Sage Journals: Discover world-class research

Abstract

Aim:

To our knowledge, the relationship between simple renal cysts, hypertension and three significant genes of the renin-angiotensin system (AGT, AT1R and ACE1) has not been studied. The present study was designed to search for possible relationships between these significant polymorphic components, hypertension and simple renal cysts in Shiraz province (Iran).

Methods:

A total of 160 participants were recruited from the Motahari Clinic at Shiraz University of Medical Sciences. The subjects were divided into four main groups. Detection of the ACE1 genotype was performed with a nested-polymerase chain reaction (PCR) protocol. Two separate restriction fragment length polymorphism-PCR assays were used to identify AGT and AT1R genotypes.

Results:

The allele frequency of AGT M235T differed significantly between group 1 (patients with simple renal cysts and hypertension) and normal individuals (p <0.05). There were no significant differences in frequency for the other genes (ACE1 and AT1R).

Conclusions:

Our findings show a relationship between the AGT-TT genotype and hypertension in patients with both hypertension and simple renal cysts. This finding suggests an additive role for the AGT gene of the renin-angiotensin system in the process of hypertension and simple renal cysts formation. Future studies are needed to elucidate the mechanisms through which this association is mediated.

Keywords

Hypertension simple renal cyst renin-angiotensin system polymorphism angiotensinogen

Introduction

Renal cysts are the most common space-occupying lesions of the kidney.¹ A classification of renal cysts on the basis of their appearance on computed tomography was introduced by Bosniak in 1986 and refined in 2003.² Whether labeled simple or complex, regardless of their radiologic characteristics, the terms used are all descriptive. Simple cysts are distinct lesions within the kidney that are typically cortical, extending outside the parenchyma. They are commonly considered as a harmless anomaly, while cases of complicated renal cysts have been reported. The majority of complications are spontaneous rupture, hemorrhage and infections. The reported overall prevalence of simple cysts is variable. Depending on the population and method of study, the prevalence ranges from 2.38% in the second to 35.29% in the seventh decade of life.³ Some potential risk factors for the appearance of simple cysts are age, sex, renal stone, serum creatinine, smoking and hypertension. Sporadically, they become symptomatic and may present with flank pain, abdominal discomfort, a palpable mass or hematuria as a result of complications, or subsequent to an enlarging cyst.^3–7

Previous studies have noted the association between simple renal cysts and hypertension, but the relationship between these cysts and hypertension has not been studied yet.^8–10 Some case reports have described patients with simple renal cysts and hypertension in whom renin released from the affected kidney was increased and blood pressure normalized after surgical removal of the cysts.¹¹

The renin-angiotensin system as a circulating or hormonal system regulates blood pressure, electrolyte and fluid homeostasis and is mainly related to the short- and long-term regulation of arterial blood pressure.^{12, 13} Studies of the renin-angiotensin system in experimental animal models have detected remarkable genetic polymorphisms chiefly involving the angiotensinogen gene (AGT), AT1 receptor gene (AT1R) and angiotensin-converting enzyme gene (ACE1). Studies that investigated hypertension in relation with the renin-angiotensin system have indicated that there are naturally occurring genetic variations within the renin-angiotensin system in animals as well as humans.¹³

In diverse genetic or environmental backgrounds, a specific gene variant might be a sign of different pathophysiological implications.^{14, 15} As a result, the present case-control study was designed as (to our knowledge) the first attempt to identify possible associations between polymorphisms of three significant genes of the renin-angiotensin system (AGT, ACE1 and AT1R) and hypertension in patients with simple renal cysts in a southern population of Iran (Shiraz).

Methods

Study subjects

A total of 160 participants were recruited from Motahari Clinic, affiliated with Shiraz University of Medical Sciences. The subjects were divided into four groups: patients with simple renal cysts and hypertension (group 1, n = 40), simple renal cysts without hypertension (group 2, n = 40) healthy individuals without any renal complications (group 3, control, n = 40) and hypertension without simple renal cysts (group 4, n = 40).

Informed consent was obtained from all participants. The protocol for this project was approved by the ethics committee of Shiraz University of Medical Sciences, Iran. In this study hypertension was defined as 140 mmHg systolic blood pressure and 90 mmHg diastolic blood pressures or the use of antihypertensive therapy. Blood pressure was measured on the right arm with an automated blood pressure monitor while the subject was seated and resting for at least 10 minutes.

DNA preparation

Blood samples (5.0 ml) were drawn from a peripheral vein into an ethylenediaminetetraacetic acid (EDTA) tube by a qualified lab technician. Genomic DNA extraction was performed with the standard salting-out protocol. The quality and quantity of extracted DNA were evaluated with a NanoDrop spectrophotometer at 260/280 nm.

Determination of ACE1, AGT and AT1R genotypes

The ACE1 genotypes (insertions and deletions) were determined with a nested polymerase chain reaction (PCR) protocol. In this method the polymorphism status was first assessed, and then to increase accuracy, another reaction was performed. The second independent reaction was run under the same PCR conditions except for annealing temperature and primer sequence (Table 1).

Table 1.

PCR cycling conditions of ACE1 are represented as temperature and time [minute (m), seconds (s) of denaturation, annealing and extension, × number of cycles].

Gene polymorphism size (bp)	Forward primer (FP) size (bp)	PCR cycling conditions	PCR product (bp)
ACE1	FP:	94°C, 1 m (1 cycle), 94°C, 30 s	490, 190
	5CTGGAGACCACCCATCCTTTCT3	58°C, 30 s
	RP:	72°C, 1 m
	5GATGTGGCCATCACATTCGTCAGAT3	72°C, 8 m
		(1 cycle) × 60
		Nested PCR
	FP:	94°C,	190, 335
	5TCGGACCACAGCGCCCGCCACTAC3	1 m
	RP:	(1 cycle)
	5CGCCAGCCCTCCCATGCCCATAA3	94°C,
		30 s
		58°C,
		30 s
		72°C,
		1 m
		72°C, 8 m (1 cycle) × 60

PCR: polymerase chain reaction; ACE1: angiotensin-converting enzyme gene; RP: reverse primer.

PCR amplification of deletions (D) and insertions (I) of ACE1 were evaluated in a 25 μl reaction mixture containing 200 ng of the template DNA, 7.5 pmol/l of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2, 2.5 µL10× buffer and 1 U Taq DNA polymerase.

The PCR amplification included 30 cycles of denaturation at 94°C (one minute), annealing at 58°C (one minute) and extension at 72°C (two minutes). The amplification reaction yielded a 335-bp product only in the presence of the I allele (ID heterozygous) whereas no product was found in DD-homozygous individuals.^{12, 13}

The PCR conditions for determining the genotype of AGT were amplification of 200–300 ng of genomic DNA in a 25 μl reaction mixture consisting of 7.5 pmol of each primer, 0.5 mM of each dNTP, 2 mM MgCl2, 2.5 µL10× buffer and 1.25 U Taq DNA polymerase.

The PCR amplification for AGT included 30 cycles at 94°C (30 seconds), 60°C (45 seconds) and 68°C (one minute). The amplified products were analyzed by electrophoresis in 1.5% agarose gel. To identify the AGT M235T genotype, 104-bp PCR products were digested with an MspI restriction enzyme. The 73-bp and 31-bp digestion products were then analyzed by electrophoresis in 3% agarose gel. AGT M235T heterozygotes showed three bands, whereas the T-type variant was not digested and showed only the 104-bp band.

The AT1R A1166C genotype was identified by restriction fragment length polymorphism-PCR amplification that was performed in a 25 μl reaction mixture containing 7.5 pmol forward primer, 7.5 pmol reverse primer, 0.2 mM of each dNTP, 2 mM MgCl2, 2.5 µL10× buffer, 1 U Taq DNA polymerase and 200 ng of the template DNA. The initial denaturation was set up for five minutes at 94°C followed by 35 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 55°C, and extension for 60 seconds at 72°C. The size of the PCR products was 540 bp for the A allele and 110 bp for the C allele. A negative control containing no genomic DNA and a positive control of known genotype were always used in the set of reactions. The PCR cycling conditions for AT1R and AGT are shown in Table 2.

Table 2.

PCR cycling conditions of AGT and AT1R are represented as temperature and time [minute (m), seconds (s) of denaturation, annealing and extension, × number of cycles].

Gene	Primers	PCR cycling	Restriction enzyme	Product size	Restriction size (bp)
AGT	FP:	94°, 5 m	Msp I	104 bp	31, 73
	5–TGACAGGATGGAAGACTGGCTGCTCCCTGC–3	(1 cycle)
	RP:	94°C 30 s
	5–AGCAGAGAGGTTTGCCTTACCTTG–3	60°C –45 s
		68°C –1 m
		72°C –
		10 m
		(1 cycle) × 30
AT1R	FP:	94 °C 5 m	Dde I	540 bp	430, 110
	5–CTG GAGACCACTCCCATCCTTTCT–3	(1 cycle)
	RP:	94°C 30 s
	5–GATGTGGCCATCACATTCAGAT–3	55°C 30 s
		72°C 60 s
		72°C 10 m
		(1 cycle) × 30

PCR: polymerase chain reaction; AGT: angiotensinogen gene; AT1R: AT1 receptor gene; FP: forward primer; RP: reverse primer.

Statistical analyses

All statistical analyses were performed with SPSS software version 14.0 for Microsoft Windows. Group findings were compared with the chi squared test. P < 0.05 was considered statistically significant.

Results

AGT genotyping

We studied 160 individuals with a mean age of 55 ± 10 years. The participants were divided into four groups. Figure 1 shows the prevalence of the different genetic polymorphisms of AGT in the four groups. None of the genotype distributions followed the Hardy-Weinberg equilibrium. The overall frequency of the M and T alleles was 52.5% and 47.5%, respectively. Table 3 compares allele frequencies for AGT, which was statistically significant between groups 1 and 2 (p value = 0.035)

Figure 1.

Genotype and allele distribution of AGT gene polymorphisms in four groups.

Table 3.

Comparison of groups 1 and 3.

Genotypes and alleles	Group 1 n (%)	Group 3 n (%)
M 235 T Genotypes
MM	11 (27.5%)	16 (40.0%)
MT	14 (35%)	19 (47.5%)
TT	15 (37.5%)	5 (12.5%)
Total	40 (100.0)	40 (100.0)
P value (95% CI)	0.035 *
OR	2.35

OR: odds ratio; CI: confidence interval.

Significant difference*

Figure 2 shows the PCR products of the AGT gene detected with gel electrophoresis.

Figure 2.

Gel electrophoresis of AGT gene (73 and 31 bp MM genotype 104, 73 and 31 bp MT genotype and 104 bp TT genotype).

AT1R genotyping

Figure 3 shows the prevalence of the different genetic polymorphisms of AT1R in the four groups. None of the genotype distributions followed the Hardy-Weinberg equilibrium. The overall frequency of the A and C alleles were 76.25% and 23.75%, respectively.

Figure 3.

Genotype and allele distribution of AT1R gene polymorphisms in four groups. There were no statistically significant differences.

ACE1 genotyping

Figure 4 shows the prevalence of the different genetic polymorphisms of ACE1 in the four groups. None of the genotype distributions followed the Hardy-Weinberg equilibrium. The overall frequency of the A and C alleles was 36.6% and 63.3%, respectively. Table 4 compares the different allele frequencies for AT1R and ACE1; none of the differences was statistically significant.

Figure 4.

Genotype and allele distribution of ACE1 gene polymorphisms in four groups. No statistically significant differences in genotype versus genotype distribution.

Table 4.

Statistical analysis of AT1R and ACE1.

Independent variant		OR	P value
AT1R	AA	1	0.421
	AC	0.838	0.760
	CC	1.348	0.627
ACE1	II	1	0.856
	ID	1.215	0.697
	DD	1.240	0.582

AT1R: AT1 receptor gene; ACE1: angiotensin-converting enzyme gene; OR: odds ratio.

Discussion

Recent advances in molecular biology have provided the genes that are responsible for a number of renal cystic diseases in adults. Some of these genes have been characterized: PKD1 and PKD2 play an important role in renal epithelial development; TSC2 and TSC1, the genes implicated in the pathogenesis of tuberous sclerosis.¹⁸

In addition, genetic variants of the renin-angiotensin-aldosterone system such as variants in the angiotensinogen gene AGT have been intensively studied in different populations with conflicting results in relation to high blood pressure.^19-21 Several studies have demonstrated a significant relationship between M235T genotype and hypertension in different human populations. For example, Jeunemaitre et al. found that the T allele was significantly more frequent in patients with hypertension than in controls.^20,21 To the best of our knowledge, this is the first study in patients with hypertension and simple renal cysts to show a significant association between the AGT-TT genotype and these clinical entities. This finding suggests an additive role for the AGT gene in the renin-angiotensin-aldosterone system and the process of hypertension and simple renal cysts formation. Moreover, no significant associations were found in the present study between the risk of hypertension (without simple cysts) and the variants of the renin-angiotensin system genes AGT, AT1R and ACE1.

In a meta-analysis of 32 case-control studies,²² hypertension or a history of hypertension was significantly associated with the T allele.²³ However, when different races were studied separately, the association with hypertension was significant only in Caucasians but not in Asians or blacks.^24–27 Because our study population is located in Asia, our results confirm these findings. This variation in the associations highlights the importance of studying these polymorphisms in diverse populations.

Conclusion

Our results, in light of earlier research, show that AGT variants in patients who have both simple renal cysts and hypertension have an important role in the pathophysiology of these clinical entities. Furthermore, the TT variant of AGT in the population of Shiraz (Iran) is an obvious genetic marker for this status, which can be used in personalized and molecular population-based medicine for both treatment and prevention. Although our results comprise the first evidence of an additive role for the TT genotype of the AGT gene in the process of hypertension and simple renal cyst formation in our study population, further studies are needed in different populations.

Footnotes

Acknowledgements

We are grateful to all faculty members and staff of the Medical Genetics Department at Shiraz University of Medical Sciences for their sincere support. We also thank K. Shashok (AuthorAID in the Eastern Mediterranean) for improving the use of English in the manuscript.

Conflict of interest

None declared.

Funding

This work was supported by a research grant from Shiraz University of Medical Sciences. All funding resources in this project were provided by the University of Medical Sciences, Shiraz, Iran.

References

Eknoyan

. A clinical view of simple and complex renal cysts. J Am Soc Nephrol 2009; 20: 1874–1876.

Israel

Bosniak

. An update of the Bosniak renal cyst classification system. Urology 2005; 66: 484—488.

Lüscher

Wanner

Siegenthaler

. Simple renal cyst and hypertension: Cause or coincidence? Clin Nephrol 1986; 26: 91–95.

Babka

Cohen

Sode

. Solitary intrarenal cyst causing hypertension. N Eng J Med 1974; 291: 343–344.

Rockson

Stone

Gunnells

Jr J.

Solitary renal cyst with segmental ischemia and hypertension. J Urol 1974; 112: 550–552.

Pedersen

Emamian

Nielsen

. Simple renal cyst: Relations to age and arterial blood pressure. Br J Radiol 1993; 66: 581–584.

Afsar

Sen

. Simple renal cysts and circadian blood pressure: Are they related to each other in patients with hypertension? Int Urol Nephrol 2011; 43: 157–165.

Pedersen

Emamian

Nielsen

. Significant association between simple renal cysts and arterial blood pressure. Br J Radiol 1997; 79: 688–691.

Chin

Lee

. The clinical significances of simple renal cyst: Is it related to hypertension or renal dysfunction? Kidney Int 2006; 70: 1468–1473.

10.

Higaki

Baba

Katsuya

. Deletion allele of angiotensin-converting enzyme gene increases risk of essential hypertension in Japanese men: The Suita Study. Circulation 2000; 101: 2060–2065.

11.

Tsai

Lai

Lin

. Renin-angiotensin system gene polymorphisms and atrial fibrillation. Circulation 2004; 109: 1640–1646.

12.

Davis

Roberts

. Molecular genetics of the renin-angiotensin system: Implications for angiotensin II receptor blockade. Pharmacol Ther 1997; 75: 43–50.

13.

Corvol

Soubrier

Jeunemaitre

. Molecular genetics of the renin-angiotensin-aldosterone system in human hypertension. Pathol Biol (Paris) 1997; 45: 229–239.

14.

Lovati

Richard

Frey

. Genetic polymorphisms of the renin-angiotensin-aldosterone system in end-stage renal disease. Kidney Int 2001; 60: 46–54.

15.

Reis

Arinsoy

Derici

. Angiotensinogen and plasminogen activator inhibitor-1 gene polymorphism in relation to chronic allograft dysfunction. Clin Transplant 2005; 19: 10–14.

16.

Zhu

Chang

Yan

. Associations between hypertension and genes in the renin-angiotensin system. Hypertension 2003; 41: 1027–1034.

17.

Jeunemaitre

Inoue

Williams

. Haplotypes of angiotensinogen in essential hypertension. Am J Hum Genet 1997; 60: 1448–1460.

18.

Katabathina

Kota

Dasyam

. Adult renal cystic disease: A genetic, biological, and developmental primer. Radiographics 2010; 30: 1509–1523.

19.

Jeunemaitre

Soubrier

Kotelevtsev

. Molecular basis of human hypertension: Role of angiotensinogen. Cell 1992; 71: 169–180.

20.

Staessen

Kuznetsova

Wang

. M235T angiotensinogen gene polymorphism and cardiovascular renal risk. J Hypertens 1999; 17: 9–17.

21.

Nishiuma

Kario

Kayaba

. Effect of the angiotensinogen gene Met235–> Thr variant on blood pressure and other cardiovascular risk factors in two Japanese populations. J Hypertens 1995; 13: 717–722.

22.

Iwai

Ohmichi

Nakamura

. DD genotype of the angiotensin-converting enzyme gene is a risk factor for left ventricular hypertrophy. Circulation 1994; 90: 2622–2628.

23.

Chiang

Hsu

Tseng

. Molecular variant M234T of the angiotensinogen gene is associated with essential hypertension in Taiwanese. J Hypertens 1997; 15: 607–611.

24.

Sato

Katsuya

Rakugi

. Association of variants in critical core promoter element of angiotensinogen gene with increased risk of essential hypertension in Japanese. Hypertension 1997; 30: 321–325.

25.

Rotimi

Morrison

Cooper

. Angiotensinogen gene in human hypertension. Lack of an association of the 235T allele among African Americans. Hypertension 1994; 24: 591–594.

26.

Rotimi

Cooper

Ogunbiyi

. Hypertension, serum angiotensinogen, and molecular variants of the angiotensinogen gene among Nigerians. Circulation 1997; 95: 2348–2350.

27.

Borecki

Province

Ludwig

. Associations of candidate loci angiotensinogen and angiotensin-converting enzyme with severe hypertension: The NHLBI Family Heart Study. Ann Epidemiol 1997; 7: 13–21.

Simple renal cysts and hypertension are associated with angiotensinogen (AGT) gene variant in Shiraz population (Iran)

Abstract

Aim:

Methods:

Results:

Conclusions:

Keywords

Introduction

Methods

Study subjects

DNA preparation

Determination of ACE1, AGT and AT1R genotypes

Statistical analyses

Results

AGT genotyping

AT1R genotyping

ACE1 genotyping

Discussion

Conclusion

Footnotes

Acknowledgements

Conflict of interest

Funding

References