Association Analysis of Chronic Kidney Disease and Its Risk Factors for Hail Region Population

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.7(2)pp69-79(2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.

Disease prevention is an important strategy for reducing the overall burden of CKD and ESRD, and the identification of markers for CKD risk is essential both for risk prediction and for potential intervention to reduce the chance of future cardiovascular events related to CKD (National Kidney Foundation 2002).
Early identification of CKD is a legitimate enterprise if it provides meaningful opportunities for effective and safe interventions that reduce the risk of death, end-stage renal disease, or complications of renal dysfunction (Richard and Christopher, 2008).Progression of CKD in the presence of definite disease, particularly in the presence of certain diseases such as micro albuminuria, can be modified by interventions with the use of inhibitors of angiotensin II; however, the evidence that such approaches can alter the progression of stage 3 CKD in the absence of other definitive features of kidney damage has not yet been proven (Richard and Christopher, 2008).Regardless of the underlying etiology of the CKD, the family physician can make a significant impact in slowing the progression of chronic kidney disease through strict blood pressure control, tight glycemic control, reduction in the degree of proteinuria, and smoking cessation.All chronic kidney disease patients are at significantly increased risk of cardiovascular events; therefore, additional cardiovascular risk factors such as hyperlipidemia shall also be managed aggressively (Murphree and Thelen, 2010).
Although genetic linkage analyses (Gharavi et al., 2005) and association studies (Hanson et al., 2007;Wetmore et.al, 2005) have implicated several loci and candidate genes in the predisposition to CKD, the genes that confer susceptibility to this condition remain to be identified definitively.In addition, given ethnic differences in lifestyle and environmental factors as well as in genetic background and renal function, it is necessary to examine genetic variants related to CKD in each ethnic group.We previously showed that genetic variants that confer susceptibility to CKD differ between individuals with or without metabolic syndrome (Yoshida et al., 2013), with or without type 2 diabetes mellitus (Yoshida et al., 2012), with or without hypertension (Yoshida et al., 2011), or with different lipid profiles (Yoshida et al., 2010).To further examine whether the association of polymorphisms with CKD is influenced by the absence or presence of hypertension or diabetes mellitus, we performed an association study for 75 polymorphisms of 176 candidate genes and CKD in 299Saudi individuals with or without hypertension or diabetes mellitus.The purpose of the present study was to identify genetic variants that confer susceptibility to CKD in individuals with or without hypertension or diabetes mellitus independently, and thereby to assess the genetic risk of CKD in such individuals separately.

Study population
The study population comprised 299 unrelated individuals (173 men, 126 women) who either visited outpatient clinics or were admitted to one of the participating hospitals (King Khaled Hospital, General Hail Hospital, Medical Centers at Baqaha and Samira in Hail region) between February till September 2015 and due to various symptoms or for an annual health checkup, or were recruited to a population-based prospective cohort study of aging and agerelated diseases in Hail regaion.
Estimated glomerular filtration rate (eGFR) was calculated with the use of the simplified prediction equation derived from a modified version of that described in the Modification of Diet in Renal Disease (MDRD) Study, as proposed by the Japanese Society of Nephrology (Matsuo et.al, 2009) The National Kidney Foundation's Kidney Disease Outcomes Quality Initiative guidelines recommend a diagnosis of CKD if the eGFR is <60 ml min −1 1.73 m −2 (National Kidney Foundation 2002).On the basis of this criterion, 299 subjects (173 men, 126 women) were diagnosed with CKD.The control subjects comprised 172 individuals (97 men, 75 women) recruited from among community-dwelling healthy individuals or patients who visited outpatient clinics regularly for treatment of various common diseases, with an eGFR ≥60 ml min −1 1.73 m −2 .Subjects with CKD and controls thus either had or did not have conventional risk factors for CKD, including hypertension (systolic blood pressure of ≥140 mmHg, diastolic blood pressure of ≥90 mmHg, or taking antihypertensive medication), diabetes mellitus (fasting blood glucose of ≥6.93 mmol/l, hemoglobin A 1c of ≥6.5%, or taking antidiabetes medication), or hypercholesterolemia (serum total cholesterol of ≥5.72 mmol/l or taking lipidlowering medication).On the basis of these criteria, 299 and 176 subjects were diagnosed with or without hypertension, respectively, CKD was identified in 70/299 (24%), of whom 27/70 (38.6%) were males and 43/70 (61.4%) were females, 49/70 (70%, P=0.005) were cases of diabetes, while 45/70 (64.3%, P=0.005) were with hypertension.
The study protocol complied with the Declaration of patients and was approved by the Committees on the Ethics of Human Research of College of Applied Medical Sciences, University of Hail and participating hospitals.Written informed consent was obtained from each participant.

Selection of polymorphisms
A total of 75 polymorphisms (data not shown) were selected by genome-wide association studies of CKD and hypertension (P-value for allele frequency <1.0×10 −7 ) with the use of the Gene Chip Human Mapping 500K Array Set (Affymetrix, Santa Clara, CA, USA) (Yamada et al., 2009).The relationship of these polymorphisms to CKD was not previously examined in our studies (Yoshida et al., 2013;Yoshida et al., 2010;Yoshida et al., 2012;Yoshida et al;2009).

Genotyping of polymorphisms
Venous blood (7 ml) was collected into tubes containing 50 mmol/l ethylenediamine-tetraacetic acid (disodium salt), and genomic DNA was isolated with a kit (Genomix; Talent, Trieste, Italy).Genotypes of the 75 polymorphisms were determined at G&G Science (Hail, Saudi) by a method that combines polymerase chain reaction and sequence-specific oligonucleotide probes with suspension array technology (Luminex, Austin, TX, USA).Primers, probes and other conditions for the genotyping of polymorphisms significantly associated with CKD are shown in Table 1.Detailed genotyping methodology was as described previously (Itoh et al., 2013).
Table 1:Primers, probes and other conditions for genotyping of polymorphisms related (P-value for allele frequency of <0.005) to chronic kidney disease.

Statistical analysis
Quantitative data were compared between subjects with CKD and controls by the unpaired Student's t-test.Categorical data were compared by the χ 2 test.Allele frequencies were estimated by the gene counting method, and the χ 2 test was used to identify departures from Hardy-Weinberg equilibrium.In the initial screen, the allele frequencies of each polymorphism were compared between subjects with CKD and controls by the χ 2 test.Polymorphisms with a P-value for allele frequency of <0.005 were further examined by multivariable logistic regression analysis with adjustment for covariates.Multivariable logistic regression analysis was thus performed with CKD as a dependent variable, and independent variables including age, gender (0, woman; 1, man), body mass index (BMI), smoking status (0, non-smoker; 1, smoker), history of hypertension, diabetes mellitus or hypercholesterolemia (0, no history; 1, positive history), and the genotype of each polymorphism.Subsequently, the P-value, odds ratio and 95% confidence interval were calculated.Each genotype was assessed according to dominant, recessive and additive genetic models.Additive models included the additive 1 (heterozygotes vs. wild-type homozygotes) and additive 2 (variant homozygotes vs. wild-type homozygotes) models, which were analyzed simultaneously using a single statistical model.We also performed a stepwise forward selection procedure to examine the effects of the genotypes as well as of other covariates on CKD.Each genotype was examined according to a dominant or recessive model on the basis of statistical significance in the multivariable logistic regression analysis.The P-levels for inclusion in and exclusion from the model were 0.25 and 0.1, respectively.Given the multiple comparisons of genotypes with CKD, we adopted the criterion of a P-value of <0.005 for statistical significance of association.For other clinical background data, a P-value of <0.05 was considered statistically significant.Statistical significance was examined by two-sided tests performed with JMP version 6.0 and JMP Genomics version 3.2 software (SAS Institute, Cary, NC, USA).

Genetic variants related to CKD in individuals with or without hypertension
The characteristics of the subjects with or without hypertension are shown in Table 2.For individuals with hypertension, age, systolic blood pressure, serum concentrations of triglycerides and low density lipoprotein (LDL)-cholesterol, blood glycosylated hemoglobin content and the prevalence of diabetes mellitus were greater, whereas BMI, the percentage of smokers, diastolic blood pressure and serum concentration of high density lipoprotein (HDL)-cholesterol were lower, in subjects with CKD than in controls.Quantitative data are the means ± SD.CKD, chronic kidney disease; LDL, low density lipoprotein; HDL, high density lipoprotein; eGFR, estimated glomerular filtration rate.
For individuals without hypertension, age, the frequency of male subjects, serum concentration of triglycerides and the prevalence of diabetes mellitus were greater, whereas diastolic blood pressure and serum concentration of HDL-cholesterol were lower, in subjects with CKD than in controls.
Comparison of allele frequencies with the χ 2 test revealed that the C→T polymorphism of F10 (rs5962) was significantly (P=0.0014)associated with CKD in individuals without hypertension, while no polymorphism was significantly associated with CKD in individuals with hypertension (data not shown).Multivariable logistic regression analysis with adjustment for age, gender, BMI, smoking status and the prevalence of diabetes mellitus and hypercholesterolemia revealed that no polymorphism was significantly (P<0.005)associated with CKD in individuals without hypertension (data not shown).A stepwise forward selection procedure was performed to examine the effects of genotypes for the polymorphism associated with CKD by the χ 2 test, as well as the effects of age, gender, BMI, smoking status and the prevalence of diabetes mellitus and hypercholesterolemia on CKD.For individuals without hypertension, age, BMI, gender and the F10 genotype (dominant model), in descending order of statistical significance, were significant (P<0.005) and independent determinants of CKD (data not shown).

Genetic variants related to CKD in individuals with or without diabetes mellitus
The characteristics of the subjects with or without diabetes mellitus are shown in Table 3.For individuals with diabetes mellitus, age, systolic blood pressure and the prevalence of hypertension were greater, whereas the percentage of smokers and serum concentration of HDL-cholesterol were lower in subjects with CKD than in controls.For individuals without diabetes mellitus, age, the frequency of male subjects, systolic blood pressure, serum concentrations of triglycerides and LDL-cholesterol, and the prevalence of hypertension were greater, whereas the percentage of smokers, diastolic blood pressure and serum concentration of HDL-cholesterol were lower in subjects with CKD than in controls.

Genetic variants related to CKD in individuals with or without hypertension and diabetes mellitus
The characteristics of the subjects with hypertension and diabetes mellitus or without these conditions are shown in Comparison of allele frequencies with the χ 2 test revealed that three or one polymorphisms were significantly (P<0.005)associated with CKD in individuals with hypertension and diabetes mellitus or without these conditions, respectively (Table 8).A stepwise forward selection procedure was performed to examine the effects of genotypes for the polymorphism associated with CKD by the χ 2 test, as well as the effects of age, gender, BMI, smoking status and the prevalence of hypercholesterolemia on CKD (Table 10).

DISCUSSION
We examined the possible relationship between 75 polymorphisms of 44 genes with the prevalence of CKD in individuals with or without hypertension or diabetes mellitus.Our association study with three steps of analysis (χ 2 test, multivariable logistic regression analysis and stepwise forward selection procedure) revealed that two polymorphisms were significantly associated with CKD: the A→G (Lys625Arg) polymorphism of CDH4 (rs6142884) in individuals without diabetes mellitus, and the C→T polymorphism of PTPRN2 (rs1638021) in individuals with hypertension and diabetes mellitus.
The cadherin 4, type 1, R-cadherin (CDH4) gene is a member of a family of cell surface glycoproteins that mediate calciumdependent cell-cell adhesion and are considered to play an important role in a wide range of cell-cell interactions (Suzuki et al., 1991) et al., 2011et al., ,Cavodeassi et al., 2015)).
In the present study, we demonstrated that the A→G (Lys625Arg) polymorphism of CDH4 (rs6142884) was significantly associated with CKD in individuals without diabetes mellitus, with the G allele protecting against this condition.The Lys625Arg polymorphism is in the cadherin repeat domain, which exists as repeats in extracellular regions thought to mediate cellcell contact when bound to calcium.The association of the A→G (Lys625Arg) polymorphism with CKD might be attributable to effects on cellular adhesion, though the mechanism responsible for this association remains to be elucidated.
The protein tyrosine phosphatase, receptor type, N polypeptide 2 (PTPRN2) gene encodes a 1015-amino acid polypeptide with a single transmembrane and one putative tyrosine phosphatase catalytic domain (Wasmeier et al., 1996).PTPRN2, which was cloned from a rat insulinoma cDNA library (Wasmeier et al., 1996), is 74% identical to the ICA512/IA-2 autoantigen of type 1 diabetes mellitus in the cytoplasmic domain, but only 29% identical in the luminal domain (Kawasaki et al., 1996).Previous reports suggest that 48 and 61% of sera from patients with new onset type 1 diabetes mellitus are positive for autoantibodies to the full-length and cytoplasmic domain of PTPRN2, respectively (Kawasaki et al., 1996).
Therefore, PTPRN2 has been considered a major autoantigen for type 1 diabetes mellitus, and is thus believed to be involved in the pathogenesis of this condition (Kawasaki et al., 1996).We have now shown that the C→T polymorphism of PTPRN2 (rs1638021) was significantly associated with CKD in individuals with hypertension and diabetes mellitus, with the T allele protecting against this condition, though the underlying mechanism remains unclear.
Studies from Saudi Arabia and other Gulf countries, have only dealt with end stage renal disease (ESRD), as well as, the risk factors associated with CKD, such as, DM, hypertension, and other cardiovascular diseases.A recent review of 44 studies, have described the epidemiology of ESRD in the countries of the Gulf Cooperation Council (GCC; which consist of Saudi Arabia, the United Arab Emirates, Kuwait, Qatar, Bahrain, and Oman), showed that the incidence of ESRD has increased while the prevalence and mortality rate of ESRD in the GCC has not been reported sufficiently (Amal et al., 2012).The modification of diet in renal disease study (Hunsicker et al., 1997) followed chronic kidney disease patients at all stages for a 2-year period and concluded that 85% of patients had a decline in their GFR, with the average rate of decline (4 ml/min) annually regardless of the baseline GFR.
Vascular disease (primarily hypertension) is the second most common cause of CKD (it causes 21% of adult CKD cases) (Duaine et al., 2010).Hypertensive nephrosclerosis is associated with addition signs of hypertensive end-organ damage, because of long periods of poorly controlled hypertension.In the present study, the frequency of hypertension was high approximately 37% and the individuals with hypertension represent 44.3% of cases of CKD.The summarized estimate of hypertension prevalence among ESRD in GCC study was 77.88% (Amal et al., 2012) Our study had several limitations: (i) we used eGFR instead of directly measured GFR to define CKD.(ii) We did not obtain information on the underlying renal disease in each subject with CKD, though such information could be obtained by detailed clinical examination, including renal biopsy; however, these diagnostic procedures are not considered feasible in a study with subjects recruited from the general population.
It is possible that one or more of the polymorphisms associated with CKD in the present study are in linkage disequilibrium with other polymorphisms in the same gene or in other nearby genes that are actually responsible for the development of this condition.(iv) The functional relevance of the identified polymorphisms to gene transcription or to protein structure or function was not determined in the present study.(v) Although we adopted the criterion of P<0.005 for association to compensate for the multiple comparisons of genotypes with CKD, it is not possible to completely exclude potential statistical errors such as false positives.(vi) Although a previous study showed smoking to be a risk factor for CKD (Orth et al., 2012), the frequency of smoking was lower in subjects with CKD than in controls in the present study.Selection bias thus could not be completely excluded in the present study.
In conclusion, our results suggest that genetic variants that confer susceptibility to CKD differ among individuals with or without hypertension or diabetes mellitus.Stratification of subjects based on hypertension or diabetes mellitus may thus be fundamental to achieving the personalized prevention of CKD with the use of genetic information.Given that our present study may be considered hypothesis generating, validation of our findings will require their replication with independent subject panels.

Table 2 :
Characteristics of subjects with chronic kidney disease and controls among individuals with or without hypertension.

Table 3 :
Characteristics of subjects with chronic kidney disease and controls among individuals with or without diabetes mellitus.
Quantitative data are the means ± SD.CKD, chronic kidney disease; LDL, low density lipoprotein; HDL, high density lipoprotein; eGFR, estimated glomerular filtration rate.

Table 4 :
Genotype distributions of SNPs significantly associated with chronic kidney disease among individuals with or without diabetes mellitus as determined by the χ 2 test.

Table 5 :
Multivariable logistic regression analysis of SNPs significantly associated with chronic kidney disease by the χ 2 test among individuals with or without diabetes mellitus.
OR, odds ratio; CI, confidenceinterval.Multivariable logistic regression analysis was performed with adjustment for age, gender, body mass index, smoking status and the prevalence of hypertension and hypercholesterolemia. P-values for allele frequency of <0.005 are shown in bol

Table 6 :
Effects of genotypes and other characteristics on chronic kidney disease among individuals with or without diabetes mellitus determined by a stepwise forward selection procedure.

Table 7 .
For individuals with hypertension and diabetes mellitus, age and systolic blood

Table 7 :
Characteristics of subjects with chronic kidney disease and controls among individuals with or without hypertension and diabetes mellitus.
Quantitative data are the means ± SD.CKD, chronic kidney disease; LDL, low density lipoprotein; HDL, high density lipoprotein; eGFR, estimated glomerular filtration rate.

Table 8 :
Genotype distributions of SNPs significantly associated with chronic kidney disease among individuals with or without hypertension and diabetes mellitus as determined by the χ 2 test.

Table 9 :
Multivariable logistic regression analysis of SNPs significantly associated with chronic kidney disease by theχ 2 test among individuals with or without hypertension and diabetes mellitus.CI, confidence interval.Multivariable logistic regression analysis was performed with adjustment for age, gender, body mass index, smoking status and the prevalence of hypercholesterolemia. P-values for allele frequency of <0.005 are shown in bold.

Table 10 :
Effects of genotypes and other characteristics on chronic kidney disease among individuals with or without hypertension and diabetes mellitus determined by a stepwise forward selection procedure.