Skip Navigation


NDT Advance Access originally published online on November 28, 2007
Nephrology Dialysis Transplantation 2008 23(2):608-611; doi:10.1093/ndt/gfm736
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/2/608    most recent
gfm736v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Courtney, A. E.
Right arrow Articles by Maxwell, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Courtney, A. E.
Right arrow Articles by Maxwell, A. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Association of functional haem oxygenase-1 gene promoter polymorphism with polycystic kidney disease and IgA nephropathy

Aisling E. Courtney1,2, Peter T. McNamee2, Shirley Heggarty3, Derek Middleton4 and A. Peter Maxwell1,2

1Nephrology Research Group, Queen's University Belfast, 2Regional Nephrology Unit, 3Genomics Core Facility, School of Medicine, Queen's University Belfast, Regional Genetics Centre, and 4Northern Ireland Histocompatibility and Immunogenetics Laboratory, Belfast City Hospital, Belfast BT9 7AB, UK

Correspondence and offprint requests to: Dr Aisling E. Courtney, Regional Nephrology Unit, Belfast City Hospital-Level 11, Lisburn Road, Belfast BT9 7AB, UK. Tel: +44-2890329241; Fax: +44-2890263535; E-mail: aecourtney{at}doctors.org.uk



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 
Background. Haem oxygenase-1 (HO-1) is a cytoprotective molecule that is reported to have a protective role in a variety of experimental models of renal injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates HO-1 gene expression; a short number of repeats (S-allele <25) increases transcription. We report the first assessment of the role of this HO-1 gene promoter polymorphism in chronic kidney disease due to autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IgAN).

Methods. The DNA from 160 patients (99% Caucasian) on renal replacement therapy (RRT) was genotyped. The primary renal disease was ADPKD in 100 patients and biopsy-proven IgAN in 60 patients.

Results. Overall, the mean age at commencement of RRT was not significantly different between patients with and without an S-allele (44.1 years versus 45.0 years, P = 0.64). In patients with ADPKD, the age at commencement of RRT was comparable regardless of the HO-1 genotype (47.7 years versus 46.7 years, P = 0.59). The same was true in patients with IgAN (38.3 years versus 42.2 years, P = 0.28).

Conclusion. This suggests that the functional HO-1 promoter polymorphism does not influence renal survival in CKD due to ADPKD or IgAN.

Keywords: gene polymorphism; haem oxygenase-1; IgA nephropathy; polycystic kidney disease; progression of kidney disease



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 
Haem oxygenase-1 (HO-1), first identified as catalyzing the conversion of haem to biliverdin [10,11], independent of the haem degradation reaction.

Experimental and clinical evidence supports an important protective role for HO-1 in renal disease. Rodent models have implicated HO-1 in the pathophysiology of specific disease entities such as polycystic kidney disease [12], obstructive nephropathy [13] and anti-glomerular basement membrane (anti-GBM) nephritis [14]. Additionally, oxidative stress is an important factor in progressive renal disease with a final common pathway of proinflammatory molecules, fibrogenic cytokines and extracellular matrix deposition irrespective of the initial insult [15]. HO-1 has anti-oxidant properties, may modulate the inflammatory response, and protect against excess transforming growth factor-β (TGF-β) activity [16].

A dinucleotide guanosine thymine (GT) repeat region is present in the proximal promoter region of the human HO-1 gene. This is a functional length polymorphism resulting in higher HO-1 expression and enzyme activity when the GT repeat length is short (S allele <25 repeats) rather than long (L allele >25 repeats) [17].

We hypothesized that reduced injury and prolonged renal survival will be observed in patients with chronic kidney disease (CKD) who carry at least one short allele. Genomic DNA from patients with end-stage renal disease (ESRD), due to two distinct disease processes, autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IgAN), was assayed to determine the HO-1 (GT)n genotype. The results were correlated with the age at entry into the renal replacement therapy (RRT) programme.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements.
 References
 
Patients
Clinical details on all patients with ESRD are prospectively recorded in a database with the primary renal diagnosis categorized according to the European Dialysis and Transplant Association (EDTA) diagnostic coding system. All patients on the renal transplant waiting list have genomic DNA material extracted from peripheral blood samples. These have been stored for all consecutive patients on the deceased donor transplant waiting list since May 1986 in the Northern Ireland Histocompatibility and Immunogenetics Laboratory, Belfast City Hospital. The first 100 patients since May 1986 with a primary diagnosis of ADPKD and the first 60 patients with ESRD due to biopsy proven IgAN that had stored genomic material were studied.

The study was performed with prior approval from Queen's University, Belfast Research Ethics Committee.

Genotyping of variable length polymorphism (GT)n
This technique has been previously described in detail [18]. Fragment length analysis involved amplification of the DNA by polymerase chain reaction (PCR) and subsequent sizing by capillary electrophoresis. PCR amplification was performed in 10-µl reactions with 0.625 units of HotStar Taq polymerase (Qiagen, Valencia, CA), using an MJ Research Tetrad Thermal cycler (MJR, Waltham, MA). Specific primers (forward: 5'-AGA GCC TGC AGC TTC TCA GA-3'; reverse: 5'-ACA AAG TCT GGC CAT AGG AC-3') were modified with a 5'-FAM label on the forward primer (Invitrogen, Carlsbad, CA).

One microlitre of diluted PCR product was added to 10 µl of HiDi formamide plus GeneScan 500 LIZ size standard (both from Applied Biosystems, Foster City, CA). Electrophoresis was performed on an ABI 3730 DNA genetic analyser (Applied Biosystems, Foster City, CA), and resultant electropherograms analysed using ABI Genemapper V 3.5 software (Applied Biosystems, Foster City, CA).

Statistics
The HO-1 (GT)n polymorphism was categorized according to allele length. Consistent with published literature, the short allele was classed as <25 GT repeats, the long allele ≥25 repeats. Genotype distribution was tested for deviation from the Hardy–Weinberg equilibrium using a chi-square test.

The mean age of entry into the RRT programme was compared between those with and without carriage of the short allele in each diagnostic group using the independent t-test. SPSS for Windows® (SPSS® Inc., Chicago, IL, USA) version 13.0 was employed and values of P < 0.05 were considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements.
 References
 
Clinical data
One hundred sixty patients were studied, >99% were Caucasian and 110 (68.8%) were male. Of these patients, 83 (51.9%) were receiving haemodialysis therapy, 71 (44.4%) peritoneal dialysis and 6 (3.4%) had pre-emptive transplantation. The primary renal diagnosis was ADPKD in 100 patients and IgAN in 60 patients.

HO-1 genotype
DNA adequate for genotyping was available for 158 (99%) patients, 98 with PKD and 60 with IgAN. The number of GT repeats ranged from 21 to 37. The distribution of the (GT)n polymorphism was bimodal in concordance with the literature, peaking at 30 repeats and 23 repeats (Figure 1).


Figure 1
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Distribution of number of GT repeats in HO-1 gene promoter.

 
The alleles were divided into two subclasses: short or S-allele with fewer than 25 GT repeats, long or L-allele with 25 or greater repeats. Various division points from 25 to 28 will give widely comparable results given the allele distribution, i.e. small numbers in the populations studied have these numbers of GT repeats. Table 1 details the frequencies of the SS, SL and LL genotypes. The genotype distribution was in Hardy–Weinberg equilibrium; 90/158 (57%) were carriers of an S-allele (heterozygotes and S-homozygotes).


View this table:
[in this window]
[in a new window]

  		Table 1: HO-1 (GT)n polymorphism genotype distribution

 
Age at commencement of RRT
Considering all patients for whom the HO-1 (GT)n genotype was available, the S-allele carriers had a mean age at RRT commencement of 44.1 years (SD 11.6), compared to 45.0 years (SD 12.1) in the L-homozygotes (P = 0.64).

In patients with ADPKD, the difference in mean age was minimal: 47.7 years (SD 9.7) in those with at least one S-allele and 46.7 years (SD 9.3) in those without an S-allele (P = 0.59). There was no significant difference in the age of commencement of RRT between males and females in this group (47.0 years and 48.0 years, P = 0.57), and inclusion of gender in the regression analysis did not alter the non-significance of the HO-1 genotype.

The carriage of an S-allele was not influential in IgAN with a mean age at RRT commencement of 38.3 years (SD 12.1) compared to 42.2 years (SD 15.6) in L-homozygotes (P = 0.28). There were only 9 (15%) females in this group but the gender did not impact on the age at RRT commencement (40.9 years in males and 33.8 years in females, P = 0.17), and in the regression analysis the S-allele remained non-significant.



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements.
 References
 
The identification of susceptibility factors and the development of therapeutic strategies to effectively retard the progression of CKD is a major challenge.

The presence of a functionally significant GT length polymorphism in the promoter region of the HO-1 gene has engendered interest in the potential role of HO-1 in a variety of disease states, based on the premise that HO-1 is induced preferentially in cells with a short number of GT repeats [2]. It is plausible that increased HO-1 production would be advantageous in chronic renal disease since it may limit progressive injury.

Renal tubular cells may be exposed to excess haem protein overtly following the disproportionate release of haem proteins from erythrocytes (as in the haematuric nephritides), or covertly during periods of oxidative stress that destabilize the intracellular environment, releasing haem proteins that are abundant in intracellular organelles. HO-1 has been demonstrated in various animal models to play a critical cytoprotective role in both situations [19,20].

HO-1 is also involved in complex negative feed-back regulatory loops with angiotensin II (AT-II), nuclear factor-{kappa}B (NF-{kappa}B), MCP-1 and TGF-β, which have been implicated in progressive renal injury [21].

In ADPKD, there is wide intra-familial variability in disease severity and additional genetic factors, separate from the germline mutation, influence disease progression. It is estimated that 43–78% of the difference in age at commencement of RRT can be attributable to inherited factors [24]. Additionally, polycystin-1 may interfere with the action of NF-{kappa}B [24]. It is therefore reasonable to hypothesize that a functionally significant HO-1 gene polymorphism will alter the progression of ADPKD.

A genetic link to IgAN is accepted. Different genes are likely to be involved in the progression rather than the pathogenesis of IgAN, and candidates for the former include TGF-β and nitric oxide synthase [25]. Increased HO-1 production may limit the progression of IgAN due to its influence as an anti-oxidant and interaction with TGF-β.

Experimental models have intrinsic limitations and confirmatory studies of the importance of HO-1 production in a clinical setting are essential. To our knowledge, this is the first report that has considered the HO-1 (GT)n length polymorphism in CKD in a clinical setting. There was no significant difference in the age at commencement of RRT between those who carried at least one S-allele and those that did not in patients with ADPKD or IgAN. Either the HO-1 genotype confers no differential effect or the effect is relatively small and masked by the milieu of factors, both environmental and genetic, that are linked to the progression of chronic renal disease.

A type 2 error (false negative result) cannot be excluded, particularly if the HO-1 polymorphism has only a subtle influence. As this is the first study of this gene polymorphism in a clinical setting of progressive CKD, the a priori effect size (if any) of the HO-1 gene polymorphism on renal survival was unknown. We could therefore not accurately calculate in advance the sample size required to achieve an adequately powered analysis.

The age at commencement of RRT, or the closely related age at ESRD, is a surrogate marker for renal survival that is widely used in studies of ADPKD. Even if the age at first referral to renal services was available, this does not provide a consistent ‘starting point’ from which ‘renal survival’ can be calculated. Data on potential confounding factors, except for gender, were unavailable and prospective studies will be required to overcome these limitations. The subpopulation of patients selected by this study, i.e. those listed for renal transplantation, is reflected in the younger age at commencement of RRT than is commonly reported in ADPKD and IgAN, but does not influence the comparison between the S-allele carriers and L-homozygotes.

Theoretically and experimentally, an increase in HO-1 ameliorates renal damage and modifies the inflammatory response to injury. This study found no convincing evidence for an association between the HO-1 (GT)n genotype and the age at commencement of RRT in two distinct renal diseases, suggesting that there is no clinically significant differential protective effect of the S-allele on renal survival in ADPKD or IgAN.



   Acknowledgements.
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
References
 
AEC received financial support from the Northern Ireland Kidney Research Fund.

Conflict of interest statement. There is no conflict of interest for any of the authors.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 

  1. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci U S A (1968) 61:748–755.[Free Full Text]
  2. Exner M, Minar E, Wagner O, et al. The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic Biol Med (2004) 37:1097–1104.[CrossRef][Web of Science][Medline]
  3. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med (2000) 6:422–428.[CrossRef][Web of Science][Medline]
  4. Peyton KJ, Reyna SV, Chapman GB, et al. Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth. Blood (2002) 99:4443–8.[Abstract/Free Full Text]
  5. Brouard S, Otterbein LE, Anrather J, et al. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med (2000) 192:1015–1026.[Abstract/Free Full Text]
  6. Pae HO, Oh GS, Choi BM, et al. Carbon monoxide produced by heme oxygenase-1 suppresses T cell proliferation via inhibition of IL-2 production. J Immunol (2004) 172:4744–4751.[Abstract/Free Full Text]
  7. Llesuy SF, Tomaro ML. Evidence of involvement of bilirubin as physiological protector against oxidative damage. Biochim Biophys Acta (1994) 1223:9–14.[Medline]
  8. Balla G, Jacob HS, Balla J, et al. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem (1992) 267:18148–18153.[Abstract/Free Full Text]
  9. Kanakiriya SK, Croatt AJ, Haggard JJ, et al. Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am J Physiol Renal Physiol (2003) 284:F546–F554.[Abstract/Free Full Text]
  10. Inguaggiato P, Gonzalez-Michaca L, Croatt AJ, et al. Cellular overexpression of heme oxygenase-1 up-regulates p21 and confers resistance to apoptosis. Kidney Int (2001) 60:2181–2191.[CrossRef][Web of Science][Medline]
  11. Woo J, Iyer S, Cornejo MC, et al. Stress protein-induced immunosuppression: inhibition of cellular immune effector functions following overexpression of haem oxygenase (HSP 32). Transpl Immunol (1998) 6:84–93.[CrossRef][Web of Science][Medline]
  12. Maser RL, Vassmer D, Magenheimer BS, et al. Oxidant stress and reduced antioxidant enzyme protection in polycystic kidney disease. J Am Soc Nephrol (2002) 13:991–999.[Abstract/Free Full Text]
  13. Kawada N, Moriyama T, Ando A, et al. Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int (1999) 56:1004–1013.[CrossRef][Web of Science][Medline]
  14. Mosley K, Wembridge DE, Cattell V, et al. Heme oxygenase is induced in nephrotoxic nephritis and hemin, a stimulator of heme oxygenase synthesis, ameliorates disease. Kidney Int (1998) 53:672–678.[CrossRef][Web of Science][Medline]
  15. Haugen E, Nath KA. The involvement of oxidative stress in the progression of renal injury. Blood Purif (1999) 17:58–65.[CrossRef][Web of Science][Medline]
  16. Haugen EN, Croatt AJ, Nath KA. Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int (2000) 58:144–152.[CrossRef][Web of Science][Medline]
  17. Sikorski EM, Hock T, Hill-Kapturczak N, et al. The story so far: molecular regulation of the heme oxygenase-1 gene in renal injury. Am J Physiol Renal Physiol (2004) 286:F425–F441.[Abstract/Free Full Text]
  18. Courtney AE, McNamee PT, Middleton D, et al. Association of functional heme oxygenase-1 gene promoter polymorphism with renal transplantation outcomes. Am J Transplant (2007) 7:908–913.[CrossRef][Web of Science][Medline]
  19. Nath KA, Haggard JJ, Croatt AJ, et al. The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol (2000) 156:1527–1535.[Abstract/Free Full Text]
  20. Shimizu H, Takahashi T, Suzuki T, et al. Protective effect of heme oxygenase induction in ischemic acute renal failure. Crit Care Med (2000) 28:809–817.[CrossRef][Web of Science][Medline]
  21. Rosenberg ME, Smith LJ, Correa-Rotter R, et al. The paradox of the renin-angiotensin system in chronic renal disease. Kidney Int (1994) 45:403–410.[Web of Science][Medline]
  22. Paterson AD, Magistroni R, He N, et al. Progressive loss of renal function is an age-dependent heritable trait in type 1 autosomal dominant polycystic kidney disease. J Am Soc Nephrol (2005) 16:755–762.[Abstract/Free Full Text]
  23. Fain PR, McFann KK, Taylor MR, et al. Modifier genes play a significant role in the phenotypic expression of PKD1. Kidney Int (2005) 67:1256–1267.[CrossRef][Web of Science][Medline]
  24. Torres VE, Harris PC, Pirson Y. Autosomal dominant polycystic kidney disease. Lancet (2007) 369:1287–1301.[CrossRef][Web of Science][Medline]
  25. Barratt J, Feehally J, Smith AC. Pathogenesis of IgA nephropathy. Semin Nephrol (2004) 24:197–217.[CrossRef][Web of Science][Medline]
Received for publication: 6. 8.07
Accepted in revised form: 19. 9.07


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/2/608    most recent
gfm736v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Courtney, A. E.
Right arrow Articles by Maxwell, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Courtney, A. E.
Right arrow Articles by Maxwell, A. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?