Decreased plasma α-Klotho predict progression of nephropathy with type 2 diabetic patients
Introduction
Diabetic nephropathy occurs in 20–40% of all patients with type 2 diabetes mellitus (Ahn et al., 2014, American Diabetes Association, 2015). It is a microvascular complication with high morbidity and mortality, as well as being the leading cause of end-stage renal disease (ESRD) (Collins et al., 2012). Although albuminuria and decreased glomerular filtration rate (GFR) are considered to be the main clinical markers for the development and progression of diabetic nephropathy, significant renal damage has already occurred by the time these signals are present (Barratt & Topham, 2007).
The Klotho gene was identified as an aging suppressor gene that extends life span when overexpressed and accelerates aging when disrupted (Akimoto et al., 2012). It is expressed as a transmembrane protein in predominantly the distal convoluted tubules in the kidney and choroid plexus in the brain (Kuro-o et al., 1997). It is also expressed in several endocrine organs such as the pituitary, pancreas, parathyroid gland, adipocyte and vascular endothelial cells (Ben-Dov et al., 2007, Chihara et al., 2006, Donate-Correa et al., 2013, Kuro-o et al., 1997). Unlike membrane Klotho, secreted Klotho is known to regulate the activity of multiple glycoproteins on cell surfaces, including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors (Kuro-o, 2010). Several studies have reported that patients with chronic kidney disease have reduced plasma and urine α-klotho levels in the early stages of kidney disease, progressively decreasing in more advanced stages (Akimoto et al., 2012, Hu et al., 2011, Kim et al., 2013).
The role of α-klotho in the pathogenesis of diabetic nephropathy has not been fully elucidated. Recently, we demonstrated that the plasma and urine soluble α-klotho levels were significantly increased in the diabetic patients with relatively preserved renal function (eGFR ≥ 60 mL/min/1.73 m2) compared to healthy control subjects, and plasma α-klotho levels decreased in proportion to urinary albumin excretion, although urine α-klotho levels were stable with increasing urinary albumin excretion in our cross-sectional study (Lee et al., 2014). However, it is unclear whether the changes in plasma and urine α-klotho precede the development of albuminuria and decline of GFR in the early development and progression of diabetic nephropathy.
The aim of this study was to evaluate the impact of plasma and urine α-klotho on the early development and progression of type 2 diabetic nephropathy and to determine whether plasma and urine α-klotho are associated with the decline in eGFR and development and/or progression of albuminuria in type 2 diabetic patients with eGFR ≥ 60 mL/min/1.73 m2.
This study analyzed subjects enrolled in a prospective observational study of early biomarkers for diabetic nephropathy (Diabetic Kidney Disease Study [DKDS]) at Pusan National University Hospital in Busan, Korea (Kim et al., 2014, Lee et al., 2014). This study was carried out in accordance with the Declaration of Helsinki and the protocol was approved by the Institutional Review Board of Pusan National University Hospital (20100024). All patients provided written informed consent before enrollment. Data are available to all interested researchers on request to the Institutional Review Board of Pusan National University Hospital.
A total of 147 Korean type 2 diabetic patients were enrolled consecutively at outpatient clinics between February 2010 and February 2012. The inclusion and exclusion criteria for eligibility have been described (Kim et al., 2014). Briefly, all enrolled patients had relatively conserved renal function (estimated glomerular filtration rate [eGFR] ≥ 60 mL/min/1.72 m2, and serum creatinine < 1.2 mg/dL). Estimated GFR of 60 is considered as the threshold value of eGFR for the current definition of chronic kidney disease (CKD) (Levey et al., 2011). In addition patients had a sufficient washout period for RAS (renin–angiotensin system) inhibitors (no history of administration of RAS inhibitors at enrollment or a washout period for these drugs of at least 2 months before enrollment). Patients who had disorders/status that affect renal function or urinary samples were excluded (active urinary tract infection; renal disease other than diabetic nephropathy; neoplastic disorders; severe liver dysfunction; active or chronic infection or inflammatory disorders; pregnancy; or a recent [within 6 months] history of acute myocardial infarction, stroke, or occlusive peripheral vascular disease).
Random spot urine and blood samples were obtained from each patient at their clinic visit. Medical histories and anthropometric measurements were also recorded at the same visit. The patients were followed up at our clinic until September 2014. Of these 147 patients, 38 were excluded during follow-up for the following reasons: 19 patients were lost during follow-up (withdrawal of study, n = 10; data not available, n = 9); 3 patients had a follow-up of relatively short duration (< 1 year); 10 patients were hospitalized for other severe acute and chronic diseases (acute myocardial infarction, acute stroke, pneumonia, rheumatoid arthritis, hemoptysis, cholangitis and iatrogenic Cushing syndrome); 5 patients were diagnosed with additional malignancies, and 1 patient died of other causes during the follow-up period. Finally, a total of 109 were enrolled in this study. Urine and blood samples were taken at intervals of 12 ± 1 (mean ± SD) months at the outpatient clinic during their follow-up period. Serum creatinine (for calculation of eGFR) and urine albumin-to-creatinine ratio (ACR) were measured at intervals of 6 ± 1 (mean ± SD) months during the follow-up period using the same method.
The eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula: MDRD = 186 × (serum creatinine [mg/dL])− 1.154 × (age in years)− 0.203 (Myers et al., 2006). A correction factor of 0.742 was used for females. The annual decline in eGFR was calculated by dividing the change in eGFR by follow-up duration. Urine ACR was measured from spot urine. Albuminuria values were defined as follows: normoalbuminuria (ACR, < 30 mg/g creatinine), microalbuminuria (ACR, 30–299 mg/g creatinine) and albuminuria (ACR, ≥ 300 mg/g creatinine). Plasma and urine concentrations of soluble α-klotho were analyzed using a commercial ELISA kit, as described (Lee et al., 2014). Briefly, plasma samples were centrifuged for 15 min at 3,000 rpm within 30 min of collection; plasma was removed and stored at − 70 °C until analysis. Urine samples were centrifuged for 10 min at 3,000 rpm to remove particulate matter and stored at − 70 °C until analysis. The plasma and urine concentrations of α-klotho were analyzed using human soluble α-klotho immunoassay kits (Immuno-Biological Laboratories, Gunma, Japan) according to the manufacturer's protocol. Samples were analyzed in duplicate and were within the range of the standard curve (93.75–6,000 pg/mL); values below the detection limit (6.15 pg/mL) were approximated using the mean value between zero and the lower limit of detection. The intra- and inter-assay coefficients of variation were less than 10%. The data on urinary α-klotho were expressed as ratios of urinary α-klotho to urinary creatinine (urine α-klotho/Cr) to assess the hydration states and renal functions of the patients.
Statistical analyses were performed using SPSS version 15.0 (SPSS, Chicago, IL, USA). We divided the diabetic patients into tertile groups according to their baseline plasma and urine α-klotho levels, respectively. Data were presented as means ± SD for normally distributed values and medians (interquartile range) for nonparametric values. Distributions of continuous variables were examined for skewness and kurtosis, and logarithm-transformed values of variables with nongaussian distribution were used for analyses. Differences between groups were analyzed by ANOVA, followed by Bonferroni's test for normally distributed values or the Kruskal–Wallis test for nonparametric values. Categorical variables were reported as frequencies and proportions. Pearson's χ2 test was employed to analyze categorical data as appropriate. Pearson correlation coefficient was used to test the correlations between individual continuous variables. We conducted multivariate regression analyses with annual rates of decline in eGFR as dependent variables and plasma and urine α-klotho as independent variables. Several models were constructed to adjust for confounding factors including age, sex, HbA1c, systolic BP, HDL cholesterol, duration of diabetes, baseline eGFR and ACR. A multivariate analysis for albuminuria persistence or progression, using an enter procedure was conducted including factors with a p value of < 0.2 in the univariate analysis. The multivariate Cox regression model for microalbuminuria/albuminuria persistence or progression was adjusted for age, HbA1c, SBP, HDL cholesterol, baseline eGFR and ACR. A p value of < 0.05 derived from the two-tailed Student's t-test was considered to indicate statistical significance.
Section snippets
Baseline clinical characteristics
Patients were divided into tertile groups according to their plasma and urine α-klotho (based on Urine α-klotho/Cr) levels, respectively (Table 1, Table 2). There were no significant differences among the three groups with respect to age, sex, BMI, duration of diabetes, C-reactive protein (CRP), and control status for SBP, glycemia and lipids. All patients had well-conserved renal function, with an eGFR of 93.0 ± 23.2 mL/min/1.73 m2, and the eGFR was not significantly different among the three
Discussion
In this study, plasma α-klotho was negatively correlated with the annual decline in eGFR and the development of albuminuria in the early stage of nephropathy of type 2 diabetic patients (eGFR ≥ 60 mL/min/1.73 m2), whereas urinary α-klotho was not. In addition, decreased levels of plasma α-klotho predicted the rapid decline of eGFR in a subgroup analysis of normo/microalbuminuric patients.
Circulating soluble α-klotho can be generated directly by alterative splicing of the α-klotho transcript, or
Acknowledgments
Ju In Kim performed ELISA of plasma and urine soluble α-klotho concentrations.
References (27)
- et al.
Decreased renal α-Klotho expression in early diabetic nephropathy in humans and mice and its possible role in urinary calcium excretion
Kidney International
(2012) - et al.
Expression of FGF23/KLOTHO system in human vascular tissue
International Journal of Cardiology
(2013) - et al.
Klotho deficiency is an early biomarker of renal ischemia-reperfusion injury and its replacement is protective
Kidney International
(2010) - et al.
Secreted Klotho protein in sera and CSF: Implication for post-translational cleavage in release of Klotho protein from cell membrane
FEBS Letters
(2004) - et al.
Soluble serum Klotho in diabetic nephropathy: Relationship to VEGF-A
Clinical Biochemistry
(2012) - et al.
Circulating α-klotho levels in CKD and relationship to progression
American Journal of Kidney Diseases
(2013) - et al.
The definition, classification, and prognosis of chronic kidney disease: A KDIGO Controversies Conference report
Kidney International
(2011) - et al.
Prevalence and determinants of diabetic nephropathy in Korea: Korea national health and nutrition examination survey
Diabetes & metabolism Journal
(2014) - et al.
Characteristics of urinary and serum soluble Klotho protein in patients with different degrees of chronic kidney disease
BMC Nephrology
(2012) Microvascular complications and foot care
Diabetes Care
(2015)
Urine proteomics: The present and future of measuring urinary protein components in disease
CMAJ
The parathyroid is a target organ for FGF23 in rats
The Journal of Clinical Investigation
Type I membrane klotho expression is decreased and inversely correlated to serum calcium in primary hyperparathyroidism
The Journal of Clinical Endocrinology and Metabolism
Cited by (51)
Klotho-derived peptide 6 ameliorates diabetic kidney disease by targeting Wnt/β-catenin signaling
2022, Kidney InternationalAstragaloside IV protects against podocyte apoptosis by inhibiting oxidative stress via activating PPARγ-Klotho-FoxO1 axis in diabetic nephropathy
2021, Life SciencesCitation Excerpt :Numerous studies have demonstrated that Klotho has multiple functions, including regulating energy metabolism, antioxidation, anti-inflammation, modulating calcium and maintaining mineral homeostasis [8,39]. Klotho is significantly downregulated in the kidneys of DN patients and experimental DN mouse models and there is a concomitant reduction of Klotho in plasma and urine of DN patients [9–14]. In addition, the decline in renal function was associated with a decrease in Klotho expression level [12].
Klotho and aging phenotypes
2021, Fibroblast Growth Factor 23The interaction between klotho protein and epigenetic alteration in diabetes and treatment options
2024, Journal of Diabetes and Metabolic DisordersPotential application of Klotho as a prognostic biomarker for patients with diabetic kidney disease: a meta-analysis of clinical studies
2023, Therapeutic Advances in Chronic DiseaseResearch progress on the role of ET-1 in diabetic kidney disease
2023, Journal of Cellular Physiology
Funding: This research was supported by a Biomedical Research Institute grant (2014–1) from Pusan National University Hospital and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2044121).
We declare that we have no conflicts of interest.
Prior Presentation: Some of the findings reported in this article were presented in poster form at the 74th Scientific Sessions of The American Diabetes Association, San Francisco, CA, June 13–17 2014.