Genomic instability in patients with autosomal-dominant polycystic kidney disease

Abstract

Objective

Autosomal-dominant polycystic kidney disease (ADPKD) is a systemic disorder affecting multiple organs that results in renal and extrarenal cysts. Patients with ADPKD may have genomic instability, making them more vulnerable to developing cancer. This study aimed to investigate latent genomic instability in patients with ADPKD, using single-cell gel electrophoresis (comet assay).

Methods

The susceptibility of peripheral blood lymphocytes to DNA damage induced by X-ray treatment (0.5 Gy) was tested in 20 patients with ADPKD using single-cell gel electrophoresis. The percentage of DNA in the comet tail (TDNA%) before and after irradiation was compared between patients with ADPKD and 20 sex- and age-matched healthy control subjects.

Results

Renal and extrarenal cysts were observed in patients with ADPKD. A significantly higher mean TDNA% was determined in patients with ADPKD compared with control subjects (8.85% versus 7.50%). After in vitro irradiation, DNA damage was significantly increased in all participants, but the increase was significantly greater in patients with ADPKD compared with control subjects.

Conclusion

These data suggest that patients with ADPKD have genomic instability, which may trigger renal and extrarenal cyst formation.

Introduction

Autosomal-dominant polycystic kidney disease (ADPKD) affects over 1.5 million people in China. ¹ The pathophysiology of ADPKD is characterized by the development and progressive enlargement of renal cysts, which typically lead to chronic renal failure in middle age. ² ADPKD is also a systemic disease that affects multiple organs, leading to the development of extrarenal cysts and serious complications that include colonic diverticulosis, inguinal hernias, cardiac valvular defects and intracranial arterial aneurysms. ³ Common pathological features of ADPKD include abnormal cell proliferation and apoptosis, disturbance of cell polarity and dedifferentiation, enhanced transepithelial fluid secretion, extracellular matrix remodelling and the development of interstitial fibrosis. ² The pathogenesis of extrarenal cysts has not been fully elucidated and few studies have investigated whether patients with ADPKD are at an increased risk of developing cancer. ⁴ Genomic instability is a term used to describe increases in the acquisition of alterations in the mammalian genome (including chromosomal destabilization, gene amplification and mutations) following cellular exposure to DNA-damaging agents. ⁵ The DNA of patients with genomic instability is often more susceptible to mutagen damage than that of the general population ⁶ and it has been proposed that genomic instability could be a driving force behind multistep carcinogenesis. ⁷

Single-cell gel electrophoresis (SCGE), also known as the comet assay, has been used to study genomic instability, and is able to demonstrate DNA damage at the single-cell level and in nonproliferating cells. ⁸ In the present study, SCGE was used to quantify baseline and γ-radiation-induced DNA damage in peripheral blood lymphocytes from patients with ADPKD. The aim was to detect the presence of latent genomic instability, as reflected by higher levels of DNA damage.

Patients and methods

Study Population

Patients with ADPKD treated at the Department of Nephrology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China between September 2007 and April 2010, were recruited sequentially to this case–control study. Diagnosis of ADPKD was based on widely used ultrasound diagnostic criteria. ⁹ Patients were excluded from study participation if they had current carcinoma, acute or chronic inflammatory disorders, immunological diseases, or severe renal or liver failure. Age- and sex-matched healthy control subjects, who were attending the First Affiliated Hospital of Soochow University for routine physical examinations, were also recruited.

The study protocol was approved by the Ethics Review Committee of the First Affiliated Hospital of Soochow University and all participants provided written or verbal informed consent.

Study Procedures

Patients and control subjects were interviewed at the time of study recruitment, using a questionnaire developed at the Department of Radiation Oncology, First Affiliated Hospital of Soochow University (S.B.Q. and J.Y.Z.), to collect data on demographics, smoking history, lifestyle, employment conditions and possible exposure to hazardous substances, which could potentially lead to increased genotoxic risk. In addition, serum creatinine concentrations were determined in each study participant, using standard methods, with an automated biochemical analyser (Hitachi Medical, Tokyo, Japan).

Whole blood samples (4 ml) were collected from each study participant by venipuncture at the time of study recruitment and were divided into two equal aliquots for analysis. To minimize extraneous DNA damage from ambient ultraviolet radiation, all steps were performed under reduced illumination. The first aliquot was collected in 1% heparinized tubes and was transported upright, at room temperature, within 3 h of collection to the Comet Assay Laboratory at the Department of Radiation Oncology, First Affiliated Hospital of Soochow University, for isolation of lymphocytes to assess DNA damage via SCGE. Blood samples were mixed with 2 ml RMPI 1640 medium (Gibco® Cell Culture, Carlsbad, CA, USA) and added to 2 ml Ficoll® separating agent (Sigma-Aldrich, St Louis, MO, USA) before centrifugation at 400  g for 20 min at room temperature. Lymphocytes were harvested, washed twice in phosphate-buffered saline (0.01 M, pH 7.4) and resuspended in 75 μl 0.8% low-melting-point agarose (LMPA; Invitrogen, Carlsbad, CA, USA) at 37°C. The second aliquot was treated identically to the first except it underwent irradiation by 6 MV X-rays at a dose of 0.5 Gy (Clinac® 23EX linear accelerator; Varian Medical Systems, Palo Alto, CA, USA) for 2.5 min as soon as possible after collection, before transportation to the Comet Assay Laboratory for DNA damage analysis.

SCGE Assay

A slightly modified version of the SCGE method described by Singh et al. ¹⁰ was used. The lymphocyte/LMPA suspension was adjusted to a final concentration of 10⁴ cells/ml before being layered onto frosted slides, previously covered with a basal layer of 110 µl of 1.0% normal-melting-point agarose (Sangon Biotechnology, Shanghai, China). The agarose was left to harden for 10 min at 4°C on ice before addition of a third layer of 0.8% LMPA (75 µl) at 37°C, which was allowed to solidify at 4°C on ice for 5 min. Slides were then immersed in cold, freshly-made lysis buffer (2.5 M sodium chloride, 100 mM ethylenediaminetetra-acetic acid [EDTA], 10 mM Tris base, 0.1 M sodium sarcosine, 1% Triton X™-100 and 10% dimethyl sulphoxide, adjusted to pH 10.0 using 4.0 M sodium hydroxide) for 1.5 h at 4°C. After lysis, slides were gently transferred into a horizontal electrophoresis box filled with alkaline buffer (1.0 mM EDTA and 300 mM sodium hydroxide, pH 12.0) for 30 min at 4°C, to allow the DNA to unwind before electrophoresis at 25 V, 300 mA for 25 min (Horizontal Gel Electrophoresis Apparatus, model DYY-6B; Beijing Liuyi Instrument Factory, Beijing, China). Following electrophoresis, slides underwent three 5-min rinses in neutralizing buffer (0.4 M Tris base, pH 7.5) at room temperature, after which they were drained and stained with 50 µl ethidium bromide (5 mg/l) for 15 min. Slides were rinsed once in chilled distilled water to remove excess ethidium bromide, dehydrated in cold 100% ethanol for 15 min and air dried prior to analysis, which was performed immediately after staining. The extent of DNA migration was analysed using a fluorescence microscope (Olympus BX51; Nikon, Melville, NY, USA) equipped with an excitation filter of 560 nm and a barrier filter of 590 nm. SCGE results in structures that visually resemble a comet (where the nucleus of the cell represents the head of the comet and the damaged DNA, which has been liberated from the nucleus by electrophoresis, represents the tail). The percentage of tail DNA (TDNA%) is the fraction of DNA that has migrated from the head. A computer-based image analysis system (Comet Assay Software Project; available at http://www.casp.of.pl) was used to estimate TDNA%. The more severe the DNA damage, the larger the value of TDNA%. ¹¹ Data were based on 50 randomly selected cells per sample: 25 cells from each of two replicate slides. Analysis was performed by the same slide reader (L.L.W.).

Statistical Analyses

All statistical analyses were performed using SAS® software, version 8.1 (SAS Institute, Cary, NC, USA). Results were expressed as mean ± SD or mean ± SE and all statistical tests were two-sided. Statistical comparison of the results before and after irradiation in patients with ADPKD and in healthy controls was performed using the paired design t-test. Comparison between patients with ADPKD and healthy control subjects was performed using one-way analysis of variance and Student–Newman–Keuls test. A P-value <0.05 was considered statistically significant.

Results

A total of 20 patients with ADPKD and 20 healthy control subjects were included in the study. No significant differences were observed between patients with ADPKD and healthy control subjects, in terms of age and other demographic characteristics (Table 1). Serum creatinine concentrations were higher in patients with ADPKD compared with control subjects and renal, hepatic or pancreatic cyst development was observed in patients with ADPKD but not in controls.

Table 1.

Demographic and baseline characteristics of patients with autosomal-dominant polycystic kidney disease (ADPKD) and healthy control subjects.

Characteristic	Control subjects n = 20	Patients with ADPKD n = 20
Age, years	43.7 ± 12.9	44.0 ± 12.7
Sex, male/female	11/9	11/9
Serum creatinine, mg/dl	1.0 ± 0.2	1.9 ± 1.4
Cysts
Renal	0 (0)	20 (100)
Renal and hepatic	0 (0)	7 (35)
Renal, hepatic and pancreatic	0 (0)	2 (10)
Smoking status, yes a	8 (40)	8 (40)
Radiation exposure, yes a	0 (0)	0 (0)
Chemical agent exposure, yes a	0 (0)	0 (0)

Data presented as mean ± SD or n (%) of patients.

a

History of smoking, radiation or chemical exposure during the previous 3 years.

At baseline, significantly higher levels of DNA damage were observed in lymphocytes from patients with ADPKD compared with those from healthy control subjects, as shown by greater TDNA% values (P < 0.05, Table 2). Irradiation (0.5 Gy) of lymphocytes was associated with a significant increase in TDNA% from baseline in both study groups (P < 0.01) but this rise was statistically significantly greater in patients with ADPKD compared with controls (P < 0.05). Representative fluorescence microscope images of typical ‘comets’ from peripheral blood lymphocytes, showing baseline and γ-radiation-induced DNA damage, are presented in Figure 1.

Table 2.

DNA damage in peripheral blood lymphocytes from patients with autosomal-dominant polycystic kidney disease (ADPKD) and healthy control subjects at baseline and after irradiation, as measured by single-cell gel electrophoresis (comet assay).

Group	TDNA%
	Baseline	After γ-radiation	Change from baseline
Patients with ADPKD n = 20	8.85 ± 0.14 b	14.84 ± 0.77a,b	5.99 ± 0.77 a
Healthy control subjects n = 20	7.50 ± 0.37	12.46 ± 0.26 a	4.96 ± 0.44

Data presented as mean ± SE.

^aP < 0.01 compared with baseline using the paired design t-test.

^bP < 0.05 compared with control subjects using one-way analysis of variance and Student–Newman–Keuls test.

TDNA%, percentage of tail DNA (fraction of damaged DNA that has been liberated from the nucleus).

Figure 1.

Single-cell gel electrophoresis and comet cell image analysis software were used to analyse DNA damage in peripheral blood lymphocytes from patients with autosomal-dominant polycystic kidney disease (ADPKD) and healthy control subjects, before and after X-ray treatment of lymphocytes. Representative fluorescence microscopy images of typical ‘comet cells’ from lymphocytes showing: (A) cells at baseline with low levels of DNA damage characterized by a small comet tail in healthy control subjects; (B) cells after irradiation (0.5 Gy) with higher levels of damaged DNA in the tail, in patients with ADPKD.

Discussion

The present study used SCGE to assess baseline and γ-radiation-induced DNA damage in peripheral blood lymphocytes, to determine whether there is genomic instability in patients with ADPKD. Genomic instability is a major driving force for tumourigenesis and is believed to be necessary for cells to accumulate the multiple mutations required for the development of carcinoma. ¹² Although the precise mechanisms for development of the renal and extrarenal manifestations of ADPKD remain unknown, both human and animal studies strongly support the concept that individual cyst formation involves a ‘two-hit’ model comprising both germline and somatic mutations of a polycystic kidney disease (PKD) gene. ¹³ The model predicts that the somatic mutation of a PKD gene is the rate-limiting step for individual cyst formation, and the frequency of somatic PKD gene mutations influences the total cyst number and disease severity in ADPKD.^14,15 In the present study, under basal conditions without in vitro irradiation, the extent of DNA damage in the peripheral blood lymphocytes of patients with ADPKD was significantly greater than that in control subjects. Taken with the elevated serum creatinine concentration and evidence of cyst formation in patients with ADPKD, these results suggest that such patients (especially those with extrarenal complications and elevated creatinine concentrations) might have an increased sensitivity to environmental agents. This hypothesis is consistent with data from patients with chronic renal failure that demonstrated an inverse relationship between renal function and genomic instability. ¹⁶

It is known that age, smoking, and physical and chemical assaults are directly associated with genomic stability. ¹⁷ Older people have increased apoptosis and basal DNA damage, and are more sensitive to the effects of oxidative damage and γ-irradiation. ¹⁸ In order to avoid such influences in the present study, the two groups under investigation were matched as closely as possible in terms of their demographic characteristics, and included equal numbers of males and females. The data demonstrate that peripheral blood lymphocytes from patients with ADPKD and healthy control subjects show significantly increased DNA damage in response to X-ray treatment; the increase in DNA damage was, however, significantly greater in those patients with ADPKD. These findings support the theory that patients with ADPKD may be subject to greater genomic instability than individuals without the disease.

Although fluid-filled cysts can arise from any tubular segment, ¹⁹ why and how mutations in PKD genes result in cyst formation remains poorly understood. Numerous models have been proposed to account for the development of cysts.^20–22 Zheleznova et al. ²³ suggested that epidermal growth factor receptor-mediated interactions are key elements in renal tubular cell proliferation, not only in the normal kidney, but also under conditions of cyst formation and enlargement. Activation of the intrinsic kinase domain results in phosphorylation of specific tyrosine residues, which in turn leads to the activation of intracellular signalling pathways. Evidence suggests that during cyst formation there are disruptions in ciliary function and/or planar cell polarity. ²⁴ Patients with ADPKD in the current study appeared to have genomic instability based on the results of the SCGE assay, suggesting that the second process in the ‘two hit’ model discussed above (i.e. somatic mutation) may have occurred and could have resulted from exposure to multiple environmental mutagenic agents and disruption of normal polycystin function. These mutations may then have ultimately predisposed the patients to cyst formation, through loss of mechanical cues in tubular epithelial cells that regulate tissue morphogenesis.

In conclusion, results of the present study support the hypothesis that patients with ADPKD have latent genomic instability and that this may trigger cyst formation in multiple organs, following exposure to a mutagenic environmental agent such as γ-radiation. Furthermore, results from the present study illustrate that the SCGE assay can be used as a tool to analyse the mechanisms underlying extrarenal manifestations in patients with ADPKD.