Clinical Biochemistry 45 (2012) 1415–1420

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Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem

Soluble serum Klotho in diabetic nephropathy: Relationship to VEGF-A

Ina Maria Kacso a, Cosmina Ioana Bondor b, Gabriel Kacso c,⁎
a
University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj Napoca, Department of Nephrology, Romania
b
University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj Napoca, Department of Informatics and Biostatistics, Romania
c
University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj Napoca, Department of Oncology, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: Both kidney expression and soluble serum Klotho are influenced by chronic kidney disease
Received 26 April 2012 (CKD) and diabetes. Serum Klotho is a yet poorly explored biomarker. We describe, for the first time to our
Received in revised form 14 June 2012 knowledge, serum Klotho in diabetic patients with CKD and its relationship to vascular endothelial growth
Accepted 15 July 2012 factor A (VEGF-A).
Available online 23 July 2012
Design and methods: We included 43 controls and 146 diabetic patients with different stages of CKD. Lab-
oratory evaluation, urinary albumin/creatinine ratio (UACR), Klotho (ELISA), VEGF-A (ELISA) were performed.
Keywords:
Albuminuria
Results: Klotho was 0.40(0.10–1.30)ng/mL in diabetic patients without CKD and 0.80(0.30–1.30)ng/mL in con-
Diabetes mellitus trols, p=0.20; VEGF-A was higher in diabetic patients 73.85(57.32–119.00)pg/mL than in controls 43.20(30.1–
Klotho protein 65.9)pg/mL, p b 0.0001. Klotho increased with CKD stage: 0.2(0.10–0.40)ng/mL in CKD 1/2, 0.60(0.20–
Glomerular filtration rate 1.1)ng/mL in CKD 3/4 and 1.45(0.425–2.90) ng/mL in dialysis patients, p b 0.0001; it also increased with
Vascular endothelial growth factor A decreasing glomerular filtration rate (GFR). Klotho was lower in albuminuric (UACR > 30 mg/g) patients
0.20(0.10–0.70) ng/mL than in normoalbuminuric (UACR b 30 mg/g) ones 0.50(0.20–1.30) ng/mL, p =
0.03; lowest Klotho was found in microalbuminuric (UACR 30–300 mg/g) patients, p = 0.07. VEGF was
lower in microalbuminuric patients but was not influenced by GFR. In diabetic patients but not in controls,
Klotho correlated to VEGF-A (r = 0.29, p = 0.0003); in multiple regression VEGF-A was the only significant
predictor of Klotho: b = 0.27, 95%CI (0.01–0.04), p = 0.001.
Conclusions: In diabetic patients, Klotho is decreased in early CKD and increases thereafter, paralleling
reduced GFR. VEGF-A is higher in diabetic patients than in controls. Both Klotho and VEGF-A are decreased
in the presence of microalbuminuria. In diabetes, Klotho strongly correlates to VEGF-A.
© 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction Little is known of Klotho in diabetic nephropathy. Diabetes acceler-

Klotho protein
Klotho is a trans-membrane protein, first described as an anti-aging
factor [1] with preponderant localization in brain and kidneys [1,2].
Membrane bound-Klotho acts as co-receptor for fibroblast-growth
factor (FGF) 23. However, the clinical and biological picture that is
found in Klotho-deficient mice involves many organs in which Klotho
is not expressed, suggesting an endocrine signaling pathway. Klotho
also exists as a soluble secreted protein, which can be identified in
blood, urine and cerebrospinal fluid [3,4], modulates ion transporters
or channels [2,5], and also plays an important antioxidant role [6].
Renal Klotho expression is decreased in chronic kidney disease
(CKD) [7–9]. Restoration of Klotho significantly ameliorates chronic
kidney injury [10–13], suggesting a possible pathogenetic link be-
tween Klotho and the severity of renal damage.
ates aging, particularly in patients with complications such as nephrop-
athy [14]. Decreased kidney expression of Klotho has been reported in
diabetic animals [7,15,16]. To our knowledge, serum levels of soluble
Klotho in diabetic patients with CKD have not been described.
Vascular endothelial growth factor A
VEGF-A is an important regulator of angiogenesis and vascular
permeability with a possible pathogenic role in diabetic nephropa-
thy [17–19]. There is preliminary evidence of an interaction between
Klotho and VEGF-A [20–22].
We aimed to describe serum Klotho in diabetic patients with and
without kidney disease and to investigate relationship of Klotho to
VEGF-A in these patients.
Material and methods

Patients
⁎ Corresponding author at: Dornei 47 Street, 400171 Cluj Napoca, Romania. Fax: +40
264 592202. We included type 2 diabetic patients from two different settings in a
E-mail address: gabi.kacso@gmail.com (G. Kacso). transversal study: consecutive outclinic patients referred for nephrologic

0009-9120/$ – see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2012.07.098
1416 I.M. Kacso et al. / Clinical Biochemistry 45 (2012) 1415–1420

evaluation to the Clinic of Nephrology Mihai Manasia Cluj and diabetic
patients from the Nephro-Care Dialysis Center Cluj. Inclusion criteria
for the predialysis patients were diagnosis of type 2 diabetes for at
least 6 months and informed consent; for the dialysis patients diagnosis
of diabetes prior to diagnosis of kidney disease and informed consent.
Exclusion criteria were the presence of urinary abnormalities except al-
buminuria for predialysis patients, other known cause of CKD, recent
(past 3 months) infections and any other serious chronic or acute dis-
ease requiring treatment. Evaluation of patients comprised: history, clin-
ical assessment, blood pressure measurement after 15 minute rest,
assessment of height, weight and waist circumference and renal ultra-
sound. Blood samples were collected in the morning after 8 h fasting
and maintained at 4 ° C for ≤4 h.
Laboratory assessment
Standard laboratory assessment was done right away; plasma and
serum were frozen at − 70 °C. Creatinine, glucose, uric acid, total and
high density lipoprotein cholesterol, triglycerides (automated colori-
metric enzymatic method), standard hematology panel (automated
flow-cytometry method), C-reactive protein (CRP), serum albumin,
glycated hemoglobin (immunoturbidymetric method) and urinary
multistick analysis (COMBUR) were performed. In non-dialysis pa-
tients, glomerular filtration rate (GFR) was estimated according to
the abbreviated Modification of Diet in Renal Disease (MDRD) formu-
la [23] and urinary albumin to creatinine ratio (UACR) was calculated
as a mean of three consecutive morning spot measurements. Renal
ultrasound was performed using Siemens Sonoline Prima B-mode ul-
trasound. Retinopathy was assessed from previous records of the pa-
tients. Serum Klotho was determined using an ELISA kit (Uscn Life
Science Inc), intra-assay variability b 10% and inter-assay variability
12%. Specificity of the assay is reported to be very good as no cross-
reactivity or interference between human Klotho and analogues
was detected. Sensitivity of the assay (lower limit of detection) was
0.063 ng/mL. All samples had detectable Klotho levels. VEGF-A was
determined by an ELISA assay (Cederlane) with minimum detection
limit 15.6 pg/mL, inter and intra assay variabilities b 10% and sensi-
tivity 15.6 pg/mL.
The study was approved by the ethical committee of our universi-
ty and is in accordance with the ethical standards of the Declaration
of Helsinki of 1975 revised in 2000; each patient signed an informed
consent.
Statistic analysis
males). Diabetic patients were classified according to the presence
of CKD-KDOQI criteria for the presence of nephropathy: 48 patients
did not have CKD; the rest had different stages of CKD, 36 of the latter
being on chronic hemodialysis.
Comparison of subjects according to presence of diabetes
Diabetic patients without CKD had somewhat lower serum Klotho
and significantly higher VEGF-A than controls (Table 1 and Fig. 1a).
Comparison of patients according to CKD
When patients are divided according to presence and stage of
CKD, we report an initial decrease of Klotho with occurrence of CKD
followed by a progressive increase. VEGF was not different in patients
with different stages of CKD (Table 2, Fig. 1b).
Comparison of patients according to GFR
When patients were compared according to GFR, serum Klotho
increased with decreasing GFR; VEGF-A was not different (Table 3,
Fig. 1c).
Comparison of patients according to UACR
Klotho was higher in normoalbuminuric (UACRb 30 mg/g creatinine)
patients — 0.50(0.20–1.30)ng/mL than in albuminuric (UACR>30 mg/g
creatinine) ones 0.20(0.10–0.70) ng/mL, p = 0.03. Microalbuminuric
subjects (UACR 30–300 mg/g) had lowest Klotho when compared to
proteinuric (UACR> 300 mg/g) and normoalbuminuric patients (p=
0.07) — Table 4, Fig. 1d.
Comparison of patients according to treatment
Ninety-two of the diabetic patients (63.01%) were on insulin treat-
ment, 43(29.45%) were treated with metformin, and 44(30.13%) with
sulfonylureas. No patient was on PPARgamma agonist. Ninety-seven
patients (66.43%) were on blockers of the renin-angiotensin axis:
75(51.36%) on angiotensin converting enzyme inhibitors and 28
(19.17%) on angiotensin receptor antagonists. Sixty patients (41.09%)
Table 1
Comparison of diabetic patients without nephropathy to controls.

Parameter Diabetes (n = 48) Controls (n = 43) p

Statistic analysis was performed using SPSS 13.0, Statistica 7.0 and
Microsoft EXCEL programs. Normal distribution was tested with Kol- Age (years) 60.71 ± 11.18 61.07 ± 11.47 0.88
Gender n (%) 30 (62.5) 20 (46.51) 0.13
mogorov–Smirnov test. Values are expressed as mean ± standard BMI (kg/m2) 30.51 (27.07–33.43) 25.68 (21.09–28.03) b0.0001
deviation for normally distributed variables, respectively as median Waist circumference (cm) 108.59 ± 13.39 94.94 ± 11.07 b0.0001
(interquartile range) for non-normally distributed variables. For com- SBP (mm Hg) 140 (125–150) 130 (115–140) 0.0003
parison of two means of independent samples, t-test or Mann–Whitney DBP (mm Hg) 80 (80–90) 80 (70–80) 0.0004
GFR (mL/min)–MDRD 95.28 ± 22.34 92.17 ± 19.88 0.49
test was used. For comparison of three means of independent samples,
GFR (mL/min)–CKD-EPI 89.28 ± 16.69 88.11 ± 15.95 0.73
ANOVA test or Kruskall–Wallis test was used, followed by post-hoc UACR (mg/g) 11.22 (1.81–16.95) 9.14 (5.11–12.08) 0.51
Scheffe or Bonferroni analysis. For comparison of qualitative variables, Hb A1C (%) 7.73 ± 1.32 4.96 ± 0.57 b0.0001
Chi-square test or Fisher exact test was employed. For identifying cor- LDL cholesterol (mmol/L) 2.68 (2.07–3.21) 3.46 (2.56–4.28) 0.001
relation between two continuous variables, Pearson's correlation co- HDL cholesterol (mmol/L) 1.12 (1.04–1.34) 1.26 (0.93–1.50) 0.30
Triglycerides (mmol/L) 1.53 (1.09–2.28) 1.52 (0.97–2.22) 0.78
efficient (r) was assessed. This was followed by multivariate linear CRP (mg/L) 2.50 (1.00–6.00) 2.70 (1.50–4.00) 0.87
regression using stepwise method. Statistic significance threshold Klotho (ng/mL) 0.40 (0.10–1.30) 0.80 (0.30–1.30) 0.20
was considered α = 0.05. VEGF-A (pg/mL) 73.85 (57.32–119.00) 43.20 (30.1–65.9) b0.0001

Results
Presentation of the cohort
We included 146 diabetic patients (mean age 63.38 ± 12.20 years,
59.58% males) and 43 healthy controls (61.07 ± 11.47 years, 46.51%
Legend: BMI — body mass index, S — systolic, D — diastolic, BP — blood pressure, GFR —
estimated glomerular filtration rate, MDRD — Modification of Diet in Renal Disease formu-
la, CKD-EPI — Chronic Kidney Disease Epidemiology Collaboration formula, UACR —
urinary albumin/creatinine ratio, HbA1C — glycated hemoglobin, LDL — low density
lipoprotein, HDL — high density lipoprotein, CRP — C reactive protein, VEGF-A —
vascular endothelial growth factor-A. Normally distributed variables are presented
as mean±standard deviation; non-normally distributed ones as median (interquartile
range).
 I.M. Kacso et al. / Clinical Biochemistry 45 (2012) 1415–1420 1417

Fig. 1. Serum Klotho levels according to a) presence of diabetes, b) chronic kidney disease, c) glomerular filtration rate, d) albuminuria. Legend: Case — diabetic patients without
CKD, CKD — chronic kidney disease, GFR — glomerular filtration rate, UACR — urinary albumin to creatinine ratio. For b) all differences are significant with the exception of patients
without CKD versus CKD 3/4 patients. For c) significant difference between CKD 5 patients versus CKD stage 1/2 and 3/4 patients respectively.

were treated with statins. When patients were divided according to use
of medication, no significant differences were found in serum Klotho
(Table 5).
Relationship to VEGF-A
We found a significant relationship between Klotho and VEGF-A
that was consistent throughout the study: in all T2D patients (r =
0.29, p = 0.0003), in normoalbuminuric (r = 0.29, p = 0.02) or
albuminuric (microalbuminuric and proteinuric) subjects (r = 0.25,
p = 0.08), in patients with GFR > 60 mL/min (r = 0.37, p = 0.0001),
GFR 15–60 mL/min (r=0.26, p=0.11) and dialysis patients (r=0.31,
p=0.07). In multiple regression analysis (variables entered: age, body
mass index, blood pressure, C-reactive protein, high density lipoprotein,
low density lipoprotein cholesterol, triglycerides, glycated hemoglobin
and for predialysis patients GFR and UACR) — the only predictor of Klotho
was VEGF-A. This holds true in all diabetic patients — coefficient b =
0.02, p = 0.001, CI(0.01–0.04) as well as in predialysis subjects —

Table 2
Comparison of diabetic patients according to CKD.

Parameter Without CKD 1/2 CKD3/4 CKD5 p

CKD (n=23) (n=39) (n=36)
(n=48)

Age (years) 60.71 ± 11.18 64.87 ± 9.17 67.77 ± 8.93 61.25 ± 8.38 0.004c,d
Male n (%) 30 (62.50) 14 (60.90) 23 (59.00) 20 (55.60) 0.93
Diabetes duration 7.50 (4–15.00) 10 (5–14) 9 (5–17) 20 (17–23.75) b0.0001c,e,f
BMI (kg/m2) 30.51 (27.07–33.46) 31.31 (26.59–34.77) 30.48 (26.67–34.36) 29.07 (24.22–32.19) 0.29
Waist circumference (cm) 108.59 ± 13.39 107.00 ± 13.37 107.59 ± 11.98 99.29 ± 16.75 0.02f
SBP (mm Hg) 140 (125–150) 140 (130–160) 140 (130–150) 130 (130–145) 0.14
DBP(mm Hg) 80 (80–90) 85 (80–90) 80 (75–90) 80 (70–90) 0.004e,f
GFR(mL/min) 95.28 ± 22.34 90.33 ± 20.50 44.03 ± 10.54 – b0.0001a,b,d
UACR (mg/g) 11.22 (1.81–16.95 99.42 (52.92–149.85) 94.26 (17.42–443.64) – b0.0001a,d
Albumin(g/L) 46.00 (43.50–48.00) 46.00 (44.0–47.00) 45.0 (43.0–47.00) 37.90 (35.00–40.60) b0.0001c,e,f
Uric acid (μmol/L) 303 ± 81 304 ± 67 391 ± 113 377 ± 78 0.001a,d,f
Hemoglobin (g/L) 138.8 ± 12.7 135.7 ± 16.3 130.4 ± 17.2 113.3 ± 15.8 b0.0001c,e,f
Hb A1C (%) 7.73 ± 1.32 7.57 ± 1.38 7.46 ± 1.97 6.75 ± 1.29 0.03f
LDL cholesterol mmol/L) 2.68 (2.07–3.21) 2.90 (1.81–3.17) 2.83 (2.18–3.57) 2.63 (2.06–3.50) 0.75
HDL cholesterol (mmol/L) 1.12 (1.04–1.34) 1.06 (0.98–1.28) 1.08 (0.90–1.19) 1.06 (0.56–1.26) 0.25
Triglycerides (mmol/L) 1.53 (1.09–2.28) 1.95 (1.43–2.31) 1.92 (1.48–2.26) 1.68 (1.23–3.34) 0.37
CRP (mg/L) 2.50 (1.00–06.8) 4.00 (2.00–10.50) 3.00 (1.00–8.00) 10.00 (5.43–22.80) b0.0001c,f
Klotho 0.40 (0.1–1.30) 0.2 (0.10–0.40) 0.60 (0.20–1.1) 1.45 (0.42–2.90) b0.0001a,b,c,e,f
VEGF-A (pg/mL) 73.85 (57.325–119) 51.9 (40.3–102.2) 71.3 (54.6–90.1) 65.30 (48.52–93.17) 0.21
Retinopathy n(%) 13 (33.30) 9 (60.00) 14 (56.00) 17 (47.20) 0.19

Legend: CKD — chronic kidney disease, BMI — body mass index, S — systolic, D — diastolic, BP — blood pressure, GFR — glomerular filtration rate, UACR — urinary albumin/creatinine ratio,
Hb — hemoglobin, HbA1C — glycated hemoglobin, LDL — low density lipoprotein, HDL — high density lipoproteins, CRP — C reactive protein, VEGF-A — vascular endothelial growth factor
A, significant difference between: a without CKD and CKD1/2; b CKD 1/2 and CKD3/4, c CKD3/4 and CKD5, dwithout CKD and CKD3/4, e CKD 1/2 and CKD 5, f without CKD and CKD5. Nor-
mally distributed variables are presented as mean±standard deviation; non-normally distributed variables as median (interquartile range).
1418 I.M. Kacso et al. / Clinical Biochemistry 45 (2012) 1415–1420

Table 3
Comparison of diabetic patients according to GFR.

Parameter GFR > 60 mL/min GFR 15–60 mL/min GFR b 15 mL/min p

(n = 71) (n = 39) (n = 36)

Age (years) 62.06 ± 10.68 67.77 ± 8.93 61.25 ± 8.38 0.005a,b

Male n (%) 44 (62) 23 (59) 20 (55.60) 0.81
Diabetes duration (years) 8 (4–15) 9 (5–17) 20 (17–23.75) b0.0001b,c
BMI (kg/m2) 30.99 30.48 29.07 0.16
(27.08–33.47) (26.67–34.37) (24.22–32.19)
Waist circumference (cm) 108.06 ± 13.31 107.59 ± 11.98 99.29 ± 16.75 0.007b,c
SBP (mm Hg) 140 (130–160) 140 (130–150) 130 (130–145) 0.41
DBP (mm Hg) 80 (80–90) 80 (75–90) 80 (70–90) 0.001c
GFR (mL/min) 93.68 ± 21.74 44.03 ± 10.54 – b0.0001
UACR (mg/g) 16.75 94.26 – 0.0002
(4.02–52.92) (17.41–443.64)
Albumin (g/L) 46.00 45.00 37.90 b0.0001b,c
(44.00–48.00) (43.00–47.00) (35.00–40.60)
Uric acid (mg/dL) 303 ± 76 391 ± 113 377 ± 78 0.0001a,c
Hemoglobin (g/L) 137.90 ± 13.80 130.40 ± 17.20 113.30 ± 15.80 b0.001b,c
Hb A1C (%) 7.68 ± 1.34 7.49 ± 1.97 6.75 ± 1.29 0.01c
LDL cholesterol(mmol/L) 2.71 (3.04–3.18) 2.83 (2.18–3.57) 3.06 (2.63–3.50) 0.56
HDL cholesterol (mmol/L) 1.11 (1.01–1.29) 1.08 (0.9–1.19) 1.06 (0.56–1.26) 0.16
Triglycerides (mmol/L) 1.64 (1.20–2.30) 1.92 (1.48–2.26) 1.68 (1.23–3.33) 0.42
CRP (mg/L) 3.00 (1.00–8.00) 3.00 (1.00–8.00) 10.00 (5.43–22.0) 0.002b,c
Klotho 0.30 (0.10–0.70) 0.6 (0.20–1.10) 1.45 (0.42–2.9) 0.0001b,c
VEGF-A(pg/mL) 66.70 71.30 65.30 0.89
(48.50–110.00) (54.60–90.10) (48.52–93.17)
Retinopathy n(%) 22 (40.70) 14 (56.00) 17 (47.20) 0.44

Legend: GFR — glomerular filtration rate, BMI — body mass index, S — systolic, D — diastolic, BP — blood pressure, UACR — urinary albumin/creatinine
ratio, HbA1C — glycated hemoglobin, LDL — low density lipoprotein, HDL — high density lipoproteins, UACR — urinary albumin to creatinine ratio, CRP — C
reactive protein, VEGF-A — vascular endothelial growth factor A. Normally distributed variables are presented as mean±standard deviation; non-normally
distributed variables as median (interquartile range).
a
Significant difference between patients with GFR>60 mL/min and those with GFR 15-60 mL/min.
b
Significant difference between and patients with GFR 15–60 mL/min and dialysis patients.
c
Significant difference between patients with GFR>60 mL and dialysis patients.

coefficient b = 0.02, p = 0.01, CI(0.005–0.04). There was no significant
correlation of Klotho to VEGF-A in controls (r= −0.09, p = 0.55).
Discussion
Serum Klotho in diabetes
One important contribution of our work is the description, for the
first time to our knowledge, of the relationship of serum Klotho to
CKD in diabetic patients.
We found lower levels of serum Klotho in diabetic patients without
nephropathy when compared to healthy controls; however the differ-
ence did not reach significance probably due to the wide distribution
of serum Klotho levels in diabetic subjects (see Fig. 1) and the relatively
limited number of patients. There is a paucity of data concerning soluble
Klotho levels in diabetic patients, and lower levels in patients with
HbA1C> 6.5% have previously been reported [24]; this also relates to
experimental evidence of reduced expression of Klotho in diabetes
[15,16]. However, no relationship of serum Klotho to blood glucose was
found in a large population-based study [25]. Further studies need to be
performed in order to clarify this issue.
Serum Klotho in CKD
In our diabetic CKD patients we found much lower soluble Klotho
levels than those from the first report [24], which were in the range
of hundreds of ng/mL; however all subsequent studies describe
values comparable to ours. [25–30]. Reduced kidney expression of
Klotho has been reported in chronic kidney disease [7–9] but the
relationship of kidney Klotho expression and soluble Klotho levels
is not yet established. Some of the nephroprotective effects of Klotho
overexpression in experimental settings seem to be related to the
soluble form of Klotho, as glomerular changes are improved [10,11]
whereas membrane bound Klotho is confined to the tubules. In ex-
perimental diabetes, exogenous addition of soluble Klotho had simi-
lar effect with Klotho overexpression [15]. Hu et al. [31] reported
that reduced blood and urine Klotho parallels decreased kidney ex-
pression and occurs early in the progression of CKD. In humans
with preserved renal function, inverse relationship of soluble Klotho
to serum creatinine has been initially reported [25], subsequent large
studies did not conform this finding in a population based study [26].
There are few studies concerning serum Klotho in CKD patients. Two
different groups reported increase in soluble Klotho in patients with
CKD and reduced GFR [24,27]. However, other authors report a
decrease in soluble Klotho in CKD patients, especially in early stages of
CKD [28,29]. There are no available data on soluble Klotho in humans
with diabetic nephropathy. In our cohort we found an initial decrease
in Klotho with the occurrence of CKD. Patients with stage1–2 CKD
have lower Klotho levels than non-CKD diabetic patients; serum Klotho
increases thereafter with CKD stage, possibly due to reduced excretion/
catabolism. We also found significantly decreased Klotho in albumin-
uric patients when compared to normoalbuminuric ones. Urinary leak-
age of this 130 kDa protein [4] is an improbable explanation for this
phenomenon as lowest Klotho is found in microalbuminuric, and not
proteinuric patients. As microalbuminuria is an early feature of diabetic
nephropathy, this is another argument for decrease of soluble Klotho in
incipient diabetic nephropathy.
Influence of treatment
Klotho expression is known to be induced by statins and angiotensin
II and reduced by PPAR gamma agonists. We studied the influence of
the presence and absence of medication that might influence serum
Klotho levels in our patients and found no significant influence of treat-
ment on serum Klotho in our cohort.
 I.M. Kacso et al. / Clinical Biochemistry 45 (2012) 1415–1420 1419

Table 4
Comparison of diabetic predialysis patients according to UACR.

Parameter UACR b 30 mg/g UACR 30–300 mg/g UACR >300 mg/g p

(n = 63) (n = 30) (n = 17)

Age (years) 62.05 ± 10.68 66.97 ± 9.50 66.53 ± 9.82 0.06

Male n (%) 39 (61.9) 20 (66.7) 8 (47.1) 0.40
Diabetes duration (years) 7 (4–14) 10 (6–17.5) 12 (35–17.5) 0.13
BMI (kg/m2) 31.14 30.86 27.69 0.32
(27.77–34.36) (26.27–34.77) (26.36–32.61)
Waist circum-ference (cm) 109.97 ± 13.10 106.23 ± 13.70 103.35 ± 8.13 0.12
SBP (mm Hg) 140(125–150) 140(130–150) 150(135–160) 0.36
DBP (mm Hg) 80(75–90) 80(75–90) 90(80–90) 0.19
GFR (mL/min) 84.67 ± 28.78 72.60 ± 26.94 50.34 ± 25.84 b0.0001b,c
UACR (mg/g) 10.52 89.01 538.62 b0.0001a,b,c
(2.260–17.41) (43.90–108.79) (420.12–1212.98)
Albumin (g/L) 46.00 46.00 44.00 0.23
(43.00–48.00) (43.50–47.50) (41.50–46.50)
Uric acid (mg/dL) 327 ± 102 339 ± 110 343 ± 54 0.69
Hemoglobin (g/dL) 137.80 ± 13.60 137.10 ± 14.00 122.00 ± 19.00 0.005b,c
Hb A1C (%) 7.59 ± 1.64 7.87 ± 1.51 7.24 ± 1.26 0.48
LDL cholesterol (mmol/L) 2.71 (3.04–3.32) 2.86 (2.10–3.19) 2.59 (2.29–2.96) 0.89
HDL cholesterol (mmol/L) 1.11 (0.93–1.24) 1.07 (0.90–1.27) 1.26 (1.01–1.46) 0.15
Triglycerides (mmol/L) 0.20 (0.14–0.26) 0.22 (0.18–0.27) 0.18 (0.14–0.26) 0.53
CRP (mg/dL) 3.00 (1.00–8.00) 3.00 (1.80–9.20) 2.00 (1.00–7.89) 0.36
Klotho 0.50 (0.20–1.30) 0.20 (0.10–0.62) 0.40 (0.10–1.00) 0.07
VEGF-A (pg/mL) 71.30 57.6 71.9 0.05a
(58.40–116.60) (42.475–86.52) (40.75–104.80)
Retinopathy n(%) 19 (30.20) 13 (43.30) 4 (23.50) 0.047a

Legend: UACR — urinary albumin/creatinine ratio, BMI — body mass index, S — systolic, D — diastolic, BP — blood pressure, GFR — glomerular filtration rate,
HbA1C — glycated hemoglobin, LDL — low density lipoproteins, HDL — high density lipoproteins, CRP — C reactive protein, VEGF-A — vascular endothelial
growth factor A. Normally distributed variables are shown as mean ± standard deviation; non-normally distributed ones as median (interquartile range).
a
Significant difference between patients with UACRb 30 mg/g and UACR 30–300 mg/g.
b
Significant difference between patients with UACR 30–300 mg/g and UACR>300 mg/g.
c
Significant difference between patients with UACRb 30 mg/g and UACR>300 mg/g.

Mechanisms of action to participate in important pathways of diabetic nephropathy, like oxi-

dative stress and endothelial dysfunction.
Since the kidney is a main site of membrane-bound Klotho expres- There is increasing evidence that Klotho deficiency promotes oxi-
sion, it is conceivable that in kidney disease a decrease Klotho expres- dative stress and Klotho overexpression confers resistance to oxida-
sion/secretion may occur. Nonetheless, it is possible for Klotho to be tive damage [8,10,32,33]. Klotho protein itself or its metabolites
not only a consequence of kidney disease, but also an active participant promote endothelial nitric oxide production and therefore alleviate
in the extent of kidney damage, as, in several experimental settings — endothelial dysfunction [6,34,35].
chronic glomerulonephritis model [10], in a model of interstitial fibrosis
[12] or hypertension induced renal disease [11], overexpression of
Klotho seems to be associated to less severe structural and functional Relationship of Klotho to VEGF-A
changes. Klotho also protects against compensatory postnephrectomy
hypertrophy [13]. In experimental diabetes, renal Klotho expression is Nitric oxide is a downstream mediator of VEGF-A induced angiogen-
reduced [15,16], and correction of hyperglycemia or antioxidant treat- esis and hyperpermeability and effects of VEGF depend on the bioavail-
ment are both associated to improved renal function paralleling in- ability of nitric oxide [20,36]. Klotho might therefore interfere with
creased expression of Klotho [16]. VEGF-dependent effects and/or be a regulator of this pathway. In fact
Secreted Klotho does not function as a soluble receptor for FGF23 be- Klotho deficiency has been associated to impaired VEGF-A dependent
cause it is the membrane Klotho-FGF receptor complex that has high af- ability of the endothelium to replicate and maintain integrity [20].
finity for FGF23 and not Klotho protein per se. Moreover, Klotho seems Also, VEGF-mediated Ca 2+ signaling is tightly regulated by interaction
with Klotho; Klotho deficient mice display increased VEGF-induced
hyperpermeability. Recently Klotho protein has been shown to directly
bind to the extracellular domain of VEGFR-2 [21]. Our study is strictly
Table 5 observational and does not permit insight on the mechanisms that in-
Comparison of serum Klotho according to treatment. terrelate Klotho to VEGF-A. Relationship of serum Klotho to serum
Serum Klotho (ng/mL) p
VEGF-A has not been described yet to our knowledge. We found a
consistent positive relationship between soluble Klotho and VEGF-A,
Not treated patients Treated patients
consistent in dialysis and predialysis patients, diabetic patients without
Converting enzyme inhibitors 0.60 (0.20–1.37) 0.50 (0.20–1.50) 0.71 nephropathy or albuminuric patients; however this relationship does
Angiotensin receptor antagonists 0.60 (0.20–1.450) 0.35 (0.10–0.85) 0.14
not exist in controls.
Converting enzyme inhibitors or 0.70 (0.20–1.37) 0.40 (0.20–1.40) 0.12
angiotensin receptor antagonists
Klotho and VEGF might be increased by the same pathogenetic
Insulin 0.30 (0.10–1.15) 0.60 (0.20–1.87) 0.46 event or there might be a reactive, counterregulatory increase (e.g. of
Metformin 0.50 (0.20–1.50) 0.40 (0.10–1.00) 0.29 Klotho in response to VEGF-A); both molecules are related to endothe-
Sulfonylureas 0.60 (0.20–1.50) 0.30 (0.10–1.00) 0.13 lial dysfunction characteristic of diabetes. Further study is needed to
Statins 0.70 (0.20–1.37) 0.40 (0.20–1.40) 0.54
address the mechanisms involved.
1420 I.M. Kacso et al. / Clinical Biochemistry 45 (2012) 1415–1420
VEGF-A in diabetic patients with CKD
In line with previous reports [37,38] we found significantly increased
levels of VEGF-A in diabetic patients compared to controls. In patients
with diabetic nephropahty, urinary reports on circulating VEGF-A are
not consistent [38–40]; we found significantly lower serum VEGF-A
in microalbuminuric patients when compared to normoalbuminuric
ones. Interestingly, in proteinuric patients, VEGF-A is also higher
than in microalbuminuric subjects; the difference is not significant,
probably due to the small size of the proteinuric cohort. This fact
can be related to changes of VEGF-A being reported early in the
course of diabetic nephropathy, with a tendency to normalize over
time [17]. GFR does not seem to influence serum VEGF-A.
Conclusion
Klotho is decreased in early CKD in diabetic patients and increases
thereafter, paralleling GFR reduction. VEGF-A is higher in diabetic
patients than in controls. Both Klotho and VEGF-A are decreased in
the presence of microalbuminuria. Klotho strongly correlates to VEGF
in diabetic patients.
Contributions
All authors have significantly contributed to the conception and de-
sign of the study, analysis and interpretation of data; drafting the article
or revising it critically for important intellectual content; final approval
of the version to be submitted. Kacso IM had main contribution in acqui-
sition of data.
Conflict of interest statement
None to declare.
Acknowledgments
This work was funded by a CNCSIS Romania Idei 1167/2008 grant.
References
[1] Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, et al. Mutation of the
mouse Klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.
[2] Hu MC, Shi M, Zhang J, Pastor J, Nakatani T, Lanske B, et al. Klotho: a novel
phosphaturic substance acting as an autocrine enzyme in the renal proximal tu-
bule. FASEB J 2010;24:3438-50.
[3] Matsumura Y, Aizawa H, Shiraki-Iida T, Nagai R, Kuro-o M, Nabeshima Y. Identifi-
cation of the human Klotho gene and its two transcripts encoding membrane and
secreted Klotho protein. Biochem Biophys Res Commun 1998;242:626-30.
[4] Imura A, Iwano A, Tohyama O, Tsuji Y, Nozaki K, Hashimoto N, et al. Secreted
Klotho protein in sera and CSF: implication for post-translational cleavage in re-
lease of Klotho protein from cell membrane. FEBS Lett 2004;565:143-7.
[5] Huang CL. Regulation of ion channels by secreted Klotho. Adv Exp Med Biol
2012;728:100-6.
[6] Rakugi H, Matsukawa N, Ishikawa K, Yang J, Imai M, Ikushima M, et al. Anti-oxidative
effect of Klotho on endothelial cells through cAMP activation. Endocrine 2007;31:82-7.
[7] Aizawa H, Saito Y, Nakamura T, Inoue M, Imanari T, Ohyama Y, et al. Downregulation
of the Klotho gene in the kidney under sustained circulatory stress in rats. Biochem
Biophys Res Commun 1998;249:865-71.
[8] Zuo Z, Lei H, Wang X, Wang Y, Sonntag W, Sun Z. Aging-related kidney damage is
associated with a decrease in Klotho expression and an increase in superoxide
production. Age (Dordr) 2011;33:261-74.
[9] Koh N, Fujimori T, Nishiguchi S, Tamori A, Shiomi S, Nakatani T, et al. Severely
reduced production of Klotho in human chronic renal failure kidney. Biochem
Biophys Res Commun 2001;280:1015-20.
[10] Haruna Y, Kashihara N, Satoh M, Tomita N, Namikoshi T, Sasaki T, et al. Ameliora-
tion of progressive renal injury by genetic manipulation of Klotho gene. Proc Natl
Acad Sci U S A 2007;104:2331-6.
[11] Wang Y, Sun Z. Klotho gene delivery prevents the progression of spontaneous
hypertension and renal damage. Hypertension 2009;54:810-7.
[12] Sugiura H, Yoshida T, Shiohira S, Kohei J, Mitobe M, Kurosu H, Kuro-O M, Nitta K,
Tsuchiya K. Reduced Klotho expression level in kidney aggravates renal intersti-
tial fibrosis. Am J Physiol Renal Physiol Feb 15 2012 [Epub ahead of print].
[13] Nagasu H, Satoh M, Kuwabara A, Yorimitsu D, Kidokoro K, Nishi Y, et al. Overexpression
of Klotho protein modulates uninephrectomy-induced compensatory renal hypertro-
phy by suppressing IGF-I signals. Biochem Biophys Res Commun 2011;407:39-43.
[14] Verzola D, Gandolfo MT, Gaetani G, Ferraris A, Mangerini R, Ferrario F, et al. Accel-
erated senescence in the kidneys of patients with type 2 diabetic nephropathy.
Am J Physiol Renal Physiol 2008;295:F1563-73.
[15] Zhao Y, Banerjee S, Dey N, LeJeune WS, Sarkar PS, Brobey R, et al. Klotho depletion
contributes to increased inflammation in kidney of the db/db mouse model of
diabetes via RelA (serine)536 phosphorylation. Diabetes 2011;60:1907-16.
[16] Cheng MF, Chen LJ, Cheng JY. Decrease of Klotho in the kidney of streptozotocin-
induced diabetic rats. J Biomed Biotechnol 2010;2010:513853.
[17] Cha DR, Kang YS, Han SY, Jee YH, Han KH, Han JY, et al. Vascular endothelial
growth factor is increased during early stage of diabetic nephropathy in type II
diabetic rats. J Endocrinol 2004;183:183-94.
[18] Flyvbjerg A, Dagnaes-Hansen F, De Vriese AS, Schrijvers BF, Tilton RG, Rasch R.
Amelioration of long-term renal changes in obese type 2 diabetic mice by a neu-
tralizing vascular endothelial growth factor antibody. Diabetes 2002;51:3090-4.
[19] Sung SH, Ziyadeh FN, Wang A, Pyagay PE, Kanwar YS, Chen S. Blockade of vascular
endothelial growth factor signaling ameliorates diabetic albuminuria in mice. J Am
Soc Nephrol 2006;17:3093-104.
[20] Shimada T, Takeshita Y, Murohara T, Sasaki K, Egami K, Shintani S, et al. Angiogen-
esis and vasculogenesis are impaired in the precocious-aging klotho mouse. Cir-
culation 2004;110:1148-55.
[21] Kusaba T, Okigaki M, Matui A, Murakami M, Ishikawa K, Kimura T, et al. Klotho is
associated with VEGF receptor-2 and the transient receptor potential canonical-1
Ca2+ channel to maintain endothelial integrity. Proc Natl Acad Sci U S A
2010;107:19308-13.
[22] Nakamura T, Sai Y, Ohyama Y, Masuda H, Sumino H, Kuro-o M, et al. Production of
nitric oxide, but not prostacyclin, is reduced in Klotho mice. Jpn J Pharmacol
2002;89:149-56.
[23] Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to
estimate glomerular filtration rate from serum creatinine: a new prediction equation.
Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461-70.
[24] Devaraj S, Syed B, Chien A, Jialal I. Validation of an immunoassay for soluble
Klotho protein: decreased levels in diabetes and increased levels in chronic kid-
ney disease. Am J Clin Pathol 2012;137:479-85.
[25] Semba RD, Cappola AR, Sun K, Bandinelli S, Dalal M, Crasto C, et al. Plasma Klotho
and cardiovascular disease in adults. J Am Geriatr Soc 2011;59:1596-601.
[26] Yamazaki Y, Imura A, Urakawa I, Shimada T, Murakami J, Aono Y, et al. Establish-
ment of sandwich ELISA for soluble alpha-Klotho measurement: age-dependent
change of soluble alpha-Klotho levels in healthy subjects. Biochem Biophys Res
Commun 2010;389:513-8.
[27] Sugiura H, Tsuchiya K, Nitta K. Circulating levels of soluble a-Klotho in patients
with chronic kidney disease. Clin Exp Nephrol 2011;15:795-6.
[28] Shimamura Y, Hamada K, Inoue K, Ogata K, Ishihara M, Kagawa T, et al. Serum
levels of soluble secreted α-Klotho are decreased in the early stages of chronic
kidney disease, making it a probable novel biomarker for early diagnosis. Clin
Exp Nephrol Mar 29 2012 [Epub ahead of print].
[29] Akimoto T, Shiizaki K, Sugase T, Watanebe Y, Yoshizawa H, Otani N, et al. Serum
levels of soluble secreted α-Klotho are decreased in the early stages of chronic
kidney disease, making it a probable novel biomarker for early diagnosis. Clin
Exp Nephrol 2012 [online first].
[30] Komaba H, Koizumi M, Tanaka H, Takahashi H, Sawada K, kakuta T, et al. Effects of
cinacalcet treatment on serum soluble Klotho levels in hemodialysis patients with
secondary hyperparathyroidism. Nephrol Dial Transplant 2012 [online first].
[31] Hu MC, Shi M, Zhang J, Quiñones H, Griffith C, Kuro-o M, et al. Klotho deficiency causes
vascular calcification in chronic kidney disease. J Am Soc Nephrol 2011;22:124-36.
[32] Yamamoto M, Clark JD, PastorV JV, Gurnani P, Nandi A, Kurosu H, et al. Regulation
of oxidative stress by the anti-aging hormone Klotho. J Biol Chem 2005;280:
38029-34.
[33] Ikushima M, Rakugi H, Ishikawa K, Maekawa Y, Yamamoto K, Ohta J, et al. Anti-
apoptotic and anti-senescence effects of Klotho on vascular endothelial cells.
Biochem Biophys Res Commun 2006;339:827-32.
[34] Nagai R, Saito Y, Ohyama Y, Aizawa H, Suga T, Nakamura T, et al. Endothelial dysfunc-
tion in the Klotho mouse and downregulation of Klotho gene expression in various
animal models of vascular and metabolic diseases. Cell Mol Life Sci 2000;57:738-46.
[35] Saito Y, Yamagishi T, Nakamura T, Ohyama Y, Aizawa H, Suga T, et al. Klotho
protein protects against endothelial dysfunction. Biochem Biophys Res Commun
1998;248:324-9.
[36] Nakagawa T. Uncoupling of the VEGF-endothelial nitric oxide axis in diabetic ne-
phropathy: an explanation for the paradoxical effects of VEGF in renal disease. Am
J Physiol Renal Physiol 2007;292:1665-72.
[37] Chiarelli F, Spagnoli A, Basciani F, Tumini S, Mezzetti A, Cipollone F, et al. Vascular
endothelial growth factor (VEGF) in children, adolescents and young adults with
Type 1 diabetes mellitus: relation to glycaemic control and microvascular compli-
cations. Diabet Med 2000;17:650-6.
[38] Kim NH, Kim KB, Kim DL, Kim SG, Choi KM, Baik SH, et al. Plasma and urinary vas-
cular endothelial growth factor and diabetic nephropathy in Type 2 diabetes
mellitus. Diabet Med 2004;21:545-51.
[39] Chaturvedi N, Fuller JH, Pokras F, Rottiers R, Papazoglou N, Aiello LP. EUCLID study
group. Circulating plasma vascular endothelial growth factor and microvascular
complications of type 1 diabetes mellitus: the influence of ACE inhibition. Diabet
Med 2001;18:288-94.
[40] Shimada K, Baba T, Neugebauer S, Onozaki A, Yamada D, Midorikawa S, et al. Plas-
ma vascular endothelial growth factor in Japanese Type 2 diabetic patients with
and without nephropathy. J Diabetes Complications 2002;16:386-90.