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.

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 moL/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 mol/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 (<1.2 mg/dL [106 mol/L]) and high (>2 mg/dL [177 mol/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
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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 L 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 37C. This is followed by 3 washes and the addition of avidin conjugated to horseradish peroxidase and incubated for 2 hours with shaking at 37C. 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 500L aliquots and stored at 20C. These samples were then
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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. 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 mol/L), comparisons were redone using age as a dependent variable.

Table 1 Intra-assay and Interassay Precision of the Klotho Assay in 20 Samples

Klotho (ng/mL) Mean Intra-assay Interassay 35.6 82.5 250.8 35.0 82.3 260.6 SD 1.7 5.2 20.4 2.9 6.8 34.5 Coefficient of Variation (%) 4.9 6.3 8.1 8.3 8.2 13.2

Table 2 Linearity and Dilution

Klotho (ng/mL) Sample 1 Expected Concentration 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 Observed Concentration 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 Recovery (%) 99 98 102 95 93 99 100 101 93 91 89 100 98 95 96 91

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. 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. 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).
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Observed Klotho (ng/mL)

600 500 400 300 200 100 0 0 200 400 600 Expected Klotho (ng/mL)

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.

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
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160.0 140.0 120.0 Klotho (ng/mL) 100.0 80.0 60.0 40.0 20.0 0.0
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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 moL/L); triglycerides (Intralipid) at 1,500, 750, and 375 mg/dL; and ascorbic acid at 400, 200 and 100 mol/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 mol/L).
450 400 350 Klotho (ng/mL) 300 250 200 150 100 50 0

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 <6.5% [0.065]); P < .001) Figure 4 and Table 3. It seems that the expression of Klotho is decreased in renal disease5-7; however, plasma levels have not been recorded. Thus, we examined Klotho levels in another set of patients with abnormal creatinine levels during the last 12 months (CKD). Previously, we have shown that FGF23 levels are significantly increased in patients with creatinine levels of more than 2 mg/dL (177 mol/L).16 We now document significantly higher levels of Klotho with kidney disease. This significance (P < .001) persisted following adjustment for the younger age of the patients with kidney disease Figure 5 and Table 4.
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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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Discussion
We report on the validation of an ELISA to successfully measure immunoreactive soluble Klotho protein in the circulation of human subjects and report significant decreases with diabetes and increases in CKD. While studies have examined Klotho in kidney tissue and in urine and there is a report of Klotho levels in humans,7-9,17 there is a paucity of data validating an assay for this biomarker and examining levels in diabetes and in subjects with renal dysfunction; thus, we modified and validated an assay for measuring secreted Klotho in human subjects. Our interassay and intra-assay precision studies demonstrated that the Klotho assay has a reasonable intra-assay precision of less than 8.3% and interassay precision of less than 13.3%; thus, it is preferable that all samples from each subject be run in duplicate to improve precision. Also, dilutional studies demonstrated that the Klotho assay was linear and demonstrated good linearity at the low end, with recoveries of more than 90% for levels as low as 15 ng/mL. The 10th percentile of our reported reference range is 27.8 ng/mL. Since common interferents such as bilirubin, lipemia, and hemolysis could affect results, we tested these common interferents. While a very high bilirubin level of 250 mg/L resulted in a significant reduction of Klotho, there was no significant reduction, even at high concentrations of the other common interferents. We and others have shown that diabetes is a proinflammatory state.18 Two groups have shown that Klotho expression is decreased in diabetic animal models.11,15 In fact, Zhao et al15 showed that soluble Klotho was anti-inflammatory and inhibited NFB activity. In the present report, we confirm these animal data in humans with diabetes with normal creatinine levels and show that Klotho is significantly decreased. Since Klotho has been shown to protect endothelial function via nitric oxide
450 400 350 Klotho (ng/mL) 300 250 200 150 100 50 0 HbA1C <6.5% HbA1C 6.5%

Figure 4 Klotho levels in diabetes. Samples were selected if they had hemoglobin (Hb)A1c levels <6.5% (n = 53) or 6.5% (n = 29), the new cutoff for diabetes. None of these subjects had abnormal creatinine levels. Klotho levels were assessed in duplicate by enzyme-linked immunosorbent assay. Values for HbA1c are given in conventional units; to convert Systme International units (proportion of total Hb), multiply by 0.01.

Table 3 Age and Laboratory Values in Diabetes*

HbA1c <6.5% (n = 53) Age (y) Creatinine (mg/dL) Glucose (mg/dL) HbA1c Klotho (ng/mL) 37 12 0.85 0.16 86 11 5.3 0.25 81 (45, 141) HbA1c 6.5% (n = 29) 43 10 0.89 0.10 157 31 8.9 1.6 35 (15, 58)

450 400 350 Klotho (ng/mL) 300 250 200 150 100 50 0 Creatinine <1.2 mg/dL Creatinine >2 mg/dL

Hb, hemoglobin. * Data are given as mean SD or median (25th percentile, 75th percentile). Values for creatinine, glucose, and HbA1c are given in conventional units; conversions to Systme International units are as follows: creatinine (mol/L), multiply by 88.4; glucose (mmol/L), multiply by 0.0555; HbA1c (proportion of total Hb), multiply by 0.01. P < .05 compared with HbA <6.5%. 1c P < .001 compared with HbA <6.5% and adjusted for age. 1c

Table 4 Age and Laboratory Values in Kidney Failure*

Creatinine <1.2 mg/dL Creatinine >2 mg/dL (n = 30) (n = 31) Age (y) Creatinine (mg/dL) Glucose (mg/dL) Klotho (ng/mL)
*

55 17 0.79 0.24 100 14 67 (43, 119)

54 17 4.23 2.49 102 14 108 (66, 182)

Figure 5 Klotho levels in chronic renal failure. Klotho levels were assessed by enzyme-linked immunosorbent assay on samples with normal (<1.2 mg/dL; n = 30) and high (>2 mg/ dL; n = 31) creatinine.

Data are given as mean SD or median (25th percentile, 75th percentile). Values for creatinine and glucose are given in conventional units; conversions to Systme International units are as follows: creatinine (mol/L), multiply by 88.4; glucose (mmol/L), multiply by 0.0555. P < .01 compared with creatinine <1.2. P < .001 compared with creatinine <1.2 and adjusted for age.

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production,13,19 this deficiency of Klotho could contribute to the endothelial dysfunction and proinflammatory state of diabetes. In future studies, we will explore the mechanism of the reduction in Klotho in diabetes, focusing on inflammation and oxidative stress. The role of Klotho as a biomarker of increased oxidative stress and inflammation in patients with diabetes needs to be validated in large cohort studies. Studies have also reported severely reduced production of Klotho in urine and kidneys (messenger RNA and protein by Western blot) of patients with renal failure.7-9,20 We report paradoxically increased levels of Klotho in patients with high compared with low creatinine levels. Previously, Klotho levels have been shown to correlate negatively with plasma creatinine levels in an apparently normal population. However, this weak correlation of 0.18 accounts for 3% of the variance to explain serum levels, suggesting that other more important unidentified factors are at play. However, there is a paucity of data examining circulating Klotho levels in patients with kidney disease, acute and chronic. In our study, it is important to note that none of the subjects had diabetes. Previously, Klotho levels have been shown to be negatively correlated with aging.17,21 Once again, the inverse correlation was 0.20, suggesting that aging contributes 4% to Klotho levels. Despite age adjustment, Klotho levels were significantly increased in our subjects with high creatinine levels and CKD compared with subjects with normal creatinine levels and correlated with creatinine levels. It is interesting that a very recent study, in support of our findings, has also reported increased levels of Klotho in patients with CKD.22 However, in that preliminary report, unlike the present study, the authors did not provide any validation of their assay and report a wide range of values in patients with CKD. While both studies report higher levels in patients with CKD, there is a wide difference in levels. This is most likely due to the different antibodies directed to different epitopes. Klotho has been shown to provide renal protection,21 and, thus, higher levels reported for the first time in patients could be plausible. One can hypothesize that since Klotho derives from many sources,3,5 as a counterregulatory mechanism, soluble Klotho is being produced by extrarenal tissue to provide renal protection via antioxidant and anti-inflammatory mechanisms since it has been shown to prevent apoptosis in acute ischemic renal injury.12 Also, one can advance that the assay is detecting the shed ectodomain and the product of splice variant, the latter accounting for the increased levels.5 Thus, in this report, we validate an assay for an exciting and relatively unknown humoral factor whose functions still need to be appreciated. However, we make the novel observation that levels are decreased in diabetes and increased in CKD. Much further research is needed to validate and standardize this biomarker in the translational arena.

From the 1Department of Pathology and Immunology, Texas Childrens Hospital and Baylor College of Medicine, Houston; and 2Laboratory for Atherosclerosis and Metabolic Research, Department of Pathology & Laboratory Medicine, University of California at Davis Medical Center, Sacramento, and VA Medical Center, Sacramento. Supported in part by a grant from the Institute of Kidney Lifescience Technologies, Ontario, CA. Address reprint requests to Dr Jialal: Laboratory for Atherosclerosis and Metabolic Research, 4635 II Ave, Research 1 Bldg, Room 3000, UC Davis Medical Center, Sacramento, CA 95817.

References
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16. Devaraj S, Duncan-Staley C, Jialal I. Evaluation of a method for fibroblast growth factor-23: a novel biomarker of adverse outcomes in patients with renal disease. Metab Syndr Relat Disord. 2010;8:477-482. 17. Yamazaki Y, Imura A, Urakawa I, et al. Establishment of sandwich ELISA for soluble alpha-Klotho measurement: age-dependent change of soluble alpha-Klotho levels in healthy subjects. Biochem Biophys Res Commun. 2010;398:513-518. 18. Devaraj S, Dasu MR, Jialal I. Diabetes is a proinflammatory state: a translational perspective. Expert Rev Endocrinol Metab. 2010;5:19-28. 19. Saito Y, Nakamura T, Ohyama Y, et al. In vivo Klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochem Biophys Res Commun. 2000;276:767-772.

20. Hu M, Kuro-o M, Moe O. Klotho and kidney disease. J Nephrol. 2010;23(suppl 16):S136-S144. 21. John GB, Cheng CY, Kuro-o M. Role of Klotho in aging, phosphate metabolism, and CKD. Am J Kidney Dis. 2011;58:127-134. 22. Sugiura H, Tsuchiya K, Nitta K. Circulating levels of soluble -Klotho in patients with chronic kidney disease [letter; published online ahead of print August 5, 2011]. Clin Exp Nephrol. 2011;15:795-796. doi:10.1007/s10157-011-0511-4.

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