https://orcid.org/0000-0002-5237-1626
Figures
Abstract
The mechanism of pathogenesis associated with APOL1 polymorphisms and risk for non-diabetic chronic kidney disease (CKD) is not fully understood. Prior studies have minimized a causal role for the circulating APOL1 protein, thus efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. Of the kidney cells reported to express APOL1, the proximal tubule expression patterns are inconsistent in published reports, and whether APOL1 is synthesized by the proximal tubule or possibly APOL1 protein in the blood is filtered and reabsorbed by the proximal tubule remains unclear. Using both protein and mRNA in situ methods, the kidney expression pattern of APOL1 was examined in normal human and APOL1 bacterial artificial chromosome transgenic mice with and without proteinuria. APOL1 protein and mRNA was detected in podocytes and endothelial cells, but not in tubular epithelia. In the setting of proteinuria, plasma APOL1 protein did not appear to be filtered or reabsorbed by the proximal tubule. A side-by-side examination of commercial antibodies used in prior studies suggest the original reports of APOL1 in proximal tubules likely reflects antibody non-specificity. As such, APOL1 expression in podocytes and endothelia should remain the focus for mechanistic studies in the APOL1-mediated kidney diseases.
Citation: Blessing NA, Wu Z, Madhavan SM, Choy JW, Chen M, Shin MK, et al. (2021) Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys. PLoS ONE 16(6): e0253197. https://doi.org/10.1371/journal.pone.0253197
Editor: Stuart E. Dryer, University of Houston, UNITED STATES
Received: February 11, 2021; Accepted: May 30, 2021; Published: June 17, 2021
Copyright: © 2021 Blessing et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was supported by the National Institutes of Health (www.nih.gov) grants DK108329 (LAB, JRS, JFO), DK095832 (JRS, JFO, LAB), and training grant DK007470 (NAB, JRS). The NIH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Merck and Company provided support in the form of salaries and laboratory resources for authors (JWC, MC, MKS, MH), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Maze Therapeutics provided support in the form of salary for the author (MH) but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are stated in the author contributions section.
Competing interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: The authors MH, JC, MC, and MKS were employees of Merck & Company, Inc. during the conduct of this study. There are no patents, products in development or marketed products to declare. The author MH was an employee of Maze Therapeutics during the conduct of this study. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Introduction
Polymorphisms in the APOL1 gene contribute significant risk for several forms of non-diabetic chronic kidney disease (CKD) [1–3]. This risk arises from a combination of recessive inheritance of variant APOL1 alleles plus exposure to an environmental stressor. The pathogenic function of the APOL1 variants and how they interact with the environmental stressor to cause CKD are not fully understood. Although APOL1 is constitutively present in the circulation, prior studies have minimized a causal role for the circulating APOL1 protein [4–7], and efforts to understand kidney pathogenesis have focused on APOL1 expressed in renal cells. The APOL1 kidney expression pattern remains unclear with published discrepancies between immunohistochemistry and mRNA in situ hybridization results, most notably the abundant APOL1 protein observed in the proximal tubule epithelium [8–10]. Since APOL1 is abundant in blood, it is unclear if APOL1 is filtered, especially in the setting of proteinuria, which could result in APOL1 protein reabsorption by the proximal tubule. Appearance of APOL1 in the proximal tubule, either by gene expression or reabsorption from filtrate, would indicate a potentially important role of the proximal tubule in APOL1-associated CKD pathogenesis.
APOL1 in circulation is bound to a 500 kDa HDL3 particle, known as trypanolytic factor 1, a 1000 kDa lipid-poor IgM complex, known as trypanolytic factor 2, and possibly other lipid-poor, high molecular complexes associated with complement factors [7, 11–13]. The proteins produced by the two CKD-associated APOL1 variant alleles, G1 and G2, bind the high molecular weight trypanolytic factors similar to the common allele G0 [14]. Although the APOL1 protein (42.5 kDa) is small enough to pass the glomerular filtration barrier size restriction limit, it is not known to circulate independent of these high molecular weight complexes [15]. However, lipoproteins and other components of HDLs can be filtered [16], and in the setting of proteinuria, larger molecular weight proteins normally restricted by the filtration barrier may appear in filtrate. It is unclear whether APOL1 or APOL1-containing complexes may be filtered in the setting of proteinuria.
To resolve these issues, we examined both APOL1 gene and protein expression in human kidney tissue and kidneys from humanized transgenic mouse models that recreate native human APOL1 expression. For these studies we validated commercial anti-APOL1 antibodies for specificity which may have contributed to prior discrepancies on kidney expression patterns. In addition, APOL1 transgenic mouse models were made proteinuric by intercrossing with a model of HIV-associated nephropathy (HIVAN), a CKD strongly associated with carriage of APOL1 risk alleles, to determine if proteinuria would change the appearance of APOL1 protein in tubular epithelial.
Materials and methods
Human tissue and mouse models
Formalin-fixed, paraffin-embedded human kidney (n = 4) and liver (n = 3) tissue from normal margins of cancer resections were obtained from the Cleveland Clinic Lerner Research Institute Biorepository. Three transgenic mouse lines expressing a 47 kb human genomic fragment in a bacterial artificial chromosome (BAC) encompassing the promoter and coding regions of the human APOL1 gene for each G0, G1, or G2 alleles have been previously described [17, 18]. Each of the BAC-APOL1 transgenic lines were ≥10 generations backcrossed to FVB/Nj, a genetic background susceptible to HIVAN. The mouse HIVAN model used to induce proteinuria was the Tg26 HIVAN4 congenic [19] that develops proteinuria and progressive focal segmental glomerulosclerosis as the parental Tg26 model (Jackson Laboratory #22354) but disease progression is slower. For all studies, kidney disease was monitored weekly after weaning by measuring proteinuria (i.e. amount of protein in spontaneously voided urine) by urinalysis using diagnostic dipsticks (Uristix, Siemens Healthcare). The IACUC-approved humane endpoint for kidney disease in this model was proteinuria reaching 4+ on dipstick. No animal reached this humane endpoint before the predetermined study endpoint, as this study required an early stage of renal dysfunction (proteinuria on dipstick of 2+ to 3+) as functioning kidneys still capable of filtration and reabsorption were needed. All animals maintained normal weights and exhibited typical grooming and activity levels, and were not in pain or distress during the study. All animals were monitored twice a week by veterinary technicians not associated with this study for overall health and well-being, and no animals required analgesics or other supportive care. Each BAC-APOL1 transgenic mouse line was intercrossed with the HIVAN4 mouse model and were sacrificed at 8–10 weeks of age when their kidney disease progressed to proteinuria levels of ≥2+ by urine dipstick (G0 n = 15, G1 n = 11, G2 n = 9). Terminal urine, blood, and tissue collections were performed under deep isoflurane anesthesia followed immediately (while still under anesthesia) by euthanasia using cervical dislocation. Albuminuria was assessed by polyacrylamide gel electrophoresis of 1μl urine, followed by Coomassie staining. Use of human tissue was reviewed and approved by the Cleveland Clinic IRB (IRB-06-050). Informed consent was waived because tissue was considered discard and no identifiable data were collected. All animal studies were reviewed and approved by Institutional Animal Care and Use Committees at the Cleveland Clinic and Case Western Reserve University (IACUC-2430).
Tissue immunofluorescence
Formalin-fixed, paraffin-embedded kidney tissue sections were subjected to antigen retrieval as previously described [8]. Antibodies used to examine APOL1 expression in this paper was a rabbit anti-human APOL1 (Sigma, HPA018885, lot E105260, 1:400 dilution). Numerous lots of this Sigma rabbit polyclonal in addition to other commercial monoclonal antibodies were also evaluated and the results are summarized in Supplemental Table 1A-1C and Supplemental Figure 1 in S1 Data. Of note, many of these polyclonal antibody lots have been exhausted and are no longer available from Sigma. Other primary antibodies include: rabbit anti-mouse APOA1 (ThermoFisher, 1:500 dilution), goat anti-mouse CD31 (R&D Systems, 1:200 dilution), guinea pig anti-nephrin (USB, 1:200), and mouse anti-GLEPP1 (gift of Roger Wiggins, 1:50). FITC-labeled Lotus tetragonolobus lectin (Vector labs) was used to label proximal tubule cells as previously described [8]. For testing of commercial antibodies against human APOL1, kidney tissues were fixed using a variety of methods and paraffin embedded for immunohistochemistry using various antigen retrieval methods. Details for each of these processing methods are provided in the Supplemental Detailed Methods in S1 Data.
APOL1 gene and protein expression
Plasma APOL1 protein concentration was determined using the Meso Scale Discovery electrochemiluminescence immunoassay as described previously [5]. Concentration was determined relative to a known liquid chromatography-mass spectrometry calibrated human high density lipoprotein solution. Serum APOL1 and APOA1 protein levels were compared using Western blotting as previously described [20].
mRNA in situ hybridization
APOL1 gene expression was examined in mouse and human kidney and liver tissue using mRNA in situ hybridization. The manual RNAScope in situ hybridization kit (ACDBio) was used for formalin-fixed, paraffin-embedded tissue following kit instructions for either single probe or dual probe detection. Probes included human APOL1 (catalog number 439871), murine nephrin (Nphs1, catalog number 433571), and murine CD31 (Pecam1, catalog number 316721). Pretreatments were 15 minute boiling and 30 minute protease digestion. The in situ hybridization signal appears as dots; one dot per ten cells is expected background.
Results
Several commercial anti-human APOL1 antibodies (Supplemental Table 1A in S1 Data) were examined for specificity to human APOL1 using tissue or protein extracts from human and mouse kidneys and cells. Since mice do not have an ortholog of human APOL1, murine cells and tissues should not be immunoreactive to antibodies against human APOL1. In Western blotting, most commercial monoclonal and polyclonal antibodies were able to detect APOL1, although several weakly detected APOL1 with stronger detection of non-specific proteins (gels bands that did not coincide with the molecular weight of APOL1, Supplemental Table 1B in S1 Data). Most antibodies detected additional non-specific proteins, which differed depending on the source of protein extracts (Supplemental Table 1B in S1 Data). Some of these non-specific gel bands could be eliminated with pretreatment of protein extracts with deglycosylating enzymes (not shown) suggesting epitope recognition was dependent on glycans. In immunohistochemistry testing, wild-type mouse kidney used as a negative control was compared with APOL1 transgenic mouse kidney (Supplemental Table 1C in S1 Data). Many of the anti-APOL1 antibodies erroneously detected proteins in wild-type mouse kidney, including very strong immunostaining in tubules (Supplemental Figure 1 in S1 Data). Based on these validation studies, we selected a Sigma polyclonal antibody lot with limited off-target detection to examine APOL1 expression patterns.
In human kidney, APOL1 protein was detected by immunofluorescence in glomeruli but not tubules (Fig 1A). Similar expression patterns in human kidney were observed using mRNA in situ hybridization. APOL1 mRNA was present in glomeruli, peritubular capillaries, and larger vessels of the kidney, but not in any tubule segment (Fig 1B). A prior study of human liver transplant recipients established that circulating APOL1 protein is largely produced by the liver [21]. In human liver, APOL1 protein and mRNA expression patterns were similar, with expression detected in hepatocytes and vascular endothelia. In mice, APOL1 expression was qualitatively lower in zone 1 and 2 hepatocytes compared to zone 3 hepatocytes, whereas in human, hepatocytes in all three zones were similar in expression level (Supplemental Figure 2 in S1 Data). The mouse liver tissues were from healthy adults, whereas the human liver tissues were normal margins from cancer resections that also had histopathologic evidence of steatosis, potentially contributing to this difference.
A. APOL1 protein expression by immunofluorescence microscopy using validated anti-APOL1 antibodies, with co-immunostaining with GLEPP1 to identify podocytes and DAPI as a nuclear stain. B. APOL1 mRNA expression by in situ hybridization. Expression was evident in podocytes (“P”) and vascular endothelia of glomerular capillaries (“E”), peritubular capillaries (“1”, arrows), and larger vessel (“2”) epithelia, but not tubular epithelia. Scale bar = 40μm.
The observed expression pattern in human tissue was confirmed in three transgenic mouse lines expressing a human BAC encompassing the entire APOL1 genomic region for either the G0, G1, or G2 alleles [17, 18]. APOL1 protein was abundant in podocytes and also was present in endothelial cells of glomerular capillaries, peritubular capillaries, and endothelia of larger vessels (Fig 2A). No APOL1 protein was detected in the proximal tubule or any other tubular segment (Fig 2B). Also similar to humans, the BAC-APOL1 transgenic mice had abundant APOL1 in blood (Fig 3), and expressed APOL1 protein and mRNA in liver hepatocytes (Supplemental Figure 2 in S1 Data). In the kidney, this circulating APOL1 protein also could be detected in blood trapped in vascular spaces (Fig 2B). There was no difference in the APOL1 expression patterns between the APOL1-G0, -G1, or -G2 expressing mice (Fig 2B), and is consistent with previous studies in human biopsies from patients with different APOL1 genotypes [8, 10, 22]. The APOL1 protein expression patterns also were confirmed using mRNA in situ hybridization (Fig 4). All three APOL1 genotypes were examined but there were no differences based on APOL1 genotype. Consistent with human kidney (Fig 1), APOL1 was expressed in podocytes (cell type confirmed with co-labeling with Nphs1) and vascular endothelia (cell type confirmed with co-labeling with Pecam1), including glomerular capillaries, peritubular capillaries, and larger vessels. APOL1 expression was not detected in proximal tubules or any other tubular segment.
A. Immunofluorescent staining for APOL1, nephrin (to identify podocytes), and CD31 (to identify endothelia cells) in the BAC-APOL1 transgenic mouse kidney (G1 mouse is shown). B. Comparison of APOL1 expression patterns in all three APOL1 transgenic lines. Proximal tubules were identified by labeling with fluorescent Lotus tetragonolobus (LT) lectin. APOL1 was present in vascular endothelia (arrow heads), in podocytes (arrows), and trapped in vascular spaces (*), but not tubular epithelia. Scale bar = 40μm.
Plasma APOL1 protein levels were measured by immunoassay in transgenic mice compared to age-matched wild-type (WT) littermates (WT, n = 10; G0, n = 6; G1, n = 7; G2, n = 10; all animals were male, approximately 21 weeks of age). APOL1 expression levels in each transgenic line were all significantly different than WT (P<0.0001). Data are mean±SD, one measurement per animal, with significance determined by one-way ANOVA.
Duplex mRNA in situ hybridization for (A-D) APOL1 and nephrin (Nphs1) gene expression and (E-K) APOL1 and CD31 (Pecam1) expression to identified podocytes or endothelial cells respectively. APOL1 expression was detected in peritubular capillaries (“1” arrows), arterioles (“2” arrows), glomerular capillaries (“3” arrows), podocytes (“4” arrows), and larger (interlobular) arteries (“5”) and veins (“6”) but not proximal tubules (“PT”) or any other tubular segment. Images shown are from BAC-APOL1-G0 and BAC-APOL1-G1 mice.
None of the BAC-APOL1 mice spontaneously developed proteinuria, which is consistent with the original description of these mouse models [17, 18], but developed heavy proteinuria when intercrossed with a model of HIV-associated nephropathy (Fig 5). Using immunofluorescence, non-specific anti-APOL1 antibody immunostaining was observed in the wildtype and HIVAN mouse kidneys, mostly in Bowman capsule (Fig 6A). In the intercrossed mice with proteinuria, APOL1 protein was evident in glomeruli, but no APOL1 protein was detected in filtrate or within proximal tubules (Fig 6B). By Western blotting, APOL1 could not be detected in voided urine of proteinuric mice (data not shown). There also was no difference in the pattern of APOL1 expression between the proteinuric APOL1-G0, -G1, or -G2 expressing mice. These same mouse kidneys were immunostained for APOA1, an apolipoprotein that is filtered, as a positive control for vascular distribution and proximal tubule reabsorption patterns. Similar to APOL1, APOA1 in plasma was readily detected in glomerular capillary lumens. However, since APOA1 is filtered it also was present in protein reabsorption droplets at the proximal tubule brush border (Fig 6C). In the setting of proteinuria, APOA1-containing reabsorption droplets increased in number and size at the proximal tubule brush border. A similar pattern in the proximal tubule was not observed with APOL1.
A. Example of proteinuria in single and dual transgenic mice assessed by gel electrophoresis of urine (Coomassie stain). Arrowhead marks albumin, and additional low molecular weight urinary proteins (asterisk) are a normal finding in mice. B. Western blot of mouse serum in the same single and dual transgenic mice showing maintenance of high levels of serum APOL1 protein in the setting of proteinuria. APOA1 Western blot as a control for a common serum protein that is freely filtered. Representative blot is shown; number of animal examined in each genotype group were: wildtype, n = 3; HIVAN4, n = 6; G0 x HIVAN4, n = 6; G1 x HIVAN4, n = 3; G2 x HIVAN4, n = 4.
A. Control immunostaining in non-transgenic (wildtype) and proteinuric HIVAN4 mice for APOL1 along with fluorescent Lotus tetragonolobus (“LT”) lectin binding to demarcate the proximal tubule brush border. Since wildtype and HIVAN4 mice do not have APOL1, the staining observed in parietal cells (arrows) is artifact. B. Immunostaining for APOL1 in proteinuric BAC transgenic mice of each APOL1 genotype (representative images are shown, number of animal examined in each genotype group were the same as for Fig 5). Images show proximal tubules at the transition with Bowman capsule. The boxed region is magnified below each panel along with the isolated fluorescent channels shown in black and white. C. Positive control immunostaining for a filtered lipoprotein, APOA1, and fluorescently-labelled Lotus tetragonolobus (“LT”) lectin. An APOL1-G0 mouse and an APOL1-G0 x HIVAN4 dual transgenic mouse with proteinuria is shown; below each respective color panel is the individual fluorescent channels (in black and white) of the boxed region for either LT lectin or APOA1. White arrows mark glomerular capillaries containing circulating APOA1 protein within capillary lumens, red arrows denote APOA1 in protein reabsorption droplets at the brush border of proximal tubules. Scale bar = 40μm.
Discussion
Antibody specificity has been recognized as one of the most significant challenges impacting reproducible research [23]. We and others had originally reported APOL1 protein is present in proximal tubules of normal and diseased subjects [8, 9]. However, our continuing work with other anti-APOL1 antibodies and using mRNA in situ hybridization indicated APOL1 was not expressed in proximal tubules [10]. A remaining possibility is that the APOL1 protein in blood is filtered and reabsorbed, resulting in the appearance of APOL1 protein in proximal tubules. Using in situ methods that do not rely on antibodies, along with the newly available human BAC-APOL1 transgenic mice, neither APOL1 protein nor APOL1 mRNA could be detected in tubular epithelia. In the setting of proteinuria, APOL1 also was not filtered. In our hands, the Sigma rabbit polyclonal antibody consistently recognizes human APOL1 but has lot-to-lot differences with recognition of other proteins. The Sigma rabbit polyclonal lot used in studies here does not replicate the strong proximal tubule staining observed in prior lots sold under the same catalog number. Although monoclonal antibodies would eliminate the variation inherent in polyclonal antibody production lots, the commercial monoclonal antibodies tested had significant non-specificity or poor sensitivity for APOL1. A recent report describing the development and validation of a large number of monoclonal antibodies against human APOL1 also did not identify proximal tubule staining in human kidney [24].
Evidence against proximal tubule expression of APOL1, or a role for the proximal tubule in the APOL1-associated CKDs, is accumulating from several other groups. A study using similar humanized APOL1 transgenic mouse models created with fosmids also did not find APOL1 expression in the proximal tubule using both protein and mRNA detection methods [25]. Use of transgenic models with inducible APOL1 expression that restrict expression to either podocytes or tubules found proteinuria and renal pathology occurs only when APOL1 is expressed in podocytes, and not in the tubule [6]. As corroborating evidence in humans, several studies surveying the human urine proteome [26–30] did not identify APOL1 protein in urine, also indicating APOL1 is not filtered in any detectable quantity.
Our studies do not rule-out the possible release of small amounts of APOL1 into the primary filtrate from podocytes either by secretion or passively release when a podocyte dies and lyses. Unlike the APOL1 expressed and secreted by hepatocytes, the major APOL1 transcript in podocytes does not have a complete signal peptide [15], and there is no conclusive evidence that podocytes secrete APOL1. Alternatively, some studies observed the disease-induced high levels of APOL1 can also produce rare alternatively spliced isoforms with different signal peptide sequences possibly permitting secretion [24, 31–33]. These potential alternative sources of APOL1 in filtrate would have been below limits of detection in the assays we and other investigators have used in kidney tissue and urine, and would be an unlikely explanation for the robust APOL1 expression previously reported in proximal tubules. In addition, it is unclear if this potential low level of podocyte-derived APOL1 in filtrate would be of physiologic significance, considering that in humans, basal levels of APOL1 expression are not associated with CKD. Our studies also cannot rule-out possible differences in APOL1 expression between humans and the BAC-APOL1 transgenic mice. The function of podocytes and proximal tubules in blood filtration and reabsorption are fundamentally similar between humans and mice, however there are acknowledged limitations of using mice with regards to replicating human glomerular kidney diseases [34].
The work presented here and other published studies have shown significant similarities between humans [8–10, 24] and transgenic mouse APOL1 expression patterns [17, 18, 24]. A reproducible and consistent observation from these combined studies is expression of APOL1 in podocytes and in endothelial cells of glomerular capillaries, peritubular capillaries, and larger blood vessels. In addition, observations here indirectly support conclusions from prior studies [4, 5] that circulating APOL1 protein is unlikely to contribute to kidney disease pathogenesis as it is not the source of kidney-localized APOL1. Evaluating the contribution of podocyte- and endothelial-expressed APOL1 is a logical focus for future studies examining the mechanism for APOL1 risk variant contributions to CKD pathogenesis.
Supporting information
S1 Raw images. Original, uncropped gel images.
https://doi.org/10.1371/journal.pone.0253197.s002
(PDF)
Acknowledgments
We thank William Baldwin and Nina Dvorina for assistance with the antibody validation studies.
References
- 1. Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, Freedman BI, et al. Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science. 2010;329(5993):841–5. pmid:20647424
- 2. Genovese G, Tonna SJ, Knob AU, Appel GB, Katz A, Bernhardy AJ, et al. A risk allele for focal segmental glomerulosclerosis in African Americans is located within a region containing APOL1 and MYH9. Kidney Int. 2010;78(7):698–704. pmid:20668430
- 3. Tzur S, Rosset S, Shemer R, Yudkovsky G, Selig S, Tarekegn A, et al. Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene. Hum Genet. 2010;128(3):345–50. pmid:20635188
- 4. Bruggeman LA, O’Toole JF, Ross MD, Madhavan SM, Smurzynski M, Wu K, et al. Plasma apolipoprotein L1 levels do not correlate with CKD. J. Am. Soc. Nephrol. 2014;25(3):634–44. pmid:24231663
- 5. Kozlitina J, Zhou H, Brown PN, Rohm RJ, Pan Y, Ayanoglu G, et al. Plasma Levels of Risk-Variant APOL1 Do Not Associate with Renal Disease in a Population-Based Cohort. J. Am. Soc. Nephrol. 2016;27(10):3204–19. pmid:27005919
- 6. Beckerman P, Bi-Karchin J, Park AS, Qiu C, Dummer PD, Soomro I, et al. Transgenic expression of human APOL1 risk variants in podocytes induces kidney disease in mice. Nat. Med. 2017;23(4):429–38. pmid:28218918
- 7. Weckerle A, Snipes JA, Cheng D, Gebre AK, Reisz JA, Murea M, et al. Characterization of circulating APOL1 protein complexes in African Americans. J Lipid Res. 2016;57(1):120–30. pmid:26586272
- 8. Madhavan SM, O’Toole JF, Konieczkowski M, Ganesan S, Bruggeman LA, Sedor JR. APOL1 localization in normal kidney and nondiabetic kidney disease. J. Am. Soc. Nephrol. 2011;22(11):2119–28. pmid:21997392
- 9. Ma L, Shelness GS, Snipes JA, Murea M, Antinozzi PA, Cheng D, et al. Localization of APOL1 protein and mRNA in the human kidney: nondiseased tissue, primary cells, and immortalized cell lines. J. Am. Soc. Nephrol. 2015;26(2):339–48. pmid:25012173
- 10. Madhavan SM, O’Toole JF, Konieczkowski M, Barisoni L, Thomas DB, Ganesan S, et al. APOL1 variants change C-terminal conformational dynamics and binding to SNARE protein VAMP8. JCI Insight. 2017;2(14):e92581. pmid:28724794
- 11. Raper J, Fung R, Ghiso J, Nussenzweig V, Tomlinson S. Characterization of a novel trypanosome lytic factor from human serum. Infection Immunity. 1999;67(4):1910–6. pmid:10085035
- 12. Smith AB, Esko JD, Hajduk SL. Killing of trypanosomes by the human haptoglobin-related protein. Science. 1995;268(5208):284–6. pmid:7716520
- 13. Vanhamme L, Paturiaux-Hanocq F, Poelvoorde P, Nolan DP, Lins L, Van Den Abbeele J, et al. Apolipoprotein L-I is the trypanosome lytic factor of human serum. Nature. 2003;422(6927):83–7. pmid:12621437
- 14. Gutierrez OM, Judd SE, Irvin MR, Zhi D, Limdi N, Palmer ND, et al. APOL1 nephropathy risk variants are associated with altered high-density lipoprotein profiles in African Americans. Nephrol Dialysis Transplant. 2016;31(4):602–8. pmid:26152403
- 15. Duchateau PN, Pullinger CR, Cho MH, Eng C, Kane JP. Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene. J Lipid Res. 2001;42(4):620–30. pmid:11290834
- 16. Yang H, Fogo AB, Kon V. Kidneys: key modulators of high-density lipoprotein levels and function. Curr Opin Nephrol Hypertens. 2016;25(3):174–9. pmid:27008596
- 17. Okamoto K, Rausch JW, Wakashin H, Fu Y, Chung JY, Dummer PD, et al. APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R. Comm. Biol. 2018;1:188. pmid:30417125
- 18. Ryu JH, Ge M, Merscher S, Rosenberg AZ, Desante M, Roshanravan H, et al. APOL1 renal risk variants promote cholesterol accumulation in tissues and cultured macrophages from APOL1 transgenic mice. PLoS One. 2019;14(4):e0211559. pmid:30998685
- 19. Prakash S, Papeta N, Sterken R, Zheng Z, Thomas RL, Wu Z, et al. Identification of the nephropathy-susceptibility locus HIVAN4. J. Am. Soc. Nephrol. 2011;22(8):1497–504. pmid:21784893
- 20. Bruggeman LA, Wu Z, Luo L, Madhavan SM, Konieczkowski M, Drawz PE, et al. APOL1-G0 or APOL1-G2 Transgenic Models Develop Preeclampsia but Not Kidney Disease. J. Am. Soc. Nephrol. 2016;27(12):3600–10. pmid:27026370
- 21. Shukha K, Mueller JL, Chung RT, Curry MP, Friedman DJ, Pollak MR, et al. Most ApoL1 Is Secreted by the Liver. J. Am. Soc. Nephrol. 2017;28(4):1079–83. pmid:27932478
- 22. Sampson MG, Robertson CC, Martini S, Mariani LH, Lemley KV, Gillies CE, et al. Integrative Genomics Identifies Novel Associations with APOL1 Risk Genotypes in Black NEPTUNE Subjects. J. Am. Soc. Nephrol. 2016 Mar;27(3):814–23. pmid:26150607
- 23. Bradbury A, Pluckthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518(7537):27–9. pmid:25652980
- 24. Scales SJ, Gupta N, De Mazière AM, Posthuma G, Chiu CP, Pierce AA, et al. Apolipoprotein L1-Specific Antibodies Detect Endogenous APOL1 inside the Endoplasmic Reticulum and on the Plasma Membrane of Podocytes. J. Am. Soc. Nephrol. 2020;31(9):2044–64. pmid:32764142
- 25. Aghajan M, Booten SL, Althage M, Hart CE, Ericsson A, Maxvall I, et al. Antisense oligonucleotide treatment ameliorates IFN-gamma-induced proteinuria in APOL1-transgenic mice. JCI Insight. 2019;4(12). pmid:31217349
- 26. Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol. 2006;7(9):R80. pmid:16948836
- 27. Li QR, Fan KX, Li RX, Dai J, Wu CC, Zhao SL, et al. A comprehensive and non-prefractionation on the protein level approach for the human urinary proteome: touching phosphorylation in urine. Rapid Comm Mass Spectrom. 2010;24(6):823–32. pmid:20187088
- 28. Marimuthu A, O’Meally RN, Chaerkady R, Subbannayya Y, Nanjappa V, Kumar P, et al. A comprehensive map of the human urinary proteome. J Proteome Res. 2011;10(6):2734–43. pmid:21500864
- 29. Santucci L, Candiano G, Petretto A, Bruschi M, Lavarello C, Inglese E, et al. From hundreds to thousands: Widening the normal human Urinome. Data Brief. 2014;1:25–8. pmid:26217681
- 30. Zhao M, Li M, Yang Y, Guo Z, Sun Y, Shao C, et al. A comprehensive analysis and annotation of human normal urinary proteome. Scientific Reports. 2017;7(1):3024. pmid:28596590
- 31. Cheatham AM, Davis SE, Khatua AK, Popik W. Blocking the 5’ splice site of exon 4 by a morpholino oligomer triggers APOL1 protein isoform switch. Scientific Reports. 2018;8(1):8739. pmid:29880816
- 32. Khatua AK, Cheatham AM, Kruzel ED, Singhal PC, Skorecki K, Popik W. Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1. Am J Physiol Cell Physiol. 2015;309(1):C22–37. pmid:25924622
- 33. Nichols B, Jog P, Lee JH, Blackler D, Wilmot M, D’Agati V, et al. Innate immunity pathways regulate the nephropathy gene Apolipoprotein L1. Kidney Int. 2015;87(2):332–42. pmid:25100047
- 34. Pippin JW, Brinkkoetter PT, Cormack-Aboud FC, Durvasula RV, Hauser PV, Kowalewska J, et al. Inducible rodent models of acquired podocyte diseases. Am J. Physiol Renal Physiol. 2009;296(2):F213–29. pmid:18784259