**4. Discussion**

In this meta-analysis, we demonstrated that serum klotho levels were significantly increased after successful KTx. While KTx recipients had lower serum klotho levels compared to unmatched healthy volunteers, serum klotho levels in kidney transplant recipients were comparable to those in eGFR-matched controls. Among kidney donors, we found a significant decrease in serum klotho levels post-nephrectomy at day 3 to day 5, which remained lower than baseline before nephrectomy at one year following kidney donation. Compared to healthy volunteers, living kidney donors had lower serum klotho levels.

The findings from our meta-analysis support that klotho is primarily synthesized in the kidneys [40], and transplanting a new kidney into ESKD patients would result in an increase in renal klotho and serum klotho levels post-KTx. In addition to the oligo-anuric state, patients with advanced CKD/ESKD have a significant reduction in klotho and progressively lose the ability to prevent phosphate retention, resulting in hyperphosphatemia, vascular calcification, and cardiovascular disease [83,84]. After successful KTx, in addition to improvement in eGFR, there is also a significant increase in klotho, altogether leading to an improvement in phosphate homeostasis. Recent studies have demonstrated that post-transplant hypophosphatemia after KTx is associated with good kidney allograft function [85,86]. Although the actual underlying mechanisms remain unclear, this is likely because excellent quality transplanted kidneys have higher eGFR and klotho expression, resulting in a reduction in phosphate levels post-KTx.

We identified two cohorts of KTx patients who received their kidneys from deceased donors; higher serum klotho levels in these donors were prognostic for good allograft function at one year after KTx [59,60]. In the ischemia-reperfusion injury (IRI), which is unavoidable to a certain degree

in all KTx surgeries, soluble klotho protects renal tubular cells from oxidative damage by inhibiting the insulin/IGF-1 signaling pathway and by inhibition of TGF-β1 for decreasing renal fibrosis [87,88], and upregulation of autophagy in renal tubular cells [3,89]. In addition, klotho is also involved in the inhibition of Wnt pathway-associated β-catenin activation, thus improving renal fibrosis [87]. Compared to patients with early graft function, a lower level of klotho is observed in implantation biopsies among patients with delayed graft function (DGF) [90]. Although data on the e ffects of klotho on long-term allograft outcomes are limited, it is well known that poor allograft function at one year after KTx and DGF is associated with renal allograft loss [91,92]. Following successful KTx, patients regain functions of klotho via FGF23-Klotho signaling, and with the previously accumulated FGF23, residual hyperparathyroidism, and the use of calcineurin inhibitors (especially cyclosporine) [93–95], post-KTx hypophosphatemia can commonly occur up to 86% [85,96,97]. Post-KTx hypophosphatemia is known to be associated with lower risks of death-censored graft failure and cardiovascular mortality [85]. The association between post-KTx hypophosphatemia and reduced cardiovascular mortality among KTx recipients could be related to the reduction of calcium phosphate product, an important factor associated with vascular calcification and cardiovascular events [98,99]. Our study demonstrated that successful KTx can result in a significant increase in serum klotho levels among KTx recipients [85]. In addition, previous literature has demonstrated trending towards normal FGF-23 levels after successful KTx [42,100]. Thus, regaining function in FGF23-Klotho signaling after KTx helps promote urinary phosphate excretion and reduced vitamin D-dependent intestinal absorption of calcium and phosphate [11,15], which might explain the association between post-KTx hypophosphatemia and reduced cardiovascular mortality. Future studies are needed to assess the impact of klotho levels on long-term cardiovascular health in KTx recipients, allograft, and patient survival.

Living donors supply approximately 40% of kidney allografts in the United States [101]. Overall, living kidney donation is considered safe and does not appear to increase long-term mortality compared with controls [102–107]. A recent systematic review of 52 studies comprising 118,426 living kidney donors reassured the safety of living kidney donations with the finding of no di fference in all-cause mortality among donors and controls [108]. In addition, a large retrospective population-based matched cohort study of 2028 kidney donors in comparison with 20,280 matched non-donor controls (followed for a median of 6.5 years) demonstrated no di fference in the rate of cardiovascular events between the two groups [109]. Although the findings of our study showed a significant reduction in serum klotho at post-operative day 3 to 5 and at one year following kidney donation, the degree of klotho reduction seemed to be attenuated at one year post-donation compared to the early post-operative period. In addition, we found no significant change in serum FGF-23 at one year post-donation. It is possible that after living kidney donation serum klotho is not severely reduced enough to stimulate the rise in serum FGF-23, which occurred in patients with advanced CKD [41,110]. Elevated FGF-23 levels have been shown to be associated with increased mortality and cardiovascular events [111–113]. Thus, no significant increase in FGF-23 levels after living kidney donation is consistent with the findings of no di fference in all-cause mortality among donors and controls in previous literature [108,109].

Despite these published reassuring findings of donor safety [108,109], a recent small multicenter study of living kidney donors and healthy controls (*n* = 124) demonstrated an association between living kidney donation and a significant increase in left ventricular mass and reduced aortic distensibility [114]. In addition to functions of klotho via FGF23-Klotho signaling, soluble klotho also has FGF23-independent e ffects, including endothelial protection from senescence, anti-fibrotic properties, cardioprotection, and prevention of vascular calcifications [84,115,116]. Klotho-deficient CKD mice have significant left ventricular hypertrophy (LVH) and cardiac fibrosis compared with wild-type mice [117]. Soluble klotho also provides cardioprotection against stress-induced exaggerated cardiac remodeling through downregulation of transient receptor potential cation channel 6 (TRPC6) [118]. Although an increased LVH and reduced aortic distensibility in living kidney donors could be related to an increased risk of hypertension post living kidney donation [102,103], future studies are required

to assess whether a reduction in serum klotho levels after living kidney donation may play a role in the higher risk of LVH, and reduced aortic distensibility observed among living kidney donors.

Our meta-analysis is subject to certain limitations. First, although there were comparative groups, all studies are observational, making them susceptible to selection bias. Second, many variables may influence klotho levels in the post-transplant period that may contribute to the heterogeneity between the included studies evaluating changes in serum klotho levels among KTx recipients. Data on medications that may a ffect endogenous klotho expression in the kidney and soluble levels such as angiotensin II inhibitors and hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors [17,21,119,120] as well as data on immunosuppression were limited in included studies. Lifestyle, diet, psychological stress, and activities such as exercises may also a ffect serum klotho levels [121–124]. Thus, future prospective studies are needed to assess the impact of changes in klotho on clinical outcomes in KTx recipients and living kidney donors. Third, the follow-up duration of included studies was limited to only one year, and future studies are required to evaluate the impacts of serum klotho levels on long-term clinical outcomes. Fourth, serum klotho is also a ffected by the aging process and declines with older age [125]. However, we demonstrated an increase in serum klotho levels after KTx at one year and a decrease in klotho levels at immediate postoperative (which is less likely to be a ffected by the aging process). Lastly, all included studies measured serum klotho levels by ELISA. Recently, immunoprecipitation-immunoblot (IP-IB) assay is shown to be superior to the ELISA and highly correlated with eGFR [126]. However, this technique requires the labor-intensive nature of the IP-IB assay, and further research is needed to evaluate the use of the IP-IB assay in KTx patients.

In conclusion, compared to patients' baseline, serum klotho levels increase early after successful KTx and decrease after living kidney donation, respectively. Future studies are required to assess the impact of serum klotho levels on risk-stratification and patient-centered outcomes in both living donors and KTx recipients.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/6/1834/s1, Table S1: Search strategy.

**Author Contributions:** Conceptualization, C.T., J.A.N., J.M., N.L., P.W.D., W.K., T.B., S.A.S., A.C., N.R.A., S.V., M.A.M. and W.C.; Data curation, C.T., N.L. and W.C.; Formal analysis, C.T. and W.C.; Funding acquisition, J.M.; Investigation, C.T., P.H., N.L., W.K. and W.C.; Methodology, C.T., J.A.N., P.H., W.K. and W.C.; Project administration, C.T. and T.B.; Resources, T.B.; Software, T.B.; Supervision, J.A.N., J.M., P.W.D., S.A.S., A.C., S.V., M.A.M. and W.C.; Validation, C.T., P.W.D., W.K., N.R.A. and W.C.; Visualization, J.A.N., P.H., J.M. and N.L.; Writing—original draft, C.T. and W.C.; Writing—review and editing, J.A.N., P.H., J.M., N.L., P.W.D., W.K., T.B., S.A.S., A.C., N.R.A., S.V., M.A.M. and W.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** None. All authors had access to the data and played essential roles in writing of the manuscript. Neyra is currently supported by an Early Career Pilot Grant from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), through Grant UL1 TR001998.

**Conflicts of Interest:** The authors deny any conflict of interest.
