localization of APO L1 in normal kidney of an African American with 2 APOL1 Vs APOL1 Endothelial marker Nuclei Lijun Ma et al. JASN 2015;26:339-348

APO L1 mrna in vessels in normal kidney Lijun Ma et al. JASN 2015;26:339-348 2015 by American Society of Nephrology

Uptake of exogenous recombinant APOL1 into human podocytes APOL1 Podocyte marker Nuclei Exogenous APOL1 uptake Lijun Ma et al. JASN 2015;26:339-348 2015 by American Society of Nephrology

Therefore, APOL1 is expressed in podocytes, tubular cells, endothelial cells and vascular wall, but is enriched in podocytes perhaps due to uptake of filtered free APOL1

How might APOL1 variants be nephrotoxic?

Known APOL1 roles: HDL structure and function

Known APOL1 roles: HDL structure and function Innate immunity (HDL-bound)

Known APOL1 roles: HDL structure and function Innate immunity (HDL-bound) Autophagy (intracellular)

Autophagy A lysosome dependent, self eating, catabolic mechanism Hartleben et al., Seminars in Nephrology, 2013

Autophagy GFP-LC3 mouse demonstrates high level of basal autophagy in podocytes Hartleben et al., Seminars in Nephrology, 2013

Autophagy Hartleben et al., Seminars in Nephrology, 2013

APOL1 in autophagy Chien-An A. Hu et al., FEBS Letters, 2012

In vitro, ApoL1 induces autophagic cell death, which can be inhibited by blockers of autophagy 2008 by American Society for Biochemistry and Molecular Biology Guanghua Wan et al. J. Biol. Chem. 2008;283:21540-21549

In vitro, APOL1 risk variants increase podocyte swelling 2014 by American Physiological Society Lan X et al. Am J Physiol Renal Physiol 2014;307:F326-F336

In vitro, APOL1 risk variants increase podocyte lysosomal membrane permeability 2014 by American Physiological Society Lan X et al. Am J Physiol Renal Physiol 2014;307:F326-F336

In vitro, APOL1 risk variants increase podocyte lysosomal membrane permeability Cathepsin L activity Lan X et al. Am J Physiol Renal Physiol 2014;307:F326-F336 2014 by American Physiological Society

Harry E. Taylor et al. J. Virol. 2014;88:592-603 What about viruses? APOL1 inhibits HIV-1production and infectivity

Exogenous APOL1Vs are toxic to not only to trypanosomes, but also human cells

However, APOL1 nephropathy is likely caused by (over)expression of APOL1Vs inside podocytes, tubular and vascular cells, and either interference with healthy autophagy or instigation of unhealthy autophagy and cell death

Clinical implications of APOL1 research Renal transplantation Clinical trials of renal diseases Search for treatments

Acknowledgements

Barry I. Freedman, M.D., John H. Felts, III, Distinguished Professor of Internal Medicine and Nephrology; Chief, Section on Nephrology Wake Forest Baptist Medical Center

Joel Topf, MD Nephrologist, St Clair Specialty Physicians; Assistant Professor, Wayne State University School of Medicine Blog: Presciousbodyfluids.com

Terry Carter, Ed D Associate Dean for Instruction and Faculty Development VCU School of Medicine Teaching in Medical Education (TiME) Course

Pokemon consultants

Nephrology Division