Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

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.

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 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 .

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 ) 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 ). Most antibodies detected additional non-specific proteins, which differed depending on the source of protein extracts (Supplemental Table 1B in ). 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 ). Many of the anti-APOL1 antibodies erroneously detected proteins in wild-type mouse kidney, including very strong immunostaining in tubules (Supplemental Figure 1 in ). 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 (). 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 (). 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 ). 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.

APOL1 protein and mRNA expression in human kidney.

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 (. No APOL1 protein was detected in the proximal tubule or any other tubular segment (). Also similar to humans, the BAC-APOL1 transgenic mice had abundant APOL1 in blood (), and expressed APOL1 protein and mRNA in liver hepatocytes (Supplemental Figure 2 in ). In the kidney, this circulating APOL1 protein also could be detected in blood trapped in vascular spaces (). There was no difference in the APOL1 expression patterns between the APOL1-G0, -G1, or -G2 expressing mice (), 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 (). All three APOL1 genotypes were examined but there were no differences based on APOL1 genotype. Consistent with human kidney (), 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.

APOL1 protein expression in BAC-APOL1 transgenic mouse kidneys.

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.

APOL1 protein in circulation in the BAC-APOL1 transgenic mouse models.

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.

APOL1 mRNA expression in the BAC-APOL1 transgenic mouse models.

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 (). Using immunofluorescence, non-specific anti-APOL1 antibody immunostaining was observed in the wildtype and HIVAN mouse kidneys, mostly in Bowman capsule (). In the intercrossed mice with proteinuria, APOL1 protein was evident in glomeruli, but no APOL1 protein was detected in filtrate or within proximal tubules (). 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 (). 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.

BAC-APOL1 transgenic mouse models developed similar proteinuria when intercrossed with the HIVAN mouse model.

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.

APOL1 does not appear in proximal tubules in proteinuric BAC-APOL1 transgenic mice.

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 ). 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.

Supplemental figures and tables.

(PDF)

Click here for additional data file.

Original, uncropped gel images.

(PDF)

Click here for additional data file.

19 Mar 2021

PONE-D-21-04749

APOL1 is not expressed in proximal tubules and is not filtered

PLOS ONE

Dear Dr. Bruggeman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands.  At the same time I note that all three reviewers were enthusiastic about the project and the conclusions.

Reviewer 2 made several specific technical critiques about the work.  I would like the authors to pay special attention to the critiques of the various co-localization studies (a point that was echoed by Reviewer 3). Those analyses are central to the argument made in this MS. I agree that it might be helpful to show more micrographs, perhaps at different magnifications and with more glomeruli in the field. The authors should also add some text to address the issue that ApoL1 expression in this somewhat artificial transgenic mouse model looks a bit different than it does in humans. It would also be helpful to address the comment about the Santa Cruz antibody mentioned by the reviewer compared to the one that was used in this study, at least in the response to critiques if not in the revised MS itself. It is also important to be transparent about all statistical methods used.  Reviewer 3 also raises some other technical issues that I think will be quite straightforward to address, and that may simply be the way the figures were prepared.  That reviewer also makes some useful suggestions on spelling and formatting.

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Reviewer #1: Bruggeman et al studied APOL1 expression in tubular cells and its filtration in the tubular lumen.

Comments:

1. These investigators were pioneer to show expression of APOL1 protein in kidney cells including tubular cells. However, they have now clarified that tubular cells do not express APOL1.

2. Interestingly, they have also shown that it is not filtered. However, a possibility of its secretion by podocytes can not be excluded.

Reviewer #2: This current study tested if ApoL1 is synthesized and/or reabsorbed by proximal tubule cells. Expression of ApoL1 mRNA and protein by renal tissues of human and ApoL1 transgenic mice were examined using in situ hybridization, immunofluorescence and immunohistochemistry techniques with commercially available antibodies. The authors concluded that ApoL1 is mainly expressed in podocytes and endothelial cells, but not by tubular cells.

Overall, the findings in this study are informative, but not novel. The manuscript is logically well written, and the data is straightforward. However, supporting evidence seems insufficient because all data except for Fig 1 was generated from a transgenic mouse model. Also, the data cited that corresponds to the conclusions made regarding the filtration and reabsorption of ApoL1 part seems weak.

Comments:

1. Most studies relied on the results from BAC-ApoL1 Tg animals, in which the expression and localization of ApoL1 most likely is different from that in humans.

Indeed, mRNA and protein expression patterns of ApoL1 in liver of human and Tg mouse (Supp F2) seem quite different (disperse vs focal). Thus, human renal tissues from healthy and diseased individuals need to be thoroughly tested using the Santa Cruz antibody (lot E105260).

2. Immunofluorescence on human kidneys (Fig 1A) shows a single glomerulus with limited views of tubules. Please show the ApoL1 protein expression pattern from more tubular regions following co-staining with proximal tubule cell markers.

3. In Fig 4 (ISH on BAC-ApoL1 Tg mouse, G0? G1? G2?), decent amounts of ApoL1 mRNA expression were also observed in Nphs1 negative cells. To make it clear that the ApoL1 signals are definitively not from PT, duplex mRNA ISH for ApoL1 and PT markers need to be performed.

4. The authors showed that ApoA1 but not ApoL1 is colocalized with LT-lectin, a tubule marker (Fig 6). However, it is still not clear if positive staining of ApoA1 observed in tubular cells resulted from reabsorption from the filtrate or if it is expressed by tubular cells. Adequate measurements need to be done to clarify this issue.

We cannot rule out the possibility that a small amount of ApoL1 is released from damaged/apoptotic podocytes and reabsorbed by the proximal tubules from the filtrate.

5. If possible, additional examination of ApoL1 protein expression in human and mouse urine samples (G0, G1, and G2 genotype) would be informative.

6. Also, in Supp F1, it would be informative if the authors show the ApoL1 staining pattern in ApoL1 Tg and normal mouse renal tissues using the Santa Cruz antibody (lot E105260).

7. The statistical method used in Fig 3 has not been described.

8. The conclusions of the paper, as stated in the title is overly quite bold. The authors fall short in providing definitive evidence for the statement that proximal tubule cells neither produce nor reabsorb ApoL1 and should therefore tone down their conclusions.

Reviewer #3: Bruggeman and colleagues report that APOL1 is not expressed in proximal tubules and is not filtered. The investigators have studied (Merck) BAC/APOL1 mice, representing each of the three genotypes, crossed to FVB/N mice for 10 generations. Their prior in situ hybridization work suggests that RNA expression is confined to podocytes and endothelial cells in glomerular and peritubular capillaries, and other vessels (reference 10).

Here, the authors have done a careful analysis of various APOL1 antibodies, particularly of the Sigma polyclonal antibody. The quality of the immunostaining and in situ hybridization images is excellent. The authors conclude that bona fide APOL1 protein expression in the BAC transgenic mice is limited to podocytes and endothelial cells. They suggest that false-positivity for APOL1 immunostaining in the proximal tubule may be due to glycosylation patterns. These finding will be of value to investigators in this field.

The paper is clear, concise and elegantly written.

1) Figure 1. To my eye, it appears that there is only partial co-localization of GLEPP1 (perhaps located in peripheral portions of glomerular capillary loops) and APOL1 (possibly in the cell bodies). I wonder whether the authors agree. I agree that both proteins are in podocytes as well as other cells. I think all this might be easier to resolve if the images were larger.

2) Figure 5, Western, is confusing, because two blots were but together, with result that order of the lanes differ and protein migration differs. A single blot would strengthen the paper.

3) After all the effort to cross the mice for 10 generations onto an FVB/N background, it would be useful to know whether the kidney phenotype become more severe (more proteinuria, more glomerulosclerosis).

Minor comments.

P4,P8, capitalize Coomassie, as it is (or rather was) a place name.

P5, capitalize Lotus, as it is a genus name

PP6-7. “APOL1 expression was not detected in proximal tubules or any other nephron segment.” This sentence appears twice. More importantly, the glomerulus is part of the nephron. Suggest “or any other tubular segment.”

P7, no need to capitalize nephrin

P8, Lotus tetragonolobus, capitalize genus, italics

Bowman’s capsule > Bowman capsule. The trend is away from the possessive, as the discoverer does not own the structure. It does sound odd to say Bowman capsule after we have been saying it differently for decades.

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Reviewer #1: Yes: Pravin C. Singhal

Reviewer #2: No

Reviewer #3: No

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11 May 2021

Editorial comments:

1. PLOS ONE style requirements are met.

2. Probe catalog numbers for in situ hybridization probes have been included.

3. Detailed information on the in vivo study is included.

4. Ethics statement has been revised and is in the cover letter and the methods section.

5. Original uncropped images are now included in the supplemental file.

6. A revised funding and competing statement is in the cover letter, and the author roles are correct in the author contributions section.

7. Reference list is complete and correct.

Reviewer critiques:

Reviewer #1

Interestingly, they have also shown that it is not filtered. However, a possibility of its secretion by podocytes cannot be excluded.

RESPONSE: We agree this remains a possibility and have included a paragraph in the discussion to address this issue.

Reviewer #2

1. Most studies relied on the results from BAC-ApoL1 Tg animals, in which the expression and localization of ApoL1 most likely is different from that in humans. Indeed, mRNA and protein expression patterns of ApoL1 in liver of human and Tg mouse (Supp F2) seem quite different (disperse vs focal). Thus, human renal tissues from healthy and diseased individuals need to be thoroughly tested using the Santa Cruz antibody (lot E105260).

RESPONSE: We believe the reviewer is referring to the Sigma – not Santa Cruz – antibody as Santa Cruz antibodies were not used and the lot number given is for the Sigma antibody. A major point of this manuscript is to highlight the limitation of antibody reagents, and one significant issue regards polyclonal antibody lots, which when exhausted are gone and studies can never be repeated or reproduced. Unfortunately, lot E105260 is now exhausted both in our lab and is no longer sold by Sigma (we have revised supplemental Table 1 to indicate this). We cannot do additional studies that require this antibody lot.

The comparison of mouse and human liver we agree there are some differences. It should be noted that the mouse livers were from normal young adults, but the human livers were “normal” margins of cancer resections and also had pathologic changes consistent with steatosis. We have revised the description of the liver expression pattern in the text and figure legends to be more specific on these issues. In general, we concur that mice and humans are different on many levels, and there may be issues studying a human gene in mice. We agree we should acknowledge this as a limitation of our study, and have included a discussion of this issue relevant to our observations.

2. Immunofluorescence on human kidneys (Fig 1A) shows a single glomerulus with limited views of tubules. Please show the ApoL1 protein expression pattern from more tubular regions following co-staining with proximal tubule cell markers.

RESPONSE: As stated above, we cannot do additional immunostaining with tubule makers, as this APOL1 antibody is no longer available.

3. In Fig 4 (ISH on BAC-ApoL1 Tg mouse, G0? G1? G2?), decent amounts of ApoL1 mRNA expression were also observed in Nphs1 negative cells. To make it clear that the ApoL1 signals are definitively not from PT, duplex mRNA ISH for ApoL1 and PT markers need to be performed.

RESPONSE: We have examined all three genotypes (G0, G1, and G2) of the BAC-APOL1 mice using ISH and have added this statement to the results section, and also indicated which genotype is shown in the figure. We have added to Figure 4 new data on duplex mRNA ISH for CD31 (Pecam1). The extra-glomerular staining is peritubular capillaries, not proximal tubules, so we used an endothelial marker as a cell-type specific marker to co-label with extraglomerular APOL1-expressing cells.

4. The authors showed that ApoA1 but not ApoL1 is co-localized with LT-lectin, a tubule marker (Fig 6). However, it is still not clear if positive staining of ApoA1 observed in tubular cells resulted from reabsorption from the filtrate or if it is expressed by tubular cells. Adequate measurements need to be done to clarify this issue. We cannot rule out the possibility that a small amount of ApoL1 is released from damaged/apoptotic podocytes and reabsorbed by the proximal tubules from the filtrate.

RESPONSE: APOA1 is expressed only by the liver, as such, we will not be able to show endogenous APOA1 expression in tubule cells, since no resident kidney cell expresses APOA1. The second point of possibly small amounts of APOL1 being released into filtrate from a dead podocyte and reabsorbed by the tubule remains plausible, however, this small amount would not account for the robust staining previously reported for the proximal tubule. We have added a paragraph to the discussion section describing other possible sources of APOL1 in the primary filtrate.

5. If possible, additional examination of ApoL1 protein expression in human and mouse urine samples (G0, G1, and G2 genotype) would be informative.

RESPONSE: We had examined urine from the HIVAN intercrossed BAC mice by Western blotting and found no evidence of APOL1 in urine. We have included this in the results section.

6. Also, in Supp F1, it would be informative if the authors show the ApoL1 staining pattern in ApoL1 Tg and normal mouse renal tissues using the Santa Cruz antibody (lot E105260).

RESPONSE: Again, we think the reviewer is referring to the Sigma (not Santa Cruz) antibody here. As per point #1, we cannot do additional studies that require lot E105260, as the supply is exhausted and is no longer sold by Sigma.

7. The statistical method used in Fig 3 has not been described.

RESPONSE: This has been added to the figure legend (ANOVA).

8. The conclusions of the paper, as stated in the title is overly quite bold. The authors fall short in providing definitive evidence for the statement that proximal tubule cells neither produce nor reabsorb ApoL1 and should therefore tone down their conclusions.

RESPONSE: We have revised the title and conclusions, taking into consideration the above comments.

Reviewer #3

1. Figure 1. To my eye, it appears that there is only partial co-localization of GLEPP1 (perhaps located in peripheral portions of glomerular capillary loops) and APOL1 (possibly in the cell bodies). I wonder whether the authors agree. I agree that both proteins are in podocytes as well as other cells. I think all this might be easier to resolve if the images were larger.

RESPONSE: We have revised Figure 1 to include higher magnification images. In our prior studies of human APOL1 expression patterns (Refs 8, 10), we demonstrated that podocyte markers such as GLEPP1 reside in a different subcellular location than APOL1. Consequently, there is not a 100% overlap in fluorescent signals. We are using proteins such as GLEPP1 for cell identity, not protein co-localization with APOL1. In addition to podocytes, some of this signal is attributable to glomerular endothelial cells which are also positive for APOL1 (CD31 as a cell identifying marker). We have also added the mRNA in situ images with CD31 (PECAM1) co-staining for endothelial cells in Figure 4.

2. Figure 5, Western, is confusing, because two blots were but together, with result that order of the lanes differ and protein migration differs. A single blot would strengthen the paper.

RESPONSE: We agree, but we are working with spot mouse urines and had limited volumes from this cohort of intercrossed mice. We have no additional material for re-running gels.

3. After all the effort to cross the mice for 10 generations onto an FVB/N background, it would be useful to know whether the kidney phenotype become more severe (more proteinuria, more glomerulosclerosis)

RESPONSE: We understand this comment, as it is well-known that many glomerular injury models are significantly different in phenotype based on the mouse genetic background. All BAC-APOL1 (G0, G1, G2) mice have no renal phenotype either on the original background (129SvJ) or the FVB/N background. There is no difference in the APOL1 phenotype on the two mouse strains in the absence of a disease-inducing stressor. To induce significant proteinuria, we intercrossed the BAC mice with the HIVAN model, and the backcrossing to the FVB/N background was done specifically to conduct the HIVAN crossbreeding. The phenotype of the HIVAN mouse model is highly dependent on the genetic strain, which we have already examined in detail (PMIDs 21784893, 19381020, 14983036). We are currently conducting a comprehensive, longitudinal study of disease phenotype comparisons based on APOL1 genotype in similar HIVAN intercrosses, but study outcomes are many months from completion, and beyond the scope of this manuscript.

Minor comments.

RESPONSE: All spelling and wording issues have been corrected as recommended.

31 May 2021

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

PONE-D-21-04749R1

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Reviewer #2: All comments have been addressed

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Reviewer #3: Yes: Jeffrey Kopp

9 Jun 2021

PONE-D-21-04749R1

Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys.

Dear Dr. Bruggeman:

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