APOL1 Renal-Risk Variants Induce Mitochondrial Dysfunction

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† †‡ | Lijun Ma,* Jeff W. Chou, James A. Snipes,* Manish S. Bharadwaj,§ Ann L. Craddock, †† Dongmei Cheng,¶ Allison Weckerle,¶ Snezana Petrovic,** Pamela J. Hicks, ‡‡ †| | Ashok K. Hemal, Gregory A. Hawkins, Lance D. Miller, Anthony J.A. Molina,§ †‡ † Carl D. Langefeld, Mariana Murea,* John S. Parks,¶ and Barry I. Freedman* §§

*Department of Internal Medicine, Section on Nephrology, †Center for Public Health Genomics, ‡Division of Public Health Sciences, Department of Biostatistical Sciences, §Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, |Department of Cancer Biology, ¶Department of Internal Medicine, Section on Molecular Medicine, **Department of Physiology and Pharmacology, ††Department of Biochemistry, ‡‡Department of Urology, and §§Center for Diabetes Research, Wake Forest School of Medicine, Winston-Salem, North Carolina

ABSTRACT APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline- inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overex- pression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016050567

The association between APOL1 G1 and G2 renal- circulating APOL1 protein concentrations are inde- risk variants and nondiabetic CKD has transformed pendent of the APOL1 genotype,7,8 and donor (but our understanding of glomerulosclerosis. HIV- not recipient) APOL1 variation contributes to poorer associated nephropathy, FSGS, hypertension- allograft survival after kidney transplantation.9–11 attributed ESRD, severe lupus nephritis, and sickle cell nephropathy reside in the APOL1-associated nephropathy spectrum and contribute to approxi- Received May 20, 2016. Accepted August 12, 2016. 1–6 mately 70% of nondiabetic ESRD in blacks. Published online ahead of print. Publication date available at The frequency of APOL1 renal-risk variants has www.jasn.org. increased likely because of the protection they pro- Correspondence: Dr. Lijun Ma or Dr. Barry I. Freedman, Section on vide from the parasite causing African sleeping Nephrology, Wake Forest School of Medicine, Medical Center Bou- sickness during natural selection.1 Intrinsic levard, Winston-Salem, NC 27157-1053. Email: [email protected] APOL1 gene expression in kidney cells appears crit- or [email protected] ical for development of nephropathy because Copyright © 2016 by the American Society of Nephrology

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APOL1 protein and mRNA are present in human podocytes, terminated at 16 hours, along with the noninduced controls. renal tubule cells, and glomerular endothelial cells.12,13 Hence, RNAs were collected to compare gene expression. Illumina kidney synthesized APOL1 risk variants likely contribute to human HT-12 v4 array-based paired analyses were performed glomerulosclerosis. as outlined in Supplemental Figure 1. Descriptions of paired To detect APOL1-associated pathways potentially involved analyses (methods and purpose) are summarized in Table 1. in nephropathy, global gene expression profiles were determined Gene expression profile changes were negligible in paired using a cell model transfected with APOL1 G0, G1, and G2 in analysis for HEK293 Tet-on EV cells with and without Dox tetracycline (Tet)-inducible HEK Tet-on 3G cell lines derived induction (data not shown). Absent Dox induction, pair-wise from HEK293 cells. These cells stably expressed APOL1 G0, analyses among HEK293 Tet-on G0, G1, and G2 cells did not G1, and G2 protein at similar levels when induced by low dose reveal significant pathways attributed to APOL1 renal-risk var- doxycycline (Dox), a derivative of Tet, whereas cell viability iants (data not shown). was minimally effected. Functional assays were used to validate HEK293 Tet-on APOL1 G0 cells with and without Dox in- bioinformatics findings. Additional experiments were per- duction revealed 1056 differentially expressed genes. These formed using nondiseased primary renal proximal tubule cells were analyzed using Cytoscape-based BiNGO software (PTCs) from black patients to determine whether the most to identify associated pathways (Supplemental Table 1), and differentially expressed genes attributed to APOL1 renal-risk Ingenuity Pathway Analysis (IPA) software to highlight top variants replicated findings from the HEK293 Tet-on cell model. canonical pathways and upstream regulators (Supplemental Tables 2 and 3). With Dox induction, key components of endoplasmic reticulum (ER) stress were significantly suppressed RESULTS (Supplemental Figure 3), and genes involved in inhibition of N-linked glycosylation (inhibited by tunicamycin) were Cell Model and Global Gene Expression downregulated (Supplemental Table 3). Unfolded protein re- As summarized in the study design (Supplemental Figure 1), global sponse (ER stress response) inhibition appeared to play an gene expression profiles of HEK293 Tet-on APOL1 (G0, G1, and important role in the biologic function of APOL1 G0. Unlike G2) cells were analyzed in paired and pattern-based analyses to in HEK293 G0 cells, the ER stress pathway was not inhibited identify affected pathways through enrichment of key transcripts. by Dox-induced APOL1 G1 and G2 variants in HEK293 Tet-on cells (compared with corresponding baseline cells without Dox Cell Viability of HEK293 Cells Stably Expressing APOL1 induction). G0, G1, and G2 Differentially expressed genes by Dox-induced APOL1 Before induction, HEK293 Tet-on APOL1 G0, G1, and G2 overexpression (versus without Dox-induction) in the variant cells (including those stably transfected with empty HEK293 G1 Tet-on cell line revealed the top associated path- pTRE2hyg vector) had similar viability. With Dox-induction, ways involved the nucleus and membrane-bound organelles, empty vector (EV), G0, G1, and G2 cells remained viable until including mitochondria (Supplemental Table 4). Differen- 16 hours, when G1 and G2 cells showed ,10% reduction in tially expressed genes by Dox-induced APOL1 overexpression viability (Supplemental Figure 2). intheHEK293G2Tet-oncellline(versuswithoutDox- induction) indicated similar pathways (Supplemental Table 5) APOL1 mRNA and Protein Levels in HEK293 Cells as HEK293 G1 cells. Paired comparisons for Dox-induced Stably Expressing G0, G1, and G2 HEK293 Tet-on APOL1 G1 versus G0 and G2 versus G0 cells Comparison of global gene expression pattern changes across were performed to assess potential biologic functions of HEK293 Tet-on APOL1 (G0, G1, and G2) cell lines relied on APOL1 renal-risk variants. On the basis of the differentially similar APOL1 expression among cell lines with Dox induction. expressed genes, Cytoscape-based BiNGO identified common By adjusting final concentrations of Dox in the culture media, the pathways related to the function of intracellular membrane- APOL1 G0 mRNA level, determined by RT-PCR, was intention- bound organelles, including mitochondria (Supplemental ally set slightly higher than G1 and G2 (Figure 1A) to ensure the Tables 6 and 7). gene expression profile changes attributed to G1 and G2 renal-risk To narrow down the differentially expressed genes attrib- variants could be convincingly detected relative to G0 at 16 hours uted to overexpression of APOL1 and its G1 and G2 risk var- postinduction. APOL1 protein levels, estimated by immunoblot iants, additional comparisons were performed for baseline and immunofluorescence (Figure 1, B and C, respectively), were HEK293 G0 versus EV, G1 versus EV, and G2 versus EV cells comparable among the three APOL1 genotypes. As anticipated, in the absence of Dox. ER components were effected for G0, APOL1 expression was low in the absence of Dox induction. G1, and G2 cells, in contrast to EV cells where APOL1 was virtually absent (data not shown). This is not unexpected Pathway Analysis of HEK293 Cells Stably Expressing because APOL1 protein is secreted; however, in contrast to G0, G1, and G2 Dox-induced HEK293 G1 and G2 cells (Supplemental Tables To maximize the effect of G1 and G2 renal-risk variants to 4 and 5), mitochondrial or nucleus pathways were not in- differential gene expression versus G0, Dox induction was volved (data not shown). These data, along with pair-wise

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Figure 1. APOL1 expression levels with Dox induction in HEK293 Tet-on G0, G1, and G2 cells are comparable. (A) Relative APOL1 mRNA levels in HEK293 Tet-on cells stably expressing G0, G1, or G2 APOL1 variants. HEK293 Tet-on cells with (+) or without (2)Dox induction were grown in complete DMEM growth media for 16 hours. Without Dox induction, APOL1 mRNA levels in HEK293 Tet-on cells were negligible compared with Dox induction. The DDCT method was used to quantify relative APOL1 mRNA expression. Fold changes were normalized to mRNA levels of G0 cells without Dox induction. Data are expressed as mean6SD. APOL1 mRNA levels in G0 cells without Dox induction set at 1 (reference). (B) Relative APOL1 levels from total protein lysates in HEK293 Tet-on cells stably expressing G0, G1, or G2 APOL1 variants. HEK293 Tet-on cells with (+) or without (2) Dox induction were grown in complete DMEM growth media for 16 hours. Four micrograms of total cell lysate protein was loaded onto a 4%–20% SDS-PAGE gel and probed with APOL1 antibody. HEK293 Tet-on empty pTRE2hyg vector cells did not express APOL1 with or without Dox induction. HEK293 Tet-on APOL1 G0, G1, and G2 cells expressed similar amounts of APOL1 with Dox induction, whereas trace amounts of APOL1 were present in HEK293 Tet-on cells without Dox induction. Data are representative of three trials of immunoblotting with similar results. (C) APOL1 immunofluorescence in HEK293 Tet-on cells stably expressing G0, G1, or G2 APOL1 variants with and without Dox induction. HEK293 Tet-on cells grown with (+) or without (2) Dox for 16 hours. After washing with PBS and fixing with 4% paraformaldehyde, cells were stained for APOL1 (red) with Epitomics anti-APOL1 antibody and counterstained with DAPI (blue). Elevated APOL1 signal intensities were comparable in HEK293 Tet-on G0, G1, and G2 cells with Dox induction, whereas APOL1 was absent in HEK293 Tet-on empty pTRE2hyg vector cells. EV, HEK293 Tet-on cell line with empty pTRE2hyg plasmid; DAPI, 49,6-diamidino-2-phenylindole. analyses among baseline HEK293 Tet-on G0, G1, and G2 cells, http://www.niehs.nih.gov/research/resources/software/ suggest the need for higher APOL1 expression to distinguish biostatistics/epig/index.cfm) to extract gene expression pro- specific pathways attributed to APOL1 renal-risk variants. files of HEK293 Tet-on APOL1 G0, G1, and G2 cells with Dox To increase power for identifying pathways attributable induction on Illumina human HT-12 v4 arrays. EPIG identified to APOL1 renal-risk variants, transcript pattern analyses 1699 significantly effected genes and 14 patterns (Figure 2A for were performed using the java-based gene expression pro- line patterns; Figure 2B for heat map) in Illumina analysis file clustering method, Extracting microarray gene expres- mode. The top three pathways for optimized patterns are sum- sion Patterns and Identifying coexpressed Genes (EPIG; marized in Table 2 on the basis of Cytoscape BiNGO; additional

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Table 1. Description of paired analysis for HEK293 Tet-on APOL1 cells using Illumina HT-12 v4 arrays Paired Analysis Purpose EV(+) versus EV(2) Clarify the effect of Dox on global gene expression

G1(2)versusG0(2) G2(2)versusG0(2) Test differential gene expression by APOL1 genotype at baseline (Dox absent) G1(2)versusG2(2)

G0(+) versus G0(2) Examine the effect of reference APOL1 (G0) when overexpressed with Dox induction G1(+) versus G1(2) Test differentially expressed genes attributed to overexpression of G1 G2(+) versus G2(2) Test differentially expressed genes attributed to overexpression of G2

G1(+) versus G0(+) Explore APOL1 G1-attributed gene expression (compared with G0) with Dox induction G2(+) versus G0(+) Explore APOL1 G2-attributed gene expression (compared with G0) with Dox induction

G0(2)versusEV(2) G1(2)versusEV(2) Narrow down the corresponding pathway alterations of interest under Dox induction G2(2)versusEV(2) Relative global gene expression levels were determined for stabilized HEK293 Tet-on APOL1 cells grown with (+) or without (2) Dox induction. EV, HEK293 Tet-on cell line with empty pTRE2hyg plasmid. informationisprovidedinSupplementalTable8.ForIllumina To verify pathways detected with Illumina HT-12 v4 arrays, analysis mode, patterns 6, 7, and 13 were enriched with mito- pattern-based comparisons were repeated on Dox-induced 2 chondrial pathways (false discovery rate [FDR] P=1.33310 27). HEK293 APOL1 Tet-on G0, G1, and G2 cell lines using the Patterns 5 and 9 appear to represent top pathways involved in Affymetrix HTA 2.0 array gene expression platform, which gene regulation by nuclear transcription factor(s) (FDR probes all exons of known transcripts. Pattern-based analysis 2 P=1.01310 14). identified ten patterns based upon 3620 significantly effected genes, using EPIG (Supplemental Figure 4 for gene expression line patterns and heat map) in Affymetrix analysis mode. Table 2 summarizes the most significant pathways detected with Cytoscape BiNGO; additional information is provided in Supple- mental Table 9. For Affymetrix analysis mode, pattern 2 highlighted mitochondrial 2 pathways as top hits (FDR P=5.19310 57). Pattern 4 contained nuclear regulation pathways via transcription factors (top 2 FDR P=3.06310 58). On the basis of similar trends for pattern 1 with 2, and pattern 3 with 4, integrated analyses were performed and results are summarized in Table 2 (detailed in Supplemental Table 9). Patterns 1, 2, 3, and 4 were verified using IPA software; this demonstrated that patterns 1 and 2 were enriched with mitochondrial dysfunction pathways (Figure 3, Supplemental Figure 5, Supplemental Table 10). In the IPA-based analysis, transfer RNA (tRNA) fi Figure 2. Illumina human HT-12 v4 arrays identify signi cantly clustered gene ex- charging surpassed the mitochondrial dys- pression patterns for HEK293 Tet-on APOL1 G0, G1, and G2 cells. From top to bottom function rating (Supplemental Table 10); all are 1699 transcripts, in order from pattern 1 to pattern 14. (A) Gene expression pat- captured genes in the tRNA charging path- terns most altered in HEK293 Tet-on G0 (circle), G1 (triangle), and G2 (diamond) cells with Dox induction. Asterisk indicates that mitochondrial pathway enrichment is pre- way encoded different aminoacyl tRNA sent (patterns 6, 7, and 13). (B) Heat map of clustered gene expression patterns: synthetases and approximately 50% were transcript expression in G0 shown in black; red and green correspond to up and mitochondria specific. This strengthened downregulation, respectively, by G1 or G2. Lighter colors denote less differential the hypothesis that APOL1 renal-risk vari- expression. ant-mediated mitochondrial dysfunction

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Table 2. Pattern-based pathway analysis on Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays using Cytoscape based BiNGO Analysis Mode Pattern GO ID Description x n X NPValue FDR P Value 2 2 Illumina 5 5634 Nucleusa 74 5183 130 17,783 3.30310 11 6.90310 08 2 2 50794 Regulation of cellular process 80 6221 130 17,783 5.50310 10 5.76310 07 2 2 50789 Regulation of biologic process 81 6551 130 17,783 3.02310 09 1.35310 06 2 2 6 5739 Mitochondriona 87 1273 342 17,780 2.80310 26 7.93310 23 2 2 44444 Cytoplasmic part 169 5147 342 17,780 7.02310 16 9.92310 13 2 2 44429 Mitochondrial parta 45 612 342 17,780 9.48310 15 8.94310 12 2 2 9 5634 Nucleusa 143 5181 317 17,767 9.40310 10 2.19310 06 2 2 44451 Nucleoplasm part 34 585 317 17,767 1.49310 09 2.19310 06 2 2 5515 Protein binding 195 8110 317 17,767 7.50310 09 5.48310 06 2 2 7 and 13 5739 Mitochondriona 34 1272 191 17,785 6.66310 07 1.34310 03 2 2 5 and 9 5634 Nucleusa 215 5180 443 17,761 2.83310 18 1.01310 14 2 2 50794 Regulation of cellular process 239 6210 443 17,761 1.04310 16 1.85310 13 2 2 50789 Regulation of biologic process 246 6540 443 17,761 4.26310 16 5.08310 13 2 2 6, 7, and 13 5739 Mitochondriona 121 1273 533 17,774 3.82310 31 1.33310 27 2 2 44444 Cytoplasmic part 248 5147 533 17,774 2.30310 18 4.01310 15 2 2 44429 Mitochondrial parta 62 612 533 17,774 3.06310 17 3.55310 14

2 2 Affymetrix 2 5739 Mitochondriona 274 1272 1357 17,736 9.68310 61 5.19310 57 2 2 43231 Intracellular membrane-bounded organellea 899 8330 1357 17,736 3.57310 50 9.58310 47 2 2 43227 Membrane-bounded organellea 899 8337 1357 17,736 5.61310 50 1.00310 46 2 2 4 5634 Nucleusa 543 5167 1020 17,727 6.00310 62 3.06310 58 2 2 10468 Regulation of gene expression 364 2920 1020 17,727 1.49310 53 3.79310 50 2 2 60255 Regulation of macromolecule metabolic process 398 3369 1020 17,727 2.30310 53 3.92310 50 2 2 1 and 2 5739 Mitochondriona 288 1272 1468 17,733 3.07310 61 1.70310 57 2 2 43231 Intracellular membrane-bounded organelle 976 8330 1468 17,733 9.56310 56 2.65310 52 2 2 43227 Membrane-bounded organelle 976 8337 1468 17,733 1.57310 55 2.90310 52 2 2 3 and 4 50794 Regulation of cellular process 728 6201 1346 17,713 2.90310 50 1.69310 46 2 2 5634 Nucleusa 640 5167 1346 17,713 8.47310 50 2.47310 46 2 2 50789 Regulation of biologic process 750 6530 1346 17,713 1.55310 48 3.01310 45 Top three pathways for each pattern with FDR P,0.01; detail in Supplemental Tables 8 and 9. GO, gene ontology; x, number of gene IDs identiﬁed in the designated pathway among input gene IDs; n, number of known gene IDs in the designated pathway; X, number of input gene IDs selected by BiNGO; N,numberof background gene IDs selected from the Gene Ontology pool by BiNGO. aHighly replicated pathway.

is a key pathway altering cellular metabolism. Patterns 3 and 4 and G2 cells (ANOVA P=0.02); relative to G0, G1 cells had represent top genes enriched in activated TGF-b signaling lower [K+](P=0.01) and G2 cells trended toward lower [K+] (Supplemental Figure 6, Supplemental Table 10) in the nuclear (P=0.07) (Supplemental Figure 7). regulation pathway in paired and pattern-based analyses (Cytoscape-based BiNGO; Supplemental Table 10, Table 2). APOL1 G1 and G2 Renal-Risk Variant Expression Mitochondrial dysfunction pathways topped common lists Results in Mitochondrial Dysfunction that were based on different analytic approaches. To assess whether mitochondrial dysfunction was attributable All relevant gene expression data performed on Illumina to APOL1 renal-risk variants in HEK293 Tet-on APOL1 cells, HT-12 v4 and Affymetrix HTA 2.0 arrays are available at http:// the 8-hour time point with Dox induction was chosen to per- www.ncbi.nlm.nih.gov/geo (accession ID: GSE85921). form mitochondrial respirometry. At 8 hours, cell viability and intracellular [K+] were similar for G0, G1, and G2 cells Intracellular Potassium Concentrations in HEK293 (Supplemental Figures 2 and 7); APOL1 expression levels were Tet-on G0, G1, and G2 Cells also comparable (Supplemental Figure 8). Immunoﬂuores- Intracellular potassium concentration [K+] was measured cence revealed that APOL1 colocalized with mitochondria in using the potassium-sensitive ﬂuorescence indicator Asante HEK293 Tet-on APOL1 cells with Dox induction by dual Potassium Green-2 AM in HEK293 Tet-on G0, G1, and G2 staining of ATP synthase 5A1 (ATP5A1, a mitochondrial cells, with and without Dox induction for 8 hours and 16 hours. marker) and APOL1 antibody (Figure 4, Supplemental Figure Intracellular [K+] was comparable in Tet-on G0, G1, and G2 9, Supplemental Video 1). Time effects on APOL1 expression cells with or without 8-hour Dox induction. After 16 hours of are shown in Supplemental Figure 10 in noninduced controls, Dox induction, intracellular [K+] differed between G0, G1, and after 4-hour and 8-hour Dox induction. With Dox

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Figure 3. APOL1 G1 and G2 renal-risk variants contribute to mitochondrial dysfunction. Affymetrix patterns 1 and 2 were analyzed using IPA for HEK293 Tet-on APOL1 G1 and G2 versus G0 cells. Results indicate marked downregulation of enzymes in mitochondrial complexes I–V. induction, respirometric assessments indicated that the max- cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) depo- imum oxygen consumption rate (ANOVA P,0.0001) and larization controls were provided for mitochondrial mem- spare respiratory capacity (ANOVA P=0.0007) were lower in brane potential in Dox-induced HEK293 Tet-on APOL1 cells HEK293 Tet-on cells overexpressing APOL1 G1 and G2 vari- (Supplemental Figure 12). ants, relative to G0 (Figure 5). The fluorescence signal intensity of mitochondrial membrane potential marker tetramethyl Differential Gene Expression by APOL1 Renal-Risk rhodamine ethyl ester (TMRE) was markedly reduced in Dox- (Two Risk Alleles) versus Nonrisk (G0G0) Genotypes in induced HEK293 Tet-on G1 and G2 cells (Supplemental Fig- Primary PTCs ure 11, A and B). However, mitochondrial content on the basis Global gene expression (mRNA) levels were examined on of immunoblot analysis of cytochrome c oxidase subunit 4 Affymetrix HTA 2.0 arrays in primary PTCs cultured from (COXIV) and voltage-dependent anion-selective channel nondiseased kidneys in 53 black patients without CKD who protein 1(VDAC1) in HEK293 Tet-on APOL1 cells was not underwent nephrectomy for localized renal cell carcinoma. In different across the G0, G1, and G2 variants (Supplemental this group, 25 had zero, 23 had one, and five had two APOL1 Figure 11C), as confirmed by comparable mRNA levels of renal-risk alleles. To detect differentially expressed gene pro- VDAC1 and COXIVacross G0, G1, and G2 cells on the Illumina files attributable to APOL1 renal-risk genotypes, only PTCs and Affymetrix arrays (data not shown). This supports that with two renal-risk alleles (n=5) and PTCs lacking renal-risk APOL1 G1- or G2-mediated mitochondrial dysfunction resul- alleles (n=25) were included in comparisons of global gene ex- ted from impaired mitochondrial respiration and membrane pression. Supplemental Table 11 lists the most differentially ex- potential, not reductions in mitochondrial mass. Carbonyl pressed genes; the fibrinogen b chain gene (FGB) topped the list.

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pattern 7 and pattern 9, respectively (Figure 2, Supplemental Table 8), representing mitochondria- and nucleus-related pathways, respectively.

DISCUSSION

Little is known regarding underlying mechanisms whereby APOL1 G1 and G2 renal-risk variants contribute to kidney disease. To assess differences in gene expression profiles relating to APOL1 variants, an HEK293 Tet-on cell model stably expressing Dox-inducible APOL1 G0, G1, and G2 was established. Relative to G0, modest overexpression of APOL1 G1 and G2 renal-risk variants led to compromised mitochondrial function. APOL1 expression was absent in parental HEK293 cells12 and EV HEK293 Tet-on cells (Figure 1), excluding interference from endogenous APOL1. Functional studies, including mitochondrial stress testing (respirometry), confirmed mitochondrial dysfunction in HEK293 cells stably overexpressing APOL1 G1 or G2 before reductions in cell viability or intracellular [K+]. Sixteen hours was chosen as the optimal time point in order to maximize the effect of G1 and G2 renal-risk variants on differential gene expression (relative to G0), because cell viability remained largely intact at that time point. At 24 hours, the viability of G0 cells began to decline, whereas that of G1 and G2 cells declined to a greater extent. Together with reduced NAPRT/NAPRT1 in primary PTCs from black patients with two APOL1 renal-risk variants (Supplemental Table 11), these findings support mitochondrial dysfunction in Dox-induced HEK293 G1 and G2 cells (versus G0) (Figure 2, Supplemental Table 8: pattern 7). Re- sults strongly suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease. The reduced NAPRT may lead to insufficient biosynthesis of NAD14 and result in mitochondrial dysfunction15 and CKD.16 Using a biotinylated membrane protein assay, Olabisi et al. reported that APOL1 was present on plasma membranes and that expression of APOL1 G1 and G2 renal-risk variants affected ion channels, decreased intracellular [K+], and resulted in cellular injury.17 We and others have shown that APOL1 resides mainly in internal organelles,12,13 typically in the ER18 and mitochondria (Figure 4, Supplemental Fig- ure 9). We note the absence of a plasma membrane pattern Figure 4. APOL1 colocalizes with mitochondria in HEK293 Tet-on for APOL1 localization compared with the report by Olabisi APOL1 cells with Dox induction. HEK293 Tet-on APOL1 cells with et al. We used monoclonal anti-APOL1 antibody (Epi- 8-hour Dox induction (stably expressing G0, G1, or G2 APOL1 var- tomics) to localize cellular APOL1. The conformation of iants) were washed with PBS and fixed with 4% paraformaldehyde. APOL1 in complexes on the cell membrane and in organ- Cells were stained for APOL1 (red) with Epitomics anti-APOL1 elles may differ because of diverse protein and lipid compo- antibody, mitochondria (green) with ATP synthase 5 A1 (ATP5A1) sitions, potentially resulting in different exposed epitopes. antibody, and counterstained with nuclear dye 49,6-diamidino-2- Therefore, antibodies that successfully recognize intracellu- phenylindole (DAPI; blue). lar APOL1 may not be able to capture plasma membrane- bound APOL1 as part of protein complexes. Importantly, In pattern-based analysis, nicotinate phosphoribosyltransferase our study found that compromised mitochondrial function (NAPRT/NAPRT1) and zinc finger E-box binding homeobox 2 occurred 8 hours after Dox induction, before G1- and G2- (ZEB2)/SMAD-interacting protein 1 (SMADIP1)appearedin induced cell death or decreases in intracellular [K+]

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Figure 5. APOL1 G1 and G2 renal-risk variants decrease mitochondrial respiration. (A) a-d: Mitochondrial stress tests in HEK293 Tet-on APOL1 cells. Cells were seeded on a V7 cell culture plate (Seahorse Bioscience) with final density 100,000 cells/well before assay. Effects of G0, G1, and G2 with (+) and without (2) Dox induction were compared using a Seahorse XF-24 extracellular flux analyzer to measure oxygen consumption rate (OCR), a measure of oxidative phosphorylation, in the presence of a series of metabolic inhibitors and uncoupling agents. The first injection (shown as vertical blue line A) was oligomycin, an inhibitor of ATP synthesis via blockade of the proton channel of ATP synthase (complex V). Oligomycin distinguishes the OCR from ATP synthesis by blocking the oxygen consumption required to overcome proton leakage across the inner mitochondrial membrane, and basal respiration rate, by blocking nonmitochondrial respiration. The second injection (shown as vertical blue line B) was FCCP, an uncoupling agent that disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the ATP synthase proton channel (complex V). Collapse of the mitochondrial membrane potential leads to rapid energy and oxygen consumption without generation of ATP. FCCP is used to calculate the spare respiration capacity of cells, defined as the quantitative difference between maximal and basal respiration rates. The third injection (shown as vertical blue line C) was a combination of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor; this combination blocks mitochondrial respiration and enables calculation of mitochondrial and nonmitochondrial cellular respiration. At least three wells were assigned for each type of HEK293 Tet-on cells with (+) or without (2) Dox induction on the same 24-well V7 cell culture plate. Data were expressed as mean6SEM of each time point, grouped by HEK293 Tet-on G0, G1, and G2 cells with or without Dox induction. (B) Bioenergetic profiles for HEK293 Tet-on APOL1 G0, G1, and G2 cells. APOL1 stably transfected HEK293 cells were incubated with (+) or without (2) Dox for 8 hours before assay on a Seahorse XF-24 extracellular flux analyzer to measure the OCR. After a 15 minute equilibration period, three measures of OCR were made for each stage, i.e., preoligomycin, between oligomycin and FCCP, between FCCP and rotenone/antimycin, and postrotenone (injections denoted as vertical blue lines, see A). The average of three measures was used to represent the OCR of the corresponding stage for each well. Data were grouped by APOL1 genotype and Dox treatment (+/2). Basal respiration rate, maximum respiration rate, and spare respiration capacity were expressed as mean6SD; n refers to replicated well number for each type of HEK293 Tet-on cells on the 24-well V7 cell culture plate. (a) For G0 cells, induction of APOL1 did not alter the basal respiration rate (t test P=0.68), but improved maximum respiration rate and spare respiration capacity (t test P values 0.005 and 0.005, respectively). (b) For G1 cells, induction of APOL1 did not alter the basal respiration rate (t test P=0.08), maximum respiration rate, and spare respiration capacity (t test P values 0.48 and 0.68, respectively). (c) For G2 cells, induction of APOL1 diminished the basal respiration rate, maximum respiration rate, and spare respiration capacity (t test P values 0.002, 0.003, and 0.01, respectively). (d) Without Dox induction, G0, G1, and G2 cells appeared to have similar basal respiration rate, maximum respiration rate, and spare respiration capacity (ANOVA P=0.78, 0.22, and 0.13, respectively). (e) With Dox induction, the basal respiration rate, maximum respiration rate, and spare respiration capacity for G1 and G2 were significantly reduced (ANOVA P=0.004, ,0.0001, and 0.0007, respectively) compared with G0. (f) Overview of bioenergetic profiles for HEK293 Tet-on APOL1 G0, G1, and G2 cells.

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Figure 5. Continued.

(measured with a fluorescence indicator in live cells; Supple- An unbiased gene expression pattern identification mental Figure 7). This suggested that mitochondrial method (EPIG) was used to detect pathways induced by dysfunction was a cause, rather than a result of cell injury APOL1 renal-risk variants by grouping transcript patterns; relating to intracellular [K+]depletion.AsinOlabisiet al., this obviated arbitrary logFC thresholds. Without Dox induc- we observed decreased intracellular [K+] but at a later time tion, few pathways were distinguishable between HEK293 point (16 hours). This would be an expected finding in APOL1 G0, G1, and G2 cells. Therefore, only Dox-induced the presence of mitochondrial dysfunction. Although HEK293 Tet-on APOL1 cells were included in pattern-based VDAC1 is also expressed in the plasma membrane, it pathway analyses. Unbiased global gene expression profiles mainly displays a cytoplasmic pattern in immunofluorescence with Dox induction were assessed in HEK293 Tet-on cells ;studies. To assess mitochondrial mass, both outer and inner on both Illumina HT-12 v4 arrays and Affymetrix HTA 2.0 mitochondrial membrane markers were chosen (VDAC1 and arrays, the latter covers all exons of transcripts in known COXIV, respectively; Supplemental Figure 11C) to quantify UCSC (University of California Santa Cruz) genes, UCSC the corresponding mitochondrial marker proteins.19 lincRNA transcripts, Ensembl, RefSeq, and Broad Institute Mitochondrial dysfunction negatively effects the activity of Human Body Map lincRNAs and TUCP (transcripts of un- plasma membrane sodium-potassium ATPase. Subsequent certain coding potential) catalog (www.affymetrix.com). Mi- reductions in intracellular ATP can result in failure to maintain tochondrial pathways appeared at the top of pattern-based transmembrane Na+/K+ gradients, leading to loss of the mem- analyses on both arrays. 2 brane potential, secondary Cl and water influx, intracellular Reductions in mitochondrial superoxide dismutase swelling, and accumulation of intracellular Ca2+, triggering (mtSOD)/superoxide dismutase 2 (SOD2) and catalase downstream enzymatic cascades that lead to cell injury.20 (CAT) transcripts were also detected in APOL1 G1 and G2 The presence of APOL1 protein in mitochondria and other expressing cells (Supplemental Figure 5; blue arrows). This membrane-bound organelles, including ER, supports poten- could exacerbate reactive oxygen species (ROS)-mediated mi- tial roles in the regulation of cellular metabolism and the stress tochondrial dysfunction. Decreases in SOD2/mtSOD may en- response,18 although the precise role of mitochondrial APOL1 hance mitochondrial oxidative damage.21,22 CAT is an enzyme remains to be elucidated. that degrades ROS. It is possible that decreased SOD2 and CAT

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Figure 5. Continued.

(pattern analysis shown in Supplemental Figure 5) worsen mi- In addition, an upregulated TGF-b signaling pathway was tochondrial oxidative stress, resulting in inhibition of cellular detected with IPA (Supplemental Figure 6, Supplemental Ta- respiration as seen in the mitochondrial stress test for APOL1 ble 10). ZEB2/SMADIP1, one of the top upregulated genes by G1 and G2 variants (Figure 5). APOL1 renal-risk variants in primary PTCs, is an essential The most significant differences in Dox-induced HEK293 component of the SMAD signaling pathway critical in trans- G1 and G2 cells, evident after addition of the respiratory chain mitting the TGF-b signal to the nucleus and for regulation of uncoupler FCCP, were the reduced maximal respiratory rate transcriptional responses.24 An activated TGF-b pathway has and spare respiratory capacity. Because uncouplers act as long been recognized as a cause of FSGS and renal fibrosis.25 protonophores across all membranes, acidifying the cytosolic Mitochondrial and TGF-b pathways are also known to inter- compartment and disrupting the function of endosomes and act. Mitochondrial ROS regulate TGF-b signaling. Increased other compartments, it is possible that the reduced maximal oxidative stress amplifies activation of the canonical TGF-b respiratory rate observed after addition of FCCP in G1 and G2 pathway, a major contributor to cellular fibrosis.26 Genetically resulted from endosomal disruption and reduced substrate disrupting mitochondrial complex III-generated ROS pro- delivery to the mitochondria (as APOL1 is not restricted to duction attenuates TGF-b –induced profibrotic gene ex- mitochondria). It is currently unclear how APOL1 renal-risk pression.27 TGF-b1 induces prolonged mitochondrial variants effect mitochondrial membrane proton permeability ROS generation through decreased complex IV activity in human cells. However, the link between APOL1-induced with senescent arrest.28 Such crosstalk may be evident in trypanolysis and apoptosis-like mitochondrial membrane our bioinformatics data. permeabilization23 may shed light on the potential mecha- Because the primary PTCs we analyzed were derived from nism of APOL1 renal-risk variant-mediated mitochondrial nondiseased kidneys and APOL1 expression levels were lower dysfunction in renal cells. than HEK293 Tet-on cells with Dox induction, effects of

10 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 www.jasn.org BASIC RESEARCH

APOL1 risk alleles were more subtle. However, two of the most Global Gene Expression and Pathway Analyses differentially expressed genes in primary PTCs corresponded Based on the study design (Supplemental Figure 1), global gene ex- to patterns in pathway analyses of HEK293 Tet-on APOL1 pression profiles were performed on HEK293 Tet-on G0, G1, G2, and cells; these represented mitochondrial dysfunction and acti- EV cells with and without Dox induction using Illumina human vated TGF-b signaling. ANOVA quantifies both “between” HT-12 v4 arrays. Another independent gene expression array sys- and “within” group variation. It is especially useful when sam- tem, Affymetrix HTA 2.0, was used to verify the results of Illumina ple sizes are small and unequal variances are present. We used arrays. Paired and pattern-based analyses were applied to detect the both fold-change and P values to define differential gene ex- mostly effected pathways because of overexpression and by APOL1 pression. The P values in Supplemental Table 11 were not genotypes. adjusted for multiple testing. However, the gene expression fold changes were marked (log2FC.1.5) and fewer than 25 Mitochondrial Respirometry top over- or underexpressed genes were included, on the basis Twenty thousand HEK293 Tet-on APOL1 cells were seeded on a V7 of the presence of two APOL1 renal-risk variants. The pathways cell culture plate (Seahorse Bioscience, Billerica, MA) for 24 hours in detected in our study are unlikely to be the sole mechanisms complete DMEM media. Dox was used to induce APOL1 expression leading to APOL1-associated nephropathy. Other stressors (concentrations provided in Supplemental Material) on the basis may amplify pathologic effects in the form of second hits,29 of the G0, G1, and G2 genotype, 8 hours before assessing cellular including ischemia,30 viral infections,31–33 interferons,34 and respiration. The final preassay density of HEK293 Tet-on APOL1 cells lupus.5,35 reached approximately 100,000/well. Cellular respiration was mea- Although no significantly altered pathways were present in sured using a Seahorse Bioscience XF-24–3 analyzer. To measure ox- the tubulointerstitial compartment of black patients with ygen consumption rate, cell culture media was replaced with assay nephrotic syndrome or FSGS in the Nephrotic Syndrome media (3 mM glucose, 1 mM sodium pyruvate, and 1.5 mM gluta- Study Network study,36 the 200 most significantly underex- mine without FBS). For bioenergetic profiling, final concentrations pressed genes (,1% of all expressed genes) on the basis of of compounds after port injections were 1.0 mM oligomycin, 0.25 mM APOL1 high risk genotypes (two renal-risk variants) in glo- FCCP, 1 mM antimycin A, and 1 mM rotenone. After respirometric meruli of patients with FSGS were nominally enriched for the assessments, cell numbers were recounted for normalization of oxygen mitochondrial pathway (FDR P=0.04; Cytoscape-based consumption rate. BiNGO analysis). A limitation of this study is that transcript-based screening Human Subjects and Primary PTC Lines may miss functional changes that relate to post-transcriptional Black patients undergoing nephrectomy for localized renal cell and post-translational modification of proteins. However, to carcinoma with preoperative eGFR.60 ml/min per 1.73 m2 (without the best of our knowledge, this is the first study that links proteinuria) were recruited to provide nondiseased kidney tissue. APOL1 renal-risk variants with mitochondrial dysfunction via Fifty three individuals (men/women, 31:22; mean6SD age, 58.36 an unbiased systems biology-based approach. Findings were 10.7 years; and serum creatinine 1.0860.33 mg/dl) were enrolled fi con rmed by mitochondrial respirometry. Increasing evi- between March 2010 and May 2015. The study was approved by dence supports mitochondrial dysfunction and the genera- the Wake Forest School of Medicine (WFSM) Institutional Review 16 tion of ROS in the pathogenesis of CKD. The mechanisms Board and all participants provided written informed consent. APOL1 fi of -associated kidney disease identi ed in this study Primary PTC lines were established using an established proto- may provide valuable insights toward developing novel col.12 DNA was isolated from peripheral blood of participants therapeutic targets for nondiabetic kidney disease in black using a Promega DNA isolation wizard kit (Promega, Madison, patients. WI) and two single nucleotide polymorphisms in the APOL1 G1 renal-risk allele (rs73885319; rs60910145) and an insertion/ deletion for the G2 renal-risk allele (rs71785313) were genotyped CONCISE METHODS using a custom assay designed at WFSM on the Sequenom platform (Sequenom Laboratories, San Diego, California).37 G1 and G2 ge- Detailed methods and information on pathway analysis are available notype calls were visually inspected for quality control. Genotyping in Supplemental Material. efficiency was 100% and 41 blind duplicates resulted in a 100% concordance rate. Establishment of HEK293 Tet-on G0, G1, G2, and EV Cells HEK293 Tet-on APOL1 cell lines were established to stably express G0, G1, G2, and empty pTRE2hyg vector, on the basis of previously ACKNOWLEDGMENTS reported methods.18 Individual clones were verified by direct sequenc- ing (ABI 3730XL, Applied Biosystems, Foster City, CA). RT-PCR and We thank Dr. Martin R. Pollak for sharing APOL1 vectors. immunoblotting were used to determine APOL1 expression levels and This work was supported by National Institutes of Health grant APOL1 protein size. R01DK070941 (to B.I.F.).

J Am Soc Nephrol 28: ccc–ccc, 2016 APOL1 and Mitochondrial Function 11 BASIC RESEARCH www.jasn.org

DISCLOSURES 12. Ma L, Shelness GS, Snipes JA, Murea M, Antinozzi PA, Cheng D, Wake Forest University Health Sciences and B.I.F. have filed for a patent Saleem MA, Satchell SC, Banas B, Mathieson PW, Kretzler M, Hemal AK, Rudel LL, Petrovic S, Weckerle A, Pollak MR, Ross MD, Parks JS, related to APOL1 genetic testing. B.I.F. receives research funding from Freedman BI: Localization of APOL1 protein and mRNA in the human Novartis (Basel, Switzerland) and B.I.F. and J.S.P. are consultants for Ionis kidney: nondiseased tissue, primary cells, and immortalized cell lines. J Pharmaceuticals (Carlsbad, CA). All other authors have nothing to disclose. Am Soc Nephrol 26: 339–348, 2015 13. Madhavan SM, O’Toole JF, Konieczkowski M, Ganesan S, Bruggeman LA, Sedor JR: APOL1 localization in normal kidney and nondiabetic REFERENCES kidney disease. 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Freedman BI, Julian BA, Pastan SO, Israni AK, Schladt D, Gautreaux 309–320, 2007 MD, Hauptfeld V, Bray RA, Gebel HM, Kirk AD, Gaston RS, Rogers J, 26. Hagler MA, Hadley TM, Zhang H, Mehra K, Roos CM, Schaff HV, Suri Farney AC, Orlando G, Stratta RJ, Mohan S, Ma L, Langefeld CD, Hicks RM, Miller JD: TGF-b signalling and reactive oxygen species drive fi- PJ, Palmer ND, Adams PL, Palanisamy A, Reeves-Daniel AM, Divers J: brosis and matrix remodelling in myxomatous mitral valves. Cardiovasc Apolipoprotein L1 gene variants in deceased organ donors are asso- Res 99: 175–184, 2013 ciated with renal allograft failure. Am J Transplant 15: 1615–1622, 2015 27. Jain M, Rivera S, Monclus EA, Synenki L, Zirk A, Eisenbart J, Feghali- 11. Freedman BI, Pastan SO, Israni AK, Schladt D, Julian BA, Gautreaux Bostwick C, Mutlu GM, Budinger GRS, Chandel NS: Mitochondrial re- MD, Hauptfeld V, Bray RA, Gebel HM, Kirk AD, Gaston RS, Rogers J, active oxygen species regulate transforming growth factor-b signaling. Farney AC, Orlando G, Stratta RJ, Mohan S, Ma L, Langefeld CD, J Biol Chem 288: 770–777, 2013 Bowden DW, Hicks PJ, Palmer ND, Palanisamy A, Reeves-Daniel AM, 28. Yoon Y-S, Lee J-H, Hwang S-C, Choi KS, Yoon G: TGF beta1 induces Brown WM, Divers J: APOL1 Genotype and Kidney Transplantation prolonged mitochondrial ROS generation through decreased complex Outcomes From Deceased African American Donors. Transplantation IV activity with senescent arrest in Mv1Lu cells. Oncogene 24: 1895– 100: 194–202, 2016 1903, 2005

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29. Freedman BI, Skorecki K: Gene-gene and gene-environment interac- regulate the nephropathy gene Apolipoprotein L1. Kidney Int 87: 332– tions in apolipoprotein L1 gene-associated nephropathy. Clin J Am Soc 342, 2015 Nephrol 9: 2006–2013, 2014 35. Leishangthem BD, Sharma A, Bhatnagar A: Role of altered mitochon- 30. Anderson BR, Howell DN, Soldano K, Garrett ME, Katsanis N, Telen MJ, dria functions in the pathogenesis of systemic lupus erythematosus. Davis EE, Ashley-Koch AE: In vivo Modeling Implicates APOL1 in Ne- Lupus 25: 272–281, 2016 phropathy: Evidence for Dominant Negative Effects and Epistasis un- 36. Sampson MG, Robertson CC, Martini S, Mariani LH, Lemley KV, Gillies der Anemic Stress. PLoS Genet 11: e1005349, 2015 CE, Otto EA, Kopp JB, Randolph A, Vega-Warner V, Eichinger F, Nair V, 31. Kopp JB, Nelson GW, Sampath K, Johnson RC, Genovese G, An P, Gipson DS, Cattran DC, Johnstone DB, O’Toole JF, Bagnasco SM, Friedman D, Briggs W, Dart R, Korbet S, Mokrzycki MH, Kimmel PL, Song PX, Barisoni L, Troost JP, Kretzler M, Sedor JR; Nephrotic Syn- Limou S, Ahuja TS, Berns JS, Fryc J, Simon EE, Smith MC, Trachtman H, drome Study Network: Integrative Genomics Identiﬁes Novel Associ- Michel DM, Schelling JR, Vlahov D, Pollak M, Winkler CA: APOL1 ge- ations with APOL1 Risk Genotypes in Black NEPTUNE Subjects. JAm netic variants in focal segmental glomerulosclerosis and HIV-associated Soc Nephrol 27: 814–823, 2016 nephropathy. J Am Soc Nephrol 22: 2129–2137, 2011 37. Freedman BI, Langefeld CD, Turner J, Núñez M, High KP, Spainhour M, 32. Divers J, Núñez M, High KP, Murea M, Rocco MV, Ma L, Bowden DW, Hicks PJ, Bowden DW, Reeves-Daniel AM, Murea M, Rocco MV, Divers Hicks PJ, Spainhour M, Ornelles DA, Kleiboeker SB, Duncan K, J: Association of APOL1 variants with mild kidney disease in the ﬁrst- Langefeld CD, Turner J, Freedman BI: JC polyoma virus interacts with degree relatives of African American patients with non-diabetic end- APOL1 in African Americans with nondiabetic nephropathy. Kidney Int stage renal disease. Kidney Int 82: 805–811, 2012 84: 1207–1213, 2013 33. Ohta A, Nishiyama Y: Mitochondria and viruses. Mitochondrion 11: 1–12, 2011 34. Nichols B, Jog P, Lee JH, Blackler D, Wilmot M, D’Agati V, Markowitz G, This article contains supplemental material online at http://jasn.asnjournals. Kopp JB, Alper SL, Pollak MR, Friedman DJ: Innate immunity pathways org/lookup/suppl/doi:10.1681/ASN.2016050567/-/DCSupplemental.

J Am Soc Nephrol 28: ccc–ccc, 2016 APOL1 and Mitochondrial Function 13 Supplemental Materials

include Supplemental Methods and References Supplemental Figures and Legends Supplemental Video Legends Supplemental Tables Supplemental Methods

Transfection and selection for stable clones of HEK293 Tet-on G0, G1, G2, and empty vector

cDNAs of APOL1 G0, G1, and G2 were inserted into the NheI- and SalI-digested pTRE2hyg vector (Clontech, Mountain View, CA).1 To obtain Tet-inducible cell lines stably expressing APOL1,

HEK 293 Tet-On 3G cell lines (Clontech, Mountain View, CA) were transfected in 100 mm dishes at 30% confluence with 5 μg pTRE2hyg plasmid containing APOL1 G0, G1, G2, and empty vector using

Lipofectamine transfection reagent (Invitrogen, Waltham, MA) based on manufacturer instructions. Cells were selected with DMEM (Invitrogen) containing 10% FBS supplemented with 400 μg/ml G418

(Cellgro, Manassas, VA) and 100 ug/ml hygromycin B (Invitrogen, Waltham, MA) 24 h post-transfection.

Selection medium was replaced every 48h for 20 days. Individual clones were isolated, expanded, and maintained in DMEM media with 10% FBS, 100 μg/ml G418 and 50 ug/ml hygromycin B (referred to as complete DMEM growth media). Eight clones were characterized for each variant; one clone per variant was selected for all experiments. Individual clones with G0, G1 and G2 APOL1 genotypes and empty pTRE2hyg vector were analyzed for inducible APOL1 expression by RT-PCR, immunoblot analysis, and immunofluorescence.

Total RNA isolation, quality control, and real-time PCR

Total RNA was isolated from HEK293 Tet-on cells using RNAeasy Mini Kit (Qiagen, Germany).

The quantity and quality of isolated RNA were determined by ultraviolet spectrophotometry and electrophoresis, respectively, on the Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and

Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

To determine APOL1 mRNA levels in cells, 200ng of RNA was reverse transcribed with random hexamer primers using the TaqMan RT kit (Applied Biosystems, Foster City, CA). Primers were designed to capture all known APOL1 splice variants. RT-PCR in the presence of SYBRGreen was performed with a Roche 480 Real-Time PCR system (Roche Diagnostics, Germany) using 18S ribosomal

RNA for normalization. Primer sequences were described previously.2 The CT method3 was used to quantify the relative levels of APOL1 mRNA. Fold-changes were normalized to mRNA levels on

HEK293 Tet-on G0 cells without Dox induction. All experiments were performed in triplicate and results expressed as mean ± SD if not specified.

After Dox dose titration experiments to induce comparable levels of APOL1 expression with each genotype, HEK293 Tet-on 3G cell lines stably expressing G0, G1, and G2 were incubated with 0.0313,

0.0625, and 0.1250 μg/ml Dox (BD Biosciences, San Jose, CA), respectively, for 8h or 16h. APOL1 G0,

G1, and G2 mRNA and protein expression was induced to similar levels in HEK293 Tet-on cell lines because inconsistent levels would complicate comparisons of downstream pathways. Dox-induced

APOL1 G0 expression levels in HEK293 Tet-on cells were introduced to a high enough level to distinguish that from non-induced cells while the cell viability remained the same for cells with and without induction. This allowed post-induction gene expression patterns for G1 and G2 vs. G0 to be more readily detectable. The cell line stably transfected with empty pTRE2hyg plasmid was incubated with

0.25 μg/ml Dox to ensure absence of APOL1 expression and serve as a control for gene expression studies.

Inclusion of HEK293 empty pTRE2hyg vector Tet-on cells excluded significant transcript level changes related to Dox induction (data not shown), and ensured that altered gene expression in HEK293 Tet-on cells with and without Dox induction were attributed to over-expression of APOL1 variants.

Cell viability assay

Stabilized HEK293 Tet-on APOL1 G0, G1, G2 and empty vector cells (20,000/well) were seeded on three 96-well plates. After 24h of growth, cells were induced with Dox for 2hr, 4hr, 8hr or 16hr in complete DMEM media; non-induced cells were incubated with corresponding DMEM media without

Dox. Cell viability was measured using Cytotox 96 LDH viability assay kit (Promega, Madison, WI) per manufacturer instructions. Each cell line (with and without Dox induction) was measured at least 6 times, with values expressed as mean ± SD on the bar graph. Full cell viability was defined as 1.0 (100% viable).

Global gene expression and pathway analyses Total RNAs were isolated from HEK293 Tet-on cell lines stably expressing APOL1 G0, G1, and

G2 (and empty vector) cell lines with and without Dox induction. All 24 RNA samples (eight groups in triplicate, see Supplemental Figure 1) were processed on Illumina human HT-12 v4 arrays in a single batch according to manufacturer instructions. Six RNA samples (G0, G1 and G2, in duplicate) were examined on exon-based Affymetrix HTA2.0 arrays to confirm the findings from Illumina bead arrays.

In addition, 53 samples of non-diseased primary proximal tubule cells (PTCs) from African Americans were processed on Affymetrix HTA2.0 arrays to determine whether the most differentially expressed genes attributed to APOL1 renal-risk variants replicated findings in the HE293 Tet-on cell model. Since

Illumina and Affymetrix arrays were done in the same batch, no batch effects were seen. The quality control analysis and normalization confirmed the absence of failed samples or outliers. Whole genome transcript intensity profiles were extracted using GenomeStudio V2011.1 for Illumina human HT-12 v4 arrays and Transcriptome Analysis Console (TAC) software for Affymetrix HTA 2.0 arrays, respectively.

4 Levels of expression were estimated by RMA normalized log2 converted probe set pixel intensity. The threshold for detectable transcripts was set at signal/noise ratio (SNR) greater than 3. The gene expression data of Illumina HT-12 v4 and Affymetrix HTA 2.0 arrays are available in the NCBI Gene Expression

Omnibus (GEO) database.

Paired analysis between HEK293 Tet-on APOL1 cell samples for differentially expressed genes were performed by empirical Bayers method5 implemented in R Biocondoctor limma package, where the selection thresholds used were |logFC| >1 and FDR (False Discovery rate) p <0.01.

EPIG6, a java-based gene expression profile clustering method was employed. It uses an un- supervised profile approach to extract patterns from whole data sets and then categorize the genes to one of the patterns if they met all of the following criteria: SNR > 3, corresponding p<0.005, absolute log2 fold change >1, and maximum Pearson correlation r value to a given pattern >0.87. This approach was applied to both Illumina and Affymetrix array data for RNA samples of Dox-induced G0, G1 and G2 cells.

The former arrays were performed in triplicate and the later in duplicate; 14 and 11 patterns were extracted, respectively. Each pattern consisted of a set of genes which were highly similar in their expression profiles, which may be co-expressed and relevant to biological co-regulation. Overall, fewer than 8% of expressed genes were selected from either array to form specific patterns, representing the high stringency of criterion settings for EPIG.

Both Cytoscape (http://www.cytoscape.org/) and Ingenuity pathway analysis (IPA)

(http://www.ingenuity.com/) were performed to identify significant pathways relevant to G1 and G2.

Paired analysis between PTCs with 2 renal-risk alleles and those without risk alleles for differentially expressed genes were performed using the Affymetrix TAC package, where the selection thresholds employed were |logFC| >1.5 and ANOVA p<0.005.

Immunoblotting

Immunoblotting was performed as reported.2 Briefly, blots were incubated overnight at 4°C with primary antibodies. The membranes were then washed three times in Tris-buffered saline containing 0.1%

Tween 20 and incubated for 1h in blocking buffer with an anti-rabbit or mouse IgG secondary antibody conjugated to horseradish peroxidase (1:20,000; Jackson ImmunoResearch Laboratories, West Grove,

PA). Detailed information on experimental conditions for primary antibodies used in immunoblot is provided in Supplemental Table 12. COX IV and VDAC1 served as mitochondrial content markers.8,9

Immunofluorescence

Immunofluorescence of APOL1 (Epitomics, Burlingame, CA) and mitochondrial marker

ATP5A1 (Invitrogen, Frederick, MD) and other markers was performed on HEK293 Tet-on cell lines using established protocols.2 Secondary antibodies (goat anti-rabbit Alexa Fluor 594 and goat anti-rabbit

Alexa Fluor 488, Invitrogen) were used to display fluorescent signals (1:500 dilution). Microscopy was performed using a Zeiss LMS 710 confocal microscope (Oberkochen, Germany).

Fifty nM of mitotracker Green (Invitrogen) and 10 nM of TMRE (Invitrogen) were incubated with HEK293 Tet-on cells for 30 min prior to signal detection. Microscopy was performed using an Olympus IX71 fluorescence microscope (Olympus Scientific Solutions Americas Corp., Waltham,

Massachusetts).

Measurement of intracellular potassium concentrations

HEK293 Tet-on APOL1 (G0, G1 and G2) cells were seeded on a 96 well plate pre-coated with

Cell-Tak (Corning, Bedford, MA) at a density of 20,000 cells/well 24 hr prior to Dox induction. After 8 hr or 16 hr of Dox induction, Dox-induced and non-induced cells were incubated with a fluorescent potassium indicator (Asante Potassium Green-2 AM, APG-2 AM, Abcam, Cambridge, MA) for 30 min in

3% BSA-PBS (final concentration 2 µM) according to the manufacturer’s instructions, followed by a 5 min gentle wash with PBS. The indicator was excited at 488 nm and fluorescent emission intensity was measured at 530 nm. Fluorescence signals were read on a BioTek Synergy-HTX multi-mode plate reader

(BioTek, Winooski, VT). A standard curve was constructed based on measurements of fluorescent intensity in HEK293 G0 cells incubated with a 50 µM of amphotericin B, an ionophore, for 15 min for each [K+] standard concentration. Ionophore treatment allowed [K+] equilibration with known standard extracellular 50, 100 and 150 mM [K+] in 3% BSA-PBS. The standards were made in cell culture buffer in which NaCl ions were iso-osmotically replaced by KCl, keeping the osmolarity 300 mOsm/L. A standard curve was obtained from a series of triplicate wells of known ionophore-balanced intracellular

[K+] on the same plate as G0, G1 and G2 cell samples, by plotting [K+] vs. fluorescent intensity and determining the line of best fit using linear regression (r2=0.99). The fluorescent signals were normalized to cell lysate total protein in corresponding wells before being used for quantification of intracellular [K+].

The measurement in each cell type (G0, G1 and G2; without and with Dox induction) was repeated in four wells, and the average of the four measurements is shown on the bar graph. P-values were assessed using ANOVA.

Supplemental References:

1. Cheng D, Weckerle A, Yu Y, Ma L, Zhu X, Murea M, Freedman BI, Parks JS, Shelness GS: Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells. J. Lipid Res. 56: 1583–1593, 2015

2. Ma L, Shelness GS, Snipes JA, Murea M, Antinozzi PA, Cheng D, Saleem MA, Satchell SC, Banas B, Mathieson PW, Kretzler M, Hemal AK, Rudel LL, Petrovic S, Weckerle A, Pollak MR, Ross MD, Parks JS, Freedman BI: Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines. J. Am. Soc. Nephrol. 26: 339–348, 2015

3. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408, 2001

4. Bolstad BM, Irizarry RA, Astrand M, Speed TP: A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinforma. 19: 185–193, 2003

5. Smyth GK: Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3: 3, 2004

6. Chou JW, Zhou T, Kaufmann WK, Paules RS, Bushel PR: Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes. BMC Bioinforma. 8: 427, 2007

7. Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinforma. 21: 3448–3449, 2005

8. De Pablo-Latorre R, Saide A, Polishhuck EV, Nusco E, Fraldi A, Ballabio A: Impaired parkin- mediated mitochondrial targeting to autophagosomes differentially contributes to tissue pathology in lysosomal storage diseases. Hum. Mol. Genet. 21: 1770–1781, 2012

9. Converso DP, Taillé C, Carreras MC, Jaitovich A, Poderoso JJ, Boczkowski J: HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism. FASEB J. 20: 1236–1238, 2006

Supplemental Figures HEK293 Tet-on APOL1 cell lines

Empty pTRE2hyg APOL1 APOL1 APOL1 vector (EV) G0 G1 G2 Baseline x3 (non-induced)

Dox-induced x3

Global gene expression pattern a) Global gene expression profile change in HEK293 Tet-on APOL1 change with and without Dox G0, G1 and G2 with and without Dox induction; induction b) Global gene expression profile and pattern changes in HEK293 Tet-on APOL1 G0, G1, and G2 cells after Dox induction

Pathway analysis using Cytoscape and Ingenuity

Verification by protein analysis and functional assays

Supplemental Figure 1 Detection of pathways most significantly impacted

p=NS p=NS p=NS p=NS p=3x10-6

Supplemental Figure 2 Supplemental Figure 2. Quantitation of serial cell viability of HEK293 Tet-on G0, G1, or G2 cells with Dox induction at time point 0, 2, 4, 8 and 16 hr.

Stabilized HEK293 Tet-on APOL1 G0, G1, G2 and empty vector (EV) cells were seeded on three 96-well plates at a density of 20,000/well. After 24h of growth, cells were induced with Dox for 2hr, 4hr, 8hr and 16hr, respectively, in complete DMEM media. Non-induced cells were incubated with corresponding DMEM media without Dox. Cell viability was measured using the Cytotox 96 LDH viability assay kit (Promega, Madison, WI) per manufacturer instructions. Each cell line (with and without Dox induction) was measured at least 6 times, with values expressed as mean ± SD on the bar graph. Full cell viability was defined as 1 (100%). NS: not significant (p>0.05).

4 Supplemental Figure 3 Supplemental Figure 3. APOL1 G0 inhibits the unfolded protein response (endoplasmic reticulum stress) Down-regulation of the endoplasmic reticulum (ER) stress pathway by Dox- induced APOL1 G0 over-expression is characterized by decreased ER stress sensor BiP, effector CHOP and chaperone HSP70/co-chaperone HSP40.

Pathway symbols are defined in Figure 3 legend. A B G0 G1 G2

1 1 * 2 * 3 ** 4 **

5 6 7 8

9 10

Supplemental 5 6 7 8 Figure 4 9 10 Supplemental Figure 4. Significantly clustered expression patterns for HEK293 Tet-on APOL1 G0, G1, and G2 cells in pattern analysis on Affymetrix HT 2.0 arrays A) Gene expression patterns most altered in HEK293 Tet-on APOL1 G0 (circle), G1 (triangle), and G2 (diamond) cells with Dox induction. *Mitochondrial dysfunction is enriched in patterns 1 and 2; **Activated TGF-β signaling is enriched in patterns 3 and 4. B) Heat map of clustered gene expression patterns from top to bottom includes 3620 transcripts, order from pattern 1 to 10. Transcript expression in G0 cells shown in black; red and green correspond to up or down regulated transcripts, respectively, in G1 or G2 cells. Lighter colors denote less differential expression. Supplemental Figure 5 Supplemental Figure 5. APOL1 G1 and G2 renal-risk variants contribute to mitochondrial dysfunction Affymetrix array patterns 1 and 2 (Supplemental Figure 4) were analyzed using IPA for HEK293 Tet-on APOL1 G1 and G2 vs. G0 cells. Results indicate marked down-regulation of enzymes in mitochondrial complexes I to V. In addition, mtSOD/SOD2 and CAT (blue arrows) were downregulated by APOL1 renal-risk variants, suggesting a role for oxidative stress in APOL1-associated nephropathy.

Pathway symbols are defined in Figure 3 legend. Supplemental Figure 6 Supplemental Figure 6. APOL1 G1 and G2 renal-risk variants contribute to activated TGF-β signaling. AFFY patterns 3 and 4 were analyzed using IPA for HEK293 Tet-on APOL1 G1 and G2 vs. G0 cells. Results indicate up-regulation of transcripts in the canonical TGF-β signaling pathway. Figure7 Supplemental

Intracellular [K+] (mmol/L)

p=0.13

p=0.17

p=0.008 p=0.065

Supplemental Figure 7. Intracellular potassium concentrations for HEK293 Tet-on G0, G1, or G2 cells without Dox and with Dox induction for 8 and 16 hr.

Intracellular [K+] was measured in quadruplicate with fluorescent potassium indicator (Asante Potassium Green-2 AM) for HEK293 Tet-on G0, G1 and G2 cells without and with Dox induction (8 hr and 16 hr). The intracellular potassium + concentration [K ] (in mmol/L) is log10 transformed on the y-axis.

No significant difference in [K+] was observed among 16hr-Dox-induced G0 cells and non-induced or 8-hr-Dox-induced G0, G1 and G2 cells (ANOVA p=0.10), the estimated average intracellular [K+] of which was 168.0±40.1 mmol/L (total n=28 in 7 groups). The estimated intracellular [K+] for G1 and G2 cells with 16 hr Dox induction was reduced to 61.2±43.2 mmol/L (n=4) and 101.8±49.8 moml/L (n=4), respectively.

14 EV(-) EV(+) G0(-) G0(+) G1(-) G1(+) G2(-) G2(+)

APOL1

β-actin

Supplemental Figure 8. Relative APOL1 expression level is comparable in HEK293 Tet-on cells with Dox induction for 8 hr. HEK293 Tet-on cells with (+) or without (-) Dox induction were grown in complete DMEM growth media for 8 hr. Four ug of total cell lysate protein was loaded onto a 4-20% SDS-PAGE gel and probed with APOL1 antibody. HEK293 Tet-on empty pTRE2hyg vector cells did not express APOL1 with or without Dox induction. HEK293 Tet-on APOL1 G0, G1, and G2 cells expressed similar amounts of APOL1 (elevated) with Dox induction, while trace or negligible amounts of APOL1 were present in HEK293 Tet-on cells without Dox induction. The data are representative of three trials of immunoblot with similar results.

Draq5 APOL1

Mitochondria Merge

Supplemental Figure 9A Supplemental Figure 9. APOL1 co-localizes with mitochondria. A) Confocal immunofluorescence microscopy (630x) on HEK293 Tet-on G0 cells with Dox induction as representative of APOL1 colocalization with mitochondria. After fixation with 4% paraformadehyde and washing with PBS, cells were stained for APOL1 (red), ATP synthase 5A1 (ATP5A1; green), a mitochondria marker, and Draq5 (blue), a nuclear stain. 1 2 3 4

5 6 7 8

9 10 11 12 Supplemental Figure 9B Supplemental Figure 9. APOL1 co-localizes with mitochondria. B) Confocal immunofluorescence microscopy (630x) reveals the photo gallery of HEK293 Tet-on G0 with Dox induction as representative of APOL1 colocalization with mitochondria. To sufficiently display the location of APOL1 and mitochondria, only the fluorescent signals of APOL1 (red) and ATP synthase 5A1 (ATP5A1; green), a mitochondria marker, are shown in the serial photo gallery (1-12) for the z-stack model (1μm depth for each scan). Fluorescent signals for APOL1 (red) are present in mitochondria (green) and beyond. A

Dox (-)

SupplementalFigure10 Dox(+) 8hr Dox (+) 4hr APOL1

mitochondria HEK293 Tet

- on EV cells on

APOL1

mitochondria

DAPI

Dox (-)

SupplementalFigure10 Dox(+) 8hr Dox (+) 4hr APOL1

mitochondria HEK293 Tet

- on G0 cells G0 on

APOL1

mitochondria

DAPI

Dox (-)

SupplementalFigure10 Dox(+) 8hr Dox (+) 4hr APOL1

mitochondria HEK293 Tet

- on G1 cells G1 on

APOL1

mitochondria

DAPI

Dox (-)

SupplementalFigure10 Dox(+) 8hr Dox (+) 4hr APOL1

mitochondria HEK293 Tet

- on G2 cells G2 on

APOL1

mitochondria

DAPI

Supplemental Figure 10. APOL1 co-localizes with mitochondria in HEK293 Tet-on APOL1 G0, G1 and G2 cells with Dox induction

HEK293 Tet-on empty vector (EV), G0, G1 and G2 cells (panels A, B, C, and D respectively) in the absence of Dox and after 4hr or 8hr Dox induction were washed with PBS and fixed with 4% paraformaldehyde. Cells were stained for APOL1 (red) with Epitomics anti-APOL1 antibody, mitochondria (green) with ATP synthase 5 A1 (ATP5A1) antibody and counterstained with nuclear dye DAPI (blue). No APOL1 was present in empty vector cells with (+) or without (-) Dox induction. Trace amounts of APOL1 were present in G0, G1 and G2 cells without (-) Dox induction; elevated APOL1 was observed in G0, G1 and G2 cells with (+) Dox induction for 4 hr; substantially elevated APOL1 was observed in G0, G1 and G2 cells with (+) Dox induction for 8 hr.

Supplemental Figure 11 Supplemental Figure 11. Mitochondrial membrane potential is reduced in Dox-reduced HEK293 Tet-on APOL1 G1 and G2 cells. A) HEK293 Tet-on cells stably expressing APOL1 G0, G1, or G2 were grown with (+) or without (-) Dox for 7.5 hr in full DMEM media. Cells were subsequently incubated for 30 min with a final concentration of 50 nM mitotracker green (Invitrogen) and 10 nM TMRE (Tetramethylrhodamine ethyl ester, Invitrogen), a live cell fluorescence marker of mitochondrial membrane potential. Mitochondrial mass did not differ across HEK293 Tet-on APOL1 cells (+/-) Dox induction, indicated by constant mitochondrial green signals. However, HEK293 Tet-on G1 and G2 cells appeared to lose mitochondrial membrane potential when induced by Dox.

B p=0.62 p=0.08 p=3.7x10-5 p=2.2x10-5

1.5

0.5

TMRE/mitotracherratio 0 EV(-) EV(+) G0(-) G0(+) G1(-) G1(+) G2(-) G2(+) (n=4) (n=4) (n=4) (n=4) (n=4) (n=4) (n=4) (n=4)

VDAC1

COX IV

β-actin

Supplemental EV(-) EV(+) G0(-) G0(+) G1(-) G1(+) G2(-) G2(+) Figure 11 Supplemental Figure 11. Mitochondrial membrane potential is reduced in Dox-reduced HEK293 Tet-on APOL1 G1 and G2 cells. B) Reduced fluorescence signal ratio of mitochondrial membrane potential (TMRE) and mitotracker green in HEK293 Tet-on G1 and G2 cells with Dox induction. Fluorescence signals of TMRE and mitotracker green were captured using an Olympus IX71 fluorescence microscope on four different fields/groups of each type of HEK293 Tet-on cells of APOL1 genotype (+/-) Dox induction. Fluorescence signals were measured by Image J (http://imagej.nih.gov/ij/). A paired t-test was used to estimate the TMRE/mitoctracker ratio difference for cells with (+) and without (-) Dox induction after correction for non-specific background. C) Mitochondrial protein expression remains constant in HEK293 Tet-on cells with and without Dox induction. Stabilized HEK293 Tet-on cells grown with (+) or without (-) Dox were lysed and fractionated by SDS-PAGE, then probed with antibodies for mitochondrial proteins COXIV and porin/VDAC1. APOL1 G1 and G2 renal-risk variants did not alter mitochondrial mass, relative to G0 (reference), with or without Dox induction. EV: HEK293 Tet-on cell line with empty pTRE2hyg plasmid.

HEK293 EV cells Mitotracker TMRE

)

- FCCP ( FCCP

FCCP FCCP 0.25 μM μM 0.25

1.0 μM FCCP FCCP 1.0 μM

Supplemental Figure 12 HEK293 G0 cells

B Mitotracker TMRE

)

- FCCP ( FCCP

FCCP FCCP

0.25 0.25 μM

1.0 μM FCCP FCCP 1.0 μM

Supplemental Figure 12

HEK293 G1 cells Mitotracker TMRE

)

- FCCP ( FCCP

0.25 μM FCCP FCCP 0.25 μM

1.0 μM FCCP FCCP 1.0 μM

Supplemental Figure 12

HEK293 G2 cells

Mitotracker TMRE

)

- FCCP ( FCCP

μM FCCP FCCP μM 0.25 0.25

1.0 μM FCCP FCCP 1.0 μM

Supplemental Figure 12

Supplemental Figure 12. Mitochondrial membrane potential declines in HEK293 Tet-on cells with FCCP incubation

Non-induced HEK293 Tet-on empty vector (EV), G0, G1 and G2 cells (panels A, B, C and D, respectively) were incubated for 30 min with a final concentration of 50 nM mitotracker green (Invitrogen) and 10 nM TMRE (Tetramethylrhodamine ethyl ester, Invitrogen), a live cell fluorescence marker of mitochondrial membrane potential, on a 96-well cell culture plate. FCCP was subsequently added to culture media in final concentrations of 0.25 µM and 1.0 µM for 30 minutes, respectively, in cells of different genotypes together with FCCP-free cells as controls. Cell images were captured by an Olympus IX71 fluorescence microscope. The mitochondrial membrane potential remained virtually intact for EV and G0 cells treated with 0.25 µM FCCP; however, reduced mitochondrial membrane potential was observed in G1 and G2 cells treated with 0.25 µM FCCP. When EV, G0, G1 and G2 cells were treated with 1.0 µM FCCP, all types cells appeared to display reduced mitochondrial membrane potential. Supplemental Video Supplemental Video 1. Co-localization of APOL1 with mitochondria in Dox-induced HEK293 Tet-on G0 cells as assessed by confocal microscopy images (z-stack model). Serial confocal immunofluorescence microscopy (630x) was performed to form the animated video of 1 μm depth per scan for HEK293 Tet-on G0 wells, as representative of all HEK293 APOL1 cells with Dox induction for 8 hr. After washing with PBS, cells were stained for APOL1 (red), mitochondria (ATP5A1; green), corresponding to Supplemental Figure 9.

Supplemental Table 1. Top 20 pathways revealed from most differentially expressed 1056 genes by Dox-induced APOL1 over-expression in the HEK293 G0 Tet-on cell line using Cytoscape BiNGO

GO-ID Description x n X N p-value FDR p-value 43231 intracellular membrane-bounded organelle 539 8338 901 17758 1.26E-15 3.72E-12 43227 membrane-bounded organelle 539 8345 901 17758 1.54E-15 3.72E-12 5634 nucleus 349 5181 901 17758 1.66E-10 1.80E-07 3676 nucleic acid binding 240 3254 901 17758 1.66E-10 1.80E-07 44424 intracellular part 643 10939 901 17758 1.86E-10 1.80E-07 34641 cellular nitrogen compound metabolic process 168 2080 901 17758 3.30E-10 2.66E-07 5622 intracellular 656 11291 901 17758 1.04E-09 7.20E-07 44237 cellular metabolic process 334 4990 901 17758 1.28E-09 7.72E-07 44260 cellular macromolecule metabolic process 250 3511 901 17758 1.95E-09 1.05E-06 43226 organelle 558 9327 901 17758 3.25E-09 1.54E-06 43229 intracellular organelle 557 9313 901 17758 3.69E-09 1.54E-06 6807 nitrogen compound metabolic process 171 2198 901 17758 3.82E-09 1.54E-06 33554 cellular response to stress 66 622 901 17758 1.04E-08 3.86E-06 44238 primary metabolic process 344 5287 901 17758 1.80E-08 5.94E-06 3677 DNA binding 176 2329 901 17758 1.84E-08 5.94E-06 90304 nucleic acid metabolic process 122 1464 901 17758 2.39E-08 7.23E-06 8152 metabolic process 379 5955 901 17758 2.63E-08 7.49E-06 44238 primary metabolic process 344 5287 901 17758 1.80E-08 5.94E-06 3677 DNA binding 176 2329 901 17758 1.84E-08 5.94E-06 90304 nucleic acid metabolic process 122 1464 901 17758 2.39E-08 7.23E-06 Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. Replicated pathways in the subsequent Ingenuity pathway analysis are shown in red. P values are shown as scientific E notation (e.g. 2.00E-5 is equivalent to 2.00x10-5) hereafter throughout the supplemental tables.

Supplemental Table 2. Top canonical pathways detected based on the most differentially expressed genes by Dox-induced APOL1 overexpression (HEK293 G0 Tet-on cell line using Ingenuity pathway analysis)

Ingenuity Canonical p-value Molecules Pathways Unfolded protein response 9.05E-07 MAP2K7,HSPA14,DDIT3,INSIG1,XBP1,CEBPB,DNAJB9,HSPA5,CEBPG,BCL2,SEL1L,EDEM1,ERO1LB ERK5 Signaling 1.50E-04 MYC,FOS,SGK1,MEF2D,GNA12,MAP3K8,RPS6KA1,RPS6KA2,CREB3L4,ATF2,MAP3K2 NRF2-mediated Oxidative 3.13E-04 MAP2K7,MAPK1,PPIB,DNAJC19,MAF,HSPB8,HERPUD1,DNAJC10,DNAJB9,MAFF,BACH1,MAFG,FO Stress Response S,AKR1A1,SOD2,JUN,MAP2K3,GSK3B,UBE2E3,DNAJB6 p38 MAPK Signaling 3.77E-04 ATF1,DDIT3,SRF,CREB3L4,MAPK12,ATF2,MYC,DUSP1,MEF2D,DUSP10,MAP2K3,RPS6KA2,EEF2K,R PS6KA1,TAB1 tRNA Charging 3.82E-04 WARS,CARS,GARS,EARS2,AARS,SARS,IARS,EPRS Replicated pathways in the prior BiNGO pathway analysis (Table S1) are shown in red.

Supplemental Table 3. Top five upstream regulators detected based on the most differentially expressed genes by Dox-induced APOL1 overexpression (HEK293 G0 Tet-on cell line using Ingenuity Pathway Analysis)

Upstream Predicted Activation p-value Target molecules in dataset Regulator Activation State z-score ATF4 Inhibited -3.56 4.53E-20 AARS,ASNS,BCAT1,CEBPB,CEBPG,CHAC1,CTH,DDIT3,DDIT4,EIF2S2,EIF4EBP1,GAR S,GDF15,HERPUD1,HSPA5,IGFBP5,JUN,KLF4,MTHFD2,PCK2,PSAT1,PSPH,PYCR1,S ARS,SEL1L,SHMT2,SIGMAR1,SLC38A2,SLC3A2,SLC6A9,SLC7A5,STC2,TNFRSF12A,T RIB3,WARS,XPOT, tosedostat Inhibited -3.184 7.46E-16 ASNS,ASS1,CARS,CBS/LOC102724560,CEBPB,CHAC1,CTH,CXCL8,DDIT3,DDIT4,PSA T1,SARS,SLC38A2,SLC7A11,STC2,TRIB3,WARS, ASNS,CDC25A,CTH,DDIT3,DDIT4,GARS,GDF15,HERPUD1,ID1,MTHFD2,PCK2,PSAT TRIB3 Activated 3.346 1.18E-14 1,PSPH,STC2,TRIB3, ASNS,ATP2A2,CCND1,CEBPB,CPT2,CREB3L2,DDIT3,DDIT4,DNAJB9,EDEM1,EDEM2 ,EGR1,ERO1LB,GADD45B,GPT2,HERPUD1,HSPA5,KLF4,LONP1,MANF,MTDH,MYC, tunicamycin Inhibited -3.464 6.04E-14 NFKBIA,PCK2,PDIA6,PLIN2,SDF2L1,SEL1L,SIGMAR1,SLC7A11,WFS1,XBP1, ATP2A2,BCL2,BET1,COPG1,CRK,CXCL8,DDIT3,DNAJB9,DNAJC10,EDEM1,EDEM2,E RO1LB,FKBP11,FKBP14,FKBP2,GOSR2,HERPUD1,HSPA5,HYOU1,KDELR3,KLF4,MYC ,PDIA4,PDIA6,PPIB,SDF2L1,SEC11C,SEC23B,SEC24D,SEC63,SERP1,SPCS3,SSR1,STX XBP1 Inhibited -4.709 1.25E-12 5,TXNDC5,WFS1, Related regulators of the ER stress pathway detected in the prior analyses (Supplemental Table 1 & 2), are shown in red. |Activation Z score|>3.

Supplemental Table 4. Top pathways revealed from most differentially expressed genes by Dox-induced APOL1 in the HEK293 G1 Tet-on cell line using Cytoscape BiNGO

GO-ID Description x n X N p-value FDR p-value Top 20 significant pathways enriched in 1357 qualified transcripts 5634 nucleus 475 5183 1140 17760 1.00E-20 5.49E-17 43231 intracellular membrane-bounded organelle 677 8338 1140 17760 2.19E-18 5.07E-15 43227 membrane-bounded organelle 677 8345 1140 17760 2.78E-18 5.07E-15 3676 nucleic acid binding 321 3253 1140 17760 2.42E-17 3.30E-14 44424 intracellular part 829 10941 1140 17760 2.34E-16 2.55E-13 5622 intracellular 848 11293 1140 17760 6.45E-16 5.87E-13 30528 transcription regulator activity 175 1507 1140 17760 2.50E-15 1.96E-12 10556 regulation of macromolecule biosynthetic process 283 2874 1140 17760 5.93E-15 4.05E-12 60255 regulation of macromolecule metabolic process 318 3374 1140 17760 2.63E-14 1.60E-11 6357 regulation of transcription from RNA polymerase II promoter 104 747 1140 17760 3.57E-14 1.95E-11 10468 regulation of gene expression 283 2925 1140 17760 5.55E-14 2.59E-11 31323 regulation of cellular metabolic process 343 3733 1140 17760 5.69E-14 2.59E-11 34641 cellular nitrogen compound metabolic process 217 2082 1140 17760 8.30E-14 3.49E-11 3677 DNA binding 235 2328 1140 17760 1.93E-13 7.52E-11 80090 regulation of primary metabolic process 326 3551 1140 17760 3.88E-13 1.41E-10 45449 regulation of transcription 256 2619 1140 17760 4.54E-13 1.46E-10 9889 regulation of biosynthetic process 288 3042 1140 17760 4.55E-13 1.46E-10 19222 regulation of metabolic process 352 3916 1140 17760 5.14E-13 1.56E-10 44237 cellular metabolic process 428 4989 1140 17760 5.49E-13 1.58E-10 43233 organelle lumen 181 1680 1140 17760 8.59E-13 2.26E-10 Top 10 significant pathways enriched in 695 qualified transcripts downregulated by G1 over-expression 5739 mitochondrion 95 1270 579 17779 1.24E-14 4.07E-11 34641 cellular nitrogen compound metabolic process 121 2081 579 17779 9.22E-11 1.51E-07 8152 metabolic process 266 5957 579 17779 1.94E-10 2.12E-07 6807 nitrogen compound metabolic process 123 2199 579 17779 7.29E-10 5.98E-07 44237 cellular metabolic process 229 4990 579 17779 9.15E-10 6.00E-07 48037 cofactor binding 29 253 579 17779 4.23E-09 2.31E-06 3824 catalytic activity 227 5093 579 17779 1.80E-08 8.44E-06 9058 biosynthetic process 100 1797 579 17779 5.41E-08 2.22E-05 42180 cellular ketone metabolic process 44 577 579 17779 1.57E-07 5.72E-05 44238 primary metabolic process 229 5288 579 17779 1.92E-07 5.72E-05 43436 oxoacid metabolic process 43 563 579 17779 2.09E-07 5.72E-05 19752 carboxylic acid metabolic process 43 563 579 17779 2.09E-07 5.72E-05 6082 organic acid metabolic process 43 570 579 17779 2.94E-07 7.41E-05 Top 10 significant pathways enriched in 662 qualified transcripts upregulated by G1 over-expression 31323 regulation of cellular metabolic process 253 3731 563 17771 1.99E-38 6.58E-35 10556 regulation of macromolecule biosynthetic process 216 2872 563 17771 3.13E-38 6.58E-35 5634 nucleus 307 5180 563 17771 1.81E-37 2.54E-34 19222 regulation of metabolic process 258 3914 563 17771 3.31E-37 3.48E-34 10468 regulation of gene expression 216 2923 563 17771 4.92E-37 4.02E-34 9889 regulation of biosynthetic process 221 3040 563 17771 5.73E-37 4.02E-34 60255 regulation of macromolecule metabolic process 235 3372 563 17771 7.44E-37 4.34E-34 30528 transcription regulator activity 147 1506 563 17771 8.26E-37 4.34E-34 31326 regulation of cellular biosynthetic process 218 3016 563 17771 5.88E-36 2.75E-33 45449 regulation of transcription 198 2618 563 17771 9.57E-35 4.03E-32 Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. Replicated pathways in the subsequent pattern-based analyses are shown in red.

Supplemental Table 5. Top pathways revealed from most differentially expressed genes by Dox-induced APOL1 in the HEK293 G2 Tet-on cell line using Cytoscape BiNGO

GO-ID Description x n X N p-value FDR p-value Top 20 significant pathways enriched in 2409 qualified transcripts 43227 membrane-bounded organelle 1240 8327 2022 17725 3.64E-43 2.45E-39 43231 intracellular membrane-bounded organelle 1238 8320 2022 17725 8.05E-43 2.71E-39 44424 intracellular part 1495 10915 2022 17725 1.26E-35 2.84E-32 5622 intracellular 1529 11268 2022 17725 7.46E-35 1.26E-31 5634 nucleus 826 5170 2022 17725 4.44E-33 5.98E-30 43229 intracellular organelle 1295 9289 2022 17725 2.21E-29 2.48E-26 43226 organelle 1296 9303 2022 17725 2.99E-29 2.88E-26 44237 cellular metabolic process 775 4985 2022 17725 2.93E-26 2.47E-23 44260 cellular macromolecule metabolic process 577 3509 2022 17725 5.16E-24 3.86E-21 34641 cellular nitrogen compound metabolic process 381 2081 2022 17725 2.56E-23 1.72E-20 8152 metabolic process 873 5947 2022 17725 6.38E-22 3.90E-19 6807 nitrogen compound metabolic process 390 2199 2022 17725 2.92E-21 1.64E-18 90304 nucleic acid metabolic process 285 1464 2022 17725 3.61E-21 1.87E-18 6139 nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 329 1790 2022 17725 2.89E-20 1.39E-17 70013 intracellular organelle lumen 307 1642 2022 17725 5.08E-20 2.28E-17 31974 membrane-enclosed lumen 317 1713 2022 17725 5.65E-20 2.38E-17 43233 organelle lumen 310 1677 2022 17725 1.89E-19 7.50E-17 5737 cytoplasm 1056 7636 2022 17725 1.01E-18 3.77E-16 44238 primary metabolic process 775 5280 2022 17725 1.41E-18 4.99E-16 3676 nucleic acid binding 513 3247 2022 17725 2.96E-17 9.97E-15 Top 10 significant pathways enriched in 1414 qualified transcripts down-regulated by G2 over-expression 5739 mitochondrion 168 1270 1169 17765 4.03E-19 1.88E-15 48037 cofactor binding 58 253 1169 17765 2.48E-17 5.76E-14 44444 cytoplasmic part 457 5139 1169 17765 7.21E-15 1.05E-11 8152 metabolic process 514 5952 1169 17765 9.02E-15 1.05E-11 55114 oxidation reduction 96 647 1169 17765 3.28E-14 3.05E-11 50662 coenzyme binding 43 181 1169 17765 9.13E-14 7.08E-11 44237 cellular metabolic process 440 4987 1169 17765 1.42E-13 9.42E-11 34641 cellular nitrogen compound metabolic process 218 2079 1169 17765 5.25E-13 3.05E-10 43231 intracellular membrane-bounded organelle 666 8336 1169 17765 6.84E-13 3.54E-10 43227 membrane-bounded organelle 666 8343 1169 17765 8.37E-13 3.89E-10 Top 10 significant pathways enriched in 995 qualified transcripts upregulated by G2 over-expression 5634 nucleus 480 5169 860 17741 5.54E-63 2.77E-59 31323 regulation of cellular metabolic process 362 3729 860 17741 1.03E-46 2.57E-43 19222 regulation of metabolic process 371 3910 860 17741 9.14E-46 1.52E-42 10468 regulation of gene expression 302 2919 860 17741 1.09E-42 1.36E-39 10556 regulation of macromolecule biosynthetic process 298 2869 860 17741 2.37E-42 2.10E-39 60255 regulation of macromolecule metabolic process 330 3368 860 17741 2.52E-42 2.10E-39 9889 regulation of biosynthetic process 304 3036 860 17741 4.18E-40 2.99E-37 80090 regulation of primary metabolic process 335 3546 860 17741 1.27E-39 7.93E-37 31326 regulation of cellular biosynthetic process 301 3013 860 17741 2.04E-39 1.13E-36 30528 transcription regulator activity 195 1505 860 17741 3.58E-39 1.79E-36 Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. Replicated pathways in the subsequent pattern-based analyses are shown in red.

Supplemental Table 6. Top pathways detected from the most differentially expressed genes with Dox-induced APOL1 G1 vs. G0 (HEK293 Tet-on cell lines based on human Illumina HT-12 v4 arrays using Cytoscape BiNGO)

GO-ID Description x n X N p-value FDR p-value Top pathway enriched in 293 qualified transcripts 6259 DNA metabolic process 19 516 211 17787 1.32E-05 2.07E-02 Top pathway enriched in 70 qualified transcripts down-regulated by G1 5739 mitochondrion 14 1271 61 17788 7.87E-05 4.43E-02 Pathway enriched in 223 qualified transcripts up-regulated by G1 None Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. Replicated pathways in the subsequent Ingenuity pathway analysis are shown in red. Since no pathway reached the nominal significance for FDR p<0.01, only the top one with FDR p<0.05 is presented. Replicated pathway(s) in the subsequent pattern-based analyses are shown in red.

Supplemental Table 7. Top pathways detected from the most differentially expressed genes with Dox-induced APOL1 G2 vs. G0 (HEK293 Tet-on cell lines based on human Illumina HT-12 v4 arrays using Cytoscape BiNGO)

GO-ID Description x n X N p-value FDR p-value Significant pathways enriched in 609 qualified transcripts 43231 intracellular membrane-bounded organelle 298 8346 490 17771 3.03E-10 6.20E-07 43227 membrane-bounded organelle 298 8353 490 17771 3.39E-10 6.20E-07 5739 mitochondrion 70 1273 490 17771 2.07E-08 2.52E-05 43229 intracellular organelle 315 9318 490 17771 5.06E-08 4.50E-05 43226 organelle 315 9332 490 17771 6.14E-08 4.50E-05 44424 intracellular part 355 10946 490 17771 2.00E-07 1.22E-04 34641 cellular nitrogen compound metabolic process 95 2078 490 17771 3.94E-07 2.07E-04 5634 nucleus 192 5184 490 17771 9.24E-07 3.85E-04 5622 intracellular 361 11300 490 17771 9.45E-07 3.85E-04 6807 nitrogen compound metabolic process 97 2196 490 17771 1.38E-06 5.05E-04 6139 nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 80 1786 490 17771 8.48E-06 2.70E-03 33554 cellular response to stress 37 618 490 17771 8.83E-06 2.70E-03 5654 nucleoplasm 48 931 490 17771 2.29E-05 6.29E-03 90304 nucleic acid metabolic process 67 1460 490 17771 2.40E-05 6.29E-03 Significant pathways enriched in 305 qualified transcripts downregulated by G2 5739 mitochondrion 59 1272 267 17786 3.59E-15 9.14E-12 5737 cytoplasm 157 7648 267 17786 1.23E-07 1.16E-04 44444 cytoplasmic part 117 5148 267 17786 1.37E-07 1.16E-04 44429 mitochondrial part 26 611 267 17786 1.87E-06 1.19E-03 46483 heterocycle metabolic process 17 337 267 17786 1.44E-05 7.04E-03 44281 small molecule metabolic process 41 1370 267 17786 1.66E-05 7.04E-03 Top 10 significant pathways enriched in 304 qualified transcripts upregulated by G2 5634 nucleus 114 5185 223 17775 3.78E-12 9.31E-09 5488 binding 192 12356 223 17775 5.37E-09 6.61E-06 50789 regulation of biological process 122 6550 223 17775 3.66E-08 2.40E-05 5515 protein binding 142 8115 223 17775 3.90E-08 2.40E-05 50794 regulation of cellular process 117 6220 223 17775 5.71E-08 2.52E-05 3676 nucleic acid binding 74 3251 223 17775 6.15E-08 2.52E-05 44451 nucleoplasm part 25 585 223 17775 9.47E-08 3.33E-05 10468 regulation of gene expression 68 2926 223 17775 1.25E-07 3.60E-05 60255 regulation of macromolecule metabolic process 75 3375 223 17775 1.32E-07 3.60E-05 3677 DNA binding 58 2328 223 17775 1.49E-07 3.66E-05 Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. Replicated pathways in the subsequent pattern-based analyses are shown in red.

Supplemental Table 8. Pathways detected in pattern-based analysis ILLU using Cytoscape-based BiNGO for 1699 qualified genes

Number FDR p- Pattern of genes GO-ID Description x n X N p-value value 1 71 2 136 3 67 4 34 5 158 5634 nucleus 74 5183 130 17783 3.30E-11 6.90E-08 50794 regulation of cellular process 80 6221 130 17783 5.50E-10 5.76E-07 50789 regulation of biological process 81 6551 130 17783 3.02E-09 1.35E-06 31323 regulation of cellular metabolic process 57 3733 130 17783 3.54E-09 1.35E-06 19219 regulation of nucleobase, nucleoside, nucleotide and 50 3012 130 17783 3.67E-09 1.35E-06 nucleic acid metabolic process 10468 regulation of gene expression 49 2925 130 17783 4.25E-09 1.35E-06 51171 regulation of nitrogen compound metabolic process 50 3038 130 17783 4.94E-09 1.35E-06 9889 regulation of biosynthetic process 50 3042 130 17783 5.17E-09 1.35E-06 60255 regulation of macromolecule metabolic process 53 3374 130 17783 6.81E-09 1.55E-06 10556 regulation of macromolecule biosynthetic process 48 2874 130 17783 7.42E-09 1.55E-06 6 393 5739 mitochondrion 87 1273 342 17780 2.80E-26 7.93E-23 44444 cytoplasmic part 169 5147 342 17780 7.02E-16 9.92E-13 44429 mitochondrial part 45 612 342 17780 9.48E-15 8.94E-12 5737 cytoplasm 212 7644 342 17780 7.71E-13 5.45E-10 43231 intracellular membrane-bounded organelle 222 8343 342 17780 1.05E-11 5.49E-09 43227 membrane-bounded organelle 222 8350 342 17780 1.16E-11 5.49E-09 44237 cellular metabolic process 147 4987 342 17780 1.72E-09 6.43E-07 5740 mitochondrial envelope 30 437 342 17780 1.82E-09 6.43E-07 43229 intracellular organelle 231 9318 342 17780 6.99E-09 2.05E-06 44424 intracellular part 260 10947 342 17780 7.24E-09 2.05E-06 7 153 8 96 9 410 5634 nucleus 143 5181 317 17767 9.40E-10 2.19E-06 44451 nucleoplasm part 34 585 317 17767 1.49E-09 2.19E-06 5515 protein binding 195 8110 317 17767 7.50E-09 5.48E-06 50794 regulation of cellular process 160 6212 317 17767 7.92E-09 5.48E-06 50789 regulation of biological process 166 6542 317 17767 9.33E-09 5.48E-06 90304 nucleic acid metabolic process 57 1460 317 17767 1.39E-08 6.79E-06 44260 cellular macromolecule metabolic process 104 3507 317 17767 2.14E-08 8.96E-06 60255 regulation of macromolecule metabolic process 100 3372 317 17767 4.66E-08 1.71E-05 16604 nuclear body 17 191 317 17767 6.03E-08 1.97E-05 19222 regulation of metabolic process 110 3914 317 17767 1.29E-07 3.40E-05 10 31 11 54 12 18 13 71 14 7 5&9 564 5634 nucleus 215 5180 443 17761 2.83E-18 1.01E-14 50794 regulation of cellular process 239 6210 443 17761 1.04E-16 1.85E-13 50789 regulation of biological process 246 6540 443 17761 4.26E-16 5.08E-13 60255 regulation of macromolecule metabolic process 152 3370 443 17761 7.76E-15 6.94E-12 31323 regulation of cellular metabolic process 161 3729 443 17761 3.97E-14 2.84E-11 19222 regulation of metabolic process 166 3912 443 17761 5.88E-14 3.51E-11 5515 protein binding 279 8107 443 17761 8.57E-14 3.69E-11 10468 regulation of gene expression 135 2922 443 17761 8.87E-14 3.69E-11 65007 biological regulation 249 6927 443 17761 9.30E-14 3.69E-11 44451 nucleoplasm part 48 585 443 17761 3.87E-13 1.39E-10 7&13 223 5739 mitochondrion 34 1272 191 17785 6.66E-07 1.34E-03 6,7 & 601 5739 mitochondrion 121 1273 533 17774 3.82E-31 1.33E-27 13 44444 cytoplasmic part 248 5147 533 17774 2.30E-18 4.01E-15 44429 mitochondrial part 62 612 533 17774 3.06E-17 3.55E-14 43231 intracellular membrane-bounded organelle 340 8341 533 17774 1.44E-15 1.17E-12 43227 membrane-bounded organelle 340 8348 533 17774 1.67E-15 1.17E-12 5737 cytoplasm 318 7643 533 17774 3.17E-15 1.84E-12 44237 cellular metabolic process 224 4986 533 17774 1.51E-12 7.54E-10 43229 intracellular organelle 356 9316 533 17774 6.00E-12 2.62E-09 43226 organelle 356 9330 533 17774 7.72E-12 2.93E-09 44424 intracellular part 401 10944 533 17774 8.39E-12 2.93E-09

Note: x, number of gene IDs identified in the designated pathway among input gene IDs; X, number of input gene IDs selected by BiNGO; n, number of known gene IDs in the designated pathway; N, number of background gene IDs selected from the GO pool by BiNGO. FDR p value of 0.01 is defined as minimum significance for each listed pattern. For multiple significant pathways, only the top ten are included for each pattern.

Pathways significant in paired and pattern-based analyses are shown in red. Supplemental Table 9. Pathways detected in pattern-based analysis AFFY using Cytoscape-based BiNGO for 3620 qualified genes

Pattern Number corr of genes GO-ID Description x n X N p-value p-value 1 222 44085 cellular component biogenesis 24 1035 116 17786 4.47E-08 3.10E-05 51276 chromosome organization 17 525 116 17786 5.20E-08 3.10E-05 6334 nucleosome assembly 8 83 116 17786 6.73E-08 3.10E-05 5634 nucleus 61 5185 116 17786 9.62E-08 3.10E-05 31497 chromatin assembly 8 87 116 17786 9.76E-08 3.10E-05 6333 chromatin assembly or disassembly 9 127 116 17786 1.45E-07 3.10E-05 44428 nuclear part 31 1741 116 17786 1.47E-07 3.10E-05 65004 protein-DNA complex assembly 8 92 116 17786 1.51E-07 3.10E-05 34728 nucleosome organization 8 93 116 17786 1.65E-07 3.10E-05 44260 cellular macromolecule metabolic process 47 3507 116 17786 2.00E-07 3.23E-05

2 1674 5739 mitochondrion 274 1272 1357 17736 9.68E-61 5.19E-57 43231 intracellular membrane-bounded organelle 899 8330 1357 17736 3.57E-50 9.58E-47 43227 membrane-bounded organelle 899 8337 1357 17736 5.61E-50 1.00E-46 44424 intracellular part 1076 10922 1357 17736 5.39E-48 7.23E-45 5622 intracellular 1091 11275 1357 17736 1.07E-44 1.15E-41 8152 metabolic process 687 5931 1357 17736 3.45E-42 3.08E-39 44237 cellular metabolic process 605 4971 1357 17736 4.81E-42 3.69E-39 34641 cellular nitrogen compound metabolic process 329 2071 1357 17736 8.65E-42 5.80E-39 44444 cytoplasmic part 613 5133 1357 17736 5.40E-40 3.22E-37 6807 nitrogen compound metabolic process 337 2189 1357 17736 6.39E-40 3.43E-37

3 238 32502 developmental process 67 3234 185 17780 4.15E-09 1.11E-05 7275 multicellular organismal development 62 2971 185 17780 1.65E-08 2.20E-05 48856 anatomical structure development 56 2656 185 17780 7.96E-08 5.94E-05 35295 tube development 16 297 185 17780 8.98E-08 5.94E-05 9653 anatomical structure morphogenesis 34 1218 185 17780 1.11E-07 5.94E-05 48731 system development 50 2422 185 17780 9.86E-07 4.39E-04 50794 regulation of cellular process 96 6219 185 17780 1.59E-06 6.07E-04 1657 ureteric bud development 7 60 185 17780 2.86E-06 9.54E-04 50789 regulation of biological process 98 6549 185 17780 5.08E-06 1.51E-03 65007 biological regulation 102 6938 185 17780 5.99E-06 1.60E-03

4 1206 5634 nucleus 543 5167 1020 17727 6.00E-62 3.06E-58 10468 regulation of gene expression 364 2920 1020 17727 1.49E-53 3.79E-50 60255 regulation of macromolecule metabolic process 398 3369 1020 17727 2.30E-53 3.92E-50 50794 regulation of cellular process 588 6202 1020 17727 2.68E-52 3.41E-49 10556 regulation of macromolecule biosynthetic process 357 2868 1020 17727 3.95E-52 4.03E-49 19222 regulation of metabolic process 433 3910 1020 17727 3.22E-51 2.73E-48 31323 regulation of cellular metabolic process 419 3727 1020 17727 7.38E-51 5.37E-48 50789 regulation of biological process 603 6532 1020 17727 4.82E-50 3.07E-47 9889 regulation of biosynthetic process 365 3035 1020 17727 9.04E-50 5.12E-47 31326 regulation of cellular biosynthetic process 362 3012 1020 17727 3.41E-49 1.74E-46

5 63

6 68

7 21

8 88 31323 regulation of cellular metabolic process 35 3734 69 17784 4.33E-08 5.66E-05 19222 regulation of metabolic process 35 3917 69 17784 1.52E-07 6.70E-05 5634 nucleus 41 5181 69 17784 1.54E-07 6.70E-05 10468 regulation of gene expression 29 2927 69 17784 4.26E-07 1.19E-04 45449 regulation of transcription 27 2621 69 17784 6.15E-07 1.19E-04 80090 regulation of primary metabolic process 32 3553 69 17784 6.69E-07 1.19E-04 60255 regulation of macromolecule metabolic process 31 3376 69 17784 7.39E-07 1.19E-04 19219 regulation of nucleobase, nucleoside, nucleotide and 29 3013 69 17784 7.94E-07 1.19E-04 6357 nucleicregulation acid of metabolic transcription process from RNA polymerase II 14 747 69 17784 8.51E-07 1.19E-04 51171 promoterregulation of nitrogen compound metabolic process 29 3039 69 17784 9.54E-07 1.19E-04

9 7

10 33

1&2 1911 5739 mitochondrion 288 1272 1468 17733 3.07E-61 1.70E-57 43231 intracellular membrane-bounded organelle 976 8330 1468 17733 9.56E-56 2.65E-52 43227 membrane-bounded organelle 976 8337 1468 17733 1.57E-55 2.90E-52 44424 intracellular part 1164 10921 1468 17733 4.77E-52 6.61E-49 5622 intracellular 1180 11274 1468 17733 2.29E-48 2.54E-45 8152 metabolic process 750 5929 1468 17733 3.45E-48 3.19E-45 44237 cellular metabolic process 661 4970 1468 17733 5.82E-48 4.61E-45 34641 cellular nitrogen compound metabolic process 360 2070 1468 17733 2.90E-47 2.01E-44 6807 nitrogen compound metabolic process 369 2188 1468 17733 2.69E-45 1.65E-42 43229 intracellular organelle 1020 9303 1468 17733 1.15E-43 6.35E-41

3&4 1665 50794 regulation of cellular process 728 6201 1346 17713 2.90E-50 1.69E-46 5634 nucleus 640 5167 1346 17713 8.47E-50 2.47E-46 50789 regulation of biological process 750 6530 1346 17713 1.55E-48 3.01E-45 10468 regulation of gene expression 426 2920 1346 17713 7.90E-47 1.15E-43 31323 regulation of cellular metabolic process 503 3726 1346 17713 9.94E-47 1.16E-43 60255 regulation of macromolecule metabolic process 468 3369 1346 17713 3.37E-46 3.28E-43 19222 regulation of metabolic process 518 3909 1346 17713 5.52E-46 4.61E-43 10556 regulation of macromolecule biosynthetic process 418 2868 1346 17713 1.18E-45 8.61E-43 9889 regulation of biosynthetic process 432 3035 1346 17713 1.10E-44 7.11E-42 31326 regulation of cellular biosynthetic process 429 3012 1346 17713 2.20E-44 1.28E-41

Pathways significant in paired and pattern-based analyses are shown in red.

Supplemental Table 10. Top pathways detected in pattern-based analysis AFFY using IPA

Pattern Top Canonical pathways FDR p-value Target molecules in dataset 1&2 tRNA Charging 8.66E-23 CARS,GARS,DARS2,NARS2,FARSB,NARS,LARS,TARS2,FARS2,KARS,DARS,EARS2,VARS,SAR S,SARS2,IARS,IARS2,RARS2,CARS2,MARS,FARSA,EPRS,WARS,RARS,YARS,AARS2,AARS Mitochondrial Dysfunction 2.49E-08 SDHB,ATP5H,NDUFA9,PSENEN,NDUFA7,COX6A1,COX8A,NDUFB5,ACO2,DHODH,NDUFB 10,NDUFS1,SOD2,PARK7,ATPAF1,NDUFS2,CASP8,ATP5F1,NDUFAF1,UCP2,ATP5O,NDUF V3,SDHC,VDAC3,ATP5C1,COX11,NDUFV2,TXN2,UQCR10,CYC1,CAT,NDUFA12,CYB5A,CO X15 Valine Degradation I 7.04E-08 BCAT1,ECHS1,BCAT2,HIBADH,DLD,DBT,ACADSB,EHHADH,ALDH6A1,BCKDHB Isoleucine Degradation I 1.15E-06 BCAT1,ACAT2,ECHS1,BCAT2,DLD,ACAT1,ACADSB,EHHADH Oxidative Phosphorylation 1.50E-06 SDHB,ATP5H,NDUFA9,NDUFA7,COX6A1,ATP5O,NDUFV3,NDUFB5,COX8A,SDHC,NDUFB1 0,ATP5C1,NDUFS1,COX11,NDUFV2,UQCR10,CYC1,ATPAF1,NDUFA12,NDUFS2,CYB5A,AT P5F1,COX15 3 & 4 Molecular Mechanisms of Cancer 2.38E-11 RAF1,AXIN1,PIK3R1,SMAD3,TAB2,GSK3A,CCND1,RHOB,GNA13,NFKBIB,HIPK2,BIRC3,CDC 25A,E2F4,CASP3,CREBBP,GNAZ,TCF3,RASGRF2,ADCY9,DAXX,CBL,GAB1,RND3,MAP2K3,N OTCH1,RAP1B,PIK3CA,BMP2,LRP6,GNA11,BMPR2,E2F3,NFKB1,BCL2,EP300,BRAF,JUN,N FKBIA,TGFB2,SMAD4,RHOF,MAP2K1,ITGB1,ARHGEF12,GNAQ,SMAD7,ADCY6,SIN3A,FZD 8,FOS,PAK2,CDKN1A,RBPJ,BCL2L11,FZD7 TGF-β Signaling 5.30E-10 RAF1,BMP2,SMAD3,CREBBP,SKI,SMAD7,BMPR2,ACVR2B,HOXC9,INHBB,EP300,SMURF1, BCL2,FOS,JUN,PIAS4,RNF111,TGFB2,SMAD4,SMURF2,MAP2K3,TFE3,MAP2K1 ERK5 Signaling 1.48E-09 IL6ST,RPS6KB1,YWHAH,SGK1,CREBBP,GNAQ,MEF2A,EP300,FOS,GAB1,MEF2D,FOXO3,F OSL1,RPS6KA1,GNA13,WNK1,MAP3K3,ELK4,MAP3K2 Glucocorticoid Receptor Signaling 2.76E-09 PBRM1,RAF1,PIK3CA,ARID1A,YWHAH,HSPA14,SGK1,NFATC3,SMAD3,PIK3R1,ARID2,NFK B1,EP300,BCL2,NFKBIA,NFAT5,JUN,CCL2,BAG1,PPP3R1,FOXO3,TGFB2,PRKAA2,SMAD4,T AF3,NFKBIB,MAP2K1,CXCL8,CREBBP,MAP3K1,HSPA2,NCOA3,HSPA8,TAF4B,FOS,POU2F1 ,TAF5,DUSP1,CDKN1A,FKBP4,NCOA1,SMARCC1,PHF10 PTEN Signaling 2.92E-09 FOXO4,RAF1,PIK3CA,YWHAH,PIK3R1,TGFBR3,BMPR2,PDPK1,GSK3A,NFKB1,CCND1,BCL2 ,FOXO3,IGF1R,MAP2K1,ITGB1,RPS6KB1,CASP3,FGFR1,FOXG1,CNKSR3,CSNK2A2,CBL,CDK N1A,INSR,BCL2L11 Pathways significant in paired and pattern-based analyses are shown in red.

Supplemental Table 11. Most differentially expressed transcripts in primary tubular cells with two APOL1 risk variants vs. homozygous G0

Gene Symbol Description Gene Fold p-value Change SEMA3A sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A -2.45 0.001082 PREX2 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 -2.15 0.001715 NAPRT/NAPRT1 nicotinate phosphoribosyltransferase; nicotinate phosphoribosyltransferase domain containing 1 -1.96 0.002019 PIK3AP1 phosphoinositide-3-kinase adaptor protein 1 -1.9 0.004585 RAB19 RAB19, member RAS oncogene family -1.89 0.000689 ATP5J ATP synthase, H+ transporting, mitochondrial Fo complex, subunit F6; ATP synthase, H+ -1.88 0.000917 transporting, mitochondrial F0 complex, subunit F6 TRIM55 tripartite motif containing 55; tripartite motif-containing 55 -1.81 0.002754 FLOT1 flotillin 1 -1.74 0.000397 SEC14L1 SEC14-like 1 (S. cerevisiae); small Cajal body-specific RNA 16; small nucleolar RNA host gene 20 -1.71 0.000408 SCARNA16 (non-protein coding); microRNA 6516; long intergenic non-protein coding RNA 338 SNHG20 MIR6516 LINC00338 LOC100133331 uncharacterized LOC100133331; long intergenic non-protein coding RNA 1001; putative -1.71 0.001442 LINC01001 uncharacterized protein encoded by LINC00174-like; uncharacterized LOC388572; novel transcript LOC101929819 LOC388572 RP11- 206L10.3 HLA-H HLA-A major histocompatibility complex, class I, H (pseudogene); major histocompatibility complex, class -1.68 0.002199 I, A SLC44A4 solute carrier family 44, member 4 -1.67 0.001366 HLA-DQB1 XXbac- major histocompatibility complex, class II, DQ beta 1; putative novel transcript -1.62 0.001076 BPG254F23.6 VARS valyl-tRNA synthetase -1.56 0.001477 BCAR3 breast cancer anti-estrogen resistance 3 -1.52 0.001427 UBALD2 UBA-like domain containing 2 -1.51 0.004272 HM13 MCTS2P histocompatibility (minor) 13; malignant T cell amplified sequence 2, pseudogene -1.5 0.004255 DNM1 dynamin 1 1.5 0.002191 ABHD13 abhydrolase domain containing 13 1.52 0.000011 ACN9 ACN9 homolog (S. cerevisiae) 1.55 0.00175 BPNT1 3(2), 5-bisphosphate nucleotidase 1; 3'(2'), 5'-bisphosphate nucleotidase 1 1.55 0.003687 PARK7 parkinson protein 7; Parkinson disease (autosomal recessive, early onset) 7 1.56 0.003173 MEST mesoderm specific transcript; mesoderm specific transcript homolog (mouse) 1.57 0.003132 TMEM45A transmembrane protein 45A 1.64 0.000339 RP13-131K19.1 novel transcript 1.64 0.002966 MIR202 microRNA 202; MIR202 host gene (non-protein coding) 1.68 0.000036 MIR202HG ZEB2 zinc finger E-box binding homeobox 2; ZEB2 antisense RNA 1 1.69 0.00312 CNN2 calponin 2 1.71 0.000316 WRB tryptophan rich basic protein 1.72 0.00135 CHIC1 cysteine-rich hydrophobic domain 1 1.76 0.001953 MEIS2 Meis homeobox 2 1.78 0.000329 FPGT-TNNI3K FPGT-TNNI3K readthrough; fucose-1-phosphate guanylyltransferase; TNNI3 interacting kinase 1.79 0.004731 ZNF253 zinc finger protein 253 1.9 0.003295 TIMM13 translocase of inner mitochondrial membrane 13 homolog (yeast) 2.03 0.000023 SLC2A9 solute carrier family 2 (facilitated glucose transporter), member 9 2.06 0.001265 FILIP1L filamin A interacting protein 1-like 2.24 0.001918 SLIT3 slit homolog 3 (Drosophila) 2.3 0.002095 LOC101927630 uncharacterized LOC101927630; novel transcript 2.32 0.002769 AC017060.1 CTSC cathepsin C 2.35 0.004052 PDE1A phosphodiesterase 1A, calmodulin-dependent 2.51 0.000152 OR2A9P OR2A20P olfactory receptor, family 2, subfamily A, member 9 pseudogene; olfactory receptor, family 2, 2.69 0.000363 OR2A1-AS1 subfamily A, member 20 pseudogene; OR2A1 antisense RNA 1; uncharacterized LOC101928605; LOC101928605 novel transcript, antisense to OR2A9P, OR2A1 and ARHGEF5 RP4-798C17.6 FGB fibrinogen beta chain 44.95 0.004883 Note: Significant threshold is set at absolute fold change greater than 1.5 and p value less than 0.005. Gene IDs shown in red are replicated transcripts identified in HEK293 Tet-on cell pattern analysis. NAPRT/NAPRT1 falls into pattern 7 and ZEB2 into pattern 9 in Supplemental Table 8. Supplemental Table 12. Primary antibodies used in immunoblot and immunofluorescence

Antibody Vendor Catalog # Host species Dilution

Immunoblot

APOL1 Lampire Custom designed ref rabbit 1:1,000 Weckerle et al., JLR

COXIV Cell Signaling 4850 rabbit 1:1,000

Porin/VDAC1 Abcam ab14734 mouse 1:1,000

β-actin Sigma A5441 mouse 1:10,000

Immunofluorescence

APOL1 Epitomics 3245-1 rabbit 1:1,000

ATP5A1 Invitrogen 439800 mouse 1:1,000