APOL1 Localization in Normal Kidney and Nondiabetic Kidney Disease

CLINICAL RESEARCH www.jasn.org

Sethu M. Madhavan,* John F. O’Toole,* Martha Konieczkowski,* Santhi Ganesan,† Leslie A. Bruggeman,* and John R. Sedor*‡

*Department of Medicine and the Rammelkamp Center for Education and Research, MetroHealth System Campus, Cleveland, Ohio; †Department of Pathology, MetroHealth Medical Center, Cleveland, Ohio; ‡Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

ABSTRACT In patients of African ancestry, genetic variants in APOL1, which encodes apolipoprotein L1, associate with the nondiabetic kidney diseases, focal segmental glomerulosclerosis (FSGS), HIV-associated nephropathy (HIVAN), and hypertensive nephropathy. Understanding the renal localization of APOL1 may provide clues that will ultimately help elucidate the mechanisms by which APOL1 variants promote nephropathy. Here, we used immunohistology to examine APOL1 localization in normal human kidney sections and in biopsies demonstrating either FSGS (n ϭ 8) or HIVAN (n ϭ 2). Within normal glomeruli, APOL1 only localized to podocytes. Compared with normal glomeruli, fewer cells stained for APOL1 in FSGS and HIVAN glomeruli, even when expression of the podocyte markers GLEPP1 and synaptopodin appeared normal. APOL1 localized to proximal tubular epithelia in normal kidneys, FSGS, and HIVAN. We detected APOL1 in the arteriolar endothelium of normal and diseased kidney sections. Unexpect- edly, in both FSGS and HIVAN but not normal kidneys, the media of medium artery and arterioles contained a subset of ␣-smooth muscle actin-positive cells that stained for APOL1. Comparing the renal distribution of APOL1 in nondiabetic kidney disease to normal kidney suggests that a previously unrecognized arteriopathy may contribute to disease pathogenesis in patients of African ancestry.

J Am Soc Nephrol 22: 2119–2128, 2011. doi: 10.1681/ASN.2011010069

Two genome-wide admixture scans identified a restricted to the genomes of humans and some non- highly significant association on chromosome human primates.10 Circulating APOL1 associates

22q12 between nondiabetic kidney disease and Af- with HDL3 particles and functions as a trypanolytic rican ancestry.1,2 The associated region contains factor in human serum.11,12 The parasite endocyto- many genes and initial investigations focused on ses APOL1-containing HDL particles, and once in- nonmuscle myosin heavy chain IIA, encoded by ternalized, APOL1 is targeted to the lysosome, MYH9, due to its podocyte expression and associa- where its colicin-like pore-forming activity causes tion with rare Mendelian giant platelet disorders osmotic swelling of the lysosome and trypanosome that are also characterized by variably penetrant death. Trypanosome species that infect humans glomerular diseases.3,4 However, attempts to iden- and cause disease have adapted to inhibit APOL1- tify the MYH9 causal variant underlying this asso- 5,6 ciation were unsucessful. Using 1000 Genomes Received January 19, 2011. Accepted June 1, 2011. Project sequence data,7 two groups demonstrated Published online ahead of print. Publication date available at that coding variants within the neighboring APOL1 www.jasn.org. 8,9 gene, which is centromeric to MYH9 on 22q12.3, Correspondence: Dr. John R. Sedor, 2500 MetroHealth Drive accounted for the association signal. APOL1 is one R415, Cleveland, OH 44109-1998. Phone: 216-778-4993; Fax: of six closely related apolipoprotein L family mem- 216-778-8248; E-mail: [email protected] ber genes clustered on chromosome 22. APOL1 is Copyright © 2011 by the American Society of Nephrology

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mediated trypanolysis. Variant APOL1, which encodes the kidney disease risk variants, can kill disease-causing trypano- somes by circumventing the parasite’s mechanism to evade lysis. The parasite-killing effect is dominant, requiring a single copy of the risk-variant APOL1 gene, whereas association with kidney disease is best fit by a recessive model.9 Since resistance to trypanosomal infection is a selective advantage in endemic regions, the kidney disease risk variants of APOL1 have been maintained in African populations. Consistent with this prem- ise, the region of chromosome 22 that contains APOL1 shows evidence for positive selection.13 Similar to sickle cell disease, the heterozygous state for APOL1 kidney-disease-associated variants is advantageous, but the homozygous state can result in disease.9 APOL1 is a 43 kD protein that is the only APOL family member with a secreted isoform. Since APOL1 transcripts are expressed in a number of tissues, including the kidney,14,15 we have characterized cellular localization of APOL1 in normal human kidney sections and biopsies from patients with focal segmental glomerulosclerosis (FSGS) and HIV-associated nephropathy (HIVAN) as a first step in understanding the association between APOL1 genetic variation and nondiabetic kidney disease.

RESULTS

APOL1 Localizes to the Glomerulus, Cortical Tubules, and Vascular Endothelium of the Normal Kidney Figure 1. APOL1 localization in the normal adult human kidney We verified the specificity of two commercially available anti- using immunoperoxidase staining. Serial, formalin-fixed, paraffin- bodies against APOL1 using multiple approaches (Supple- embedded sections of normal human kidney specimens were mentary Figures 1, 2, and 3) and examined formalin-fixed, immunostained with rabbit polyclonal APOL1 antibody (A, C, E, & G) or purified rabbit polyclonal IgG as control (B, D, F, & H). (A) paraffin-embedded human kidney sections without renal pa- Low magnification image showing predominant APOL1 staining thology to determine the normal cellular distribution of APOL1 in the renal cortex, with relative sparing of medulla. (C) APOL1 in renal parenchyma. Using immunohistochemistry, we found expression is seen in a glomerulus and some cortical tubules. (E) that all APOL1 immunoreactivity at low magnification was in the Magnified image from boxed region in (C) shows APOL1 staining renal cortex of normal human kidneys (Figure 1A) relative to in a cell with anatomical position and morphologic characteristics control sections, where only faint background resulting from of a podocyte, abutting the outer lamina of the glomerular base- endogenous peroxidase activity was detected (Figure 1 B). ment membrane with red blood cells (asterisk) contained in a Higher magnification views revealed robust staining in a subset capillary loop. (G) APOL1 staining is also seen in extraglomerular of cortical tubules that appeared to be proximal tubules by arterial endothelium (arrow). Comparable images of normal kid- morphology (Figure 1 C). The localization of APOL1 within ney sections (B, D, F, & H) incubated with nonimmune purified proximal tubules was confirmed using the lectin lotus tet- rabbit polyclonal IgG have minimal staining consistent with residual endogenous peroxidase activity. Scale bars: 300 ␮m(A&B), ragonolobus (Supplementary Figure 4). APOL1-postive tu- 50 ␮m (C, D, G, & H). bules were distinct from tubules positive for uromodulin, a marker of thick ascending limb of Henle. Less intense glomerular staining was observed in a subset of cells (Figure 1 C), APOL1 Colocalizes with Podocyte Markers in the which was not evident in control sections (Figure 1 D). High Normal Glomerulus magnification views of the glomerulus demonstrated peroxi- We used immunofluorescent confocal microscopy to colocalize dase staining of cells that are consistent with podocytes with APOL1 with markers for podocytes and mesangial and endothe- respect to anatomical position and morphology (Figure 1 E). lial cells in normal human kidney sections (Figure 2). The cytosol At similar magnification, glomerular cells in control sections of cells within glomeruli staining for APOL1 was completely showed no immunostaining (Figure 1 F). In addition, signal outlined by GLEPP1, a podocyte transmembrane receptor was detected in the endothelium of small arteries (Figure 1 G), phosphatase (Figure 2 A–F), suggesting localization of which was not seen in control sections (Figure 1 H). APOL1 within the podocyte. This pattern was consistent in

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express ␣-SMA, which did not overlap with APOL1 staining in cells that are endothelia by their location (Figure 2 G–I). Staining with antibodies against APOL1 and CD31, an endothelial marker, did not overlap in the glomerular capillaries, with the CD31 signal clearly on the opposite side of the glomerular basement membrane from the APOL1 signal (Figure 2 J–L). In contrast, CD31 and APOL1 antibodies identified the same cells in arterial vessels.

APOL1 Risk–variant Genotypes of FSGS and HIVAN Biopsy Samples We obtained deidentified, archival renal biopsy specimens demonstrating FSGS (n ϭ 8) or HIVAN (n ϭ 2), along with patient ages, genders, and self-reported races, and determined the genotype at the G1 and G2 loci with Sanger sequencing of DNA extracted from an additional section cut from tissue blocks9 (Table 1). The risk genotype was successfully determined in nine of the 10 samples examined in this study; Supplementary Figure 6 shows the sequencing data from each patient. Although the G1 risk variant is more common than the G2 risk variant, it was not found in the biopsies examined. The G2 risk allele was homozygous in two Afri- can American patients and was heterozygous in one African American and one Figure 2. Colocalization of APOL1 with cell markers in the normal adult human kidney. Caucasian patient, likely reflecting African Confocal immunofluorescent microscopy demonstrates (A, D, G, & J, green) APOL1 admixture in this individual. Specimens staining in the glomerulus of the normal kidney. (B & E, red) GLEPP1, a podocyte were reviewed to confirm that pathology transmembrane protein tyrosine phosphatase, demarcates podocyte plasma membranes. (C & F) Merged images show that the APOL1 signal (green) is completely was characteristic of FSGS and HIVAN, circumscribed by GLEPP1 staining (red) and appears to be present in the same cells. (D–F) and images of representative renal pathol- Magnified view of the indicated region in (C) shows that APOL1 (green) localizes to two ogy specimens, using standard histochem- cell bodies (arrowheads) outlined by GLEPP1 staining (red), and nucleated endothelial ical stains, are shown as supplementary cells lining the adjacent capillary loop (asterisks) show no APOL1 staining. (H) Cells within data (Supplementary Figure 7). normal adult human glomeruli do not express ␣-SMA (red); however, positive ␣-SMA staining is seen in an adjacent arterial wall (double arrows). (I) Merged image shows APOL1 Is Increased in the APOL1 staining (green) in the adjacent arterial endothelium (arrow) but not within the Vasculature and Decreased in arterial wall. (K & L, red) CD31 staining of the glomerular endothelium does not colocalize Podocytes in FSGS with APOL1 (green). The inset in (L) is a magnified image of the indicated region in that Since genetic variants in APOL1 have panel. (C, F, I, & L, blue) Nuclei were visualized with TOTO-3 staining. Scale bars: 50 ␮m been associated with nondiabetic kidney (A–C & G–L), 5 ␮m (D–E). diseases in African Americans, we determined if the renal distribution of APOL1 additional fields stained for GLEPP1 and also when anti- varied with respect to risk genotype in the biopsies. FSGS sam- bodies to another podocyte marker (synaptopodin) were ples were examined for glomerular APOL1 expression using used (Supplementary Figure 5). We were unable to detect immunohistochemistry (Figure 3 A–C). The overall immu- significant glomerular expression of ␣-smooth muscle actin nohistochemical staining for APOL1 appeared to be re- (␣-SMA) in the mesangium of the normal kidney. Mural duced in the FSGS biopsy specimens with fewer APOL1- cells in arterial vessels near the vascular pole of the glomeruli did positive tubules as seen in the low power view (Figure 3A).

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Table 1. APOL1 risk genotypes in FSGS and HIVAN samples those observed in the FSGS samples. Risk rs136177a There appeared to be fewer APOL1 pos- Disease Age Gender Race Comment Genotype Genotype itive tubules (Figure 5A), less glomerular FSGS 28 F AA wild type A/G signal (Figure 5 B), and de novo appear- FSGS 33 M AA Hom G2 A/A ance in the vessel wall of renal arteries FSGS 44 M AA wild type A/G (Figure 5 C). When present in glomeruli, FSGS 45 M C Het G2 A/A APOL1 remained colocalized with podo- FSGS 49 M C wild type A/G cyte markers (Figure 5 D–F) but not with FSGS 56 F C failed Failed Unable to isolate DNA the endothelial marker CD31 (Figure 5 FSGS 61 M C wild type A/A H–J). ␣-SMA and APOL1 did not colo- FSGS 74 F C wild type A/A calize within the glomeruli (Supplemen- HIVAN 33 M AA Hom G2 A/A tary Figure 10 A–C) but, similar to the HIVAN 47 M AA Het G2 A/A FSGS biopsies, did colocalize within the AA, African American; C, Caucasian; Hom, homozygous; Het, heterozygous. ars136177 is a synonymous SNP that is independent of the G1 and G2 risk variants but is contained vascular smooth muscle cells of small re- within the amplicon used to genotype the variants. Three patients, with homozygous wild type alleles at nal arteries (Supplementary Figure 10 the G1 and G2 loci, had heterogeneous genotypes at rs136177, verifying that both alleles were D–F). APOL1 localization within the en- sequenced in these individuals. dothelium of renal arteries in HIVAN biopsies (Supplementary Figure 10 D–F) The number of cells staining for APOL1 within the FSGS appeared similar in intensity and distribution to that identified glomeruli appeared to be less numerous compared with in biopsies of FSGS and normal sections (Figure 4). normal glomeruli (Figures 3B, 1C). In contrast to normal kidneys, a robust signal was detected in vessel walls at the APOL1 Is Expressed in Renal Cell Lines and Induced glomerular vascular pole (Figure 3 A,B) and small arteries in by Inflammatory Mediators the renal parenchyma (Figure 3C), which had not been ob- To examine whether APOL1 is endogenously expressed in the served in normal kidney sections (Figure 1G). kidney or absorbed from the circulation, we prepared lysates To further characterize changes in APOL1 glomerular dis- from human podocyte, proximal tubular, and vascular endo- tribution of FSGS samples, we used the same cell lineage mark- thelial cell lines grown in culture. Lysates were immunoblotted ers described above to examine mildly affected (Figure 3 D–L) for APOL1 and a single band was detected at the expected and severely affected glomeruli (Supplementary Figure 8). The molecular weight (Figure 6A). RT-PCR was performed on overall fluorescence intensity of APOL1 signal in the glomer- RNA extracted from a human podocyte cell line and the tran- uli of FSGS and HIVAN samples was diminished relative to script of expected size was detected (Figure 6 B). Based on prior glomeruli from samples without renal pathology (Supple- reports, APOL1 expression can be induced by inflammatory mentary Figure 9). Residual APOL1 signal remained colo- mediators such as recombinant tumor necrosis factor (TNF) calized with GLEPP1 but some podocytes that still ex- and lipopolysaccharide (LPS). Stimulation of human micro- pressed GLEPP1 has diminished or absent APOL1 vascular endothelial cells with TNF or LPS induced expression expression (Figure 3 D–F). Glomerular staining of ␣-SMA in of APOL1 relative to tubulin (Figure 6 C–D). FSGS samples was increased, labeling reactive mesangium, but did not colocalize with APOL1 (Figure 3 G–I). However, APOL1 did colocalize with ␣-SMA staining in the vascular DISCUSSION smooth muscle cells of arterioles at the glomerular vascular pole (Figure 3 G–I). CD31 staining of the glomerular endothe- We have characterized the cellular localization of APOL1 in lium did not colocalize with APOL1 (Figure 3 J–L). Interest- sections from a single healthy kidney and from biopsies dem- ingly, the FSGS samples demonstrated a striking increase of onstrating FSGS and HIVAN. The key results demonstrate APOL1 in the vascular wall of the small renal arteries outside that, in the absence of pathology, APOL1 is detected in podo- glomeruli, which was not found in the normal kidney (Figure 4). cytes, proximal tubules, and in medium-sized arterial and ar- As in the vessel wall of the glomerular vascular pole (Figure 3 G–I), teriolar endothelial cells. In HIVAN and FSGS, the cellular APOL1 colocalized with ␣-SMA staining in some arterial wall distribution pattern evolves. The number of APOL1-positive cells (Figure 4). Endothelial cells continued to stain for APOL1 in podocytes is diminished before a decrease of the podocyte dif- FSGS biopsy specimens (Figure 4). APOL1 staining patterns were ferentiation markers GLEPP1 and synaptopodin. Tubular consistent in all biopsies, regardless of APOL1 G2 genotypes. APOL1 also diminishes and there is a de novo appearance of APOL1 within cells of the arterial medial wall. Alterations in APOL1 Localization Are Consistent An immediate question is whether renal localization of between FSGS and HIVAN APOL1 reflects de novo synthesis or uptake from circulation.

While only two HIVAN samples were available for evaluation, APOL1 is a component of HDL3 particles and its presence overall the alterations in APOL1 distribution were similar to within kidney cells could reflect uptake of this subset of HDL

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particles. Proximal tubules express CD36, which binds unmodified HDL,16 and the punctate pattern of APOL1 staining in proximal tubules may indi- cate uptake by an endocytic process. En- dothelial cells take-up HDL through scavenger receptor B1,17 and the dyslipi- demia associated with nephrotic syndrome is associated with elevated vascular deposition of various lipoprotein complexes.18 In advanced chronic kidney disease, however, HDL production is typically repressed and thus the dynam- ics of renal exposure to circulating APOL1-containing HDLs during the course of the disease may be complex, especially in kidney diseases caused by infection such as HIVAN. We were surprised that APOL1 had such robust expression within specific kidney domains and hypothesized that APOL1 may be endogenously synthesized by renal tissue. Although origi- nally cloned as a pancreas-specific apolipoprotein,19 subsequent publications showed APOL1 transcripts in a number of tissues, including in the kidney, consistent with intrarenal APOL1 synthesis.14,15 Our studies have not conclu- sively established whether the APOL1 detected in the various renal parenchy- mal cells was synthesized de novo or was produced elsewhere and taken up Figure 3. Altered renal localization of APOL1 in focal segmental glomerulosclerosis by renal cells. However, based on our ob- (FSGS). Immunoperoxidase (A–C) and confocal immunofluorescent images (D–L) of servations in cultured cells and in con- kidney biopsy specimens from patients with FSGS stained using anti-APOL1 antibody. junction with published expression array (A) Reduced APOL1 staining is seen in glomeruli and cortical tubules compared with the normal kidney. In contrast, glomerular vascular tuft demonstrates prominent APOL1 expres- studies, APOL1 synthesis within the kid- sion (arrowheads). (B) Selected glomerulus from (A) shows diminished APOL1 staining (ar- ney is possible. Both de novo expression rows); however, the juxtaglomerular arteriole wall is prominently APOL1 positive (arrow- and extracellular uptake may uniquely or head). (C) In contrast to the normal kidney, APOL1 also appears in the vessel wall of synergistically contribute to pathogene- the renal arterioles. (D & F, green) APOL1 signal is diminished in the glomerulus, but sis. glomerular GLEPP1 expression (E & F, red) is relatively preserved in early FSGS. The Another important point to consider merged images (F) highlight these differences, where all APOL1 (green) positive cells in the context of renal APOL1 expression stain for GLEPP1 (red) but not all GLEPP1 expressing cells stain for APOL1. (G–I) is its intracellular trafficking and secre- Residual glomerular APOL1 signal (G & I, green) in early FSGS does not colocalize tion. Although all APOL1 isoforms have ␣ with the -SMA staining (H & I, red) of reactive mesangium. However, APOL1 signal signal peptides, regulatory mechanisms (G & I, green) is observed in the wall of the arteriole at the vascular pole of the of APOL1 synthesis and intracellular glomerulus (asterisk) and colocalizes with ␣-SMA (H & I, red), which labels vascular smooth muscle cells in the extraglomerular arteriolar wall (asterisk). (J–L, green) trafficking have not been characterized APOL1 remains diminished in early FSGS and does not colocalize with glomerular in metazoa. An obvious approach to de- anti-CD31 staining (K & L, red), which identifies endothelial cells, but does colocalize fine the relative importance of circulating in the extraglomerular vascular endothelium (arrowheads). (L) Merged image shows and renal-expressed APOL1 in the patho- overlap of APOL1 (green) and CD31 (red) staining in the extraglomerular vascular genesis of nondiabetic kidney diseases endothelium (arrow) but not within the glomerulus. (F, I, & L, blue) Nuclei were would be the examination of transplant visualized with TOTO-3 staining. Scale bars: 100 ␮m (A), 50 ␮m (B–L). registries to determine if APOL1 risk variants in the recipient and/or donor organ

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study has limitations. Biopsy sample size is small, and population frequencies for the G1 and G2 APOL1 alleles cannot be accurately quantified from nine patient samples. Although technical difficulties in genotyping from biopsies may ac- count for failure to identify patients with the G1 variant, our sequence chromato- grams were of high quality (Supplemen- tary Figure 6). The presence of APOL1 in the podocyte within the glomerular compartment is consistent with the prevailing notion that the podocyte plays a central role in the pathogenesis of FSGS and HIVAN. Since APOL1 is lost from podocytes before GLEPP1 and synaptopodin in FSGS, Figure 4. De novo localization of APOL1 to the renal arterial wall in FSGS. Confocal we reason that its disappearance is not a immunofluorescence imaging of medium sized renal arterioles from the normal human consequence of generalized podocyte kidney (A–C) and from FSGS (D–F). (A & C, green) APOL1 signal is identified in cells dedifferentiation. Rather, we suspect anatomically consistent with endothelium of medium-sized renal arterioles (arrowheads) in the normal adult human kidney and adjacent tubular segments but does not colocalize that the loss of APOL1 from podocytes is with vascular smooth muscle cells of renal arterioles stained with anti-␣-SMA antibody (B either an early marker of dedifferentia- & C, red). APOL1-positive cells are located in the luminal wall of the blood vessel with tion or that preserved APOL1 function is surrounding ␣-SMA positive vascular smooth muscle cells, consistent with endothelial required to maintain a differentiated localization. (D & F, green) Representative cross-section of a renal arteriole from an FSGS podocyte phenotype. In vitro work in biopsy demonstrates persistent endothelial APOL1 signal and de novo appearance other cell types has shown that overex- within the vessel wall compared with the normal arteriole (A). (E & F, red) Anti-␣-SMA pressed APOL1 sequesters phosphatidic antibody staining identifies APOL1-positive cells as vascular smooth muscle (F, green). acid and cardiolipin and promotes au- The vascular endothelium remains positive for APOL1 staining (arrowhead). (C & F, blue) tophagocytic cell death.23,24 However, ␮ Nuclei were visualized with TOTO-3 staining. Scale bars: 50 m (A–F). these observations on APOL1 overex- pression do not readily provide insight confer increased susceptibility to graft loss after transplantation into APOL1-regulated kidney disease pathogenesis. HIVAN through recurrent or de novo pathologies associated with APOL1 and FSGS biopsy findings do not demonstrate marked changes risk variants. in APOL1 abundances within individual podocytes compared The presence of APOL1 in the normal kidney suggests that with normal kidney sections, although fewer cells express it is serving a functional role in the normal kidney. As a con- APOL1 within the disease glomeruli. If APOL1, at normal ex-

stituent of HDL3 particles, it has been assumed, but not dem- pression levels, does regulate autophagy, variant APOL1 may onstrated, that APOL1 participates in cholesterol metabolism. cause disease by dysregulating autophagic pathways, similar to In addition, APOL1 forms ion channels in lipid bilayers20,21 studies in mice with podocyte deletion of autophagy-related 5 and may disrupt membranes,22 but these studies, while infor- (ATG5) caused glomerulopathy in aging mice.25 mative about APOL1’s trypanolytic activity, do not illuminate The vascular distribution of APOL1 was restricted to the function within normal cells. Currently, the function of endothelium of arterioles in normal kidney sections, and we APOL1 in any of the resident cells of the kidney is entirely were unable to detect APOL1 in the glomerular capillary speculative. Although we detected a clear change from the nor- endothelium of normal or diseased kidney sections. Previ- mal distribution of APOL1 in both FSGS and HIVAN biop- ous reports have shown in situ expression of APOL1 and sies, the patterns did not correlate with kidney-disease- APOL3 transcripts in aortic endothelial cells.26,27 The endo- associated genotypes. Similar cellular distribution changes thelial localization of APOL1 in the arteriolar endothelium were demonstrated in patients homozygous, heterozygous, remained in the FSGS and HIVAN samples, and this expres- or null for the G2 risk variant, suggesting that the kidney- sion may be increased in disease. With a role in combating disease-associated variants do not cause disease by altering Trypanosome infection, APOL1 is considered an acute APOL1 trafficking or protein levels. Rather, the G2 risk variant phase reactant, and like other acute phase reactants, its pro- of APOL1 likely results in dysregulation of APOL1’s functional duction is regulated by cytokines that mediate immune re- role within the kidney cells where it is localized. None of our sponses such as TNF and type I interferons.26,27 Our in vitro patients had the G1 risk variants, so we cannot comment on the studies similarly found that vascular endothelial cells in- effect of this variant on APOL1 localization in the kidney. Our duced APOL1 expression in response to TNF and bacterial

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small sample size, we have shown that APOL1 is present in the normal kidney and may be endogenously expressed, and that the cell compartments in which it appears change in HIVAN and FSGS. The cellular distribution patterns do not correlate with kidney- disease-associated APOL1 variants. While the podocyte is an attractive fo- cus for further work, given the evidence for podocyte dysfunction in glomerular disease, the abundant APOL1 expression in cortical tubules may contribute to the pathogenesis of the tubu- lointerstitial disease that characterizes both HIVAN and FSGS. The de novo appearance of APOL1 in renal arteries and arterioles is intriguing and suggests that vascular wall remodeling may also contribute to FSGS and HI- VAN pathogenesis.

CONCISE METHODS

Figure 5. APOL1 redistribution is consistent between FSGS and HIVAN samples. Patient Samples Immunoperoxidase (A–C) and confocal immunofluorescent images (D–I) of kidney biopsy We obtained formalin-fixed, paraffin-em- specimens from patients with HIVAN stained using anti-APOL1 antibody. (A) Reduced bedded normal human kidney sections (Im- APOL1 staining in glomeruli and cortical tubules compared with the normal kidney. (B) genex Corp, San Diego, CA) and deidentified, Selected glomerulus from (A) shows diminished APOL1 staining (arrowhead). (C) In archival, formalin-fixed, paraffin-embedded contrast to the normal kidney, APOL1 also appears in vessel wall of the renal arterioles. (D & F, green) APOL1 signal is diminished in the glomerulus, but remains colocalized with human kidney biopsy specimens assigned a GLEPP1 expression (E & F, red), which is also diminished in HIVAN. (H & J, green) histopathologic diagnosis of FSGS or HIVAN Glomerular APOL1 signal does not overlap with CD31 staining (I & J, red), which by a clinical pathologist. Age, self-reported identifies glomerular capillary endothelium. (F & I, blue) Nuclei were visualized with race, gender, and histopathologic diagnosis TOTO-3 staining. Scale bars: 100 ␮m (A), 50 ␮m (B–I). were obtained for each biopsy sample. Sam- ples were otherwise deidentified. All studies endotoxin, consistent with prior observations with aortic involving human tissues were performed with approval of the insti- endothelia cells.26,27 tutional review board of the MetroHealth System campus, Case West- The de novo appearance of APOL1 in the arterial vessel wall ern Reserve University. Eight samples with FSGS and two samples of was unexpected. In contrast to the normal kidney, APOL1 was HIVAN were obtained for these studies. dramatically increased in the vascular smooth muscle cells of the medium size and arteriolar vessel walls in the FSGS and Cell Lines HIVAN biopsies. The origin of these APOL1-positive cells is A conditionally immortalized, human podocyte cell line has been not clear but the partial colocalization of APOL1 and ␣-SMA described previously.32 Human proximal tubule cells immortalized suggests the cell is of vascular smooth muscle lineage. Arterial by adeno-12/SV40 transformation were obtained from the laboratory vascular wall remodeling is a characteristic of many disease of Dr. Jeffrey Schelling and were cultured in DMEM:F12 1:1 with 10% processes, and the vascular smooth muscle cell exhibits pheno- FBS and 1% PCN/Strep at 37 °C in 5% CO2. Primary human micro- typic plasticity.28 Alternatively, APOL1-positive cells could re- vascular endothelial cells from lung (hMVECs; Lonza Walkersville, flect migration of activated adventitial cells into the medial Inc., Walkersville, MD) were cultured in EBM-2 basal media with layer of the arteriole.29 Prior reports have demonstrated that EGM-2 MV supplements (Lonza Walkersville, Inc.) at 37 °C with 5% vascular sclerosis is worse in biopsies from African American CO2 and used at the seventh passage number. For experiments exam- compared with Caucasian30,31 patients, suggesting the hypoth- ining the stimulation of APOL1 expression in hMVECs, cells were esis that the de novo appearance of variant APOL1 in small grown in culture for 48 h to confluence, then treated for an additional renal arteries contributes to kidney disease pathogenesis. 24 h before protein extraction with recombinant human TNF (R&D Although our conclusions should be tempered by the Systems, Minneapolis, MN) at [20 ng/ml], with LPS from the O55:B5

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tagged APOL1, generated as previously published33 (Supplementary Figure 3).

Immunohistochemistry Formalin-fixed, paraffin-embedded human kidney sections of 4-␮m thickness on standard microscopy slides were heated overnight at 40 °C and then at 62 °C for 1 hr. Paraffin was cleared with xylenes, and the section was rehydrated in a graded ethanol series and im-

mersed in ddH2O. Slides were transferred to a pressure boiler containing boiling antigen retrieval solution (10 mM trisodium citrate di- hydrate, pH ϭ 6.0, 0.05% tween-20), sealed and heated an additional 4.5 min. Slides were cooled to 25 °C over 1.5 h, rinsed in PBS, pH ϭ 7.4, and washed in PBS, pH ϭ 7.4, with 0.2% tween-20 (PBST). Endogenous peroxidase activity was blocked for 30 min at room Figure 6. APOL1 is expressed in cultured human kidney cells and is induced in vascular ϭ endothelial cells with TNF/LPS treatment. (A) APOL1 is detectable by immunoblotting in temperature with 3% H2O2 in PBS, pH 7.4, cultured human podocytes, proximal tubule (HPRT) cells, and CHO cells transfected with then washed in PBST and blocked for 1 hr at an APOL1 expression construct. The blot is representative of three independent exper- room temperature in 5% serum in PBST. Sec- iments. (B) APOL1 transcript is detectable in total RNA isolated from cultured human tions were then incubated overnight at 4 °C in podocytes. The blot is representative of three independent experiments. (C, D) In vitro 1% serum, PBS with a polyclonal rabbit anti- expression of APOL1 was detected in unstimulated and TNF- or LPS-stimulated human APOL1 primary antibody (Sigma, St. Louis, microvascular endothelial cells (hMVECs) by immunoblotting and normalized by the MO) at 1:500 or equivalent purified rabbit expression of tubulin (C). APOL1 expression was quantified by densitometry in unstimu- polyclonal IgG (Millipore, Billerica, MA). An ϭ lated hMVECs and hMVECs stimulated with TNF or LPS (n 3) (D). Results were anti-rabbit immunoperoxidase labeling sys- normalized to tubulin expression and expressed as the mean Ϯ SE; p values were tem, the Vectastain ABC kit, was used accord- calculated with ANOVA, followed by t test, to make intergroup comparisons. *P Ͻ 0.05. ing to manufacturer’s instructions (Vector Laboratories, Burlingame, CA), washed in strain of Escherichia coli (Sigma) at [1 ng/ml], or no additions as an PBST, and immersed in PBS. Peroxidase activity was monitored after unstimulated control. the addition of substrate using a DAB kit (Vector Laboratories) according to manufacturer’s instructions. Sections were counterstained

Reagents and Anti-APOL1 Characterization with Hematoxylin, rinsed in tap H20, dehydrated in a graded ethanol Reagents used in these studies were obtained (Fisher Scientific, Pitts- series then xylenes, and mounted with permount (Fisher Scientific, burgh, PA) unless otherwise stated. The specificity of two commer- Pittsburg, PA). Sections were viewed using an Olympus Bx51 micro- cially available anti-APOL1 antibodies was characterized in cell scope and images captured with Olympus DP71 camera with DP con- culture and kidney tissue (Supplementary Figures 1–3). We found troller software (Olympus America, Inc., Center Valley, PA). Images the polyclonal anti-APOL1 antibody, HPA018885 (Sigma, St. were prepared for publication using Adobe CS4 software (Adobe Sys- Louis, MO), raised in rabbit using a recombinant fusion protein tems, Inc., San Jose, CA). containing amino acids 263 to 398 of human APOL1 isoform 1, NM_003661.3, to be superior to the polyclonal anti-APOL1 antibody Immunofluorescence Microscopy (Abcam, Cambridge, MA) raised in goat using a synthetic peptide Formalin-fixed, paraffin-embedded sections were prepared as described corresponding to amino acids 386 to 397 of human APOL1 isoform 1. for immunohistochemistry through the antigen-retrieval protocol. Then The rabbit polyclonal APOL1 antibody (Sigma) was used throughout the sections were washed in PBST, blocked with 5% serum in PBST, and these studies and specifically recognized APOL1 but not APOL3 (Sup- incubated overnight at 4 °C in PBS, pH ϭ 7.4, 1% serum, 0.1% tween-20 plementary Figure 1). We did not determine cross-reactivity with with appropriate primary antibodies, normal purified rabbit polyclonal other APOL family members, which have similarity to APOL1. Mouse IgG, or exclusion of primary antibody. APOL1 was detected using a poly- does not express an APOL1 orthologue, and kidney sections incu- clonal rabbit antibody (Sigma, St. Louis, MO) at 1:50. Podocytes were bated with the rabbit anti-APOL1 antibody showed no immuno- labeled with a mouse monoclonal anti-GLEPP1 antibody (a gift from Dr. reactivity (Supplementary Figure 2). In contrast, the goat APOL1 Roger Wiggins) at 1:50 or a mouse monoclonal anti-synaptopodin anti- antibody demonstrated significant, nonspecific immunostaining. body, clone G1D4 (Meridian Life Science, Inc., Saco, ME) at 1:5. Endo- Immunoreactivity in human kidney sections was abrogated by thelial cells were labeled with a mouse monoclonal anti-CD31 antibody, preincubating the rabbit anti-APOL1 with recombinant, HIS- clone JC70A (Dako, Carpinteria, CA) at 1:20. Smooth muscle was labeled

2126 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 2119–2128, 2011 www.jasn.org CLINICAL RESEARCH with a mouse monoclonal ␣-SMA antibody, clone 1A4 (Sigma, St. Louis, biopsy cores that had been sectioned onto glass slides. A biopsy sec- MO) at 1:300. After incubation with primary antibody or normal puri- tion from each of the samples used for microscopic analysis was re- fied rabbit polyclonal IgG sections were washed with PBST, incubated moved from the slide using a sterile scalpel blade, and DNA was ex- with species specific Fluorophore conjugated secondary antibody (Mo- tracted using RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion, lecular Probes, Eugene, OR) at 1:400 in PBS, and washed with PBST. Cell Inc., Austin, TX) according to manufacturer’s instructions. Isolated nuclei were visualized with TOTO-3 iodide (Molecular Probes, Eugene, genomic DNA was quantitated using a Nanodrop-1000 spectropho- OR) staining. Sections were mounted with Vectashield (Vector Labora- tometer (Nanodrop Products, Wilmington, DE) and 50–100ng of tories, Burlingame, CA) and confocal images captured using a Leica TCS genomic DNA was used as template in a PCR reaction amplifying a SP2 confocal system (Leica Microsystems, Wetzlar, Germany). single 380 bp amplicon containing the known risk variants (prim- ers: forward-5Ј- AGACGAGCCAGAGCCAATC-3Ј, reverse-5Ј- Total RNA Isolation and RT-PCR CTGCCAGGCATATCTCTCCT-3Ј). PCR products were evaluated A human podocyte cell line32 was cultured and total RNA prepared as using agarose gel electrophoresis with ethidium bromide; the remain- described previously.34 Reverse transcription was done using random der of the reaction was purified using a PCR purification kit (Marligen hexamers and Superscript III First-Strand Synthesis Supermix (Invit- Biosciences, Inc., Ijamsville, MD) and sequenced using a 3730xl DNA rogen, Carlsbad, CA).32 Sample cDNA (4 ␮l) was used to perform Analyzer (Applied Biosystems, Carlsbad, CA) with the PCR forward PCR for APOL1 with the HotStarTaq Master Mix Kit (Qiagen, Valen- primer. Sequences were evaluated using Sequencher® Software (Gene cia, CA) using the following conditions: 95 °C for 15 min; 94 °C for Codes, Ann Arbor, MI). 15 s, 54 °C for 30 s, 72 °C for 30 s (36 cycles); 72 °C for 5 min, 4 °C hold. Primer pairs for APOL1: Forward 5Ј-CAGCAGTACCATGGAC- TACGG-3Ј and Reverse 5Ј-CCGCTCCAGTCACAGTTCTTGGTC- ACKNOWLEDGMENTS 3Ј. For GAPDH message, sample cDNA (1.2 ␮l) was used as template in the PCR reaction with an annealing temperature of 57 °C and the This work was supported, in whole or in part, by National Institutes of Ј Ј following primer pair: Forward 5 -GGAGCCAAACGGGTCATC-3 Health grants DK-064719 and DK-071108. and Reverse 5Ј-TGTTGCTGTAGCCGTATTCAT-3Ј. PCR products were visualized in 1% TBE agarose gel with 0.5 ␮g/ml ethidium bromide and imaged using a Fotodyne FOTO/analyst archiver worksta- DISCLOSURES tion (Fotodyne Incorporated, Hartland, WI). None.

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