|Year : 2012 | Volume
| Issue : 5 | Page : 993-999
|Expression of angiotensin-converting enzyme mRNA gene in the kidneys of patients with glomerulonephrites
Alsayed Ahmed Alnahal1, Usama Ahmed Khalil1, Magada Alsayed Diab1, Ali Fahmy Zanaty2
1 Department of Internal Medicine and Nephrology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
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|Date of Web Publication||13-Sep-2012|
| Abstract|| |
A little is known about the behavior of the renin-angiotensin system (RAS) in glomerulo-nephritis (GN), although it is activated in other models of injury. To study renal angiotensin-converting enzyme (ACE) messenger ribonucleic acid (mRNA) gene expression in patients with GN to determine its role in the disease process and other factors that may influence the course of the disease and the prognosis, e.g. treatment with ACE inhibitor (ACEI) drugs, we studied 20 patients with GN allocated to two groups: ten patients received an ACEI drug and ten patients did not receive ACEI in addition to a control group of ten healthy subjects. Routine and special laboratory investigation, histopathological studies and quantitative polymerase chain reaction analysis for renal ACE mRNA were done for both the study and the control groups. There was a statistically significant increase in ACE mRNA gene expression in the GN groups than in control group, but no statistically significant difference in ACE mRNA gene expression between the patients group that received and the group that did not receive ACEI. A significant correlation was found between the ACE mRNA gene expression and the mean blood pressure, serum creatinine, blood urea nitrogen and 24-h urinary protein. In conclusion, a higher level of ACE mRNA gene expression in patients suffering from GN may suggest a role of the RAS in the process of GN, perhaps contributing to glomerular hypertrophy and matrix overproduction. The use of ACEI drugs possibly slows the rate of progression of renal failure and plays a role in controlling the pathophysiology.
|How to cite this article:|
Alnahal AA, Khalil UA, Diab MA, Zanaty AF. Expression of angiotensin-converting enzyme mRNA gene in the kidneys of patients with glomerulonephrites. Saudi J Kidney Dis Transpl 2012;23:993-9
|How to cite this URL:|
Alnahal AA, Khalil UA, Diab MA, Zanaty AF. Expression of angiotensin-converting enzyme mRNA gene in the kidneys of patients with glomerulonephrites. Saudi J Kidney Dis Transpl [serial online] 2012 [cited 2018 May 24];23:993-9. Available from: http://www.sjkdt.org/text.asp?2012/23/5/993/100881
| Introduction|| |
Glomerulonephritis (GN) is a group of diseases with complex etiology, pathogenesis, morphological features and clinical course. The renin-angiotensin system (RAS) genes are an important group of candidate genes involved in the pathogenesis of chronic renal diseases.  Clinical disease states such as renal artery stenosis, hypertension, diabetes and non-diabetic nephropathies may result from over activity of the RAS. ,
The RAS tissue system is possibly involved in the long-term pathophysiology of hypertension,  and local angiotensin II formation accounts for the autocrine-paracrine effect of the RAS.  All the components of the RAS are found in the vascular tissue, including the coronary vessels;  local production is the major source of angiotensin I and angiotensin II found in the veins. ,, Angiotensin II may contribute to the pathogenesis of glomerulosclerosis via stimulation of cell proliferation and secretion of matrix components.  In chronic glomerular diseases, mesangial cell proliferation and extracellular matrix expansion precede glomerulosclerosis. 
Molecular biology has supplied new tools for research in three main areas. First, primary structure of the two main enzymes renin and angiotensin-converting enzyme (ACE); second, the different combined deoxy ribonucleic acids (cDNAs) that can be used as probes to detect and evaluate the expression of the genes in different types of cells; third, molecular biology as a powerful tool for genetics studies to detect sequence variations of genomic DNA. ,
The most promising immediate clinical application for patient care is the identification of messenger ribonucleic acid (mRNA) expression patterns that help to characterize path physiological phenomenon in diseased organs  and their correlation with diagnosis, prognosis and responsiveness to the different available therapies. 
To date, the most conclusive information available for diagnostic and therapeutic decisions in clinical nephrology come from analysis of fine needle biopsy and histology.  The quantitative measurement of mRNA levels encoding functionally relevant molecules will add important information to this powerful diagnostic procedure in nephrology.  mRNA expression screening experiments on human glomeruli have already identified novel regulatory pathways active in renal disease. 
We aimed, in this study, to determine the expression of ACE in patients suffering from GN of different etiologies and comparing their levels with that of healthy persons using the polymerase chain reaction (PCR) technique in addition to ruling out any change in the expression of the ACE gene in hypertensive patients receiving ACE inhibitors (ACEI) compared with those not receiving them.
| Patients and Methods|| |
The study included 30 subjects who were divided into two main groups:
All subjects of this group were selected clinically free from other conditions or states that might affect the level of RAS mRNA other than renal disease, and also non-diabetic, free from hepatic and cardiovascular diseases. In addition, the females were not on contraceptive pills or other hormonal therapy or diuretics, and none of the patients was on dietary restriction before the renal biopsies or had contraindication to renal biopsy.
- Group I: (Control group) that includes 10 subjects (six males and four females). Their ages ranged from 20 to 55 years, with a mean of 49 ± 4.99 years. They were selected either with traumatic nephrectomy (completely healthy before the trauma) or with early renal masses (early renal cell carcinoma), and all of them had no clinical, laboratory or histopathological evidence of GN. About 5-10 g of the neighboring unaffected tissue of the removed kidneys were sent for histopathological study and examination and only those with proven to be normal renal tissues were used as controls. They were selected to be free from diabetes, hypertension and cardiovascular, hepatic or renal diseases. In addition, they were not under any drugs that affect the ACE gene expression (e.g., diuretics or ACEI).
- Group II: (the GN group) included 20 subjects (eight male and 12 female). Their age ranged from 17 to 55 years, with a mean ± SD of 34.3 ± 13.3 years. They were diagnosed to have GN by clinical, laboratory (e.g., hypertension, edema, proteinuria and/or hematuria) and kidney biopsies. There were nine cases of focal segmental glomerulosclerosis, rapidly progressive post-streptococcal crescentic GN (three cases), minimal change GN (three cases) and stage II lupus nephrites (three cases) (stage III lupus nephrites (two cases). Sixteen patients had normal kidney function, estimated GFR (eGFR) 60 mL/min/1.73 min). Their mean blood pressure ranged from 96.7 to 118 mmHg, with mean ± SD of III ± 6.16 mmHg.
This group was further subdivided into Group II-A (+ve ACEI group), which comprised ten patients who received ACEI (captopril, in a dose varying from 25 to 50 mg/day) for at least one week prior to renal biopsy. There were five males and five females, and their mean age ± SD was 34.6 ± 9.22 years and their mean blood pressure ± SD was 109 ± 6.82 mmHg. Group II-B (-ve ACEI group) comprised 10 patients who did not receive ACEI as treatment before renal biopsy. There were three males and seven females, and their mean age ± SD was 33.6 ± 14.9 years and their mean blood pressure ± SD was 113 ± 4.9 mmHg.
All subjects of the study were submitted to history and clinical examination and investigations including urinalysis, liver function tests, complete blood count, sedimentation rate, resting 12-lead ECG, blood urea nitrogen (BUN), serum creatinine, fasting and 2-h postprandial blood glucose levels, C-reactive protein and antistreptolysin O liter, plain chest X-ray, creatinine clearance, 24-h urinary Na excretion, 24-h urinary protein excretion, serum electrolytes (Na+, K+, Ca+) and lupus profile: anti-double stranded DNA, c 3 and antinuclear antibodies.
Ultrasound-guided percutaneous renal biopsies by disposable biopsy gun devices were performed, and the glomeruli were also counted per biopsy sample under the microscope and then placed in pre-sterilized (by ultraviolet rays) reaction tubes (Eppendorf, Hamburg, Germany) frozen in liquid nitrogen and stored at -80°C until used (informed consent was taken). Quantification of ACE mRNA was performed using the competitive method "Internal Standard," which is defined as concentration of human ACE-cDNA. It contained a deletion between the primer binding sites of the primers used for amplification of endogenous ACE gene product. Kits used were High pure TM RNA tissue kits for isolation of total RNA from tissue, Boehringer Mannheim, Germany. The kits are stable at room temperature. The test principle is as follows:
Flow chart for the test:
- Isolating intact RNA is a prerequisite for analysis of gene expression.
- This isolated RNA is submitted to reverse transcriptase PCR (RT-PCR).
- To add 0.5 volume ethanol abs.
- Lysale supernatant (tissue homogenezed in lysis buffer).
- Mix and apply lysate (max 700 uL at a time) to high pure M T [filter tube, centrifuge at 13000 xg for 30 s (repeat if the lysate volume is more than 700 uL)].
- Add 90 uL Dnase incubalion- Incubate 15 min, at room temp.
- Buffer 110 ul Dnase working solution.
- Discard flow through.
- Centrifuge at 8000 xg for 15 s.
- Add 500 ul wash buffer.
- Discard flow through.
- Centrifuge at 8000 xg for 15s.
- Add 500 ul wash buffer.
- Discard flow through.
- Centrifuge at 13000 xg for 2 min.
- Add 300 ul wash buffer.
- Centrifuge at 8000 xg for 1 min.
- Add 100 ul elution buffer.
- Pure total RNA.
| Statistical Analysis|| |
Analysis of data using SPSS version 13. Data were expressed as mean ± standard deviation (SD). One way-analysis of variance (ANOVA), independent sample student "t" test, calculation of the least significant difference and Pearson correlation coefficient studies were used as deemed appropriate.
| Results|| |
The mean values ± SD of tissue total RNA (μg/mL) in the different groups of the study were 1669 ± 781, 1476 ± 323 and 898 ± 240 μg/mL in control, patients in group II A who received ACEI and patients in group II B who did not receive the ACEI, respectively [Table 1].
|Table 1: Simple analysis of variance (ANOVA) of total RNA, ACE mRNA, ACE mRNA/total RNA, ACE mRNA/no. of glomeruli.|
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Post hoc test was applied to test the significance of the difference in the mean values of the total RNA among the different groups of the study (P <0.01). Least significance difference (LSD) revealed a significant decrease in the total RNA (μg/mL) in group IIB when compared with group I and group IIA (P <0.01 and P <0.05, respectively). However, there was no significant decrease in the total RNA (μg/mL) in group I when compared with group IIA.
[Table 2] shows a statistically negative correlation between total RNA versus each of serum Na and 24-h urinary protein (P <0.05 and P <0.01, respectively), while there was no significant correlation between total RNA versus age, serum urea, creatinine and mean blood pressure.
|Table 2: The correlation coefficient (r) between ACE mRNA, total RNA ACE mRNA/total RNA, ACE mRNA/total RNA and ACE mRNA per glomerulus versus each of other parameters in all studied groups (n = 30 subjects).|
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The mean values ± SD of ACE mRNA (μg/ mL) in the different studied groups were 5.60 ± 2.95, 33.8 ± 16.3 and 27.9 ± 20.6 (μg/mL) in the control, diseased group IIA and group IIB, respectively [Table 2].
ANOVA was applied to test the significance of difference in the mean values of ACE mRNA (μg/mL) among the different groups of the study; a highly significant difference was revealed among the three groups (P <0.01).
LSD showed a significant increase in ACE mRNA (μg/mL) in the two study groups when compared with the control group (P <0.01 and P <0.01), while there was no significant increase in the ACE mRNA (μg/mL) in group IIA when compared with group IIB (P >0.05).
[Table 2] shows that there was a significant positive correlation between the ACE mRNA and serum Na, mean blood pressure, serum creatinine, BUN, 24-h urinary protein, ACE mRNA/total RNA and ACE mRNA/no. of glomeruli (r = 0.815 and P <0.01) (r = 0.4003 and P = 0.05), (r = 0.408 and P <0.05), (r = -0.475 and P <0.01), (r = 0.655 and P <0.01), (r = 0.505 and P <0.01) and (r = 0.895 and P <0.01), respectively. However, no significant correlation could be found between ACE mRNA and age, urinary Na, creatinine clearance and total RNA.
[Table 1] shows the mean values ± SD of ACE mRNA/total RNA (μg/mL) in the different study and control groups. ANOVA showed a significant difference among the studied groups (P <0.05), and revealed a statistically significant increase in the ACE mRNA/total RNA in the group IIA when compared with the control group, while there was no statistically significant increase in ACE mRNA/total RNA in group IIB when compared with the control and group IIA.
There was a significant positive correlation between ACE mRNA/total RNA and each of the mean blood pressure, serum creatinine, BUN, 24-h urinary protein, ACE mRNA and ACE mRNA/no. of glomeruli (r = 0.432 and P <0.01), (r = 0.422 and P <0.05), (r = 0.395 and P <0.05), (r = 0.400 and P <0.05), (r = 0.505 and P <0.01) and (r = 0.427 and P <0.01), respectively. Moreover, there was a significant negative correlation between ACE mRNA/total RNA and creatinine clearance (r = 0.418 and P <0.05), while no significant correlation could be found between the ACE mRNA/total RNA and each of age, serum Na, urinary Na and total RNA.
The mean values ± SD of ACE mRNA (μg/mL) per glomerulus in the different study groups were: 0.50 ± 0.18, 3.01 ± 2.27 and 2.20 ± 1.61 in the control group and subgroup IIA and subgroup IIB, respectively. ANOVA showed a significant increase among the three groups of the study (P <0.01) [Table 1]. LSD showed a significant increase in the ACE mRNA (μg/ mL) per glomerulus when we compared the two study groups and the control group (P <0.01 and P <0.05), but no significant difference was found between the study groups.
A significant positive correlation was found between ACE mRNA (μg/mL) per glomerulus and each of BUN, 24-h urinary protein and ACE mRNA/total RNA (r = 0.369 and P = 0.05), (r = 0.591 and P = 0.01), (r = 0.895 and P <0.01) and (r = 0.427 and P <0.01), respectively [Table 2].
| Discussion|| |
This study was designed to determine the ACE mRNA gene expression in patients suffering from GN of different etiologies and to compare their levels with those of healthy individuals. In addition, we studied the expression of the total RNA and the ratio between ACE mRNA and total RNA and ACE mRNA per glomerulus and other laboratory tests done to find out factors that may influence the ACE mRNA levels and whether treatment with ACEI drugs may influence the local expression of ACE mRNA.
In our study, we found that the renal total RNA was significantly decreased in the study groups when compared with the control group.
The levels of the total renal RNA were lowest in the study group IIB who did not receive ACEI drugs; the decreased levels of total renal RNA could be explained by the decrease in the levels of renin mRNA as found by others.  Moreover, the increased activity of RAS causes renal scarring and decreases the number of functioning nephrons thus reducing the total RNA. , The levels of total renal RNA were decreased in our patients who received ACEI, which may have ability to control the pathophysiology of GN. In this context, Cohen and Kretzler found that in diseased kidneys, the mRNA expression is often differentially regulated in discrete nephron segments. 
We found a significant negative correlation between the levels of the total mRNA and serum Na. However, no correlation could be found between the total mRNA and urinary Na. This negative correlation could be explained by the increased activity of RAS, which results in increased anti-natriuretic effect of RAS with subsequent increased serum Na. 
In our study, we found a significant increase in the ACE mRNA levels in both study groups compared with the control group. The levels of ACE mRNA were highest in those who received ACEI (group IIA); a similar finding was detected by Ibrahim et al.  This elevation in the level of ACE mRNA could explain the role of RAS in the pathogenesis process of GN and support the role of RAS in the development of glomerular scarring and fibrosis. ,
Although in our study the ACE mRNA level was higher in the study group that received ACEI, this elevation was not significant when compared with those who did not receive ACEI. This finding was in contrast with the findings of Steven and Suzanne.  They found that ACEIs increase the ACE mRNA in endothelial cells, and this effect was both concentration and time dependant. The levels began to increase within 30 min of exposure to ACEI, and showed early peak at 2 hours and a higher delayed peak at 48 h. This contrast of results could be explained by the fact that our study was performed on the renal tissue and not on pulmonary artery endothelial cells. Moreover, the disease process itself may affect the response of the tissues to the ACEIs. This concept was supported by the findings of Costerousse et al, who demonstrated that the ACE concentrations increase during chronic treatment with enalapril, an ACEI, in a dose that ranged from 0.3 to 10 mg/kg/day. There was a dose- and time-dependant increase in both plasma ACE levels up to two- to three-times the control values. Furthermore, there was a significant increase in the steady state of the ACE mRNA in the lung, duodenum and aorta, but there was no significant increase in the ACE mRNA expression in the kidneys.  Another factor that we did not study here may be the genotyping of ACE gene, whether DD and D I or II types as the response to treatment with ACEI is affected by the genotyping as was found by Essen et al. 
In our study, there was a positive correlation between the levels of ACE mRNA and the mean blood pressure. The increased levels of ACE mRNA increase the ACE, which in turn converts more AngII with the resultant vasoconstriction and salt and water retention that result in elevation of the systolic blood pressure; we found a positive correlation between the levels of ACE mRNA and serum sodium as well. Moreover, angiotensin II, by inducing local pro-inflammatory and profibrotic signaling together with elevated blood pressure, can cause renal scarring, loss of the functional renal unites and, subsequently, elevation of both BUN and serum creatinine, which positively correlated with the levels of ACE mRNA.
We can conclude that higher levels of ACE mRNA gene expression in patients suffering from GN may suggest a role of RAS in the development of glomerular hypertrophy and matrix overproduction. The use of ACEI drugs possibly seem to slow the rate of progression of renal failure and to play a role in controlling the pathophysiology.
Larger studies are needed to study the relation between regulation of RAS, activity and progression or development of GN, both at the level of gene expression and in the circulating components of this hormonal system and the potential benefits of early intervention with ACEIs or angiotensin receptor blockers in the progression of GN and the survival of patients.
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Alsayed Ahmed Alnahal
Department of Internal Medicine and Nephrology, Zagazig University, Zagazig
[Table 1], [Table 2]
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