University of Groningen
Klotho in vascular biology
Mencke, Rik
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Publication date: 2018
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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437
Chapter 14
Associations of Klotho allele variants with
graft survival after kidney transplantation
R. Mencke J.L. Hillebrands M.H. de Borst
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Abstract
Kidney transplantation is the renal replacement therapy of choice for patients with end-stage renal disease (ESRD). Long-term outcomes of kidney transplantation are complicated by various pathological processes, including interstitial fibrosis and tubular atrophy (IFTA) and transplant vasculopathy, which ultimately lead to graft failure. The renal protein Klotho has protective effects on the kidney, vasculature, while also exerting immunomodulatory effects, making Klotho a potential target for the improvement of kidney transplantation outcomes. To assess whether Klotho could be relevant in graft survival after kidney transplantation, we assessed whether 11 SNPs in the Klotho gene are associated with graft survival in a cohort of 1271 donor-recipient pairs.
We found that the minor alleles of rs575536, rs540153, rs577912, and rs2320763, and the major allele of rs211243 were more frequently found in kidney transplant recipients compared to donors, potentially reflecting a higher risk of ESRD. Furthermore, the minor alleles of rs577912 and rs553791 in recipients were associated with a significantly lower graft survival (HR 2.366 [1.158-4.836] and 1.984 [1.234-3.191], respectively).
We speculate that the relevance of recipient SNPs in the Klotho gene in the long-term outcomes of kidney transplantation lies in the regulation of Klotho expression in recipient immune cells. Klotho could prove relevant for long-term outcomes after kidney transplantation and recipient genotyping for rs577912 and rs553791 may be important for clinical follow-up after transplantation.
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Introduction
Kidney transplantation is the renal replacement therapy of choice for patients with end-stage renal disease, a condition with a growing prevalence. The long-term success of kidney transplantation is curbed by several factors that limit graft survival, including interstitial fibrosis and tubular atrophy (IFTA), transplant vasculopathy, and immune cell infiltration (1). The protective effects of the renal ageing suppressor Klotho have formed a recent focus in nephrology research. Klotho knockout mice develop a phenotype resembling human ageing
(2) and mice overexpressing Klotho exhibit an extended lifespan (3, 4). The most potent effects of Klotho include its protective effects on the kidney (5-10) and its direct and indirect protective effects on the vasculature (11-13). More precisely, Klotho has been shown to exert anti-fibrotic effects in various models of renal disease (7, 8, 14), Klotho can prevent neointima formation (15, 16), and it may exert immunomodulatory effects (17). This combination of characteristics makes Klotho a promising therapeutic target in order to improve kidney graft survival. Furthermore, Klotho deficiency induces arteriolar hyalinosis, suggesting that Klotho could possibly inhibit the development of calcineurin inhibitor-induced arteriolar hyalinosis in renal transplant recipients.
While it is certainly tempting to speculate that Klotho may harbour the potential of improving kidney graft survival, there is currently no direct experimental evidence to support this hypothesis. To explore whether there is any merit to this notion, as a first step, we assessed whether SNPs in the Klotho gene are associated with kidney graft survival in renal transplant recipients.
Materials and Methods
Patient cohort
This study was performed in the REGaTTA cohort, which includes all kidney transplantation donor-recipient pairs transplanted between 1993 and 2008. Time to graft failure was defined as return to dialysis or re-transplantation. Graft failure was censored for death with a functioning graft. Patient data were retrieved from the available medical records. The study protocol was approved by the Institutional Review Board of the University Medical Center Groningen (UMCG), the Netherlands. Informed consent was obtained from living donors and recipients, while consent to organ donation from deceased donors includes consent to related research projects. This study was conducted in accordance with the principles of the Declaration of Helsinki.
440 Genotyping
DNA was extracted from peripheral whole blood or, in deceased donors, from lymph nodes/splenic lymphocytes. DNA was purified, followed by genotyping of several Klotho SNPs (rs562020, rs495392, rs385564, rs575536, rs211243, rs9526961, rs541053, rs9526984, rs577912, rs2320763, and rs557391), using the Illumina VeraCode GoldenGate assay (Illumina, USA), according to manufacturer instructions.
Statistical analysis
Normally distributed data are reported as mean ± standard deviation (SD) and non-normally distributed data are reported as median [interquartile range]. Normality was assessed using the Kolmogorov-Smirnov test. Differences between categorical variables were tested using the χ² test. Linkage disequilibrium in the GBR population was assessed using LDlink (https://analysistools.nci.nih.gov/LDlink/). Survival analysis was performed using Kaplan-Meier plots and differences between genotypes were assessed using the log-rank test. Cox regression analysis was used to generate hazard ratios [95% confidence interval] using the homozygous major allele genotype as the reference. Statistical analyses were performed using SPSS version 23 (IBM, USA) and GraphPad Prism version 5 (GraphPad, USA). A p value < 0.05 was considered statistically significant.
Results
We genotyped 1271 kidney donor-recipient pairs, with the donors having a median age of 46.0 [35.0-55.0] years, of which 53% were male, and 61.9% were brain-dead donors. Recipients were 50.3 [38.2-58.5] years old and 58% were male. Patient characteristics are listed in Table 1.
We genotyped the donor-recipient pairs for 11 SNPs, which were all located in intron 1 of the Klotho gene. The linkage disequilibrium structure of these SNPs in the GBR population is depicted in Supplemental Figure 1. We first assessed the allele frequencies for these 11 SNPs (Table 2). Notably, rs575536 A allele carriers, rs540153 A allele carriers, rs577912 A allele carriers, and rs2320763 G allele carriers were significantly more frequent in kidney transplant recipients compared to kidney donors, suggesting that these genotypes are associated with the development of chronic kidney disease. The rs211243 G allele, conversely, was less frequent in kidney transplant recipients, suggesting that this genotype could have a protective effect.
441 Table 1. Patient characteristics.
Transplantation pairs (N = 1271) Donor age (years) 46.0 [35.0-55.0]
Donor sex (% male) 53%
Donor type living (%) brain-dead (%) NHB (%) 282 (22.2) 787 (61.9) 202 (15.9) Recipient age (years) 50.3 [38.2-58.5] Recipient sex (% male) 58%
Primary recipient disease IgA nephropathy (%) Nephrotic syndrome (%) Membranoproliferative GN (%) Membranous nephropathy (%) Glomerulonephritis (%) Rapidly progressive GN (%) Pyelonephritis/interstitial nephritis (%) Tubulointerstitial nephritis (%) Drug-induced nephropathy (%) Cystic kidney disease (%) Diabetes mellitus (%) Congenital renal disease (%) Renal vascular disease (%) Multi-system disease (%)
CKD, other or unknown etiology (%)
98 (7.7) 10 (0.8) 38 (3.0) 10 (0.8) 168 (13.2) 16 (1.3) 148 (11.6) 5 (0.4) 15 (1.2) 208 (16.4) 51 (4.0 54 (4.2) 145 (11.4) 49 (3.9) 256 (20.1) Cold ischemia time (hours) 20 [16-24] Total warm ischemia time (min) 40 [33-51] Delayed graft function (%) 415 (32.7) Death-censored graft failure (%) 205 (16.1) Death with functioning graft (%) 191 (15.0)
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Assessing the associations between the Klotho SNPs and graft survival, we found that the rs577912 AA genotype in recipients was associated with a significantly increased risk of graft loss (HR 2.366 [1.158-4.836] compared to the CC reference genotype). Similarly, the TT and TG genotypes for the rs553791 SNP in recipients were associated with a significantly increased risk of graft loss as well (HR 1.984 [1.234-3.191] and 1.337 [1.001-1.785], respectively). Interestingly, these genotypes in the donors do not affect graft survival. The other SNPs were not associated with graft survival when present in either donors or recipients. Cox regression analyses are summarized in Table 3. Kaplan-Meier survival analyses depicted in Figure 1 illustrate the associations between rs577912 and rs553791, and graft survival.
Figure 1. Kaplan-Meier analysis for graft survival. (A) The rs577912 AA genotype in recipients is associated with significantly lower graft survival. (B) The rs577912 AA, AC, and CC genotypes in donors are not differentially associated with graft survival. (C) The rs553791 TT genotype in recipients is associated with significantly lower graft survival. (D) The rs553791 TT, TG, and GG genotypes in donors are not differentially associated with graft survival.
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Table 2. Klotho gene SNP frequencies among kidney graft donors and recipients.
SNP Donor genotypes Donor minor allele frequency Recipient genotypes Recipient minor allele frequency χ² p value Rs562020 TT TC CC 155 550 556 T: 0.341 143 571 557 T: 0.337 0.087 Rs495392 TT TG GG 98 498 672 T: 0.274 101 506 660 T: 0.279 0.506 Rs385564 CC CG GG 97 571 602 C: 0.301 147 519 605 C: 0.319 0.474 Rs575536 AA AG GG 82 544 628 A: 0.284 105 517 644 A: 0.287 0.024 Rs211243 GG AG AA 213 630 424 G: 0.417 219 559 91 G: 0.393 0.006 Rs9526961 GG GC CC 25 308 916 G: 0.143 21 284 935 G: 0.131 0.813 Rs541053 AA AG GG 248 638 378 A: 0.449 308 586 376 A: 0.473 0.003 Rs9526984 GG GA AA 4 191 1071 G: 0.079 11 179 1078 G: 0.079 0.054 Rs577912 AA AC CC 12 331 873 A: 0.146 27 370 928 A: 0.160 <0.001 Rs2320763 GG TG TT 177 635 454 G: 0.391 223 588 459 G: 0.407 0.021 Rs553791 TT TG GG 83 520 664 T: 0.271 90 513 668 T: 0.273 0.818
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Table 3. Cox regression analysis for Klotho gene SNPs and kidney graft survival.
SNP Donor SNP hazard ratio p value Recipient SNP hazard ratio p value
Rs562020 TT TC CC 1.508 [0.995-2.284] 1.252 [0.928-1.689] 1 0.053 0.141 0.113 1.008 [0.644-1.579] 0.906 [0.677-1.213] 1 0.972 0.507 0.776 Rs495392 TT TG GG 1.220 [0.740-2.013] 1.118 [0.838-1.492] 1 0.435 0.449 0.625 0.932 [0.551-1.574] 0.946 [0.708-1.264] 1 0.791 0.710 0.917 Rs385564 CC CG GG 0.793 [0.445-1.415] 0.976 [0.735-1.296] 1 0.433 0.867 0.735 1.014 [0.648-1.585] 1.018 [0.760-1.363] 1 0.953 0.907 0.993 Rs575536 AA AG GG 1.162 [0.663-2.038] 1.159 [0.871-1.542] 1 0.600 0.312 0.578 1.149 [0.685-1.926] 1.221 [0.917-1.626] 1 0.599 0.171 0.387 Rs211243 GG AG AA 0.880 [0.651-1.190] 0.852 [0.564-1.289] 1 0.407 0.449 0.639 0.686 [0.446-1.054] 0.811 [0.604-1.090] 1 0.086 0.116 0.158 Rs9526961 GG GC CC 1.213 [0.497-2.955] 0.880 [0.631-1.228] 1 0.672 0.453 0.669 0.919 [0.293-2.882] 1.143 [0.827-1.579] 1 0.885 0.418 0.706 Rs541053 AA AG GG 1.191 [0.794-1.786] 1.200 [0.863-1.667] 1 0.398 0.279 0.531 1.364 [0.926-2.010] 1.332 [0.945-1.879] 1 0.116 0.102 0.200 Rs9526984 GG GA AA 1.681 [0.235-12.001] 0.946 [0.639-1.402] 1 0.605 0.783 0.838 1.292 [0.321-5.210] 1.225 [0.844-1.779] 1 0.719 0.285 0.538 Rs577912 AA AC CC 1.285 [0.318-5.190] 1.075 [0.791-1.459] 1 0.724 0.645 0.853 2.366 [1.158-4.836] 1.153 [0.858-1.548] 1 0.018 0.345 0.051 Rs2320763 GG TG TT 1.234 [0.815-1.866] 1.051 [0.776-1.425] 1 0.320 0.747 0.606 1.032 [0.705-1.511] 0.837 [0.617-1.137] 1 0.872 0.255 0.407 Rs553791 TT TG GG 1.227 [0.725-2.075] 0.938 [0.702-1.253] 1 0.446 0.664 0.612 1.984 [1.234-3.191] 1.337 [1.001-1.785] 1 0.005 0.049 0.009
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Discussion
We found that several Klotho SNPs are associated with chronic kidney disease, and that recipients bearing the rs577912 AA and rs553791 TT genotypes in particular are affected by a significantly lower graft survival. These findings raise several questions.
First of all, our data indicate that Klotho is likely relevant in modulating kidney graft survival. Although we cannot draw conclusions of causality or even of effect directions from this study, we hypothesize that Klotho has protective effects on kidney grafts and may positively influence graft survival.
Secondly, although the present study indicates that certain Klotho allele variants affect graft survival, we currently do not know what the molecular effect of these SNPs is. Since all SNPs investigated in this study are intronic SNPs, the expectation is that Klotho protein function is unaffected, but that perhaps Klotho gene expression is increased or decreased via cis-acting elements. Alternatively, intronic SNPs like rs577912 and rs553791 could be in perfect linkage disequilibrium with other (non-investigated) SNPs present in the coding sequence or in the promoter region.
Thirdly, it is particularly interesting that the presence of certain SNPs in recipients, rather than in donors, is associated with an increased risk of graft loss. Klotho is highly expressed in the kidney and the kidney generates virtually all of the Klotho present in the systemic circulation
(18). In addition, patients with end-stage renal disease are pan-Klotho-deficient (11, 19, 20). Therefore, it would be intuitive to expect an effect of donor SNPs that affect Klotho gene expression and therefore Klotho protein production in the transplanted kidney graft. The relevance of Klotho SNPs in recipients, who are severely if not completely Klotho-deficient, therefore poses a conundrum. One possibility would be that not renal or systemic Klotho is relevant in this matter, but local Klotho constitutively expressed in immune cells, modulating the immune response. There are recent indications that low levels of local Klotho expression are relevant to the functions of various cell types (21-23). As an example, mice with Duchenne-type muscular dystrophy have decreased Klotho expression in skeletal muscle (where Klotho is expressed at a level about 1000-fold lower than in the kidney and which was unaffected by the mdx mutation) (24), the restoration of which has been shown to ameliorate the mdx phenotype (25). Additionally, recent data indicate that Klotho expression in local macrophages constitute an important mediator of this protective effect (26). Perhaps a similar mechanism is at play here, with the newly transplanted kidney eliminating the uremia-induced down-regulation of Klotho in PBMCs (27), which in turn allows for a low level of Klotho expressed in recipient immune cells to modulate the interaction between the recipient immune system and the kidney graft. Future studies are required to determine whether such a mechanism exists and whether it is relevant to Klotho effects on renal and vascular cells in transplantation.
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The discrepancy between donor and recipient SNP effects found in this study echoes findings by other authors, like a study by Ozdem et al., in which a Klotho SNP in kidney graft recipients was associated with bone loss after transplantation (28). If our findings can be replicated in other kidney transplantation cohorts, it should also be considered whether pre-transplantation genotyping for rs577912, rs553791, and perhaps other Klotho SNPs we have not assessed could prove to be a useful tool in clinical care, since patients carrying a risk allele may warrant closer monitoring during follow-up.
In short, we found that certain Klotho SNPs are more frequent in ESRD patients and that rs577912 and rs553791 in recipients are associated with an increased risk of graft loss, which we speculate may be due to an effect of these SNPs on Klotho gene expression in the immune system of recipients. Further studies are necessary to establish whether there could be a role for Klotho in the treatment or diagnostics of kidney transplant recipients.
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