Validation of an Immunoassay for Soluble Klotho Protein Decreased

Clinical Chemistry / Klotho Assay and Levels in Diabetes

Validation of an Immunoassay for Soluble Klotho Protein Decreased Levels in Diabetes and Increased Levels in Chronic Kidney Disease Sridevi Devaraj, PhD, DABCC,1 Basir Syed,2 Alexander Chien,2 and Ishwarlal Jialal, MD, PhD, DABCC, FRCPath2 Key Words: Klotho; Fibroblast growth factor; Diabetes; Chronic kidney disease; Aging; Inflammation DOI: 10.1309/AJCPGPMAF7SFRBO4

Abstract The Klotho gene has been identified as an aging suppressor gene that encodes a transmembrane protein, which is expressed primarily in renal tubules. There are 2 forms of Klotho, membrane and secreted. However, there is a paucity of data on levels of soluble Klotho in diseases like diabetes and kidney disease. We validated an enzyme-linked immunosorbent assay for Klotho and quantitated Klotho levels separately in patients with diabetes and also in patients with chronic kidney disease (CKD). The Klotho assay showed good precision and was linear down to 19 ng/mL. There were no significant effects on Klotho levels with the addition of common interferents such as ascorbate, triglycerides, or hemolysis; only bilirubin (250 mg/L) significantly reduced Klotho levels (P < .05). There was a significant reduction in Klotho levels in samples with glycated hemoglobin (HbA1c) levels of 6.5% or more compared with control samples (HbA1c < 6.5%; P < .001). We also documented significantly higher levels of Klotho with CKD. Thus, we validated an assay for Klotho and made the novel observation that levels are decreased in diabetes and increased in CKD.

The Klotho gene has been identified as an aging suppressor gene. It encodes a transmembrane protein, which is expressed primarily in renal tubules. Mice defective in Klotho gene expression develop a premature aging phenotype, whereas transgenic mice overexpressing Klotho live longer than “wild-type” mice.1,2 In addition, the major function of Klotho is in mineral metabolism. There seem to be 2 forms of Klotho protein, membrane and secreted. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate coreceptor for the phosphatonin FGF23.3-5 The secreted form of Klotho seems to function as a humoral factor and seems to regulate multiple glycoproteins on the cell surface, including ion channels and growth factors, and potentially has been implicated in insulin/insulin-like growth factor receptor function.5 The extracellular domain of Klotho protein is clipped on the cell surface by ectodomain shedding via membrane-anchored proteases.6 This generates a secreted form of Klotho protein with a molecular mass of between 120 and 130 kDa that is detectable in body fluids, including blood, urine, and cerebrospinal fluid.7 Secreted Klotho does not function as a soluble receptor for FGF23 because it is the Klotho-FGF receptor complex that has high affinity for FGF23 and not Klotho protein per se. The functions of circulating Klotho protein have not been identified. Klotho expression in the kidneys is significantly decreased in streptozotocin-induced diabetic rats and in chronic kidney disease (CKD) and animal models of acute and chronic kidney diseases.8-11 Also, soluble Klotho seems to be anti-inflammatory and inhibits apoptosis and stimulates endothelial nitric oxide synthase.12,13 However, there is a paucity of data on levels of soluble Klotho in disease states such as diabetes and kidney disease. To validate an assay for

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Klotho for further translational research, we looked at classical interferents (bilirubin, hemolysis, and lipemia). In addition, we quantitated Klotho levels in patients with diabetes and also in patients with CKD. We report on the validation of an assay for immunoreactive Klotho and also report on Klotho levels in patients with diabetes mellitus and in patients with CKD.

Materials and Methods Assay We evaluated the human Klotho enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO Biotech, Newark, DE) with some modifications. The modifications included the following: (1) 1:2,000 dilution of the sample; (2) incubation of samples/standards with antibody-coated plate overnight, instead of a 2-hour incubation; and (3) inclusion of a wash step after overnight incubation of sample/standard with the coated plate. This assay is a sandwich ELISA precoated with an antibody specific to recombinant or natural human Klotho protein. Standards or samples (200 μL of 1:2,000 dilution of serum in sample buffer) are added to the coated plate wells overnight at room temperature, followed by 3 washes and the addition of a biotin-conjugated antibody preparation specific for Klotho for 2 hours at 37°C. This is followed by 3 washes and the addition of avidin conjugated to horseradish peroxidase and incubated for 2 hours with shaking at 37°C. Then a TMB (3,3',5,5'-tetramethylbenzidine) substrate solution is added to each well, and the reaction is terminated by the addition of a sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Klotho in the samples is determined from the standard curve. All assays were run according to the procedure described in the original kit manual, except the sample incubation was changed to overnight at room temperature and a wash step after sample incubation was added. The assay was linear in the range between 7.8 and 500 pg/mL, and, thus, standards with these concentrations were used in the standard curve. This assay recognizes recombinant and natural human Klotho. Absorbance values were measured with a BioTek Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). All samples and standards were assayed in duplicate. Values are expressed in nanograms per milliliter. Precision Studies The Clinical and Laboratory Standards Institute protocol for method evaluation was used for interassay and intra-assay precision studies. Serum samples with 3 concentrations of Klotho (low, medium, and high), were divided into 500μL aliquots and stored at –20°C. These samples were then 480 480

Am J Clin Pathol 2012;137:479-485 DOI: 10.1309/AJCPGPMAF7SFRBO4

assayed on 20 separate occasions during the next several days to determine interassay precision. Serum samples with the 3 different concentrations of Klotho were run in replicates of 20 to determine intra-assay precision. Linearity and Dilution Samples with high concentrations of Klotho were diluted with the sample buffer, and the linearity of each of the assays was determined. Recovery was calculated by dividing the result obtained by the expected value. Also, recombinant Klotho (from Prospec Bio, East Brunswick, NJ) was spiked with the samples at 2 different concentrations, and recovery was determined. Interference Studies A pool of serum samples was divided into aliquots. Aliquots were then individually spiked with different concentrations of free hemoglobin, ascorbic acid, bilirubin, and triglycerides, substances known to frequently interfere with similar assays. Hemoglobin was added at 100, 40, and 20 g/ dL, values far higher than what would be expected in normal plasma or hemolyzed plasma. Bilirubin was added at 250, 125, and 62.5 mg/L (428, 214, and 107 μmoL/L; 25, 12.5, and 6.25 mg/dL); these values are higher than what would be expected even in severe liver disease with high levels of bilirubin. Triglycerides (Intralipid) were added at 1,500, 750, and 375 mg/dL, levels that would be expected from a dyslipidemic patient or from highly lipemic plasma samples. Ascorbic acid was added at 300, 200, and 100 μmol/L; these values are far higher than what would be expected with supplementation. Each spiked plasma sample was assayed in duplicate; results were compared with those from an aliquot of native plasma. Patient Comparison Studies All procedures were in accord with the Helsinki Declaration of 1975. Following waiver of institutional review board consent, 2 sets of serum samples were obtained from the University of California Davis Medical Center (UCDMC, Sacramento) Clinical Pathology laboratories that were accessioned in for evaluation of glycated hemoglobin (HbA1c) or creatinine levels. Procedures followed were in accord with the ethical standards established by UCDMC and the Helsinki Declaration of 1975. Samples were selected if they had HbA1c values of less than 6.5% (0.065) or 6.5% or more (≥0.065; n = 53 and n = 29, respectively), which is the new cutoff for diabetes.14 None of these subjects had abnormal creatinine levels. We also tested the Klotho levels on another set of samples with normal (2 mg/dL [177 μmol/L]) creatinine levels (n = 30 and n = 31, respectively) because previous studies in animal models suggest that CKD results in impaired renal Klotho expression. All samples with increased creatinine had persistently increased levels for at © American Society for Clinical Pathology

Clinical Chemistry / Original Article

least 12 months, consistent with CKD. HbA1c and creatinine levels on the samples were measured in the clinical pathology laboratory at UCDMC using routine methods, and the estimated glomerular filtration rate was calculated using the IDMS-traceable MDRD (isotope dilution mass spectrometry– traceable Modification of Diet in Renal Disease) equation.

❚Table 1❚ Intra-assay and Interassay Precision of the Klotho Assay in 20 Samples Klotho (ng/mL)

Intra-assay

Results Precision Studies For the precision studies, serum samples with 3 concentrations of Klotho were studied. Intra-assay and interassay precision results are provided in ❚Table 1❚. For intra-assay precision, the Klotho assay showed good precision at low, medium, and high levels with a CV of 8.2% or less. Interassay precision was also good, with a CV of less than 8.4% at low and medium levels of Klotho and a CV of 13.2% at high levels of Klotho.

Interassay

SD

Coefficient of Variation (%)

35.6 82.5 250.8 35.0 82.3 260.6

1.7 5.2 20.4 2.9 6.8 34.5

4.9 6.3 8.1 8.3 8.2 13.2

❚Table 2❚ Linearity and Dilution Klotho (ng/mL) Sample 1

2

3

Observed Klotho (ng/mL)

Statistics Intra-assay and interassay precision were evaluated by determining means, SD, and coefficient of variation (CV). Differences in interference studies were evaluated by paired t tests. Analysis of variance was performed followed by paired t tests and Wilcoxon tests for parametric and nonparametric data, respectively. Linear regression analysis was used to correlate the assays with glucose and HbA1c, creatinine, and estimated glomerular filtration rate calculated by using the modified MDRD equation. For samples with less than 1.2 vs more than 2 mg/dL creatinine (106 vs 177 μmol/L), comparisons were redone using age as a dependent variable.

Mean

Expected Concentration

Observed Concentration

Recovery (%)

487.5 243.7 126.4 63.2 31.66 15.83 265.7 132.8 66.4 33.2 16.6 357.8 178.9 89.5 44.7 22.3

487.5 241.6 123.4 64.5 30.2 14.7 265.7 134.1 61.5 30.2 14.8 357.8 175.9 84.7 43.1 20.3

99 98 102 95 93 99 100 101 93 91 89 100 98 95 96 91

600 500 400

Linearity and Dilution Samples with high concentrations of Klotho were diluted to determine linearity. The assay showed very good linearity down to a level of 15 ng/mL ❚Table 2❚. ❚Figure 1❚ shows excellent linearity of the assay with a plot of expected vs observed Klotho levels giving an r 2 of 0.99, an intercept of 0.01, and a slope of 1.003. Also, recombinant Klotho was added at 2 concentrations, 50 and 100 ng/mL, and recovery was determined. When recombinant Klotho at a concentration of 100 ng/mL was added to samples with Klotho levels of 35 and 48 ng/mL, the recoveries were 91% and 95%, respectively.

❚Figure 1❚ Observed and expected Klotho levels. Samples were diluted 1:1. Linear regression of data: y = 1.003x + 0.01; r 2 = 0.99.

Interference Studies The results of the interference studies are provided in ❚Figure 2❚. There were no significant effects on Klotho levels with the addition of ascorbate, triglycerides, or hemolysis. However, a very high bilirubin level (250 mg/L) resulted in a significant reduction in Klotho levels (P < .05).

Reference Range The reference range of 4.7 to 437.6 ng/mL for the Klotho assay was derived from samples from 57 healthy control subjects with an age range of 27 to 49 years. Klotho levels decreased with aging (r = –0.45; P = .081). As shown

300 200 100 0 0

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Devaraj et al / Klotho Assay and Levels in Diabetes

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❚Figure 2❚ Interference studies for the Klotho enzyme-linked immunosorbent assay. A pool of serum samples was divided into aliquots. Aliquots were then individually spiked with different concentrations of free hemoglobin, ascorbic acid, bilirubin, and triglycerides as described in “Materials and Methods”. Hemoglobin was added at 100, 40, and 20 g/dL; bilirubin at 250, 125, and 62.5 mg/L (428, 214, and 107 μmoL/L); triglycerides (Intralipid) at 1,500, 750, and 375 mg/dL; and ascorbic acid at 400, 200 and 100 μmol/L. Each spiked plasma sample was assayed in duplicate; results were compared with those from an aliquot of native plasma. Data are presented as mean (bars) and SD (error bars). * P < .05 compared with unspiked sample.

in ❚Figure 3❚, while the mean level was 103 ng/mL (SD, 95 ng/mL), the median Klotho level was 76 ng/mL, with the following percentiles: 5th, 16 ng/mL; 10th, 28 ng/mL; 25th, 40 ng/mL; 75th, 130 ng/mL; 90th, 229 ng/mL; and 95th, 275 ng/mL. All volunteers had a glucose level of less than 126 mg/dL (7.0 mmol/L) and a creatinine level of less than 1.2 mg/dL (106 μmol/L). 450 400 Klotho (ng/mL)

350 300 250 200 150 100 50 0

❚Figure 3❚ The reference range for the Klotho assay was derived from 57 healthy control subjects with an age range of 27-49 years, who were otherwise healthy as described in “Materials and Methods.”

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Patient Comparison Studies Because 2 groups have shown lower expression of Klotho in animal models of diabetes,11,15 we examined levels in human samples from people with and without diabetes. Klotho levels were first assessed according to values for HbA1c. Samples were divided into those with HbA1c levels less than 6.5% (0.065) vs those with levels of 6.5% or more (≥0.065), which is the accepted criterion for diabetes.14 Both groups had creatinine levels in the reference range. There was a significant reduction in Klotho levels in samples with HbA1c levels of 6.5% or more (≥0.065) compared with control samples (HbA1c