Kidney Disease-Associated APOL1 Variants Have Dose-Dependent, Dominant Toxic Gain-of-Function

Abstract

BackgroundTwo coding renal risk variants (RRVs) of the APOL1 gene (G1 and G2) are associated with large increases in CKD rates among populations of recent African descent, but the underlying molecular mechanisms are unknown. Mammalian cell culture models are widely used to study cytotoxicity of RRVs, but results have been contradictory. It remains unclear whether cytotoxicity is RRV-dependent or driven solely by variant-independent overexpression. It is also unknown whether expression of the reference APOL1 allele, the wild-type G0, could prevent cytotoxicity of RRVs.MethodsWe generated tetracycline-inducible APOL1 expression in human embryonic kidney HEK293 cells and examined the effects of increased expression of APOL1 (G0, G1, G2, G0G0, G0G1, or G0G2) on known cytotoxicity phenotypes, including reduced viability, increased swelling, potassium loss, aberrant protein phosphorylation, and dysregulated energy metabolism. Furthermore, whole-genome transcriptome analysis examined deregulated canonical pathways.ResultsAt moderate expression, RRVs but not G0 caused cytotoxicity in a dose-dependent manner that coexpression of G0 did not reduce. RRVs also have dominant effects on canonical pathways relevant for the cellular stress response.ConclusionsIn HEK293 cells, RRVs exhibit a dominant toxic gain-of-function phenotype that worsens with increasing expression. These observations suggest that high steady-state levels of RRVs may underlie cellular injury in APOL1 nephropathy, and that interventions that reduce RRV expression in kidney compartments may mitigate APOL1 nephropathy.

A glomerular transcriptomic landscape of APOL1 in Black patients with focal segmental glomerulosclerosis

“…There was significant enrichment for terms such as “mitochondrial inner membrane,” “oxidative phosphorylation,” “mitochondrial respiratory chain complex I,” “mRNA splicing,” and “ribonucleoprotein” ( Table S8 ). Previous studies have implicated mitochondrial dysfunction, abnormal energetics and reduced transcription in APOL1 associated FSGS 14-16, 21, 48, 49 . Given its large size and its enrichment for genes, subcellular structures, and pathways previously implicated in APOL1 FSGS, we focused on more deeply characterizing the Pink module and its submodules.…”

“…A major distinction between HR and LR FSGS related to differences in expression and/or co-expression of genes localized to mitochondrial compartments and/or processes. In the past decade, many experimental studies using inducible cell lines have linked increased HR APOL1 expression to mitochondrial dysfunction 14-16, 21, 48, 49 . Here, we extend these in vitro findings to patients with FSGS, increasing the relevancy and importance of dissecting these pathways.…”

“…While APOL1 is absent outside of humans and some primates, transgenic animals and cell lines have provided insights into disease pathogenesis 7-9 . Insights include; (1) increased APOL1 expression by immune activation 10 (2) toxicity associated with the RV and increased expression 8, 11 , (3) APOL1 ion channel formation and function dependent on complex intracellular trafficking and pH changes 12, 13 , and (4) multiple pathways implicated in cellular damage including mitochondrial dysfunction 14-16 , inflammasome activation 17 , ER stress 18, 19 , increased cation transport 13, 20 , and global protein synthesis inhibition 21,22 .…”

APOL1 genotype-associated morphologic changes among patients with focal segmental glomerulosclerosis

“…The characteristics of the participants recruited and samples collected in the NEPTUNE study provided us with a patient population and set of data with which to ask some fundamental questions about the relationship between APOL1 genotype, glomerular expression, and kidney tissue damage in unique ways: (1) the median age of the cohort at the time of biopsy was 16 years old (54% pediatric onset overall) which gave us a chance to study the morphologic consequences of APOL1 in a cohort younger than previously reported; (2) we restricted our cohort to only those of self-reported Black race or African genetic ancestry, which allowed us to make comparisons between risk genotypes and expression among these patients only. This prevented confounding by overall differences in genetic ancestry or by societal differences between races (e.g., structural racism against Black individuals) [28]; (3) the spectrum of patients with different number of risk alleles and available expression data gave us the opportunity to study whether single copies of risk variants and/or increased glomerular expression of APOL1 was associated with kidney damage, as reported in other studies [9,[29][30][31]; (4) This is a case-case study, which allows us to ask questions about how APOL1 may be causing a unique form of nephrotic syndrome, rather than a case-control study comparing APOL1-associated NS tissue to normal tissue; (5) the availability of 83 validated histologic and ultrastructural descriptors from light and electron microscopy created and measured as part of the NEPTUNE protocol allowed a level of morphologic granularity beyond that typically reported in clinical biopsy reports.…”

The Relationship between APOL1 Structure and Function: Clinical Implications

“…69 Hence it is possible that APOL1-G0 related protective cellular processes are "activated" in response to an external second stress, which explains why not all individuals with two copies of APOL1 G1 and/or G2 variants develop kidney disease. However, APOL1-G1 and -G2 appears to change cellular localization and oligomerization pattern 36 with associated cytotoxicity, which was not rescued by APOL1-G0 70 in in vitro studies suggesting that a dominant gain-of-function could also mediate CKD pathogenesis.…”