Apolipoprotein L genes are novel mediators of inflammation in beta cells.

AIMS/HYPOTHESIS: Inflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage. METHODS: We used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-ßH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells. RESULTS: APOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus. CONCLUSIONS/INTERPRETATION: APOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes. DATA AVAILABILITY: scRNAseq data generated by our laboratory and used in this study are available in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/ ), accession number GSE218316.

Emerging Viral Infections and the Potential Impact on Hypertension, Cardiovascular Disease, and Kidney Disease.

Viruses are ubiquitous in the environment and continue to have a profound impact on human health and disease. The COVID-19 pandemic has highlighted this with impressive morbidity and mortality affecting the world's population. Importantly, the link between viruses and hypertension, cardiovascular disease, and kidney disease has resulted in a renewed focus and attention on this potential relationship. The virus responsible for COVID-19, SARS-CoV-2, has a direct link to one of the major enzymatic regulatory systems connected to blood pressure control and hypertension pathogenesis, the renin-angiotensin system. This is because the entry point for SARS-CoV-2 is the ACE2 (angiotensin-converting enzyme 2) protein. ACE2 is one of the main enzymes responsible for dampening the primary effector peptide Ang II (angiotensin II), metabolizing it to Ang-(1-7). A myriad of clinical questions has since emerged and are covered in this review. Several other viruses have been linked to hypertension, cardiovascular disease, and kidney health. Importantly, patients with high-risk apolipoprotein L1 (APOL1) alleles are at risk for developing the kidney lesion of collapsing glomerulopathy after viral infection. This review will highlight several emerging viruses and their potential unique tropisms for the kidney and cardiovascular system. We focus on SARS-CoV-2 as this body of literature in regards to cardiovascular disease has advanced significantly since the COVID-19 pandemic.

IFI16 Is Indispensable for Promoting HIF-1α-Mediated APOL1 Expression in Human Podocytes under Hypoxic Conditions.

Genetic variants in the protein-coding regions of APOL1 are associated with an increased risk and progression of chronic kidney disease (CKD) in African Americans. Hypoxia exacerbates CKD progression by stabilizing HIF-1α, which induces APOL1 transcription in kidney podocytes. However, the contribution of additional mediators to regulating APOL1 expression under hypoxia in podocytes is unknown. Here, we report that a transient accumulation of HIF-1α in hypoxia is sufficient to upregulate APOL1 expression in podocytes through a cGAS/STING/IRF3-independent pathway. Notably, IFI16 ablation impedes hypoxia-driven APOL1 expression despite the nuclear accumulation of HIF-1α. Co-immunoprecipitation assays indicate no direct interaction between IFI16 and HIF-1α. Our studies identify hypoxia response elements (HREs) in the APOL1 gene enhancer/promoter region, showing increased HIF-1α binding to HREs located in the APOL1 gene enhancer. Luciferase reporter assays confirm the role of these HREs in transcriptional activation. Chromatin immunoprecipitation (ChIP)-qPCR assays demonstrate that IFI16 is not recruited to HREs, and IFI16 deletion reduces HIF-1α binding to APOL1 HREs. RT-qPCR analysis indicates that IFI16 selectively affects APOL1 expression, with a negligible impact on other hypoxia-responsive genes in podocytes. These findings highlight the unique contribution of IFI16 to hypoxia-driven APOL1 gene expression and suggest alternative IFI16-dependent mechanisms regulating APOL1 gene expression under hypoxic conditions.

Apolipoprotein L1 (APOL1) cation current in HEK-293 cells and in human podocytes.

Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.

Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers based on five-year survival rates. Genes contributing to chemoresistance represent novel therapeutic targets that can improve treatment response. Increased expression of ANGPTL4 in tumors correlates with poor outcomes in pancreatic cancer. METHODS: We used statistical analysis of publicly available gene expression data (TCGA-PAAD) to test whether expression of ANGPTL4 and its downstream targets, ITGB4 and APOL1, were correlated with patient survival. We measured the impact of ANGPTL4 overexpression in a common pancreatic cancer cell line, MIA PaCa-2 cells, using CRISPRa for overexpression and DsiRNA for knockdown. We characterized global gene expression changes associated with high levels of ANGPTL4 and response to gemcitabine treatment using RNA-sequencing. Gemcitabine dose response curves were calculated on modified cell lines by measuring cell viability with CellTiter-Glo (Promega). Impacts on cell migration were measured using a time course scratch assay. RESULTS: We show that ANGPTL4 overexpression leads to in vitro resistance to gemcitabine and reduced survival times in patients. Overexpression of ANGPTL4 induces transcriptional signatures of tumor invasion and metastasis, proliferation and differentiation, and inhibition of apoptosis. Analyses revealed an overlapping signature of genes associated with both ANGPTL4 activation and gemcitabine response. Increased expression of the genes in this signature in patient PDAC tissues was significantly associated with shorter patient survival. We identified 42 genes that were both co-regulated with ANGPTL4 and were responsive to gemcitabine treatment. ITGB4 and APOL1 were among these genes. Knockdown of either of these genes in cell lines overexpressing ANGPTL4 reversed the observed gemcitabine resistance and inhibited cellular migration associated with epithelial to mesenchymal transition (EMT) and ANGPTL4 overexpression. CONCLUSIONS: These data suggest that ANGPTL4 promotes EMT and regulates the genes APOL1 and ITGB4. Importantly, we show that inhibition of both targets reverses chemoresistance and decreases migratory potential. Our findings have revealed a novel pathway regulating tumor response to treatment and suggest relevant therapeutic targets in pancreatic cancer.

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation.

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel's pore-lining region to the C-terminal domain (residues 332-398) and identified a membrane-insertion domain (MID, residues 177-228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368-395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.

Apolipoprotein L1 is increased in frontotemporal lobar degeneration post-mortem brain but not in ante-mortem cerebrospinal fluid.

AIMS: Frontotemporal Dementia (FTD) is caused by frontal-temporal lobar degeneration (FTLD), characterized mainly by brain protein aggregates of tau (FTLD-Tau) or TDP-43 (FTLD-TDP). The clinicopathological heterogeneity makes ante-mortem diagnosis of these pathological subtypes challenging. Our proteomics study showed increased Apolipoprotein L1 (APOL1) levels in CSF from FTD patients, which was prominently expressed in FTLD-Tau. We aimed to understand APOL1 expression in FTLD post-mortem brain tissue and to validate its potential as a CSF biomarker for FTD and its pathological subtypes. METHODS: APOL1 levels were analyzed in the frontal cortex of FTLD (including FTLD-Tau and FTLD-TDP) and non-demented controls by immunohistochemistry (FTLD total = 18 (12 FTLD-Tau and 6 FTLD-TDP); controls = 9), western blot (WB), and a novel prototype ELISA (FTLD total = 44 (21 FTLD-Tau and 23 FTLD-TDP); controls = 9). The association of APOL1 immunoreactivity with phosphorylated Tau (pTau) and TDP-43 (pTDP-43) immunoreactivity was assessed. CSF APOL1 was analyzed in confirmed FTD patients (n = 27, including 12 FTLD-Tau and 15 FTLD-TDP) and controls (n = 15) using the same ELISA. RESULTS: APOL1 levels were significantly increased in FTLD post-mortem tissue compared to controls as measured by immunohistochemistry, WB, and ELISA. However, no differences between the pathological subtypes were observed. APOL1 immunoreactivity was present in neuronal and glial cells but did not co-localize with pTau or pTDP-43. CSF APOL1 levels were comparable between FTD patients and controls and between pathological subtypes. CONCLUSION: APOL1 is upregulated in FTLD pathology irrespective of the subtypes, indicating a role of this novel protein in FTD pathophysiology. The APOL1 levels detected in brain tissue were not mirrored in the CSF, limiting its potential as a specific FTD biofluid-based biomarker using our current immunoassay.

Treatment potential in APOL1-associated nephropathy.

PURPOSE OF REVIEW: More than 5 million African-Americans, and millions more in Africa and worldwide, possess apolipoprotein L1 gene (APOL1) high-risk genotypes with an increased risk for chronic kidney disease. This manuscript reviews treatment approaches for slowing the progression of APOL1-associated nephropathy. RECENT FINDINGS: Since the 2010 discovery of APOL1 as a cause of nondiabetic nephropathy in individuals with sub-Saharan African ancestry, it has become apparent that aggressive hypertension control, renin-angiotensin system blockade, steroids and conventional immunosuppressive agents are suboptimal treatments. In contrast, APOL1-mediated collapsing glomerulopathy due to interferon treatment and HIV infection, respectively, often resolve with cessation of interferon or antiretroviral therapy. Targeted therapies, including APOL1 small molecule inhibitors, APOL1 antisense oligonucleotides (ASO) and inhibitors of APOL1-associated inflammatory pathways, hold promise for these diseases. Evolving therapies and the need for clinical trials support the importance of increased use of APOL1 genotyping and kidney biopsy. SUMMARY: APOL1-associated nephropathy includes a group of related phenotypes that are driven by the same two genetic variants in APOL1. Clinical trials of small molecule inhibitors, ASO, and inflammatory pathway inhibitors may improve outcomes in patients with primary forms of APOL1-associated nephropathy.

A Four Autophagy-Related Gene-Based Prognostic Signature for Pancreatic Cancer.

This study aimed to screen autophagy-related genes (ARGs) that affect the prognosis of pancreatic cancer patients based on The Cancer Genome Atlas (TCGA) gene expression data and genotype-tissue expression (GTEx) databases. The expression data of pancreatic cancer and normal pancreas were downloaded from TCGA and GTEx databases. Human ARGs list was obtained through the Human Autophagy Database (HADB) and GeneCards database. The Wilcox test was performed to screen differentially expressed ARGs. Differentially expressed ARGs were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. The CIBER-SORT algorithm was utilized to analyze immune cell infiltration in samples. A total of 21 up-regulated ARGs and 11 down-regulated ARGs were screened in the TCGA-GTEx integrated data set. The enrichment analysis of GO and KEGG showed that 32 differentially expressed ARGs were significantly enriched in autophagy-related pathways. Univariate Cox regression analysis showed that 12 candidate ARGs were significantly related to the prognosis of pancreatic cancer patients. Multivariate Cox regression analysis found that ATG16L2, GNAI3, APOL1, and PTK6 genes may be the key ARGs affecting the prognosis of pancreatic cancer patients. Based on these four key ARGs, a prognostic risk assessment model was constructed, and pancreatic cancer patients were classified into the high-risk and low-risk group according to the risk value. Survival analysis and ROC analysis confirmed that the prognostic risk assessment model can accurately predict the prognosis of patients with pancreatic cancer. Immune infiltration analysis found that B cells naive, B cells memory, plasma cells, T cells CD8, T cells CD4 memory resting, monocytes and macrophages M0 were significantly different in tissue samples of pancreatic cancer patients in the high and low risk groups. Pearson's correlation coefficient showed that the four key ARGs may affect the development of pancreatic cancer by affecting immune cell components in the tumor micro-environment. In conclusion, ATG16L2, GNAI3, APOL1, and PTK6 may be related to the prognosis of pancreatic cancer patients. The prognostic risk assessment model constructed based on these four key ARGs could accurately predict the prognosis of pancreatic cancer patients.

Knockdown of lncRNA MALAT1 ameliorates acute kidney injury by mediating the miR-204/APOL1 pathway.

BACKGROUND: Acute kidney injury (AKI) was characterized by loss of renal function, associated with chronic kidney disease, end-stage renal disease, and length of hospital stay. Long non-coding RNAs (lncRNAs) participated in AKI development and progression. Here, we aimed to investigate the roles and mechanisms of lncRNA MALAT1 in AKI. METHODS: AKI serum samples were obtained from 129 AKI patients. ROC analysis was conducted to confirm the diagnostic value of MALAT1 in differentiating AKI from healthy volunteers. After hypoxic treatment on HK-2 cells, the expressions of inflammatory cytokines, MALAT1, miR-204, APOL1, p65, and p-p65, were measured by RT-qPCR and Western blot assays. The targeted relationship between miR-204 and MALAT1 or miR-204 and APOL1 was determined by luciferase reporter assay and RNA pull-down analysis. After transfection, CCK-8, flow cytometry, and TUNEL staining assays were performed to evaluate the effects of MALAT1 and miR-204 on AKI progression. RESULTS: From the results, lncRNA MALAT1 was strongly elevated in serum samples from AKI patients, with the high sensitivity and specificity concerning differentiating AKI patients from healthy controls. In vitro, we established the AKI cell model after hypoxic treatment. After experiencing hypoxia, we found significantly increased MALAT1, IL-1ß, IL-6, and TNF-α expressions along with decreased miR-204 level. Moreover, the targeted relationship between MALAT1 and miR-204 was confirmed. Silencing of MALAT1 could reverse hypoxia-triggered promotion of HK-2 cell apoptosis. Meanwhile, the increase of IL-1ß, IL-6, and TNF-α after hypoxia treatment could be repressed by MALAT1 knockdown as well. After co-transfection with MALAT1 silencing and miR-204 inhibition, we found that miR-204 could counteract the effects of MALAT1 on HK-2 cell progression and inflammation after under hypoxic conditions. Finally, NF-κB signaling was inactivated while APOL1 expression was increased in HK-2 cells after hypoxia treatment, and lncRNA MALAT1 inhibition reactivated NF-κB signaling while suppressed APOL1 expression by sponging miR-204. CONCLUSIONS: Collectively, these results illustrated that knockdown of lncRNA MALAT1 could ameliorate AKI progression and inflammation by targeting miR-204 through APOL1/NF-κB signaling.

ApoL1 induces kidney inflammation through RIG-I/NF-κB activation.

The genetic variations of the apolipoprotein L1 (APOL1) gene are associated with non-diabetic kidney diseases. However, very little is known about the role of ApoL1 in glomerular damage. Here, we aimed to identify the function and mechanism of ApoL1 in glomerular damage. The mice were randomly divided into two groups: one group was intraperitoneally injected with phosphate buffer saline (PBS), while the other group was intraperitoneally injected with recombinant ApoL1 every other day for 3 months. Hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining were used to demonstrate the effects of ApoL1 on kidney inflammation and injury. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses revealed that ApoL1-treated mice exhibited enhanced expression of various inflammation markers in the kidney and serum compared to the PBS-treated mice. Immunofluorescence staining revealed that ApoL1 accumulated in kidney podocytes. Treatment with ApoL1 dose-dependently increased the expression of inflammation markers and apoptotic markers. The abnormal gene expression associated with ApoL1-mediated podocyte inflammation was evaluated using microarray analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the upregulated genes were enriched in the inflammation-related processes, such as the RIG-I/NF-κB signaling pathway. Consistently, the knockdown of RIG-I significantly mitigated the ApoL1-induced upregulation of inflammatory and apoptotic markers in the human podocytes. Additionally, the ApoL1-induced glomerular damage was attenuated in AAV-shRIG-I mice. Therefore, the effects of ApoL1 on glomerular damage may be, at least partially, through inducing abnormal expression of inflammatory molecules, which may have important implications for treatment of kidney diseases.

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1.

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, 'inhibitor of cysteine peptidase', had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum.

APOL1-Associated Collapsing Focal Segmental Glomerulosclerosis in a Patient With Stimulator of Interferon Genes (STING)-Associated Vasculopathy With Onset in Infancy (SAVI).

Apolipoprotein L1 (APOL1) risk variants G1 and G2 are known to result in risk for kidney disease in patients of African ancestry. APOL1-associated nephropathy typically occurs in association with certain environmental factors or systemic diseases. As such, there has been increasing evidence of the role of interferon (IFN) pathways in the pathogenesis of APOL1-associated collapsing glomerulopathy in patients with human immunodeficiency virus (HIV) infection and systemic lupus erythematosus, 2 conditions that are associated with high IFN levels. Collapsing glomerulopathy has also been described in patients receiving exogenous IFN therapy administered for various medical conditions. We describe a patient with a genetic condition that results in an increased IFN state, stimulator of IFN genes (STING)-associated vasculopathy with onset in infancy (SAVI), who developed collapsing glomerulopathy during a flare of his disease. The patient was found to have APOL1 G1 and G2 risk variants. This case supports the role of IFN in inducing APOL1-associated collapsing glomerulopathy.

An Acidic Environment Induces APOL1-Associated Mitochondrial Fragmentation.

BACKGROUND: Apolipoprotein L1 gene (APOL1) G1 and G2 kidney-risk variants (KRVs) cause CKD in African Americans, inducing mitochondrial dysfunction. Modifying factors are required, because a minority of individuals with APOL1 high-risk genotypes develop nephropathy. Given that APOL1 function is pH-sensitive and the pH of the kidney interstitium is <7, we hypothesized the acidic kidney interstitium may facilitate APOL1 KRV-induced mitochondrial dysfunction. METHODS: Human embryonic kidney (HEK293) cells conditionally expressing empty vector (EV), APOL1-reference G0, and G1 or G2 KRVs were incubated in media pH 6.8 or 7.4 for 4, 6, or 8 h. Genotype-specific pH effects on mitochondrial length (µm) were assessed using confocal microscopy in live cells and Fiji derivative of ImageJ software with MiNA plug-in. Lower mitochondrial length indicated fragmentation and early dysfunction. RESULTS: After 6 h doxycycline (Dox) induction in pH 6.8 media, G2-expressing cells had shorter mitochondria (6.54 ± 0.40) than cells expressing EV (7.65 ± 0.72, p = 0.02) or G0 (7.46 ± 0.31, p = 0.003). After 8 h Dox induction in pH 6.8 media, both G1- (6.21 ± 0.26) and G2-expressing cells had shorter mitochondria (6.46 ± 0.34) than cells expressing EV (7.13 ± 0.32, p = 0.002 and p = 0.008, respectively) or G0 (7.22 ± 0.45, p = 0.003 and p = 0.01, respectively). Mitochondrial length in cells incubated in pH 7.4 media were comparable after 8 h Dox induction regardless of genotype. APOL1 mRNA expression and cell viability were comparable regardless of pH or genotype after 8 h Dox induction. CONCLUSION: Acidic pH facilitates early mitochondrial dysfunction induced by APOL1 G1 and G2 KRVs in HEK293 cells. We propose that the acidic kidney interstitium may play a role in APOL1-mediated mitochondrial pathophysiology and nephropathy.

APOL1 renal risk variants exacerbate podocyte injury by increasing inflammatory stress.

BACKGROUND: Apolipoprotein L1, APOL1, is a trypanosome lytic factor present in human and certain other primates. APOL1 gene variants, present in individuals of recent sub-Saharan African descent, increase risk for glomerular disease and associate with the disease progression, but the molecular mechanisms have not been defined. OBJECTIVES: We focus on the mechanism how APOL1 variant proteins enhance podocyte injury in the stressed kidney. METHODS: First, we investigated the expression of APOL1 protein isoform and the localization of APOL1 protein in the kidney. Next, we examined the role of APOL1 in the podocyte stress and the inflammatory signaling in the kidney after hemi-nephrectomy. RESULTS: We identified a novel RNA variant that lacks a secretory pathway signal sequence and we found that the predicted APOL1-B3 protein isoform was expressed in human podocytes in vivo and by BAC-APOL1 transgenic mice. APOL1-B3-G2 transgenic mice, carrying a renal risk variant, manifested podocyte injury and increased pro-IL-1ß mRNA in isolated glomeruli and increased IL-1ß production in the remnant kidney after uninephrectomy. APOL1-B3 interacted with NLRP12, a key regulator of Toll-like receptor signaling. CONCLUSIONS: These results suggest a possible mechanism for podocyte injury by which one of the APOL1 protein isoforms, APOL1-B3 and its renal risk variants, enhances inflammatory signaling.

Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu.

We hypothesized that a functional apolipoprotein LI (APOL1)-miR193a axis (inverse relationship) preserves, but disruption alters, the podocyte molecular phenotype through the modulation of autophagy flux. Podocyte-expressing APOL1G0 (G0-podocytes) showed downregulation but podocyte-expressing APOL1G1 (G1-podocytes) and APOL1G2 (G2-podocytes) displayed enhanced miR193a expression. G0-, G1-, and G2-podocytes showed enhanced expression of light chain (LC) 3-II and beclin-1, but a disparate expression of p62 (low in wild-type but high in risk alleles). G0-podocytes showed enhanced, whereas G1- and G2-podocytes displayed decreased, phosphorylation of Unc-51-like autophagy-activating kinase (ULK)1 and class III phosphatidylinositol 3-kinase (PI3KC3). Podocytes overexpressing miR193a (miR193a-podocytes), G1, and G2 showed decreased transcription of PIK3R3 (PI3KC3's regulatory unit). Since 3-methyladenine (3-MA) enhanced miR193a expression but inhibited PIK3R3 transcription, it appears that 3-MA inhibits autophagy and induces podocyte dedifferentiation via miR193a generation. miR193a-, G1-, and G2-podocytes also showed decreased phosphorylation of mammalian target of rapamycin (mTOR) that could repress lysosome reformation. G1- and G2-podocytes showed enhanced expression of run domain beclin-1-interacting and cysteine-rich domain-containing protein (Rubicon); however, its silencing prevented their dedifferentiation. Docking, protein-protein interaction, and immunoprecipitation studies with antiautophagy-related gene (ATG)14L, anti-UV radiation resistance-associated gene (UVRAG), or Rubicon antibodies suggested the formation of ATG14L complex I and UVRAG complex II in G0-podocytes and the formation of Rubicon complex III in G1- and G2-podocytes. These findings suggest that the APOL1 risk alleles favor podocyte dedifferentiation through blockade of multiple autophagy pathways.

APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

The mechanism that explains the association of APOL1 variants with nondiabetic kidney diseases in African Americans remains unclear. Kidney disease risk is inherited as a recessive trait, and many studies investigating the intracellular function of APOL1 have indicated the APOL1 variants G1 and G2 are associated with cytotoxicity. Whether cytotoxicity results from the absence of a protective effect conferred by the G0 allele or is induced by a deleterious effect of variant allele expression has not be conclusively established. A central issue hampering basic biology studies is the lack of model systems that authentically replicate APOL1 expression patterns. APOL1 is present in humans and a few other primates and appears to have important functions in the kidney, as the kidney is the primary target for disease associated with the genetic variance. There have been no studies to date assessing the function of untagged APOL1 protein under native expression in human or primate kidney cells, and no studies have examined the heterozygous state, a disease-free condition in humans. A second major issue is the chronic kidney disease (CKD)-associated APOL1 variants are conditional mutations, where the disease-inducing function is only evident under the appropriate environmental stimulus. In addition, it is possible there may be more than one mechanism of pathogenesis that is dependent on the nature of the stressor or other genetic variabilities. Studies addressing the function of APOL1 and how the CKD-associated APOL1 variants cause kidney disease are challenging and remain to be fully investigated under conditions that faithfully model known human genetics and physiology.

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

HDL in CKD-The Devil Is in the Detail.

The picture of HDL cholesterol (HDL-C) as the "good" cholesterol has eroded. This is even more surprising because there exists strong evidence that HDL-C is associated with cardiovascular disease (CVD) in the general population as well as in patients with impairment of kidney function and/or progression of CKD. However, drugs that dramatically increase HDL-C have mostly failed to decrease CVD events. Furthermore, genetic studies took the same line, as genetic variants that have a pronounced influence on HDL-C concentrations did not show an association with cardiovascular risk. For many, this was not surprising, given that an HDL particle is highly complex and carries >80 proteins and several hundred lipid species. Simply measuring cholesterol might not reflect the variety of biologic effects of heterogeneous HDL particles. Therefore, functional studies and the involvement of HDL components in the reverse cholesterol transport, including the cholesterol efflux capacity, have become a further focus of study during recent years. As also observed for other aspects, CKD populations behave differently compared with non-CKD populations. Although clear disturbances have been observed for the "functionality" of HDL particles in patients with CKD, this did not necessarily translate into clear-cut associations with outcomes.

Apolipoprotein L1 confers pH-switchable ion permeability to phospholipid vesicles.

Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases. ApoL1 has been hypothesized to function like a pore-forming colicin and has been reported to have permeability effects on both intracellular and plasma membranes. Here, to gain insight into how ApoL1 may function in vivo, we used vesicle-based ion permeability, direct membrane association, and intrinsic fluorescence to study the activities of purified recombinant ApoL1. We found that ApoL1 confers chloride-selective permeability to preformed phospholipid vesicles and that this selectivity is strongly pH-sensitive, with maximal activity at pH 5 and little activity above pH 7. When ApoL1 and lipid were allowed to interact at low pH and were then brought to neutral pH, chloride permeability was suppressed, and potassium permeability was activated. Both chloride and potassium permeability linearly correlated with the mass of ApoL1 in the reaction mixture, and both exhibited lipid selectivity, requiring the presence of negatively charged lipids for activity. Potassium, but not chloride, permease activity required the presence of calcium ions in both the association and activation steps. Direct assessment of ApoL1-lipid associations confirmed that ApoL1 stably associates with phospholipid vesicles, requiring low pH and the presence of negatively charged phospholipids for maximal binding. Intrinsic fluorescence of ApoL1 supported the presence of a significant structural transition when ApoL1 is mixed with lipids at low pH. This pH-switchable ion-selective permeability may explain the different effects of ApoL1 reported in intracellular and plasma membrane environments.