Localization of APOL1 Protein and mRNA in the Human Kidney: Nondiseased Tissue, Primary Cells, and...

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Localization of APOL1 Protein and mRNA in theHuman Kidney: Nondiseased Tissue, Primary Cells,and Immortalized Cell Lines

Lijun Ma,* Gregory S. Shelness,† James A. Snipes,* Mariana Murea,* Peter A. Antinozzi,‡

Dongmei Cheng,† Moin A. Saleem,§ Simon C. Satchell,| Bernhard Banas,¶

Peter W. Mathieson,§ Matthias Kretzler,** Ashok K. Hemal,†† Lawrence L. Rudel,†

Snezana Petrovic ,*‡‡ Allison Weckerle ,† Martin R. Pollak ,§§ Michael D. Ross ,||

John S. Parks ,†‡ and Barry I. Freedman *

*Internal Medicine-Nephrology, †Pathology-Lipid Sciences, and ‡Biochemistry, Wake Forest School of Medicine,Winston-Salem, North Carolina; §Children’s Renal Unit, Bristol Royal Hospital for Children, University of Bristol, Bristol,United Kingdom; |Learning and Research Southmead Hospital Bristol, University of Bristol, Bristol, United Kingdom;¶Internal Medicine II—Nephrology/Transplantation, University Medical Center, Regensburg, Germany; **Departmentof Internal Medicine-Nephrology, University of Michigan at Ann Arbor Medical School, Ann Arbor, Michigan;††Departments of Surgical Sciences-Urology and ‡‡Physiology and Pharmacology, Wake Forest School of Medicine,Winston-Salem, North Carolina; §§Division of Nephrology, Department of Medicine, Beth Israel Deaconess MedicalCenter and Harvard Medical School, Boston, Massachusetts; and ||Renal Division, Department of Medicine, Brigham andWomen’s Hospital, Boston, Massachusetts

ABSTRACTAlthough APOL1 gene variants are associated with nephropathy in African Americans, little is known aboutAPOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examinedAPOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immuno-fluorescencemicroscopyperformedonnondiseasednephrectomycryosections frompersonswithnormal kidneyfunction revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin andWilms’ tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridizationdetectedAPOL1mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenoussynthesis in these cell types.When these analyses were extended to renal-derived cell lines, quantitative RT-PCRdid not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were ob-served inproximal tubule cells andglomerular endothelial cells,with lower expression inpodocytes.Westernblotanalysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancybetweenthemarkedabundanceofAPOL1protein inkidneypodocytesobserved incryosectionsversus the lesserabundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily byhuman podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, orproximal tubule cells.Wehypothesize that thehigher levels ofAPOL1protein in humancryosectionedpodocytesmay reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

J Am Soc Nephrol 26: 339–348, 2015. doi: 10.1681/ASN.2013091017

Received September 25, 2013. Accepted May 16, 2014.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Lijun Ma or Dr. Barry I. Freedman, InternalMedicine-Nephrology, Medical Center Boulevard, Wake Forest

School of Medicine, Medical Center Boulevard, Winston-Salem,NC 27157-1053. Email: [email protected] or [email protected]

Copyright © 2015 by the American Society of Nephrology

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Molecular genetics is transforming our understanding of thepathogenesis in nondiabetic nephropathy in populations ofAfrican ancestry.1,2 HIV-associated collapsing glomerulopathy,idiopathic FSGS, severe lupus nephritis, and hypertension-attributed nephropathy are strongly associated with apolipo-protein L1 (APOL1) G1 andG2 gene variants on chromosome22q13.1 in African Americans.1–3 APOL1 is a minor apolipo-protein component of HDL, and APOL1 nephropathy var-iants are associated with HDL subfraction concentrations inAfrican Americans.4 Circulating APOL1 has the ability to killTrypanosoma brucei rhodesiense, a cause of African sleepingsickness.5 APOL1 protein is present in plasma,6 but its intra-cellular effects on kidney function remain unknown. To betterunderstand potential links between APOL1 and renal disease,localizing APOL1 protein and mRNA in the healthy kidneytissue of African Americans and Americans of European de-scent remains an important goal.

Although APOL1 transcripts are expressed in many tissues,including the kidney,7,8 the specific renal cell types that engagein APOL1 transcription are not known. It is uncertain whichkidney cells are enriched in APOL1 protein because mRNAabundance does not necessarily correlate with cellular proteinabundance. It is also unclear how renal abundance of APOL1compares with levels in liver and serum. Madhavan et al. re-ported, using formalin-fixed paraffin-embedded (FFPE) kidneysections and heat-induced epitope retrieval methods, thatAPOL1 was present in tubule cells and with lower signal inten-sity in podocytes.9 We tested whether these findings would bereplicated in kidney cryosections using another antibody recog-nizing native APOL1 protein, and we extended these analyses toprimary and immortalized renal cell models to further explorecell-specific localization of APOL1 and to address the relativecontribution of endogenous synthesis versus exogenous uptaketo APOL1 protein localization and abundance. These studiescomprehensively assess sites of renal APOL1 synthesis and lo-calization and suggest that glomerular localization of APOLImay be a consequence of both endogenous synthesis and podo-cyte uptake ofAPOL1 from the circulation or glomerularfiltrate.

RESULTS

Specificity of Rabbit anti-APOL1 AntibodiesThe specificity of a commercially available rabbit anti-APOL1monoclonal antibody (3245–1; Epitomics, Burlingame, CA)was established. The Epitomics antibody successfully recog-nized circulating APOL1 in its native form in human serum byimmunoprecipitation (data not shown).When used in immu-nofluorescence microscopy, the antibody detected APOL1 inAPOL1-transfected, but not empty vector–transfected, Chinesehamster ovary cells (Supplemental Figure 1). When used tostain kidney cryosections, abundant glomerular staining wasobserved, which was blocked upon pretreatment with theAPOL1 immunogen blocking peptide (Supplemental Figure 2),further confirming the specificity of this antibody.

APOL1 Protein Localization in Human NondiseasedKidney CryosectionsKidney cryosections from European Americans and AfricanAmericans with preoperative eGFRs calculated with Modifi-cation of Diet in Renal Disease (MDRD) equation10 .60 ml/min per 1.73 m2 and who underwent nephrectomy for renalcell carcinomawere examined to establish APOL1 distributionin nondiseased parenchyma. On the basis of immunofluores-cence at3100 magnification, APOL1 was present in the renalcortex of nondiseased kidney from European Americans (datanot shown). Higher magnification (3200 and3400) revealedrobust glomerular podocyte APOL1 staining (Figure 1). Lo-calization of APOL1 in podocytes was confirmed by costainingwith Wilms’ tumor 1 suppressor 1 (WT1; Figure 1) and

Figure 1. Glomerular enrichment of APOL1 on kidney cryo-sections. Immunofluorescence localization of APOL1 and WT1 innondiseased adult kidney cryosections from a European Ameri-can: Kidney cryosections were stained for APOL1 (red) and WT1(green), and counterstained with 49,6-diamidino-2-phenylindole(DAPI) (blue). APOL1 signals are enriched in glomeruli, withweaker expression in cortical tubules (A–D; original magnifica-tion, 3200). APOL1 and WT1, a cytoplasmic podocyte marker,display similar patterns (E and F; original magnification, 3400);overlay suggests APOL1 and WT1 colocalize in podocytes (G andH; original magnification, 3400).

340 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 339–348, 2015


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synaptopodin (SYNPO; Figure 2). Although the glomerularAPOL1 fluorescent signal did not overlap with CD31, an en-dothelial cell marker (Figure 2B) or a-smooth muscle actin(a-SMA), a mesangial cell marker (Figure 2D), APOL1 couldstill be present in low abundance in these cell types if it wasobscured by the robust signal in podocytes. In addition,APOL1 fluorescence was observed in renal tubules but withconsiderably lower fluorescence intensity than in podocytes(Figures 1 and 2C).

Podocyte APOL1 enrichment was also observed in non-diseased kidney cryosections fromAfricanAmericans basedoncostaining withWT1 (Figure 3, A–P). Interestingly, an AfricanAmerican compound-heterozygote participant with twoAPOL1 nephropathy variants (G1/G2) displayed marked glo-merular APOL1 beyond the podocyte (Figure 3, O and P),possibly reflecting protein within glomerular endothelial cells(GECs) based on colocalization with CD31 (Figure 3, Q–V).

APOL1 Protein Distribution in Primary Human RenalCell LinesPrimary proximal tubule cells (PTCs), GECs, and podocyteswere prepared as described in the Supplemental Methods.

Characterization of primary cells was based on the presenceand absence of appropriate cell-specific markers as assessed byimmunofluorescence (data not shown). As shown in Figure 4,APOL1 was detected in podocytes, GECs, and PTCs; however,the extent of expression in each cell varied, possibly reflectingtheir nonsynchronous state with respect to cell cycle. In addi-tion, many cells displayed heminuclear localization of APOL1,consistent with Golgi localization and secretory protein traf-ficking. Although we were unable to culture primary

Figure 2. Enrichment of APOL1 in glomerular podocytes onkidney cryosections of a European American. Colocalization ofAPOL1 with renal cell marker proteins in nondiseased adult kidneycryosections from a European American: Kidney cryosectionswere stained for APOL1 (red) and the indicatedmarker cell protein(green) and counterstained with DAPI. (A) APOL1 colocalizes withSYNPO (podocyte marker)-positive cells. (B) APOL1 does notcolocalize with CD31 (endothelial cell marker)-positive cells. (C)APOL1 is not enriched in dipeptidyl peptidase 4 (proximal tubulecell membrane marker)-positive cells. (D) APOL1 signal does notoverlap with a-SMA (mesangial cell marker)-positive cells. (For all:original magnification, 3400.)

Figure 3. Glomerular enrichment of APOL1 on kidney cryo-sections of African Americans with various APOL1 genotypes.Immunofluorescence localization of APOL1 and WT1 in non-diseased adult kidney cryosections from African Americans: Kid-ney cryosections were stained for APOL1 (red) and WT1 (green)and counterstained with DAPI (blue). (A–D) Patient homozygousfor wild-type APOL1. (E–H) Patient heterozygous for the APOL1G1 variant. (I–L) Patient heterozygous for the APOL1 G2 variant.(M–P) Patient with two nephropathy variants (compound heterozy-gote; G1/G2) revealing APOL1 signal beyond podocytes. (Q–V) InG1/G2 kidney cryosections, APOL1 colocalizes with CD31-positivecells (see boxes in Q–S). (T–V) Zoomed images corresponding toboxed areas above. For A–S: original magnification, 3400.

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mesangial cells, immunolocalization using an immortalizedhuman mesangial cell (HMC) line failed to reveal APOL1(Supplemental Figure 3E). In contrast to the finding in kidneycryosections, where APOL1 was predominantly localized topodocytes, APOL1 signal intensity was not significantly higherin the primary podocyte cell lines compared with primaryGECs and PTCs.

Quantification of APOL1 Protein in Human Tissues andCell LinesAPOL1 protein was measured in various renal and nonrenaltissues andcell lines byWesternblot analysis.Wewereunable toculture enough primary podocytes or HMCs to harvest RNAandprotein, andwe lacked ahighly specificmarker for isolatingprimary HMCs. Therefore, we relied on immortalized podo-cyte and HMC cell lines for these analyses. While APOL1 wasbarely detectable in predifferentiated podocytes, it was ex-pressed weakly after differentiation (Figure 5, lanes 1 and 2).Consistent with immunofluorescence, APOL1 was also detec-ted in primary GECs and PTCs, with protein abundanceslightly exceeding that observed in the podocyte cell line andapproaching levels observed in the liver, kidney cortex, andHepG2 human hepatoma cell line. A 45-kD band that waspresent on the Western blot for human liver (Figure 5, lane 7)was probably nonspecific because several other antibodiesspecific for human APOL1 failed to react with this band (datanot shown). The amount of APOL1 in 1 ml of serum is con-siderably higher than that observed in 70 mg of the kidney orliver extracts. When quantified with internal standards pro-duced in Escherichia coli, serum APOL1 concentration was

estimated to be approximately 15 mg/ml(data not shown), suggesting that the cir-culation provides an abundant reservoirof APOL1 for potential renal cell uptakepathways.

APOL1 mRNA Expression Patterns inRenal Cell Lines and TissuesQuantitative RT-PCR was used to verifypatterns of APOL1 expression. Among re-nal cells and cell lines, the highest APOL1mRNA levels were found in primary PTCsandGECs; these levels were similar to thoseobserved in HepG2 cells and liver tissue(Figure 6). Consistent with Western blotanalysis, APOL1 mRNA abundance in po-docyte cells was lower; however, no differ-ences were observed between pre- andpostdifferentiated cells.

The distribution of sites of APOL1 syn-thesis was explored further inwhole kidneycortex via florescence in situ hybridization(FISH) (Figure 7). The staining patterns forAPOL1 and SYNPO mRNA in the glomer-ulus were similar but did not overlap per-

fectly (Figure 7, A and B). This indicates that APOL1 mRNAsmay be present in glomerular cells other than podocytes. Fur-ther analysis showed that APOL1 mRNA was also present inglomerular and interstitial endothelial (CD31-positive) cells(Figure 7, C and D) but did not overlap in glomeruli witha-SMA (Figure 7E). As expected, APOL1 and a-SMAmRNAscolocalized in kidney interstitial arterial smooth muscle cells(Figure 7F). APOL1 mRNA was distributed unevenly amongtubule cells and was not limited to proximal tubules. The rel-ative abundance of APOL1 mRNA in tubule cells was similarto that observed in glomerular podocytes (Figure 7, G and H),which contrasted with the marked podocyte enrichment ofAPOL1 protein seen on kidney cryosections (Figures 1 and2). The reliability of RNA FISH and its imaging was demon-strated by absence of signal when using either no probe set or aprobe set specific for insulin, which is not present in kidney(Supplemental Figure 4).

Uptake of Recombinant APOL1 Protein intoDifferentiated Human Cell LinesTo evaluate potential mechanisms underlying the higherAPOL1 protein abundance in podocytes from nondiseasedkidney cryosections relative to primary podocytes, the differ-entiated human immortalized podocyte cell line was exposedto serum-free basic growth media containing purified re-combinant C-terminal 63-His tagged APOL1 for 6 hours. Onthe basis of immunofluorescence, with anti-APOL1 antibody,fluorescent signal above background was observed in podo-cytes incubated with as little as 0.1 mg of APOL1 per milliliter(Supplemental Figure 3N). At higher concentrations (APOL1

Figure 4. APOL1 protein is not enriched in primary human podocytes. Localization ofAPOL1 in primary human podocytes, glomerular endothelial cells, and proximal tubule cells:Indicated primary cells were stained for APOL1 and dual stained for WT1, CD31, or DPP4.Primary podocytes (A–D), primary GECs (E–H), and primary PTCs (I–L) all demonstrateAPOL1 staining; APOL1 is not enriched in primary podocytes compared with primary GECsand PTCs. Primary podocytes, GECs, and PTCs were all positive for their specific markers,WT1, CD31, and DPP4, respectively. (For all: original magnification, 3200.)






0.5 and 1.0 mg/ml), intracellular punctate deposits were de-tectable (Supplemental Figure 3, O and P). Uptake of APOL1was further confirmed byWestern blot analysis (SupplementalFigure 5) and confocal microscopy, which revealed a cyto-plasmic localization (Figure 8, Supplemental Figure 6,Supplemental Video 1). Finally, immunofluorescence usinganti-His tag antibody confirmed that the uptake involved

transport of exogenous APOL1 as opposed to induction ofendogenous accumulation (Supplemental Figure 7). APOL1was not taken up readily by GEC or HMC (SupplementalFigure 3, A–H), although it appeared to precipitate in the ex-tracellular spaces between cells at higher APOL1 concentra-tions, suggesting poor solubility under these conditions.APOL1 was visualized on both cell membranes and in thecytoplasm of PTC lines, but only at the higher concentrationsof APOL1 (Supplemental Figure 3, K and L). Hence, the PTCAPOL1 uptake pathway may be less efficient than that in po-docytes. To explore whether the APOL1 uptake pathway isclathrin dependent, we repeated the experiments in the pres-ence of potassium-deficient media, which disrupts clathrin-coated pit formation.11,12 APOL1 uptake decreased markedlywhen podocytes were incubated for 2 hours with APOL1,0.1 mg/ml, in potassium-deficient buffer compared with thepotassium-replete control (Supplemental Figure 8). Hence, weconclude that clathrin-mediated endocytosismay be responsiblefor the uptake of APOL1, although othermechanisms could alsoplay a role. Finally, we explored the consequences of APOL1uptake into podocytes by assessing cell viability and markersof cell proliferation and differentiation. On the basis of a lactatedehydrogenase release assay, exogenous APOL1 did not affectcell viability (Supplemental Figure 9). Likewise, we observed nochange in cellular cytoskeletal structure, as assessed by stainingof F-actin with phalloidin (Supplemental Figure 10), or altera-tions in the expression of proliferation and differentiationmark-ers (Supplemental Figure 11). Hence, the uptake of APOL1 didnot lead to a detectable change in cellular physiology, under theconditions tested.

DISCUSSION

The cellular distribution and abundance of APOL1protein andmRNA were studied in nondiseased kidney tissues, primaryrenal cells, and immortalized renal cell lines. APOL1 mRNAand APOL1 protein abundance in primary PTCs and GECs,differentiated immortalized human podocyte cell lines, andHepG2 cells were similar, a finding inconsistent with our ob-servation that APOL1 protein was highly enriched in podo-cytes on kidney cryosections. Podocyte APOL1 enrichmentrelative to other cell types could be the result of uptake ofcirculating APOL1, a concept supported by in vitro studiesdemonstrating uptake of recombinant APOL1 by immortal-ized differentiated podocytes but not other renal cell types.Although the APOL1 concentration in podocyte growth me-dium was low (,1% of mean serum APOL1 concentrations),uptake by the podocyte cell line was nonetheless rapid. This isconsistent with the prevailing concept that podocytes play acentral role in the pathogenesis of FSGS.

In contrast to these studies, Madhavan et al. reported9 thatAPOL1 was present in podocytes and tubule cells, with lowersignal intensity in podocytes. However, that report mainlyevaluated diseased kidney tissue (FSGS), where the APOL1

Figure 5. APOL1 protein levels in immortalized podocytes arenot higher than in primary GECs and PTCs. Relative APOL1protein levels in human cells, cell lines, and tissues were assessedby Western blot analysis. Seventy micrograms of cell lysate pro-tein (lanes 1–10) and 1 ml human serum (lane 11) were fraction-ated by 4%–20% SDS-PAGE and transferred to nitrocellulosemembrane. The membrane was probed with Sigma APOL1 an-tibody. Lane 1, Podo line (-) (predifferentiated podocyte cell line);lane 2, Podo line (+) (differentiated podocyte cell line); lane 3,primary GEC (European American); lane 4, primary PTC (AfricanAmerican); lane 5, primary PTC (European American); lane 6,HMC cell line; lane 7, liver (European American); lane 8, kidneycortex (European American); lane 9, kidney cortex (AfricanAmerican); lane 10, HepG2 cells; lane 11, human serum (EuropeanAmerican). Data are representative of three independent Westernblot experiments.

Figure 6. APOL1 mRNA levels in immortalized podocytes arenot higher than in primary GECs and PTCs. Relative APOL1mRNA expression levels in human cells, cell lines, and tissueswere assessed by quantitative RT-PCR. 1, Podo line (-) (pre-differentiated podocyte cell line); 2, Podo line (+) (differentiatedpodocyte cell line); 3, primary GEC (European American); 4, pri-mary PTC (European American); 5, HMC line; 6, liver (AfricanAmerican); 7, liver (European American); 8, HEK293 cells; 9,HepG2 cells. Relative mRNA levels were normalized to HepG2cell values. Data are mean6SD, n=3.
















signal could have been dampened by podo-cyte injury. The divergent outcomes mayalso be due to use of different APOL1 anti-bodies and analytical methods. We con-firmed the observations of Madhavanet al. using the same APOL1 antibody(HPA018885; Sigma-Aldrich) and heat-induced epitope retrieval on FFPE kidneysections (data not shown). However, thisantibody failed to produce a detectable sig-nal during immunofluorescence on kidneycryosections and also failed to recognize na-tive APOL1 during immunoprecipitation(data not shown). Hence, for our studieswe selected an antibody that reacted withnative human APOL1 (Epitomics, catalogno. 3245–1). This rabbit anti-humanAPOL1 monoclonal antibody displayed af-finity for native APOL1 based on a strongfluorescent signal during immunofluores-cence and effectiveness in immunoprecipi-tation. The Epitomics APOL1 antibody alsoallowed performance of immunofluores-cence on both primary and immortalizedrenal cell lines, which, along with mRNAexpression data, offered a generalized viewof the distribution of endogenous APOL1protein and mRNA abundance in cells thathad not been exposed to the systemic circu-lation. Because the kidney has a rich bloodsupply, circulating lipid-free/lipid-poorAPOL1 protein could affect the renalAPOL1 cell type distribution in frozen orfixed kidney tissue.

Endogenous APOL1 protein andmRNAwere present in primary PTCs, GECs, andimmortalized human podocyte cell lines

Figure 7. APOL1 mRNA is present in human kidney podocytes, GECs, and tubuleswith similar abundance on non-diseased human kidney FFPE sections. (A) APOL1mRNA (green) colocalizes with SYNPO mRNA (red; podocyte marker) in a glomerulusand is also present beyond podocytes. (B) APOL1 mRNA is present in tubules.

(C) APOL1 mRNA colocalizes with CD31mRNA (red; GEC marker) in a glomerulus andis also present beyond endothelial cells. (D)APOL1 mRNA localizes to renal blood vesselendothelial cells (CD31-positive). (E) APOL1mRNA does not overlap with a-SMA mRNA(red) in the glomerulus. (F) APOL1 mRNA ispresent in renal arterial smooth muscle cells(a-SMA positive) and renal tubule cells. (G)APOL1 mRNA does not colocalize with DPP4mRNA (red; proximal tubule cell marker) in theglomerulus. (H) APOL1 overlaps with DPP4 intubules; however, APOL1 is not limited toproximal tubules and may extend to other tu-bule cells. (For all: original magnification,3630.)



(Figures 5 and 6), whereas they were largely absent in mesangialcells based on Western blot, immunofluorescence, RT-PCR,and RNA FISH. The pattern of mRNA and protein abundancein these cell lines was similar, consistent with the predictionthat protein abundance reflects endogenous synthesis. Becausewe had limited numbers of primary podocytes from study par-ticipants, we were unable to isolate sufficient RNAor protein toexamine APOL1 expression. No APOL1 protein or RNA signalcolocalized with a-SMA on kidney sections, demonstratingthat mesangial cells do not synthesize or accumulate APOL1.Unlike podocytes, mesangial cells are not felt to be primarilyinvolved in the pathogenesis of APOL1-associated forms ofglomerulosclerosis; hence, we elected not to isolate primaryHMCs. Nevertheless, we did not observe higher APOL1 con-centrations in primary podocytes relative to primary PTCs andGECs based on immunofluorescence signal intensity (Figure4). The consistency between mRNA and protein levels, how-ever, was not observed in the kidney, where disproportionatelyhigh levels of APOL1 proteinwere present in podocytes relativeto mRNA levels. These data, along with the in vitro uptakeexperiments, suggest that the disproportionate abundance ofAPOL1 in podocytes observed in kidney cryosectionsmay arisefrom an exogenous source. Additional in vivo evidence sup-porting an extracellular source of APOL1 protein in podocytescame fromRNAFISH.Here, theAPOL1mRNA signal on FFPEkidney sections was not as enriched in podocytes as was ob-served for APOL1 protein on kidney cryosections. Indeed,there was no obvious difference in APOL1 mRNA signal in-tensity between SYNPO-positive podocytes and CD31-positiveglomerular endothelial cells. This study did not explore howAPOL1 genotype might affect APOL1 expression levels becausewe currently have insufficient numbers of participants with tworisk alleles to achieve statistical power. However, human tissuesample collection is ongoing, which should enable us to addressthis important question in future studies.

The solubility of APOL1 protein (Sino Biologic), the identityofwhichwas confirmed byWestern blot analysis (SupplementalFigure 12), appeared to be limited without serum in the growthmedia but was much higher in 10% FBS media (data notshown). This is probably due to the absence of HDL, whichnormally transports APOL1 in plasma.13 Nonetheless, smallamounts of non–lipoprotein-bound APOL1 may exist in hu-man serum and could potentially reach podocytes and renaltubule cells. Nonbound APOL1 is small enough (42 kD) to befiltered across the glomerular barrier andwould be available forreabsorption by kidney cells, similar to albumin (67 kD), whichhas also been detected in podocytes14 and PTCs.15 The APOL1concentrations in media to which our cell lines were exposedwere far lower than those previously reported by Duchateau inserum (5–10mg/ml)16 and estimated in our studies (15mg/ml).Assuming that #5% of serum APOL1 exists in a lipid-freeform, as is the case for APOA1,17,18 we estimate that approxi-mately 0.75 mg/ml APOL1 could potentially cross the glomer-ular filtration barrier. This concentration is within the rangeused in our in vitro study, where we observed significant uptakeof APOL1 by podocytes (Figure 8, Supplemental Figures 3, 5–7), but not by endothelial and mesangial cells, and is consistentwith the observed enrichment of podocyte APOL1 signal inkidney cryosections. APOL1 uptake into podocytes did notappear to affect cell viability (Supplemental Figure 9) or cyto-skeletal structure (Supplemental Figure 10). It also appearedthat there were no significant changes in mRNA levels of pro-liferation or differentiation markers in podocytes treated withand without APOL1 (Supplemental Figure 11). Hence, the ef-ficient uptake of APOL1 by podocytes might serve an as yetunknown physiologic function.9

Because the only direct evidence for podocyte uptake ofAPOL1wasobtainedusing cell lines, onemight argue that renalcells or cell lines in vitro may behave differently from cells invivo with respect to both expression and uptake of APOL1.However, the validity of studies using primary and immortal-ized renal cells was supported by the demonstration that theyexpress the same patterns of cell-specific markers (WT1,SYNPO, CD31, a-SMA, and DPP4) observed in cryosection(Figures 1–4 and 7). Furthermore, the podocyte uptake ofAPOL1 was specific for this cell type and was not observedto any great extent in mesangial, endothelial, or tubule cells.Hence, we suggest that the behavior observed in vitro by thesecell lines, coupled with the mRNA and protein distributionobserved in vivo, are consistent with an exogenous proteinuptake pathway contributing to the enrichment of APOL1 inpodocytes. Because nondiseased kidney tissue was used forimmunofluorescence and FISH, it is not known whether theintracellular APOL1 protein contributes directly to the path-ogenesis of FSGS and related nondiabetic kidney diseases. Aswas proposed by Madhavan et al.,9 APOL1 may be functionalin renal tissue and hence, reductions of APOL1 in podocytes ofpatients with FSGS andHIV-associated nephropathy could be aconsequence of disrupted intracellular APOL1 synthesis or re-duced cellular uptake. Data obtained from cultured primary

Figure 8. Uptake of exogenous recombinant APOL1 into humanpodocytes. Confocal immunofluorescence microscopy reveals (A)less APOL1 in differentiated human podocytes not incubated withAPOL1 compared with (B) podocytes incubated with 0.5 mg/mlAPOL1 for 6 hours in serum-free RPMI media. APOL1-containingmedia were removed, and cells were washed with PBS. Cellswere fixed and immunostained for APOL1 (red) and podocalyxin(green; podocyte marker). (For all: original magnification, 3630.)










proximal tubule cell lines from 10 African Americans with dif-ferentAPOL1 genotypes and normal kidney function indicatedthatAPOL1mRNA levels were positively associated withCDK4(R=0.84; P=0.003) (Supplemental Figure 13), a cell cycle G1phase-specific proliferation marker,19 but not with other pro-liferation markers (MCM2, MKI67, and PCNA1) (Supplemen-tal Table 1–1). As was observed in a previous report,20 MCM2,MKI67, and PCNA1 mRNA levels were correlated with eachother (Supplemental Tables 1 and 2). On the other hand, theclear pathogenicity associated with the G1 and G2 disease al-leles suggests that while wild-type APOL1 may promote cellsurvival, consistent with the long half-life of podocytes, thesedisease variants may have acquired a deleterious gain of func-tion, which becomes manifest in combination with other ge-netic and/or environmental inputs. On the basis of kidneytransplant studies, in which donor but not recipient APOL1genotypes affected graft survival,21,22 some would concludethat circulating APOL1 protein is less likely than intrinsic renalgene expression to be involved in the pathogenesis of APOL1-associated nephropathy. However, we caution against applyingthe transplant model to the pathogenesis of native kidneydisease, in part because APOL1-associated kidney disease is adecades-long process whereas transplant failure is a short-termoutcome confounded by many issues, including cold ischemiaand use of nephrotoxic immunosuppressive medications.

In conclusion, we provide evidence that APOL1 in humankidney cortexmay be derived from both endogenous synthesisandextracellular sources.Renal toxicity fromcirculating (non–lipoprotein-bound) APOL1may contribute toAPOL1-associatednephropathy, particularly as contributed by theG1 andG2diseasevariants. Enrichment of APOL1 protein in podocytes was evi-dent in nondiseased kidney cryosections in EuropeanAmericansand African Americans with different APOL1 genotypes (WT,heterozygous G1/WT, heterozygous G2/WT, and a G1/G2compound risk heterozygote), suggesting that APOL1 mayserve a functional role in the kidney.9Madhavan et al.9 reportedthat APOL1 protein was localized in the media of medium-sized arteries and arterioles in the kidneys of patients withFSGS and HIV-associated nephropathy. We extend this by ob-serving APOL1 mRNA in the media of medium-sized arteriesin persons without kidney disease (Figure 7F). Podocytes maynot be the only renal cells affected by APOL1; glomerular andextraglomerular endothelial cells and PTCs may also be af-fected by variant APOL1 proteins. This finding may explainthe widespread pattern of glomerulosclerosis with vascularchanges and interstitial fibrosis universally present in all formsof APOL1-associated nephropathy.

CONCISE METHODS

Nephrectomy SpecimensPatients scheduled to undergo radical or partial nephrectomy were

identified and recruited. Genomic DNAwas isolated from peripheral

lymphocytes and stored at280°C. Age, sex, presence of hypertension,

diabetes, cardiovascular disease, family history of kidney disease, and

medications were recorded. Preoperative BUN, serum creatinine,

eGFR, urinalysis, and urine protein-to-creatinine ratio were ob-

tained. Only patients with preoperative MDRD eGFR.60 ml/min

per 1.73 m2 were enrolled. The Institutional Review Board of Wake

Forest School of Medicine approved the study, and all patients pro-

vided written informed consent.

After removal of the affected kidney, the tissue was immediately

dissected to obtain the unaffected pole (radical nephrectomy) and

nondiseased tissue (partial nephrectomy). Nondiseased tissue was

immediately washed with sterile HBSS (Lonza, Walkersville, MD);

sectioned; and either (1) rapidly frozen in optimal cutting tempera-

ture gel (Sukaru FineTek Inc., Torrance, CA) for cryosection immu-

nofluorescence studies, snap frozen for protein andRNApreparation,

placed in a biopsy cassette (Fisher HealthCare, Houston, TX), and

immersed in 4% paraformaldehyde for preparation of paraffin-

embedded kidney tissue blocks or (2) kept in F12/DMEM (Invitrogen,

Grand Island, NY) media for primary cell isolation.

Immortalized Kidney Cell LinesThe immortalized human mesangial cells and conditionally immor-

talized humanproximal tubule, podocyte, and glomerular endothelial

cell lines were cultured in basic growth medium as described.23–26

Appropriate material transfer agreements were signed and approved

by all involved institutional review boards.

Uptake of Exogenous APOL1 into Human Podocyte,Mesangial, Endothelial, and Proximal Tubule Cell LinesTo examine whether exogenous APOL1 can be taken up by the HMC

line and differentiated human podocyte, GEC, and PTC lines,

recombinant humanAPOL1 (13910-H08B; Sino Biologic) wasmixed

in serum-free basic growthmedia and cultured with the cell lines for 6

hours in four-well chamber slides (BD BioSciences, San Jose, CA).

Immunofluorescence for APOL1 and corresponding cell markers

(CD31 for GEC line,27 DPP4 for PTC line,28 a-SMA for HMC line,29

and WT1 or podocalyxin for the podocyte cell line30,31) was used to

dually label the cells. Fluorescent signals were detected by conven-

tional and confocal fluorescence microscopy. The effect of APOL1

uptake into podocytes on cell viability was assessed by lactate dehy-

drogenase release assay (CytoTox96; Promega). Cytoskeletal structure

was assessed with Alexa Fluor 488 phalloidin (Invitrogen). The de-

pendence of APOL1 uptake on a clathrin-mediated pathway was as-

sessed using potassium-deficient media, as described elsewhere.11,12

Immunofluorescence Microscopy on Cultured Cells andKidney Tissue CryosectionsImmunofluorescence of APOL1 and markers was performed on

immortalized human renal cell lines, APOL1-transfected and empty

vector–transfected Chinese hamster ovary cell lines, and human pri-

mary renal cells using established protocols.32 Supplementary meth-

ods contain isolation, culture, and characterization protocols for the

primary renal cells. Human kidney cryosections were sliced at 6-mm

intervals using a cryotome (Leica Biosystems, Buffalo Grove, IL). In-

formation on the primary antibodies and antibody dilutions for im-

munofluorescence are listed in Supplemental Table 2. To demonstrate








the specificity of the Epitomics anti-APOL1 antibody, it was preincu-

bated overnight at 4°C with a 20-fold molar excess of the APOL1 pep-

tide (3245-P; Epitomics) that was used to immunize rabbits. The

preabsorbed antibody was then incubated with kidney cryosections,

and the staining pattern compared with that obtained with nonpreab-

sorbed primary antibody. Secondary antibodies (goat anti-rabbit Alexa

Fluor 594, catalogno.A11037, andgoat anti-mouseAlexaFluor 488, catalog

no. A11001) were used to display fluorescent signals (1:500 dilution).

Western BlotWestern blot was performed as described previously.33 Blots were

incubated overnight at 4°C with anti-APOL1 antibody (1:1,000,

HPA018885; Sigma-Aldrich). The membranes were then washed

three times in Tris-buffered saline containing 0.1% Tween 20 and

incubated for 1 hour in blocking buffer with anti-rabbit IgG conju-

gated to horseradish peroxidase (1:20,000; Jackson ImmunoResearch

Laboratories, West Grove, PA).

RNA FISHFFPE human kidney tissue blocks were sectioned at 5-mm intervals.

The following QuantiGene View RNA FISH “double Z” type 1 oli-

gonucleotide probe sets were obtained from Affymetrix (Santa Clara,

CA): i.e., SYNPO (Accession: NM_007286; probe ID VA1–13234),

CD31 (Accession: NM_000442; probe ID VA1–10872), a-SMA (Ac-

cession: NM_001613; probe ID VA1–10300), DPP4 (Accession:

NM_001935; probe ID VA1–14933), and INS (Accession:

NM_000207, probe ID VA1–10099). They were used according to

the manufacturer’s instructions. An APOL1 type 6 oligo-probe set was

custom designed by Affymetrix Panomics (Supplemental Table 3). To

assure probe specificity, blocking probes were added to the probe set to

mask regions highly homogenous with other APOL family members.

RT-PCRTotal RNAwas isolated from kidney and liver tissue using the RNAeasy

Tissue Mini Kit (Qiagen, Valencia, CA) and from immortalized and

primary cells using RNAeasy Mini Kit (Qiagen). The quantity and

quality of isolated RNA were determined by ultraviolet spectropho-

tometry and electrophoresis, respectively, using the Nanodrop 2000

(Thermo Fisher Scientific, Wilmington, DE) and Agilent 2100

Bioanalyzer (Agilent Technologies, Santa Clara, CA). One microgram

of RNAwas reverse transcribed with random hexamer primers using

theTaqManRTkit (AppliedBiosystems,FosterCity,CA).Primerswere

designed to capture all of the knownAPOL1 splice variants. RT-PCR in

thepresenceofSYBRGreenwasperformedwithanABI7500-FastReal-

Time PCR system (Applied Biosystems) using 18S ribosomal RNA as

normalization standard. Primer sequences are listed in Supplemental

Table 4. TheDelta-Delta CTmethod34 was used to quantify the relative

levels of APOL1 mRNAs across all study samples. Fold-changes were

normalized to mRNA levels on HepG2 cells, which were set to 1. All

experiments were performed in triplicate and expressed as mean6SD.

ACKNOWLEDGMENTS

This study was supported byNational Institutes of Health grants R01-

DK070941 and R01-DK084149 (PI: B.I.F.).

DISCLOSURESNone.

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This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013091017/-/DCSupplemental.





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