Most ApoL1 Is Secreted by the Liver

Abstract

Two coding sequence variants in the APOL1 gene (G1 and G2) explain much of the increased risk for FSGS, HIV-associated nephropathy, and hypertension-attributed ESRD among people of recent African ancestry. The ApoL1 protein is expressed in a wide variety of cell tissues. It has been assumed that the majority of circulating ApoL1 is produced by the liver, but this has not been shown. Using mass spectrometry, we genotyped and quantified the circulating ApoL1 in two liver transplant recipients whose native APOL1 genotype differed from the genotype of the deceased donors, allowing us to differentiate liver- from nonliver-produced ApoL1. Our findings confirm that the liver is indeed the main source of circulating ApoL1. However, the liver is not the sole source of circulating ApoL1, because we found that residual amounts of native ApoL1 continued to circulate in the blood, even after the liver transplant.

Two coding sequence variants in the APOL1 gene (called G1 and G2) explain much of the increased risk for FSGS, HIV-associated nephropathy, and hypertension-attributed ESRD among people of recent African ancestry.^1,2 Since the initial observations of this association, this basic finding has been widely replicated.^3,4

ApoL1 was first described in 1997 by Duchateau et al.⁵ as an HDL-bound protein expressed in the pancreas and later shown to be expressed in multiple tissues.⁶ Although most studies report that ApoL1 circulates as part of HDL complexes, recent work by Weckerle et al.⁷ suggests that most circulating ApoL1 resides in lipid–poor multiprotein complexes, which largely represent Trypanosome lytic factors. The relationship between ApoL1 and HDL has led to the assumption that the circulating ApoL1 is mainly produced in the liver.⁸ However, to date, this has not been explicitly shown.

Here, we take advantage of an in vivo natural experiment that tests this assumption by using a mass spectrometric assay that distinguishes between and quantifies the different ApoL1 protein variants. We analyzed the circulating concentrations of all three ApoL1 protein variants in the serum of two patients who had received liver transplants from deceased donors with APOL1 haplotypes different from their own. Because any ApoL1 protein variant found in the circulation that differs from the donor liver’s expressed variants must derive from the recipient’s extrahepatic tissues, this analysis is poised to answer the question of the origin of circulating ApoL1.

To address this, we performed a study under the aegis of a protocol approved by the institutional review board at Beth Israel Deaconess Medical Center (BIDMC). To find suitable subjects, we analyzed the APOL1 genetic haplotypes of DNA samples collected between 1990 and 2008 from black liver transplant donors and recipients. These DNA samples had been isolated from the liver explants of transplant recipients and their donors at the time of surgery, labeled with coded research identifiers, and stored in the Hepatology Laboratory at Massachusetts General Hospital.

From these samples, we identified the APOL1 variant haplotypes using real time PCR (7300 Real Time PCR System; Applied Biosystems, Foster City, CA). We analyzed the haplotypes of 20 transplant recipients, 16 of which were G0G0 haplotypes, three of which were G0G1, and one of which was G1G1. We contacted the three recipients with G0G1 haplotypes, and two of these patients consented to participate (subject A and subject B). Both of these subjects had received livers from deceased white donors. Genetic testing of the donor livers by DNA sequencing (Genewiz Inc., Cambridge, MA) revealed that subject A received a liver with G0G0 genotype and that subject B received a liver with G0G2 genotype. Serum was collected from subjects after their consent and stored frozen at −20°C until processing for mass spectrometric analysis. An additional 20 discarded and deidentified serum samples originally collected from hospital outpatients for clinical purposes were used to characterize typical concentrations of ApoL1 in subjects with and without liver disease. Categorization of subjects’ hepatic function was on the basis of subjects’ medical records showing a previous diagnosis of chronic liver disease and also, current serum albumin concentrations <3.5 g/dl. The samples and their associated clinical data were deidentified after collection and stored at −80°C until the time when they were thawed for ApoL1 measurement. These samples and their associated clinical data were collected under the aegis of a human subjects study protocol approved by the BIDMC Center for Clinical Investigation Institutional Review Board.

Genotyping and quantifying the circulating ApoL1 using tandem mass spectrometry was first reported by Zhou et al.⁹ On the basis of their previously described method, we adapted a similar method for measurement of ApoL1 G0, G1, and G2 protein variants. Briefly, 8 μl serum was mixed with isotopic peptide standards, digested with trypsin, and analyzed by high-performance chromatography and tandem mass spectrometry using multiple reaction monitoring (MRM). The mass spectrometer was programmed to measure tryptic peptides specific for each protein variant, including LNILNNNYK for G0, LNMLNNNYK for G1, and LNILNNK for G2. To measure total ApoL1 protein, the assay also monitored peptide ALDNLAR, a tryptic peptide common to all three variants.

Using our liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, we measured the concentrations of total ApoL1 and each protein variant in the serum of our two transplanted subjects as well as the serum of healthy control subjects homozygous and heterozygous for each of three variants (Figure 1). (LC-MS/MS assay and methods for quantitation of ApoL1 protein variants is described in the concise methods section and in Supplemental Table 2).

Figure 1.

Comparison of serum ApoL1 concentrations amongst test subjects. Scatterplot of serum total ApoL1 concentrations measured in individual subjects grouped by genotype and liver disease status and in transplanted subject A and subject B.

The proportional concentrations that each ApoL1 protein variant found in circulation normally depend on whether all variants are secreted in equal proportions and whether they have equivalent half-lives in circulation. By measuring ApoL1 variant concentrations in nontransplanted control subjects known to be heterozygous for G0, G1, and G2, we found that G2 variants circulate at slightly lower concentrations compared with G0 and G1 on average (Table 1). The proportionately lower concentrations of G2 are consistent with recent published findings by other groups.¹⁰ After liver transplantation, however, the proportional concentrations depend on how much of total ApoL1 derives from the new liver versus other tissues. Our tandem mass spectrometry results showed subject A (originally G0G1 and received G0G0 liver) to have mainly G0 ApoL1 in circulation (92% of the total ApoL1), with a small amount of G1 ApoL1 (8% of total ApoL1). Subject B (originally G0G1 and received a G0G2 liver) had mainly G0 and G2 ApoL1 in circulation (43% G0 and 54% G2), with a trace amount of G1 ApoL1 (3%) (Figure 2).

Table 1.

Absolute concentrations of total ApoL1 protein and median relative concentrations of ApoL1 protein variants in subject A, subject B, and the genotype control patient

Genotype	N	Median Concentration ±SD (μg/ml)	G0, Median %	G1, Median %	G2, Median %
G0/G0 (healthy)	14	7.0±4.3	100
G0/G0 (liver disease)	10	3.5±2.7	100
G0/G1	4	5.9±2.6	48	52
G0/G2	4	20.9±15.9	58		42
G1/G2	4	6.5±4.7		59	41
G1/G1	1	7.7		100
G2/G2	1	8.9			100
Subject A		4.0	92 (63.5)a	8 (5.5)	0 (nd)
Subject B		29.8	43 (220)	3 (15)	54 (276)

N, number of subjects; nd, not detected.

^aNormalized concentrations of ApoL1 variants (in micrograms per milliliter), calculated on the basis of variant-to-total peptide ratios, are shown in parentheses with their percentage fraction of total ApoL1.

Figure 2.

APOL1 genotypes and ApoL1 serum concentrations. Proportional concentrations of circulating ApoL1 protein variants after transplantation. Note that subject A’s transplanted liver was failing at the time of sample collection.

Both subjects were originally heterozygous for G0 and G1 variants, and both received livers that did not express G1 variants. It is interesting, therefore, that small but significant amounts of G1 were detected in both subjects after transplantation, and the only interpretation of these findings is that the trace concentrations of G1 found come from nonhepatic sources. It is also interesting in Table 1 that the relative amount of G1 variant was proportionately greater relative to total ApoL1 in subject A (G0G0 liver > G0G1 host) compared with subject B (G0G2 liver > G0G1 host; 8% versus 3%); this finding may have been due to the fact that subject A’s new transplanted liver was failing and likely producing significantly less ApoL1 than a healthy liver (as demonstrated in Table 1, ApoL1 concentrations in subject A were similar to those in subjects with histories of chronic liver disease). As a result, the relative amount of the native ApoL1 (G1) in subject A (coming from extrahepatic sources) constituted a greater percentage of total circulating ApoL1. Alternatively, the low proportion of endogenous G1 variant in subject B may be due to the relatively high concentrations of liver–derived G0 and G2 variants found in this patient after transplantation with a G0G2 liver. It is also interesting that the other G0G2 control subjects also displayed the highest average ApoL1 concentrations compared with the other genotypes. Although we must be cautious about inferring any conclusions on the basis of these two patients and a small number of controls, these finding raise the possibility of significant differences in circulating ApoL1 concentrations between different combinations of variants.

Through LC-MS/MS technology, we were able to genotype and quantify the circulating ApoL1 in the serum of two liver transplant recipients and determine the origin of the circulating ApoL1. On the basis of our findings, the liver is indeed the main source of the circulating ApoL1; however, other organs seem to contribute a minor fraction. The earliest papers describing ApoL1 showed mRNA expression in the liver as well as kidneys, pancreas, lung, spleen, vasculature, and other tissues.⁶ Although the kidney and other organs may express ApoL1 mRNA, our findings suggest that the liver is the primary secretor of circulating ApoL1 protein. These results raise the question: “which other organs contribute to circulating ApoL1?” Furthermore, if ApoL1 mRNA is expressed in the liver and kidneys, but the majority of ApoL1 protein is secreted by the liver, why is the kidney the primary target for ApoL1 toxicity, and why is the liver spared this effect? Maybe future studies investigating the cellular biology of ApoL1 secretion as well as clinical studies of recipients of APOL1 variant kidneys, pancreas, lung, or kidney/liver transplants will provide further insight into these questions. Note that our study has limitations deserving mention. First and foremost is its relatively small sample size, which limits our ability to draw strong conclusions regarding differences in circulating variant concentrations in different genotypes.

The role of circulating ApoL1 variants in the development of APOL1-associated nephropathy and the significance of APOL1 variants in transplanted donor livers are still being investigated. Recent work by Dorr et al.¹¹ showed that deceased donor APOL1 genotype does not significantly affect outcomes in liver transplantation. To the contrary, Freedman et al.¹² observed that renal allografts from black deceased donors harboring two APOL1 risk variants failed more rapidly after renal transplantation than those with zero or one risk variant, suggesting that it is the ApoL1 specifically expressed by the kidneys that is the source of APOL1-associated nephropathy. These findings do not exclude a contribution of the circulating ApoL1 to human phenotypes, however, especially that podocytes take up circulating ApoL1.¹³ Moreover, some studies have suggested an increased risk of cardiovascular disease in people with high–risk APOL1 genotypes independent of kidney disease, raising the possibility that circulating ApoL1 has other roles in human disease.¹⁴ The toxic effects of ApoL1 protein reported in several studies^8,15,16 suggest that this is an important topic for further exploration.

Concise Methods

Subjects

We genotyped the APOL1 variants of 20 DNA samples received from the Hepatology Laboratory at Massachusetts General Hospital using real time PCR (7300 Real Time PCR System). The transferred clinical samples were labeled with coded research identifiers (no patient identifiers), which were linked to a protected electronic research database containing transplant recipients’ clinical data and identifying information. From this pool of 20 candidates, we contacted four liver recipients found to be carriers for G1 or G2 APOL1 variants through their hepatologist. Two of the candidates contacted consented to participate in the study (subject A and subject B), and both were heterozygous for G0 and G1. Samples of isolated DNA of the donor livers transplanted into subject A and subject B were obtained from Massachusetts General Hospital, and DNA sequencing revealed that subject A received a liver with G0G0 genotype and that subject B received a liver with G0G2 genotype. ApoL1 was also measured in archived samples from 10 additional healthy G0/G0 control subjects with no history of liver disease as well as 10 G0/G0 subjects with history of chronic liver disease and serum albumin concentrations <3.5 g/dl.

LC-MS/MS Measurement of Total ApoL1 and Individual ApoL1 Protein Variants

Specimen Processing

Eight microliters serum was diluted with 136 μl buffer (50 mM ammonium bicarbonate [pH 8], 80 nM mixture of isotopic internal standard peptides, and 1% sodium deoxycholate). The samples were then reduced with 4 μl dithiothreitol for 45 minutes at 60°C, alkylated with 4 μl iodoacetamide for 60 minutes at room temperature in the dark, and digested for 1 hour with 12 μl Trypsin (Sigma-Aldrich, Columbia, MO) at 37°C. After 60 minutes, an additional 3 μl Trypsin was added and returned to 37°C for 30 minutes (during assay validation experiments, we discovered that 60 minutes was sufficient for complete digestion and that longer digestion times were associated with significant deamidation of peptide asparagine residues, and therefore, digestion was limited to 90 minutes to minimize loss of yield) (Supplemental Figure 1). Deoxycholate was removed by precipitation with 8 μl 50% formic acid and centrifugation at 14,000 rpm for 10–15 minutes, and the peptide digestion in the supernatant was cleaned by solid-phase extraction on an Oasis HLB 96-Well µSPE Plate (Waters Corp., Milford, MA) and eluted with 50% methanol (which did not interfere with chromatographic resolution). After solid-phase extraction 50 μl purified digest was injected onto a Kinetex C8 Reverse-Phase Column (100×4.6 mm, 2.6-μm bead size; Phenomenex Inc., Torrence, CA). Peptides were resolved on an Shimadzu HPLC with a 0%–100% acetonitrile plus 0.1% formic acid gradient and analyzed on an API 5000 Triple Quadruple Mass Spectrometer; we monitored for peptides L*NILNNNYK, L*NMLNNNYK, and LNIL*NNK that correspond to the G0 (wild type), G1, and G2 peptide variants, respectively, and total ApoL1 was quantified by monitoring for peptide ALDNL*AR, which is common to all of the variants (L* indicates the position of the isotopic leucine). Two MRM transitions were monitored for each peptide for quantification and confirmation (Supplemental Table 2 shows mass spectrometer settings).

Isotopic Peptide Synthesis

Isotopically labeled Fmoc-leucine (¹³C₆, ¹⁵N; Anaspec Inc., Freemont, CA) was purchased and used for the synthesis of all four of the internal standard peptides. Isotopic peptides were synthesized from Fmoc–protected amino acids and purified by reverse-phase chromatography by PrimmBiotech Inc. (Cambridge, MA).

Total ApoL1 Protein Quantification

To quantify total ApoL1, we measured the area under the curve (AUC) for the MRM peak of ApoL1 tryptic peptide ALDNLAR (a peptide common to all three ApoL1 variants); the AUC for the ALDNLAR peptide was normalized for assay yield by dividing by the AUC for its isotopic peptide standard to obtain the normalized AUC. A standard curve was generated by measuring diluted calibrator standards made from diluted purified recombinant ApoL1 protein purchased from Origene (Rockville, MD), and the calibrator concentrations were on the basis of the manufacturer’s stated concentration on the label. Normalized AUC values were converted into absolute ApoL1 concentrations (in micrograms per milliliter) from this standard curve. All samples were digested and analyzed in duplicate (except for those from subject A and subject B, which were measured in eight replicates), and the average coefficient of variance of duplicate measurements was 28%. The specific protein identity of the ApoL1 protein standard was verified by both LC-MS/MS and Western blot analysis. Note that a previous study that used LC-MS/MS quantification reported that digestion efficiency of the ALDNAR peptide required overnight digestion and was altered depending on whether ApoL1 was the G0, G1, or G2 variant.⁹ During our assay development and validation experiments, we found that 90 minutes was sufficient for complete digestion using our own methods and reagents and that longer digestion times were associated with significant deamidation of peptide asparagine residues, and therefore, digestion was limited to 90 minutes to minimize loss of yield (Supplemental Figure 1). Furthermore, we found that digestion yield of ALDNLAR peptide after 90 minutes was equivalent to yield of another ApoL1 tryptic peptide, VAQELEEK, validating the efficiency of digestion using our procedures (Supplemental Figure 2).

Measurement of Relative Concentrations of Apol1 Protein Variants

Purified protein standards for ApoL1 G1 and G2 variants are not commercially available, and thus, to determine concentrations of each specific variant in subjects’ samples on the basis of measurement of the ApoL1 variant–specific peptides, we used serum samples from G0/G0, G1/G1, and G2/G2 homozygous subjects as calibration standards. Total ApoL1 was first quantified in the G0G0, G1G1, and G2G2 homozygous subjects using the ALDNLAR quantification peptide as described above at the same time, and the normalized AUC values for the ApoL1 variant–specific peptides LNILNNNYK, LNMLNNNYK, and LNILNNK were simultaneously quantified in these samples. Variant–specific peptide AUC values were then plotted against their absolute concentrations (in micrograms per milliliter) to generate calibration curves for each ApoL1 variant, and these standard curves were used to quantify the absolute concentrations of each variant in the other subjects’ assay measurements. Using this approach, concentrations of G0, G1, and G2 could be measured independently in subjects with multiple circulating variants.

Disclosures

D.J.F. and M.R.P. have filed patents related to ApoL1–associated kidney disease and own equity in Apolo1 Bio, LLC (Boxford, MA).