Basic Research

Localization of APOL1 Protein and mRNA in the Human 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
JASN February 2015, 26 (2) 339-348; DOI: https://doi.org/10.1681/ASN.2013091017

Abstract

Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms’ tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.

Molecular genetics is transforming our understanding of the pathogenesis in nondiabetic nephropathy in populations of African ancestry.1,2 HIV-associated collapsing glomerulopathy, idiopathic FSGS, severe lupus nephritis, and hypertension-attributed nephropathy are strongly associated with apolipoprotein L1 (APOL1) G1 and G2 gene variants on chromosome 22q13.1 in African Americans.1–3 APOL1 is a minor apolipoprotein component of HDL, and APOL1 nephropathy variants are associated with HDL subfraction concentrations in African Americans.4 Circulating APOL1 has the ability to kill Trypanosoma brucei rhodesiense, a cause of African sleeping sickness.5 APOL1 protein is present in plasma,6 but its intracellular effects on kidney function remain unknown. To better understand potential links between APOL1 and renal disease, localizing APOL1 protein and mRNA in the healthy kidney tissue of African Americans and Americans of European descent remains an important goal.

Although APOL1 transcripts are expressed in many tissues, including the kidney,7,8 the specific renal cell types that engage in APOL1 transcription are not known. It is uncertain which kidney cells are enriched in APOL1 protein because mRNA abundance does not necessarily correlate with cellular protein abundance. It is also unclear how renal abundance of APOL1 compares with levels in liver and serum. Madhavan et al. reported, using formalin-fixed paraffin-embedded (FFPE) kidney sections and heat-induced epitope retrieval methods, that APOL1 was present in tubule cells and with lower signal intensity in podocytes.9 We tested whether these findings would be replicated in kidney cryosections using another antibody recognizing native APOL1 protein, and we extended these analyses to primary and immortalized renal cell models to further explore cell-specific localization of APOL1 and to address the relative contribution of endogenous synthesis versus exogenous uptake to APOL1 protein localization and abundance. These studies comprehensively assess sites of renal APOL1 synthesis and localization and suggest that glomerular localization of APOLI may be a consequence of both endogenous synthesis and podocyte uptake of APOL1 from the circulation or glomerular filtrate.

Results

Specificity of Rabbit anti-APOL1 Antibodies

The specificity of a commercially available rabbit anti-APOL1 monoclonal antibody (3245–1; Epitomics, Burlingame, CA) was established. The Epitomics antibody successfully recognized circulating APOL1 in its native form in human serum by immunoprecipitation (data not shown). When used in immunofluorescence microscopy, the antibody detected APOL1 in APOL1-transfected, but not empty vector–transfected, Chinese hamster ovary cells (Supplemental Figure 1). When used to stain kidney cryosections, abundant glomerular staining was observed, which was blocked upon pretreatment with the APOL1 immunogen blocking peptide (Supplemental Figure 2), further confirming the specificity of this antibody.

APOL1 Protein Localization in Human Nondiseased Kidney Cryosections

Kidney cryosections from European Americans and African Americans with preoperative eGFRs calculated with Modification of Diet in Renal Disease (MDRD) equation10 >60 ml/min per 1.73 m2 and who underwent nephrectomy for renal cell carcinoma were examined to establish APOL1 distribution in nondiseased parenchyma. On the basis of immunofluorescence at ×100 magnification, APOL1 was present in the renal cortex of nondiseased kidney from European Americans (data not shown). Higher magnification (×200 and ×400) revealed robust glomerular podocyte APOL1 staining (Figure 1). Localization of APOL1 in podocytes was confirmed by costaining with Wilms’ tumor 1 suppressor 1 (WT1; Figure 1) and synaptopodin (SYNPO; Figure 2). Although the glomerular APOL1 fluorescent signal did not overlap with CD31, an endothelial cell marker (Figure 2B) or α-smooth muscle actin (α-SMA), a mesangial cell marker (Figure 2D), APOL1 could still be present in low abundance in these cell types if it was obscured by the robust signal in podocytes. In addition, APOL1 fluorescence was observed in renal tubules but with considerably lower fluorescence intensity than in podocytes (Figures 1 and 2C).

Figure 1.
Figure 1.

Glomerular enrichment of APOL1 on kidney cryosections. Immunofluorescence localization of APOL1 and WT1 in nondiseased adult kidney cryosections from a European American: Kidney cryosections were stained for APOL1 (red) and WT1 (green), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). APOL1 signals are enriched in glomeruli, with weaker expression in cortical tubules (A–D; original magnification, ×200). APOL1 and WT1, a cytoplasmic podocyte marker, display similar patterns (E and F; original magnification, ×400); overlay suggests APOL1 and WT1 colocalize in podocytes (G and H; original magnification, ×400).

Figure 2.
Figure 2.

Enrichment of APOL1 in glomerular podocytes on kidney cryosections of a European American. Colocalization of APOL1 with renal cell marker proteins in nondiseased adult kidney cryosections from a European American: Kidney cryosections were stained for APOL1 (red) and the indicated marker cell protein (green) and counterstained with DAPI. (A) APOL1 colocalizes with SYNPO (podocyte marker)-positive cells. (B) APOL1 does not colocalize with CD31 (endothelial cell marker)-positive cells. (C) APOL1 is not enriched in dipeptidyl peptidase 4 (proximal tubule cell membrane marker)-positive cells. (D) APOL1 signal does not overlap with α-SMA (mesangial cell marker)-positive cells. (For all: original magnification, ×400.)

Podocyte APOL1 enrichment was also observed in nondiseased kidney cryosections from African Americans based on costaining with WT1 (Figure 3, A–P). Interestingly, an African American compound-heterozygote participant with two APOL1 nephropathy variants (G1/G2) displayed marked glomerular 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).

Figure 3.
Figure 3.

Glomerular enrichment of APOL1 on kidney cryosections of African Americans with various APOL1 genotypes. Immunofluorescence localization of APOL1 and WT1 in nondiseased adult kidney cryosections from African Americans: Kidney cryosections were stained for APOL1 (red) and WT1 (green) and counterstained with DAPI (blue). (A–D) Patient homozygous for wild-type APOL1. (E–H) Patient heterozygous for the APOL1 G1 variant. (I–L) Patient heterozygous for the APOL1 G2 variant. (M–P) Patient with two nephropathy variants (compound heterozygote; G1/G2) revealing APOL1 signal beyond podocytes. (Q–V) In G1/G2 kidney cryosections, APOL1 colocalizes with CD31-positive cells (see boxes in Q–S). (T–V) Zoomed images corresponding to boxed areas above. For A–S: original magnification, ×400.

APOL1 Protein Distribution in Primary Human Renal Cell Lines

Primary proximal tubule cells (PTCs), GECs, and podocytes were prepared as described in the Supplemental Methods. Characterization of primary cells was based on the presence and absence of appropriate cell-specific markers as assessed by immunofluorescence (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 reflecting their nonsynchronous state with respect to cell cycle. In addition, many cells displayed heminuclear localization of APOL1, consistent with Golgi localization and secretory protein trafficking. Although we were unable to culture primary mesangial cells, immunolocalization using an immortalized human mesangial cell (HMC) line failed to reveal APOL1 (Supplemental Figure 3E). In contrast to the finding in kidney cryosections, where APOL1 was predominantly localized to podocytes, APOL1 signal intensity was not significantly higher in the primary podocyte cell lines compared with primary GECs and PTCs.

Figure 4.
Figure 4.

APOL1 protein is not enriched in primary human podocytes. Localization of APOL1 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 demonstrate APOL1 staining; APOL1 is not enriched in primary podocytes compared with primary GECs and PTCs. Primary podocytes, GECs, and PTCs were all positive for their specific markers, WT1, CD31, and DPP4, respectively. (For all: original magnification, ×200.)

Quantification of APOL1 Protein in Human Tissues and Cell Lines

APOL1 protein was measured in various renal and nonrenal tissues and cell lines by Western blot analysis. We were unable to culture enough primary podocytes or HMCs to harvest RNA and protein, and we lacked a highly specific marker for isolating primary HMCs. Therefore, we relied on immortalized podocyte and HMC cell lines for these analyses. While APOL1 was barely detectable in predifferentiated podocytes, it was expressed weakly after differentiation (Figure 5, lanes 1 and 2). Consistent with immunofluorescence, APOL1 was also detected in primary GECs and PTCs, with protein abundance slightly exceeding that observed in the podocyte cell line and approaching levels observed in the liver, kidney cortex, and HepG2 human hepatoma cell line. A 45-kD band that was present on the Western blot for human liver (Figure 5, lane 7) was probably nonspecific because several other antibodies specific for human APOL1 failed to react with this band (data not shown). The amount of APOL1 in 1 µl of serum is considerably higher than that observed in 70 μg of the kidney or liver extracts. When quantified with internal standards produced in Escherichia coli, serum APOL1 concentration was estimated to be approximately 15 µg/ml (data not shown), suggesting that the circulation provides an abundant reservoir of APOL1 for potential renal cell uptake pathways.

Figure 5.
Figure 5.

APOL1 protein levels in immortalized podocytes are not higher than in primary GECs and PTCs. Relative APOL1 protein levels in human cells, cell lines, and tissues were assessed by Western blot analysis. Seventy micrograms of cell lysate protein (lanes 1–10) and 1 μl human serum (lane 11) were fractionated by 4%–20% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with Sigma APOL1 antibody. 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 (African American); lane 5, primary PTC (European American); lane 6, HMC cell line; lane 7, liver (European American); lane 8, kidney cortex (European American); lane 9, kidney cortex (African American); lane 10, HepG2 cells; lane 11, human serum (European American). Data are representative of three independent Western blot experiments.

APOL1 mRNA Expression Patterns in Renal Cell Lines and Tissues

Quantitative RT-PCR was used to verify patterns of APOL1 expression. Among renal cells and cell lines, the highest APOL1 mRNA levels were found in primary PTCs and GECs; these levels were similar to those observed in HepG2 cells and liver tissue (Figure 6). Consistent with Western blot analysis, APOL1 mRNA abundance in podocyte cells was lower; however, no differences were observed between pre- and postdifferentiated cells.

Figure 6.
Figure 6.

APOL1 mRNA levels in immortalized podocytes are not higher than in primary GECs and PTCs. Relative APOL1 mRNA expression levels in human cells, cell lines, and tissues were assessed by quantitative RT-PCR. 1, Podo line (-) (predifferentiated podocyte cell line); 2, Podo line (+) (differentiated podocyte cell line); 3, primary GEC (European American); 4, primary PTC (European American); 5, HMC line; 6, liver (African American); 7, liver (European American); 8, HEK293 cells; 9, HepG2 cells. Relative mRNA levels were normalized to HepG2 cell values. Data are mean±SD, n=3.

The distribution of sites of APOL1 synthesis was explored further in whole kidney cortex via florescence in situ hybridization (FISH) (Figure 7). The staining patterns for APOL1 and SYNPO mRNA in the glomerulus were similar but did not overlap perfectly (Figure 7, A and B). This indicates that APOL1 mRNAs may be present in glomerular cells other than podocytes. Further analysis showed that APOL1 mRNA was also present in glomerular and interstitial endothelial (CD31-positive) cells (Figure 7, C and D) but did not overlap in glomeruli with α-SMA (Figure 7E). As expected, APOL1 and α-SMA mRNAs colocalized in kidney interstitial arterial smooth muscle cells (Figure 7F). APOL1 mRNA was distributed unevenly among tubule cells and was not limited to proximal tubules. The relative abundance of APOL1 mRNA in tubule cells was similar to that observed in glomerular podocytes (Figure 7, G and H), which contrasted with the marked podocyte enrichment of APOL1 protein seen on kidney cryosections (Figures 1 and 2). The reliability of RNA FISH and its imaging was demonstrated by absence of signal when using either no probe set or a probe set specific for insulin, which is not present in kidney (Supplemental Figure 4).

Figure 7.
Figure 7.

APOL1 mRNA is present in human kidney podocytes, GECs, and tubules with similar abundance on non-diseased human kidney FFPE sections. (A) APOL1 mRNA (green) colocalizes with SYNPO mRNA (red; podocyte marker) in a glomerulus and is also present beyond podocytes. (B) APOL1 mRNA is present in tubules. (C) APOL1 mRNA colocalizes with CD31 mRNA (red; GEC marker) in a glomerulus and is also present beyond endothelial cells. (D) APOL1 mRNA localizes to renal blood vessel endothelial cells (CD31-positive). (E) APOL1 mRNA does not overlap with α-SMA mRNA (red) in the glomerulus. (F) APOL1 mRNA is present in renal arterial smooth muscle cells (α-SMA positive) and renal tubule cells. (G) APOL1 mRNA does not colocalize with DPP4 mRNA (red; proximal tubule cell marker) in the glomerulus. (H) APOL1 overlaps with DPP4 in tubules; however, APOL1 is not limited to proximal tubules and may extend to other tubule cells. (For all: original magnification, ×630.)

Uptake of Recombinant APOL1 Protein into Differentiated Human Cell Lines

To evaluate potential mechanisms underlying the higher APOL1 protein abundance in podocytes from nondiseased kidney cryosections relative to primary podocytes, the differentiated human immortalized podocyte cell line was exposed to serum-free basic growth media containing purified recombinant C-terminal 6×-His tagged APOL1 for 6 hours. On the basis of immunofluorescence, with anti-APOL1 antibody, fluorescent signal above background was observed in podocytes incubated with as little as 0.1 μg of APOL1 per milliliter (Supplemental Figure 3N). At higher concentrations (APOL1 0.5 and 1.0 μg/ml), intracellular punctate deposits were detectable (Supplemental Figure 3, O and P). Uptake of APOL1 was further confirmed by Western blot analysis (Supplemental Figure 5) and confocal microscopy, which revealed a cytoplasmic localization (Figure 8, Supplemental Figure 6, Supplemental Video 1). Finally, immunofluorescence using anti-His tag antibody confirmed that the uptake involved transport of exogenous APOL1 as opposed to induction of endogenous accumulation (Supplemental Figure 7). APOL1 was not taken up readily by GEC or HMC (Supplemental Figure 3, A–H), although it appeared to precipitate in the extracellular spaces between cells at higher APOL1 concentrations, suggesting poor solubility under these conditions. APOL1 was visualized on both cell membranes and in the cytoplasm of PTC lines, but only at the higher concentrations of APOL1 (Supplemental Figure 3, K and L). Hence, the PTC APOL1 uptake pathway may be less efficient than that in podocytes. To explore whether the APOL1 uptake pathway is clathrin dependent, we repeated the experiments in the presence of potassium-deficient media, which disrupts clathrin-coated pit formation.11,12 APOL1 uptake decreased markedly when podocytes were incubated for 2 hours with APOL1, 0.1 µg/ml, in potassium-deficient buffer compared with the potassium-replete control (Supplemental Figure 8). Hence, we conclude that clathrin-mediated endocytosis may be responsible for the uptake of APOL1, although other mechanisms could also play a role. Finally, we explored the consequences of APOL1 uptake into podocytes by assessing cell viability and markers of cell proliferation and differentiation. On the basis of a lactate dehydrogenase release assay, exogenous APOL1 did not affect cell viability (Supplemental Figure 9). Likewise, we observed no change in cellular cytoskeletal structure, as assessed by staining of F-actin with phalloidin (Supplemental Figure 10), or alterations in the expression of proliferation and differentiation markers (Supplemental Figure 11). Hence, the uptake of APOL1 did not lead to a detectable change in cellular physiology, under the conditions tested.

Figure 8.
Figure 8.

Uptake of exogenous recombinant APOL1 into human podocytes. Confocal immunofluorescence microscopy reveals (A) less APOL1 in differentiated human podocytes not incubated with APOL1 compared with (B) podocytes incubated with 0.5 µg/ml APOL1 for 6 hours in serum-free RPMI media. APOL1-containing media were removed, and cells were washed with PBS. Cells were fixed and immunostained for APOL1 (red) and podocalyxin (green; podocyte marker). (For all: original magnification, ×630.)

Discussion

The cellular distribution and abundance of APOL1 protein and mRNA were studied in nondiseased kidney tissues, primary renal cells, and immortalized renal cell lines. APOL1 mRNA and APOL1 protein abundance in primary PTCs and GECs, differentiated immortalized human podocyte cell lines, and HepG2 cells were similar, a finding inconsistent with our observation that APOL1 protein was highly enriched in podocytes on kidney cryosections. Podocyte APOL1 enrichment relative to other cell types could be the result of uptake of circulating APOL1, a concept supported by in vitro studies demonstrating uptake of recombinant APOL1 by immortalized differentiated podocytes but not other renal cell types. Although the APOL1 concentration in podocyte growth medium was low (<1% of mean serum APOL1 concentrations), uptake by the podocyte cell line was nonetheless rapid. This is consistent with the prevailing concept that podocytes play a central role in the pathogenesis of FSGS.

In contrast to these studies, Madhavan et al. reported9 that APOL1 was present in podocytes and tubule cells, with lower signal intensity in podocytes. However, that report mainly evaluated diseased kidney tissue (FSGS), where the APOL1 signal could have been dampened by podocyte injury. The divergent outcomes may also be due to use of different APOL1 antibodies and analytical methods. We confirmed the observations of Madhavan et al. using the same APOL1 antibody (HPA018885; Sigma-Aldrich) and heat-induced epitope retrieval on FFPE kidney sections (data not shown). However, this antibody failed to produce a detectable signal during immunofluorescence on kidney cryosections and also failed to recognize native APOL1 during immunoprecipitation (data not shown). Hence, for our studies we selected an antibody that reacted with native human APOL1 (Epitomics, catalog no. 3245–1). This rabbit anti-human APOL1 monoclonal antibody displayed affinity for native APOL1 based on a strong fluorescent signal during immunofluorescence and effectiveness in immunoprecipitation. The Epitomics APOL1 antibody also allowed performance of immunofluorescence on both primary and immortalized renal cell lines, which, along with mRNA expression data, offered a generalized view of the distribution of endogenous APOL1 protein and mRNA abundance in cells that had not been exposed to the systemic circulation. Because the kidney has a rich blood supply, circulating lipid-free/lipid-poor APOL1 protein could affect the renal APOL1 cell type distribution in frozen or fixed kidney tissue.

Endogenous APOL1 protein and mRNA were present in primary PTCs, GECs, and immortalized human podocyte cell lines (Figures 5 and 6), whereas they were largely absent in mesangial cells based on Western blot, immunofluorescence, RT-PCR, and RNA FISH. The pattern of mRNA and protein abundance in these cell lines was similar, consistent with the prediction that protein abundance reflects endogenous synthesis. Because we had limited numbers of primary podocytes from study participants, we were unable to isolate sufficient RNA or protein to examine APOL1 expression. No APOL1 protein or RNA signal colocalized with α-SMA on kidney sections, demonstrating that mesangial cells do not synthesize or accumulate APOL1. Unlike podocytes, mesangial cells are not felt to be primarily involved in the pathogenesis of APOL1-associated forms of glomerulosclerosis; hence, we elected not to isolate primary HMCs. Nevertheless, we did not observe higher APOL1 concentrations in primary podocytes relative to primary PTCs and GECs based on immunofluorescence signal intensity (Figure 4). The consistency between mRNA and protein levels, however, was not observed in the kidney, where disproportionately high levels of APOL1 protein were present in podocytes relative to mRNA levels. These data, along with the in vitro uptake experiments, suggest that the disproportionate abundance of APOL1 in podocytes observed in kidney cryosections may arise from an exogenous source. Additional in vivo evidence supporting an extracellular source of APOL1 protein in podocytes came from RNA FISH. Here, the APOL1 mRNA signal on FFPE kidney sections was not as enriched in podocytes as was observed for APOL1 protein on kidney cryosections. Indeed, there was no obvious difference in APOL1 mRNA signal intensity between SYNPO-positive podocytes and CD31-positive glomerular endothelial cells. This study did not explore how APOL1 genotype might affect APOL1 expression levels because we currently have insufficient numbers of participants with two risk alleles to achieve statistical power. However, human tissue sample collection is ongoing, which should enable us to address this important question in future studies.

The solubility of APOL1 protein (Sino Biologic), the identity of which was confirmed by Western blot analysis (Supplemental Figure 12), appeared to be limited without serum in the growth media but was much higher in 10% FBS media (data not shown). This is probably due to the absence of HDL, which normally transports APOL1 in plasma.13 Nonetheless, small amounts of non–lipoprotein-bound APOL1 may exist in human serum and could potentially reach podocytes and renal tubule cells. Nonbound APOL1 is small enough (42 kD) to be filtered across the glomerular barrier and would be available for reabsorption by kidney cells, similar to albumin (67 kD), which has also been detected in podocytes14 and PTCs.15 The APOL1 concentrations in media to which our cell lines were exposed were far lower than those previously reported by Duchateau in serum (5–10 µg/ml)16 and estimated in our studies (15 µg/ml). Assuming that ≤5% of serum APOL1 exists in a lipid-free form, as is the case for APOA1,17,18 we estimate that approximately 0.75 µg/ml APOL1 could potentially cross the glomerular filtration barrier. This concentration is within the range used in our in vitro study, where we observed significant uptake of APOL1 by podocytes (Figure 8, Supplemental Figures 3, 5–7), but not by endothelial and mesangial cells, and is consistent with the observed enrichment of podocyte APOL1 signal in kidney cryosections. APOL1 uptake into podocytes did not appear to affect cell viability (Supplemental Figure 9) or cytoskeletal structure (Supplemental Figure 10). It also appeared that there were no significant changes in mRNA levels of proliferation or differentiation markers in podocytes treated with and without APOL1 (Supplemental Figure 11). Hence, the efficient uptake of APOL1 by podocytes might serve an as yet unknown physiologic function.9

Because the only direct evidence for podocyte uptake of APOL1 was obtained using cell lines, one might argue that renal cells or cell lines in vitro may behave differently from cells in vivo with respect to both expression and uptake of APOL1. However, the validity of studies using primary and immortalized renal cells was supported by the demonstration that they express the same patterns of cell-specific markers (WT1, SYNPO, CD31, α-SMA, and DPP4) observed in cryosection (Figures 1–4 and 7). Furthermore, the podocyte uptake of APOL1 was specific for this cell type and was not observed to any great extent in mesangial, endothelial, or tubule cells. Hence, we suggest that the behavior observed in vitro by these cell lines, coupled with the mRNA and protein distribution observed in vivo, are consistent with an exogenous protein uptake pathway contributing to the enrichment of APOL1 in podocytes. Because nondiseased kidney tissue was used for immunofluorescence and FISH, it is not known whether the intracellular APOL1 protein contributes directly to the pathogenesis of FSGS and related nondiabetic kidney diseases. As was proposed by Madhavan et al.,9 APOL1 may be functional in renal tissue and hence, reductions of APOL1 in podocytes of patients with FSGS and HIV-associated nephropathy could be a consequence of disrupted intracellular APOL1 synthesis or reduced cellular uptake. Data obtained from cultured primary proximal tubule cell lines from 10 African Americans with different APOL1 genotypes and normal kidney function indicated that APOL1 mRNA levels were positively associated with CDK4 (R=0.84; P=0.003) (Supplemental Figure 13), a cell cycle G1 phase-specific proliferation marker,19 but not with other proliferation markers (MCM2, MKI67, and PCNA1) (Supplemental Table 1–1). As was observed in a previous report,20 MCM2, MKI67, and PCNA1 mRNA levels were correlated with each other (Supplemental Tables 1 and 2). On the other hand, the clear pathogenicity associated with the G1 and G2 disease alleles suggests that while wild-type APOL1 may promote cell survival, consistent with the long half-life of podocytes, these disease variants may have acquired a deleterious gain of function, which becomes manifest in combination with other genetic and/or environmental inputs. On the basis of kidney transplant studies, in which donor but not recipient APOL1 genotypes affected graft survival,21,22 some would conclude that circulating APOL1 protein is less likely than intrinsic renal gene expression to be involved in the pathogenesis of APOL1-associated nephropathy. However, we caution against applying the transplant model to the pathogenesis of native kidney disease, in part because APOL1-associated kidney disease is a decades-long process whereas transplant failure is a short-term outcome confounded by many issues, including cold ischemia and use of nephrotoxic immunosuppressive medications.

In conclusion, we provide evidence that APOL1 in human kidney cortex may be derived from both endogenous synthesis and extracellular sources. Renal toxicity from circulating (non–lipoprotein-bound) APOL1 may contribute to APOL1-associated nephropathy, particularly as contributed by the G1 and G2 disease variants. Enrichment of APOL1 protein in podocytes was evident in nondiseased kidney cryosections in European Americans and African Americans with different APOL1 genotypes (WT, heterozygous G1/WT, heterozygous G2/WT, and a G1/G2 compound risk heterozygote), suggesting that APOL1 may serve a functional role in the kidney.9 Madhavan et al.9 reported that APOL1 protein was localized in the media of medium-sized arteries and arterioles in the kidneys of patients with FSGS and HIV-associated nephropathy. We extend this by observing APOL1 mRNA in the media of medium-sized arteries in persons without kidney disease (Figure 7F). Podocytes may not be the only renal cells affected by APOL1; glomerular and extraglomerular endothelial cells and PTCs may also be affected by variant APOL1 proteins. This finding may explain the widespread pattern of glomerulosclerosis with vascular changes and interstitial fibrosis universally present in all forms of APOL1-associated nephropathy.

Concise Methods

Nephrectomy Specimens

Patients scheduled to undergo radical or partial nephrectomy were identified and recruited. Genomic DNA was isolated from peripheral lymphocytes and stored at −80°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 obtained. 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 provided 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 temperature gel (Sukaru FineTek Inc., Torrance, CA) for cryosection immunofluorescence studies, snap frozen for protein and RNA preparation, 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 Lines

The immortalized human mesangial cells and conditionally immortalized human proximal 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 Lines

To examine whether exogenous APOL1 can be taken up by the HMC line and differentiated human podocyte, GEC, and PTC lines, recombinant human APOL1 (13910-H08B; Sino Biologic) was mixed in serum-free basic growth media 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 α-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 conventional and confocal fluorescence microscopy. The effect of APOL1 uptake into podocytes on cell viability was assessed by lactate dehydrogenase release assay (CytoTox96; Promega). Cytoskeletal structure was assessed with Alexa Fluor 488 phalloidin (Invitrogen). The dependence of APOL1 uptake on a clathrin-mediated pathway was assessed using potassium-deficient media, as described elsewhere.11,12

Immunofluorescence Microscopy on Cultured Cells and Kidney Tissue Cryosections

Immunofluorescence 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 primary renal cells using established protocols.32 Supplementary methods contain isolation, culture, and characterization protocols for the primary renal cells. Human kidney cryosections were sliced at 6-μm intervals using a cryotome (Leica Biosystems, Buffalo Grove, IL). Information on the primary antibodies and antibody dilutions for immunofluorescence are listed in Supplemental Table 2. To demonstrate the specificity of the Epitomics anti-APOL1 antibody, it was preincubated overnight at 4°C with a 20-fold molar excess of the APOL1 peptide (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 nonpreabsorbed primary antibody. Secondary antibodies (goat anti-rabbit Alexa Fluor 594, catalog no. A11037, and goat anti-mouse Alexa Fluor 488, catalog no. A11001) were used to display fluorescent signals (1:500 dilution).

Western Blot

Western 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 conjugated to horseradish peroxidase (1:20,000; Jackson ImmunoResearch Laboratories, West Grove, PA).

RNA FISH

FFPE human kidney tissue blocks were sectioned at 5-μm intervals. The following QuantiGene View RNA FISH “double Z” type 1 oligonucleotide 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), α-SMA (Accession: 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-PCR

Total RNA was 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 spectrophotometry and electrophoresis, respectively, using the Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). One microgram of RNA was reverse transcribed with random hexamer primers using the TaqMan RT kit (Applied Biosystems, Foster City, CA). Primers were designed to capture all of the known APOL1 splice variants. RT-PCR in the presence of SYBR Green was performed with an ABI 7500-Fast Real-Time PCR system (Applied Biosystems) using 18S ribosomal RNA as normalization standard. Primer sequences are listed in Supplemental Table 4. The Delta-Delta CT method34 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 mean±SD.

Disclosures

None.

Acknowledgments

This study was supported by National Institutes of Health grants R01-DK070941 and R01-DK084149 (PI: B.I.F.).

Footnotes

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Localization of APOL1 Protein and mRNA in the Human 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, Barry I. Freedman
JASN Feb 2015, 26 (2) 339-348; DOI: 10.1681/ASN.2013091017
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