Home About us Current issue Back issues Submission Instructions Advertise Contact Login   

Search Article 
  
Advanced search 
 
Saudi Journal of Kidney Diseases and Transplantation
Users online: 2603 Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size 
 

Table of Contents   
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 24  |  Issue : 5  |  Page : 959-964
Role of expression of endothelin-1 and angiotensin-II and hypoxia-inducible factor-1α in the kidney tissues of patients with Diabetic Nephropathy


Department of Nephrology, First Affiliated Hospital of Zhengzhou University, Zheng Zhou, Henan, China

Click here for correspondence address and email

Date of Web Publication12-Sep-2013
 

   Abstract 

The objective of this study was to detect the expression of Angiotensin-II (Ang-II), Hypoxia-inducible factor-1α (HIF-1α) and Endothelin-1 (ET-1) in the kidneys of patients with diabetic nephropathy (DN) and to investigate their relationship with renal interstitial fibrosis (RIF). A total of 47 paraffin specimens of patients with DN and six controls were enrolled in this study, and all were diagnosed by histopathology. We studied the expressions of Ang-II, HIF-1α and ET-1 by immuno-histochemical staining and the level of RIF by Masson staining. The following results were found: (a) RIF existed in the kidneys of patients with DN, (b) the expressions of Ang-II, HIF-1α and ET-1 were lower in the control group but increased significantly in the DN group, (c) the expression of Ang-II, HIF-1α and ET-1 in tubular epithelial cells directly correlated with RIF (r s = 0.659, 0.633, 0.716, P <0.01) and (d) the expression of Ang-II, ET-1 and HIF-1α in the kidneys of patients with DN positively correlated with serum creatinine (Scr) levels (r s = 0.391, 0.594, 0.531, P <0.01) but they did not correlate with the 24-h urinary protein, blood glucose and serum albumin levels. These results provide new insights suggesting that over-expression of Ang-II, HIF-1α and ET-1 promote the progression of RIF in DN. Thus, targeting reduction in the expression of Ang-II, HIF-1α and ET-1 can delay RIF in DN. Further studies are needed to validate this observation.

How to cite this article:
Sagar SK, Zhang C, Guo Q, Yi R, L. Role of expression of endothelin-1 and angiotensin-II and hypoxia-inducible factor-1α in the kidney tissues of patients with Diabetic Nephropathy. Saudi J Kidney Dis Transpl 2013;24:959-64

How to cite this URL:
Sagar SK, Zhang C, Guo Q, Yi R, L. Role of expression of endothelin-1 and angiotensin-II and hypoxia-inducible factor-1α in the kidney tissues of patients with Diabetic Nephropathy. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2019 Nov 15];24:959-64. Available from: http://www.sjkdt.org/text.asp?2013/24/5/959/118098

   Introduction Top


Renal interstitial fibrosis (RIF) is an important pathological basis of the progression of diabetic nephropathy (DN). [1] The degree of RIF is a better predictor of glomerular filtration rate (GFR) and long-term prognosis than the severity of glomerular damage in DN. [2] Angiotensin-II (Ang-II) and Endothelin-1 (ET-1) mediate inflammation and stimulate the synthesis of extra-cellular matrix and inhibit its degradation [3] thus participating in the pathogenesis of RIF. Over-expression of ET-1 is sufficient to induce structural and functional changes, including glomerular and tubule interstitial fibrosis, because ET-1 causes hypoxic injury due to the constriction of peritubular capillaries and stimulates mesangial cell proliferation. [4] Chronic ischemia and hypoxia that exist in the presence of RIF increase the expression of hypoxia-inducible factor-1α (HIF-1α); Ang-II can also increase the expression of HIF-1α in renal tubular epithelial cells. [5] In recent years, the role of the three cytokines in the pathogenesis of DN has been receiving much attention and, till now, research on the expression of these substances in the kidney has not been reported yet. This study adopts immuno-histochemical techniques to detect the expression and distribution of Ang-II, ET-1 and HIF-1α in the kidneys of patients with DN and discusses their relationship with the degree of RIF and their clinical significance to further clarify the pathogenesis of DN. It is hoped that this data will provide a theoretical basis for targeting future therapeutic interventions to halt the progression of DN.


   Materials and Methods Top


Clinical data

This study was conducted in the Nephrology Department of the First Affiliated Hospital of Zhengzhou University from January 2008 to June 2010. Study material included paraffin specimens of 47 patients with DN (male: 20 and female: 27) aged 29-70 years and six controls (male: three, female: three). The clinical data of patients with DN and the control group are given in [Table 1].
Table 1

Click here to view


Experimental material

Ang-II, HIF-1α mouse anti-human monoclonal antibody, ET-1 rabbit anti-human polyclonal antibody, PV-9001 immuno-histochemical detection kit and DAB color kit were purchased from the Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.

Experimental methods

The immuno-histochemical method adopted was in strict accordance with the instructions of the reagent box; the working concentration of Ang-II, ET-1 and HIF-1α's primary antibody was 1:100. We observed the expression of Ang-II, ET-1 and HIF-1α under light microscopy (×200 times). The criteria of immuno-histochemical results included the following: Nucleus, cytoplasm or cell membrane stained with brown, tan, granular materials were considered positive. Staining levels were classified as follows: Unstained on background = zero points; light staining = one point; moderate staining = two points; deep staining = three points. The percentage of stained cells was classified as follows: Negative = zero points, staining range ≤25% = one point; 26-50% = two points; 51-75% = three points; >75% = four points. The final points were calculated by multiplying the staining level score of each section by the range score, and were as follows: ≤1 were negative (-); >1 but ≤3 were weakly positive (1+); >3 but ≤5 were moderately positive (2+) and >5 was strongly positive (3+). The tubulo-interstitial fibrosis lesions were observed under light microscopy with Masson staining. The results were analyzed automatically by the IDA-2000 high-resolution digital microscopic image analysis system and the area of RIF was calculated. Ten views were collected excluding the glomerulus and artery from the renal cortex (×200 times) and the ratio of the green area of the interstitium on Masson staining was measured and compared with the entire field of vision.


   Statistical Analysis Top


Measurement and comparison of data was made between the DN group and the control group with the ± s. T-test was used to compare the immuno-histochemistry and Masson staining results by using non-parametric statistical methods and correlation analysis was made using Spearman's rank correlation points. P <0.05 was considered as statistically significant, and those with P <0.01 had a significantly distinct meaning statistically. SPSS 17.0 statistical software was used for the analysis.


   Results Top


Light microscope Masson staining of renal tissue [Figure 1]
Figure 1: Changes in the kidney tissue in the control group and the DN group (Masson × 200)

Click here to view


In the healthy control group, no significant tubulo-interstitial changes were observed but in the DN group, obvious tubulo-interstitial atrophy and fibrosis and other pathological changes were seen.

Ang-II, ET-1 and HIF-1α expression in renal tissues [Figure 2]
Figure 2: Expressions of Ang-II, ET-1 and HIF-1α in the kidney tissues of the control group and the diabetic nephropathy group (immuno-histochemical × 200)

Click here to view


Immuno-histochemistry showed that in the normal control group there was almost no expression of Ang-II in the kidney; there were expressions of ET-1 and HIF-1α in small amounts and their main distribution was in the renal tubular epithelial cells. In the DN group, Ang-II and ET-1 expressions concentrated in the cytoplasm of the tubular epithelial cells, and their positive expression rates were 76.60% and 85.11%, respectively; there was also expression of HIF-1α in the cytoplasm and nuclei of the tubular epithelial cells and its positive expression rate was 82.98%.

Ang-II, ET-1 and HIF-1α and RIF

We found that the greater the severity of tubulo-interstitial fibrosis, more obvious was the presence of the above three cytokines in the renal tissue of patients with DN. Thus, the degree of RIF in the kidney tissue of patients with DN and the expression levels of the three cytokines showed a positive correlation. The correlation coefficients with the RIF were r s = 0.659, 0.716, 0.633, P-value <0.01.

Ang II, ET-1 and HIF-1α and clinical relevance of biochemical analysis

There was a positive correlation between Ang-II, ET-1 and HIF-1α and serum creatinine (Scr) levels in patients with DN (r s = 0.391, 0.594, 0.531), P-value < 0.01; however, there was no correlation between Ang-II, ET-1 and HIF-1α and 24-h urine protein levels (r s = 0.114, 0.270, 0.152), P >0.05; blood sugar (rs = -0.266, -0.260, -0.109), P >0.05; or serum albumin levels (r s = 0.149, 0.160, 0.222), P-value >0.05.


   Discussion Top


Renal tubulo-interstitial fibrosis is a final common pathway to end-stage renal failure in DN. [6] The renin-angiotensin system (RAS) and the endothelin systems are in an active state in DN. [7] Ang-II is the major effector peptide of the RAS and ET-1 has the most active biological effects on the endothelin system. Ang-II and ET-1 are not only potent vasoconstrictor substances but also important factors inducing inflammation and promoting fibrosis. [8] It has been demonstrated that Ang-II can promote interstitial fibrosis. This explains the protective effect of angiotensin converting enzyme inhibitors (ACEI) in preventing and/or slowing the progression of RIF in DN.

This study focuses on the expression of Ang-II and ET-1 in the kidney tissue of patients with DN and their relationship with RIF. The results show that in the control group, there was almost no expression of Ang-II in the kidney while there was expression of ET-1 in a small amount in the renal tubular epithelial cells. In the DN group, expression of Ang-II and ET-1 concentrated in the cytoplasm of renal tubular epithelial cells, and their positive expression rates were 76.6% and 85.11%, respectively. Greater the severity of RIF, more obvious was their expression in the kidneys of patients with DN, and their expression levels correlated positively with the degree of RIF. These data suggest that over-expression of Ang-II and ET-1 may be involved in the development of RIF in DN.

In the pathogenesis of RIF, production of Ang-II and ET-1 is increased, which causes a prolonged period of vasoconstriction, leading to tissue ischemia and hypoxia. [9] Chronic hypoxia is one of the main factors leading to RIF, and the role of hypoxia in renal injury is mainly mediated by HIF-1α. [10]

In this study, we also detected the expression of HIF-1α protein level in kidney tissue. Immuno-histochemistry showed that HIF-1α expressed in a small amount in the kidney tissue of the control group but, in the DN group, HIF-1α expressed both in the cytoplasm and in the nucleus of renal tubular epithelial cells, and its positive expression rate increased significantly, reaching 82.98%. The results suggest that the over-expression of HIF-1α correlates with RIF in DN.

The study also showed the expression of Ang-II, HIF-1α and ET-1 in the kidneys of patients with DN positively correlated with Scr, suggesting that they may be involved in the occurrence and development of RIF in DN.

The measures to be taken to prevent and delay the occurrence and development of RIF in DN are an enormous challenge that clinicians face. This study suggests that increased expression of Ang-II, HIF-1α and ET-1 is involved in the development of RIF in DN. However, the specific molecular mechanisms in the development of RIF and the possibility of delaying the progression of RIF in DN by reducing the expression of Ang-II, HIF-1α and ET-1 need further research.

These results provide new insights suggesting that over-expression of Ang-II, HIF-1α and ET-1 are involved in the development of RIF in DN. Thus, targeting reduction in the expression of Ang-II, HIF-1α and ET-1 can delay the occurrence of RIF in DN. Further studies are needed to validate this observation

 
   References Top

1.Li J, Qu X, Yao J, et al. Blockade of endothelial-mesenchymal transition by a Smad3 inhibitor delays the early development of streptozotocin-induced diabetic nephropathy. Diabetes 2010;59:2612-24.  Back to cited text no. 1
[PUBMED]    
2.Thomas MC, Burns WC, Cooper ME. Tubular changes in early diabetic nephropathy. Adv Chronic Kidney Dis 2005;12:177-86.  Back to cited text no. 2
[PUBMED]    
3.Garncarczyk A, Jurzak M, Gojniczek K. Characteristic of the endogenous peptidesendothelins and their role in the connective tissue fibrosis. Wiad Lek 2008;61:126-34.  Back to cited text no. 3
[PUBMED]    
4.Richter CM. Role of endothelin in chronic renal failure-developments in renal involvement. Rheumatology (Oxford) 2006 ;45:Siii3 6-8.  Back to cited text no. 4
    
5.Wang Z, Tang L, Zhu Q, et al. Hypoxia-inducible factor-1α contributes to the profibrotic action of angiotensin II in renal medullary interstitial cells. Kidney Int 2011;79:300-10.  Back to cited text no. 5
[PUBMED]    
6.Liu Y. Renal fibrosis: New insights into the pathogenesis and therapeutics. Kidney Int 2006;69:213-7.  Back to cited text no. 6
[PUBMED]    
7.Zanatta CM, Canani LH, Silveiro SP, Burttet L, Nabinger G, Gross JL. Endothelin system function in diabetic nephropathy. Arq Bras Endocrinol Metabol 2008;52:581-8.  Back to cited text no. 7
    
8.Seccia TM, Maniero C, Belloni AS et. al. Role of angiotensin II, endothelin-1 and L-type calcium channel in the development of glomerular, tubulointerstitial and perivascular fibrosis. J Hypertens 2008;26:2022-9.  Back to cited text no. 8
    
9.Meng L, Qu L, Xiaomei L. Astragulus & Angelica mixture enhances nitric oxide production and reduces angiotensin II in the kidney of UUO rats. Chinese J N 2006;22:94-9.  Back to cited text no. 9
    
10.Nangaku M, Inagi R, Miyata T, Fujita T. Hypoxia and hypoxia inducible factor in renal disease. Nephron Exp Nephrol 2008;110:e1-7.  Back to cited text no. 10
[PUBMED]    

Top
Correspondence Address:
Lin- Tang
Department of Nephrology, First Affiliated Hospital of Zhengzhou University, Zheng Zhou, Henan 450052
China
Login to access the Email id


DOI: 10.4103/1319-2442.118098

PMID: 24029261

Rights and Permissions


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]

This article has been cited by
1 Reprint of: miRNA-1 regulates endothelin-1 in diabetes
Biao Feng,Yanan Cao,Shali Chen,Michael Ruiz,Subrata Chakrabarti
Life Sciences. 2014;
[Pubmed] | [DOI]
2 miRNA-1 regulates endothelin-1 in diabetes
Biao Feng,Yanan Cao,Shali Chen,Michael Ruiz,Subrata Chakrabarti
Life Sciences. 2014;
[Pubmed] | [DOI]



 

Top
   
 
 
    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
    Email Alert *
    Add to My List *
* Registration required (free)  
 


 
    Abstract
   Introduction
    Materials and Me...
   Statistical Analysis
   Results
   Discussion
    References
    Article Figures
    Article Tables
 

 Article Access Statistics
    Viewed2182    
    Printed31    
    Emailed0    
    PDF Downloaded637    
    Comments [Add]    
    Cited by others 2    

Recommend this journal