The recent and exclusively in humans and a few other higher primates expressed APOL1 (Apolipoprotein L1) gene is linked to African human trypanosomiasis (also known as African sleeping sickness) as well as to different forms of kidney diseases. Whereas APOL1’s role as a trypanolytic factor is well established, pathobiological mechanisms explaining its cytotoxicity in renal cells remain unclear. In this study, we compared the APOL family members using a combination of evolutionary studies and cell biological experiments to detect unique features causal for APOL1 nephrotoxic effects. We investigated available primate and mouse genome and transcriptome data to apply comparative phylogenetic and maximum likelihood selection analyses. We suggest that the APOL gene family evolved early in vertebrates and initial splitting occurred in ancestral mammals. Diversification and differentiation of functional domains continued in primates, including developing the two members APOL1 and APOL2. Their close relationship could be diagnosed by sequence similarity and a shared ancestral insertion of an AluY transposable element. Live cell imaging analyzes showed that both expressed proteins show a strong preference to localize at the endoplasmic reticulum (ER). However, glycosylation and secretion assays revealed that—unlike APOL2—APOL1 membrane insertion or association occurs in different orientations at the ER, with the disease-associated mutants facing either the luminal (cis) or cytoplasmic (trans) side of the ER. The various pools of APOL1 at the ER offer a novel perspective in explaining the broad spectrum of its observed toxic effects.

Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons control blood filtration, as well as resorption and recycling of inorganic and organic ions, sugars, amino acids, peptides, vitamins, water and further metabolites of endogenous and exogenous origin. All nephron segments consist of polarized cells and express high levels of Pals1. Mice that are functionally haploid for Pals1 develop a lethal phenotype, accompanied by heavy proteinuria and the formation of renal cysts. However, on a cellular level, it is still unclear if reduced cell polarization, incomplete cell-cell contact formation, or an altered Pals1-dependent gene expression accounts for the renal phenotype. To address this, we analyzed the transcriptomes of Pals1-haploinsufficient kidneys and the littermate controls by gene set enrichment analysis. Our data elucidated a direct correlation between TGFβ pathway activation and the downregulation of more than 100 members of the solute carrier (SLC) gene family. Surprisingly, Pals1-depleted nephrons keep the SLC’s segment-specific expression and subcellular distribution, demonstrating that the phenotype is not mainly due to dysfunctional apicobasal cell polarization of renal epithelia. Our data may provide first hints that SLCs may act as modulating factors for renal cyst formation.

The evolutionarily conserved Crumbs (CRB) polarity complex, which consists of the core components CRB3a, PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. To address the role of PALS1 at the cellular level, we generated PALS1 knockout MDCKII cell lines using the CRISPR/Cas9 system. The loss of PALS1 resulted in increased paracellular permeability indicative of an epithelial barrier defect. This barrier defect was associated with a redistribution of several tight junction-associated proteins from bicellular cell-cell contacts to tricellular junctions. The regulation of tight junction protein localization at bicellular junctions by PALS1 was dependent on its interaction with PATJ. Together, our data uncover a critical role of PALS1 in the correct positioning of tight junction proteins to bicellular junctions.