Peripheral blood leukocyte telomere length is associated with age but not renal function: A cross-sectional follow-up study

Abstract

Objectives

We aimed to evaluate the relationship between baseline renal function and changes in telomere length in Han Chinese.

Methods

The telomere restriction fragment (TRF) length of leukocytes in the peripheral blood was measured in healthy volunteers recruited in 2014. The estimated glomerular filtration rate (eGFR) was calculated based on serum creatinine (Scr) and serum cystatin C (CysC)-eGFRcys and eGFRScr-cys through the Cockcroft-Gault formula (eGFRC-G) or the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI / eGFRCKD-EPI) equation. The correlation between telomere length changes over time and renal function was analyzed.

Results

Leukocyte TRF lengths were negatively correlated to age (r = -0.393, p < 0.001) and serum CysC (r = -0.180, p < 0.01), while positively associated with eGFRCKD-EPI, eGFRC-G, eGFRcys, and eGFRScr-cys (r = 0.182, 0.122, 0.290, and 0.254 respectively, p < 0.01). The 3-year change of telomere length was 46 bp/years. When adjusted for age, the associations between telomere length changes and baseline, subsequent TRF lengths, and serum CysC were no longer present. No association was observed between TRF length changes and renal function.

Conclusion

The rate of telomere length changes was affected by age and baseline telomere length. The telomere length changes might be important markers for aging.

Introduction

Sarcopenia is a serious health problem for older adults sinTelomeres lie at the end of eukaryotic chromosomes and consist of highly conserved repeat nucleotide sequences (G-rich) with specific proteins (1). Telomeres stabilize chromosome structure via their location at the chromosome ends and by avoiding DNA degradation. The average lengths of human telomeres are 5 to 15 kb (1, 2); they are maintained primarily by telomerase. Telomere length gradually decreases with cell division. Frequency of cell division and telomerase activity determine the telomere length (3, 4). However, telomerase activity is limited due to its low expression level in somatic cells of humans (5, 6).

Studies have found that telomere length is closely associated with renal function, but the relationship is inconsistent (7–10). Moreover, whether an association exists between telomere length and renal function, particularly between telomere length changes over time and renal function, is unknown. More importantly, whether telomere length can be a marker for estimating renal progression and patient prognosis is controversial.

The current study aimed to investigate the relationship between telomere length and changes in renal function in a cohort of healthy people in Beijing, China (11); to observe the relationship between changes to short telomere length and changes in renal function over time, and to analyze whether telomere length can be a potential marker of renal function as well as aging.

Materials and Methods

Sample screening

In 2011, we evaluated telomere lengths in 138 healthy individuals (11). In 2014, we enrolled another cohort of 471 healthy volunteers of Han Chinese ethnicity from 1207 candidate volunteers (mean age 35 to 90 years) in Beijing, China—80 of these individuals were also examined in 2011. The volunteers did not take any medications and had not been hospitalized between 2011 and 2014. All participants provided written informed consent.

The inclusion criteria followed the study flow chart previously described in 2010 (12). This study was approved by the Ethics Committee on Human Experimentation of the Chinese PLA General Hospital (Beijing, China) and was conducted in accordance with the Declaration of Helsinki and subsequent amendments.

Clinical and biochemical parameter examined

Clinical features—including age, gender, body height, body weight, waist circumference, hip circumference, and blood pressure (systolic blood pressure [SBP], diastolic blood pressure [DBP], pulse pressure, and pulse pressure index)— were all recorded. Laboratory parameters—including blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA), triglycerides, total cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL), urine routine analyses, and serum cystatin C (CysC)—were assayed. Estimated glomerular filtration rate (eGFR) was derived using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation and the Cockcroft-Gault (CG) formula.

Determination of telomere restriction fragment (TRF) length

Genomic DNA was isolated by using a genomic DNA extraction kit [Tiangen Biotech (Beijing) Co Ltd] and the TRF length was measured using the Telo TTAGGG telomere length assay (Roche, Mannheim, Germany). Briefly, genomic DNA (1.5µg) was digested with the restriction enzymes (Rsa I and Hinf I) for 2 hours at 37°C. After digestion, DNA fragments were separated in a 0.8% agarose gel by electrophoresis, denatured, neutralized, transferred to a nylon membrane, and cross-linked with UV light (UVP, Upland, CA).

Blotted DNA fragments were then incubated with a telomeric probe (digoxigenin) (DIG) 3'-end labeled 5'-[CCCTAA]3) at 42°C for 3 hours, followed by incubation with a DIG-specific antibody covalently coupled to alkaline phosphatase. Binding sites of the telomere probe were visualized using a highly sensitive chemiluminescence substrate that metabolizes alkaline phosphatase.

TRF lengths were then compared with molecular weight markers (Roche, Mannheim, Germany) (2), and mean TRF lengths were estimated using Quantity One® 1-D analysis software (Bio-Rad Laboratories, Inc., Hercules, CA) (2).

Statistical analysis

Data were expressed as mean ± standard deviation, and significance was set at a p value < 0.05. A linear regression model was used to detect independent relationship between variables of interest. All statistical calculations were performed using SPSS v17.0 software (SPSS, Inc., Chicago, IL).

Result

In 2014, 471 healthy volunteers were enrolled and divided into five age groups: 35 to 44 years, 45 to 54 years, 55 to 64 years, 65 to 74 years, and > 75 years (Supplementary File 1). The mean age was 59.5 ± 14.4 years, and 50.3% were women. The mean leukocyte TRF lengths were 6.84 ± 0.74 kb. Telomere length was negatively correlated with age (r = -0.393, p < 0.001) (Supplementary File 2), and female participants had significantly longer telomere lengths than male participants (p = 0.011). A comparison between age groups showed that leukocyte TRF lengths in the 55 to 64 years, 65 to 74 years, and > 75 years age groups were significantly lower than those in 35 to 44 years age group (Figure 1). TRF length was negatively associated with CysC while positively associated with eGFR measured by CKD-EPI equation, CG formula, CysC-based formula, and Scr/CysC-based formula (p < 0.01) (Supplementary File 3).

Figure 1.

Telomere restriction fragment (TRF) lengths among different age groups (* p < 0.05)

The mean age of the 80 participants with data in 2011 and 2014 was 61 ± 13 years, and 55% were female. Their mean TRF lengths in 2011 (6.89 ± 0.64 kb) differed significantly from those examined in 2014 (6.76 ± 0.57 kb) (t = 6.738, p < 0.01). Both TRF lengths at baseline or after follow-up exhibited significantly negative associations (for baseline levels in 2011, r = -0.571, p < 0.001; for follow-up levels in 2014, r = -0.590, p < 0.001, Figure 2).

Figure 2.

Correlation plot between age and telomere restriction fragment (TRF) length at baseline and after follow-up

We defined changes in TRF length ≥1% as a significant increase in TRF lengths, changes between -1% and 1% as decrease in TRF lengths. Among the 80 participants, the mean length of telomere wear was 138 bp, equivalent to an average annual wear of 46 bp; 73% of the 80 participants showed decreased TRF lengths, 11% showed unchanged TRF lengths, and 16% showed slightly increased TRF lengths. We further divided these 80 participants into four groups according to their TRF length in 2011 (quartile 1, 8.74–7.41 kb; quartile 2, 7.4–6.94 kb; quartile 3, 6.91–6.49 kb; and quartile 4, 6.48–5.21 kb).

Comparing between different quartiles, we found that the changes in TRF lengths in quartile 1 were significantly higher than those in the other three quartiles; on the other hand, TRF length changes in quartile 2 was significantly higher than those in quartile 4 (Figure 3). We also found that TRF lengths in 2014 were significantly lower than those in 2011 among participants of quartile 1, quartile 2, and quartile 3 (all p < 0.01), while TRF lengths in participants of quartile 4 did not differ between data in 2014 and those in 2011 (p = 0.684) (Table 1).

Figure 3.

Changes in telomere restriction fragment (TRF) lengths among different age groups

Table 1.

Comparison between telomere lengths before and after follow-up among different age groups of the 80 selected cases

Group	2011 telomere length	2014 telomere length	t	p
quartile 1 (8.739–7.409kb)	7.668±0.332	7.390±0.320	8.614	<0.001**
quartile 2 (7.403–6.943kb)	7.131±0.142	6.990±0.212	4.395	<0.001**
quartile 3 (6.908–6.490kb)	6.722±0.147	6.607±0.199	3.581	0.002**
quartile 4 (6.484–5.208kb)	6.052±0.305	6.033±0.356	0.413	0.684

Variables are expressed as mean ± standard deviation; * p < 0.05, ** p < 0.01.

Among the 80 participants, TRF lengths in 2011 displayed significant correlation with eGFR measured by CKD-EPI equation, CG formula, and Scr/CysC-based formula (Table 2); similarly, TRF lengths in 2014 showed significant correlation with eGFR measured by CKD-EPI equation, CG formula, CysC-based formula, and Scr/CysC-based formula (Table 2).

Table 2.

Correlation analysis between telomere lengths at baseline and renal function results at follow-up among the 80 selected participants

Renal function assessment methods	2011 annual	2011 telomere length		2014 annual	2014 telomere length
	average			average
		r	p		r	p
cysC (mg/l)	0.78±0.13	-0.219	0.051	0.85±0.15	-0.433	0.000**
Scr (mg/dl)	0.76±0.13	-0.039	0.728	0.81±0.14	-0.112	0.324
eGFRC-G (ml/min/1.73m2)	50.08±15.77	0.328	0.003**	52.21±16.56	0.296	0.008**
eGFRcys (ml/min/1.73m2)	107.91±24.20	0.174	0.123	96.58±21.28	0.440	0.000**
eGFRScr-cys (ml/min/1.73m2)	100.46±18.07	0.313	0.005**	90.63±18.68	0.445	0.000**
eGFRCKD-EPI (ml/min/1.73m2)	95.43±19.77	0.420	0.000**	87.55±19.53	0.436	0.000**

Variables are expressed as mean ± standard deviation; ** p < 0.01; Abbreviations: Scr, serum creatinine; cysC, cystatin C; eGFRC-G, Cockcroft-Gault equation; eGFRcys, CKD-EPI Chronic Kidney Disease Epidemiology Collaboration cysC equation; eGFRC-G, CKD-EPI Chronic Kidney Disease Epidemiology Collaboration cysC-Scr equation; eGFRCKD-EPI, CKD-EPI Chronic Kidney Disease Epidemiology Collaboration Scr equation.

Changes in TRF lengths during follow-up were significantly associated with TRF lengths in 2011 and 2014; however, after adjusting for age, changes in TRF lengths were significantly associated with TRF lengths in 2011 (Table 3). Changes in TRF lengths were associated with serum CysC in 2014 only (Table 4), while TRF lengths in 2011 and in 2014 were both significantly associated with changes in serum CysC, eGFRcys, and eGFRScr-cys (Table 5).

Table 3.

Correlation analysis between changes in telomere lengths, telomere length in 2011 and in 2014

Related factors	<TRF
	Uncalibrated age		Calibration age
	r	p	r	p
2011 TRF	-0.495	0.000**	-0.502	0.000**
2014 TRF	-0.234	0.037*	-0.179	0.114

Variables are expressed as mean ± standard deviation; * p < 0.05, ** p < 0.01; Abbreviation: TRF, telomere restriction fragment.

Table 4.

Correlation analysis between changes in telomere lengths, renal function, and changes in renal function

Assessment of renal function mode				<TRF
	2011 kidney function		2014 kidney function		Changes in renal function
	r	p	r	p	r	p
cysC (mg/l)	0.202	0.072	0.232*	0.038*	0.068	0.552
Scr (mg/dl)	0.122	0.281	0.124	0.273	0.013	0.913
eGFRC-G (ml/min/1.73m2)	0.045	0.694	0.000	1.000	-0.083	0.468
eGFRcys (ml/min/1.73m2)	-0.213	0.057	-0.200	0.076	0.045	0.692
eGFRScr-cys (ml/min/1.73m2)	-0.196	0.081	-0.178	0.115	0.021	0.852
eGFRCKD-EPI (ml/min/1.73m2)	-0.115	0.310	-0.074	0.517	0.082	0.468

Variables are expressed as mean ± standard deviation; * p < 0.05; Abbreviation: TRF, telomere restriction fragment.

Table 5.

Correlation analysis between changes in telomere lengths and renal function

Changes in renal function	2011 TRF		2014 TRF
	r	p	r	p
cysC (mg/l)	-0.331	0.003**	-0.348	0.002**
Scr (mg/dl)	-0.162	0.151	-0.178	0.115
eGFRC-G (ml/min/1.73m2)	-0.087	0.445	-0.124	0.278
eGFRcys (ml/min/1.73m2)	0.264	0.018*	0.310	0.005**
eGFRScr-cys (ml/min/1.73m2)	0.250	0.026*	0.286	0.010*
eGFRCKD-EPI (ml/min/1.73m2)	-0.028	0.803	-0.005	0.962

Variables are expressed as mean ± standard deviation;

^*p < 0.05

^**p < 0.01; Abbreviation: TRF, telomere restriction fragment.

Discussion

The average length of human telomere DNA is 5 to 15 kb; its main role is to stabilize human genomes through gradual shortening as cells divide. When telomere length is reduced excessively, cell proliferation ceases and senescence or apoptosis follows. Telomere length is thus called the «splitting clock» of cells (13). Telomere length differs individually, and is also influenced by the detection methods used. Compared to other methods, Southern blotting has been considered the most classic approach, due to its technical maturity, accuracy, and reliability of results.

In this study, we used a digoxin-labeled probe telomere length assay kit (Telo TAGGG Telomere Length Assay) among the 471 healthy volunteers, and found that leukocyte TRF lengths gradually decreased with aging (r = -0.393, p < 0.001). This relationship existed for both baseline and follow-up telomere length. These findings were also reported by most previously published studies, thus suggesting a correlation coefficient of about -0.3 between telomere length and age (14). This is consistent with our previous finding in 139 cases of healthy volunteers (r = -0.314, p <0.001) (15).

Existing studies yield different results regarding the relationship between telomere length and gender (16). We found that female volunteers had significantly longer telomere lengths than males (p = 0.011), which is consistent with prior observations showing that women generally have longer life expectancy and better survival than men. This gender difference might be related to genetic heterogamety, body size, oxidative stress, and telomere maintenance (17).

After 3 years of follow-up, we found a significant decrease in telomere lengths, thus further supporting the notion that telomere length could be a marker of aging (18). However, the degree of telomere shortening can vary individually, and not all participants show a decrease in leukocyte telomere length after follow-up (19, 20). Indeed, we discovered that after 3 years about 27% of the participants had unchanged or increased TRF; this might be explained by their differences in dietary habits, lifestyle factors, or the inadequate duration of follow-up in this study.

We discovered that changes in telomere lengths were more prominent in volunteers with longer baseline telomere lengths, although telomere lengths at baseline and after follow-up all exhibited significant associations with changes in telomere length. However, this relationship disappeared after adjustment for age, except for telomere length at baseline—indicating that baseline telomere length plays a more important role in determining telomere length changes. These findings were also reported by Ehrlenbach et al (20) and Aviv et al (21), and might result from preferential action of telomerase on short telomeres, or from the lower frequency of division for aging cells, thus reducing the risk of telomere shortening.

Existing studies did not yield consistent findings between telomere lengths and renal function, although most studies suggest an association exists between the two factors (7–10). Plausible reasons to explain this inconsistency include the influence of genetic and environmental factors on telomere lengths, such as the ethnicity of the tested population, economic status and habits of the participants (22), and the differences in pathogenesis of renal diseases, since disease severity may also affect telomere lengths (7, 23). In addition, telomere length may differ between tissues (24); for example, telomere lengths in renal cortical tissues were significantly longer than those in blood cells, while telomere lengths in renal carcinoma cells were significantly shorter than those in blood cells and the renal cortex (25). Clinically speaking, renal tissue is frequently inaccessible for measuring telomere lengths, except in patients undergoing surgery or biopsy, and studies often use peripheral leukocyte telomere lengths as surrogates for telomere lengths in renal tissues (8).

Finally, the type of assays for telomere lengths also poses influence on the results of evaluation. Differences in quantitative or semi-quantitative available assays including Southern blotting (26), quantitative polymerase chain reaction (Q-PCR) (27), and fluorescent in situ hybridization (FISH) (28) may affect the detection results; the differences in analytic software for output results and in operators may also play a role in affecting detection results. For example, the software for calculating the TRF length from results of Southern blotting, include quantitative Bio-Rad Imaging Densitometer Model GS-670 (7), Image Gauge software (29), Aida software (30), and Quantity One® 1-D analysis software (11). However, available studies are mostly cross-sectional with small sample sizes, and the data of these studies might not completely conform to normal distribution. Further improvement and standardization of the methods for detecting telomere length and for enhancing the reliability of test results is therefore needed.

Our results also suggested that telomere lengths were negatively associated with serum CysC levels but positively associated with eGFR results using the CKD-EPI equation, CG formula, Scr/cysC-based formula, and CysC-based formula (p < 0.01). This indicates that telomere lengths in peripheral leukocytes are closely associated with renal function. We further found that among the 80 participants being assessed twice changes in telomere lengths were significantly associated with serum CysC after follow-up (p = 0.038), but after adjusting for age, this relationship no longer existed (p = 0.119).

This finding is also supported by other researchers who discovered that short telomere lengths are associated with low glomerular filtration rate (r = 0.147, p < 0.0001), but after adjusting for age and sex, this relationship disappears (r = 0.024, p = 0.10) (31). Bansal et al. derived similar results in a longitudinal study of 954 cases, which showed that renal function decline in patients with coronary heart disease was significantly associated with short baseline telomere length and rapid shortening of telomere length, but after adjustment for age, this correlation did not exist. However, other studies clearly showed that shorter leukocyte telomere length in patients with chronic heart failure is closely associated with renal function decline (32). Moreover, a study focusing on 866 patients of chronic heart failure patients identified that telomere lengths were weakly correlated with glomerular filtration rate (r = 0.123, p < 0.001) after adjusting for age and sex (10). Thus, telomere length changes might not correlate with changes in renal function. Although telomere shortening and renal function decline often coexist in individuals, the main driving force for change is increasing age, and after accounting for the influence of aging, individual-specific traits may also be underrecognized.

This study has some strengths. Namely, we used a classic, widely used approach (Southern blotting) to measure the telomere length of peripheral blood leukocytes from healthy volunteers. The results we derived are reliable, since we used six different formulae to assess renal function, and further utilized longitudinal follow-up to evaluate the relationship between changes in telomere lengths and renal function. However, the present study also has some limitations. Namely, the number of participants with longitudinal follow-up is modest, and genomic DNA was extracted from peripheral blood leukocyte instead of renal tissues.

In summary, we found that there was no relationship between decreased renal function and telomere length shortening during the 3-year follow-up period. Given the limited sample size of this study, larger studies with a longer follow-up period are needed to verify our conclusions.

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical standard

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Electronic supplementary material

Supplementary material is available for this article at https://doi.org/10.1007/s12603-017-0905-4 and is accessible for authorized users.

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