Acidic Environment Facilitates Mitochondrial Fragmentation Induced by APOL1 Renal‐Risk‐Variants in Kidney Cells

Abstract

African Americans frequently carry G1 and G2 renal‐risk variants of the apolipoprotein L1 (APOL1) gene. Two copies of G1 and/or G2 confer 5–29 fold higher risk of chronic kidney disease (CKD); however, only 20% of those carrying risk‐genotype develop CKD. This indicates that an additional stressor may facilitate development of CKD in those carrying G1/G2 risk variants. Considering that a) G1/G2 variants elicit mitochondrial dysfunction in renal cells, b) APOL1 function is pH sensitive and c) renal interstitial pH is <7, we hypothesized that acidic interstitial environment in the kidney may amplify mitochondrial dysfunction induced by G1/G2 variants. To test the hypothesis, we incubated human embryonic kidney (HEK293) cells expressing APOL1 in pH=6.8 or 7.4; we estimated early mitochondrial dysfunction by measuring mitochondrial network morphology with confocal microscopy (Fig.1). HEK293 cells stably expressing APOL1 G0, G1, G2 or empty vector (EV) under doxycycline (Dox) control were exposed to Dox for 4, 6 or 8hr, while cultured in media pH=6.8 or pH=7.4. Confocal images were acquired at 1000 magnification in live cells kept in an environmentally‐controlled chamber at each time point; FIJI software was used to quantify relative mitochondrial length (ratio of “rods” vs. “network‐branches”). We also ascertained that a) APOL1 expression was comparable regardless of pH or APOL1 genotype for up to 8hr post Dox‐induction using real‐time PCR and b) cell viability was comparable under all conditions using lactate‐dehydrogenase‐based cytotoxicity assay. Without Dox, relative mitochondrial length was similar regardless of APOL1 genotype or pH. However, after 6hr of Dox‐induction in media pH=6.8, G2‐expressing cells had shorter mitochondrial length (6.54±0.40) vs. EV (7.65±0.72, p=0.02) or G0 cells (7.46±0.31, p=0.003), indicating early mitochondrial fragmentation. After 8hr of doxycycline‐induction in media pH=6.8, mitochondrial length in G1 (6.21±0.26) and G2 cells (6.46±0.34) was lower than EV (7.13±0.32, p=0.002 and p=0.008, respectively) and G0 cells (7.22±0.45, p=0.003 and p=0.01, respectively; Fig. 2). At media pH 7.4, mitochondrial length was comparable up to 8hr of Dox‐induction, regardless of APOL1 genotype. We concluded that acidic pH may facilitate early mitochondrial dysfunction induced by G1/G2 APOL1 risk‐variants in renal cells. We propose that an acidic interstitial pH may facilitate adverse effects of APOL1 renal‐risk variants in the kidney, but not other organs expressing APOL1.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.