α-Klotho: An Early Risk-Predictive Biomarker for Acute Kidney Injury in Patients with Acute Myocardial Infarction

Background Acute kidney injury (AKI) was a common and serious complication in patients with acute myocardial infarction (AMI). Novel biomarkers and therapies were deficient and imperative for AKI's early diagnosis and therapy after AMI. α-Klotho was considered as an early biomarker and potential therapy for AKI recently. Previous studies reported that the expression of α-Klotho was decreased in AKI rodents, and supplement of α-Klotho alleviated kidney injury. Nevertheless, its effect has not been studied in patients presenting with AMI. Methods A total of 155 consecutive diagnosed with AMI at emergency department whose eGFR >60 ml/min ∗ 1.73 m2 were enrolled in this prospective observational cohort study which conducted between May 2016 and April 2019 in Peking University People's Hospital. AKI was defined according to the KDIGO criteria in 2012. At admission, the clinical data of patients were collected and serum α-Klotho was tested by ELISA. The relationship between α-Klotho, serum creatinine, eGFR, systolic pressure, BNP, LVEF, and Hgb of AKI were analyzed and their discrimination performances were compared. The association variables were calculated (adjusted odds ratio) with a confidence interval (CI) of 95% by binary logistic regression. And, we followed up the incidence of CKD and rehospitalization after patients' discharge in one year. Our study was approved by the ethics committee (no. 2016PHB042-01). Results AKI incidence was 17.4% (27/155) during hospitalization. Compared to patients without AKI, the AKI group had obviously higher mortality and was more female and had higher incidence of chronic kidney disease, worse cardiac function, more cardiac complications, larger doses of diuretics, and less use of angiotensin-converting enzyme inhibitors/angiotensin receptor blocker. By contrary to previous animal experiments, we found serum α-Klotho levels were increased significantly in AKI patients (740.2 ± 306.8 vs. 419.0 ± 272.6 pg/mL, p < 0.001). And, the areas under the receiver operating curves indicated serum α-Klotho levels had a superior discriminative power for predicting AKI after AMI compared with other risk factors (0.792, 95% CI, 0.706–0.878, p < 0.001). Meanwhile, logistic regression model indicates extensive anterior myocardial infarction, Killip classification ≥2 grade, α-Klotho ≥516.9 pg/mL, eGFR (decrease per 10 ml/min ∗ 1.73 m2), Hgb, and nonuse of ACEI/ARB were the risk factors of AKI after AMI. Moreover, one-year follow-up presented AMI patients developed CKD had higher α-Klotho levels (739.7 ± 315.2 vs. 443.8 ± 292.5 pg/mL, p = 0.001), but no significant difference in rehospitalization. And, patients with α-Klotho ≥516.9 pg/ml was 6.699 times more likely to develop CKD than those with α-Klotho <516.9 pg/ml (relative risk 6.699, 95% CI 1.631–27.519, p = 0.007). Conclusion Compared with traditional cardiac and renal biomarkers, serum α-Klotho could be a more appropriate predict biomarker for AKI after AMI in patients' eGFR >60 ml/min ∗ 1.73 m2. Higher α-Klotho levels are related to the development of AKI during hospitalization and suggest a higher prevalence of CKD after discharge. By contrary to animal experiments, whether the increased expression of α-Klotho could be a protective factor secreted by AKI after AMI, is remained to be further studied.


Introduction
Acute kidney injury (AKI) is well recognized as a common and critical complication in patients with acute myocardial infarction (AMI), which is associated with an increased risk of recurrent myocardial infarction, heart failure, chronic kidney disease (CKD), dialysis, rehospitalization, inhospital, and long-term mortality.Previous studies have indicated that the incidence of AKI ranged from 7.1 to 29.3% in AMI patients and with poor prognosis [1][2][3].Multiple guidelines have emphasized AKI prediction and prevention as a major healthcare priority, for therapeutic options are defcient once AKI occurs [4,5].
According to 2012 KDIGO criteria of AKI defnition [4], serum creatinine and urine output are widely used in the diagnosis of AKI; however, both of them are insensitive.Accordingly, there is an urgent need for better biomarkers for AKI prediction in order to improve the prognosis after AMI.On the other side, searching the novel potential renal protective factors has always been a research hotspot for clinical scientists.Recently, α-Klotho has been reported as both a biomarker and replacement therapy for AKI [6,7].α-Klotho, referred as Klotho, is a 130 kD transmembrane protein with two types: shed and secreted [7,8].Te extracellular domain of transmembrane Klotho could be cleaved by proteases and released from the kidney into circulation, both as a full-length protein or as Klotho1 and Klotho2 fragments [7,8].Tese circulating forms of Klotho protein are collectively called "soluble" Klotho, playing a biological efect on multiple organs.Presently the research of Klotho mainly focuses on antiaging, antioxidation, regulating bone, calcium, and phosphorus metabolism, inhibiting cell apoptosis, inducing autophagy, and cell senescence [7,8].
Klotho is confrmed to be mainly expressed in renal distal convoluted tubule and also found in the proximal convoluted tubule, although at lower levels [9].In various AKI rodent experiments including ischemia-reperfusion injury, cisplatin induced AKI and postrenal unilateral ureteral obstruction models, all indicate a systemic Klotho defciency state, and applying Klotho supplement possess a considerable potential therapeutic efect [10][11][12].Studies in clinical studies also have shown low levels of α-Klotho in chronic kidney disease (CKD) and end stage renal disease (ESRD), and low Klotho is reported to be associated with cardiovascular events in hemodialysis patients recently [7,13,14].However, in our previous study, we have reported α-Klotho increased signifcantly in AMI patients without ESRD induced AKI, which with poor early predictive value [15].Consequently, in order to eliminate the infuence of renal dysfunction, we conducted a prospective study in AMI patients with estimated glomerular fltration rate (eGFR) ≥60 ml/min * 1.73 m 2 to explore the role of α-Klotho as AKI develop.

Research Subjects.
From May 2016 to April 2019, 155 consecutive patients admitted to Peking University People's Hospital in China with a diagnosis with AMI frstly in the emergency department were enrolled in this prospective study.AMI was diagnosed according to the standard criteria [15,16].Exclusion criteria were as follows: patients with eGFR <60 ml/min * 1.73 m 2 , septic shock, unable to provide informed consents and who died or were discharged within 48 h.Our study was approved by the hospital ethics committee (No. 2016PHB042-01) and all patients signed informed consent.

Defnition.
AMI was defned based on a typical chest pain, diagnostic electrocardiographic changes, elevation of troponin I (TNI), and wall motion abnormalities on an ultrasonic cardiogram [16].AKI was diagnosed according to the criteria of KDIGO criteria, by an increase in serum creatinine of 0.3 mg/dl within 48 h, an elevation of 1.5 fold from the baseline level within the frst seven days [4].eGFR was calculated using modifcation of diet in renal disease equation for Chinese patients [17].Te AKI stage 1 is defned as an increase in serum creatinine of more than or equal to 0.3 mg/dL or increase to more than or equal to 1.5∼2 fold from baseline.Stage 2 is an increase in serum creatinine to more than 2∼3 fold from baseline.Stage 3 is an increase in serum creatinine to more than 3 fold from baseline, greater than or equal to 4.0 mg/dL, or initiation of renal replacement therapy [4].Te diagnose and development of CKD is defned as abnormalities of kidney structure or function, present for 3 months according to KDIGO 2012 Clinical Practice Guideline [18].

Baseline Characteristics.
Clinical data recorded include: age, gender, comorbidities (hypertension and diabetes mellitus), smoking history, heart rate, blood pressure, STsegment elevation myocardial infarction (STEMI) or non-ST-segment elevation myocardial infarction (USTEMI), cardiac function and complications, contrast volume, laboratory tests, and therapies about AMI.Te maximum daily doses of intravenous loop diuretics were counted and converted to furosemide, expressed as 1 mg bumetanide ≈ 20 mg torsemide ≈ 40 mg furosemide.Te peak of TNI, creatine kinase-MB (CK-MB), and brain natriuretic peptide (BNP) levels were used in the statistical analysis.All patients were treated with standard anticoagulation and antiplatelet therapies.

Measurement of α-Klotho.
Once patients were diagnosed with AMI in emergency, serum samples were collected immediately for α-Klotho measurements.Serum creatinine was measured, respectively, at admission, third, and seventh day.All samples were centrifuged at 1500 rpm for 10 minutes and then stored at −80 °C until detection.All biomarkers were measured in duplicate by a single enzymelinked immunosorbent assay (ELISA) according to the instruction of manufacturer.α-Klotho ELISA kit was purchased from R & D Systems (DY5334-05).
2.5.One-Year Followup.After patients' discharge, we carried on a one-year followup to observe CKD's appearance and rehospitalization rate through telephone enquiry.Te reason for readmission included unstable angina, recurrent myocardial infarction and heart failure.
2.6.Statistical Analysis.All variables were tested for normal distribution by the Kolmogorov-Smirnov test and the descriptive statistics were summarized and displayed as the mean ± standard deviation or the median (25∼75%).Continuous variables and normal distribution data were 2 International Journal of Clinical Practice compared using independent sample t-tests.Categorical data were tested by the Chi-square test or Fisher's exact test.
During hospitalization, all data were compared between the AKI group and non-AKI group.Binary logistic regression was generated using the Enter mode, and the association variables were calculated (adjusted odds ratio) with a confdence interval (CI) of 95%.For the followup, we compared the α-Klotho levels of CKD group with those of non-CKD group, and the rehospitalization group with nonrehospitalization group.A p value <0.05 was defned statistical signifcantly.Discrimination was assessed based on the area under a receiver operating characteristic curve (AUROC).And, the AUROC analysis was conducted to calculate the cut-of values, sensitivity, and specifcity, and the cut-of points were estimated by determining the best Youden index.All analyses were performed by SPSS 23.0 software.

Results
3.1.Baseline Characteristics.155 patients were enrolled totally, 135 men and 20 women participated respectively in this study.Among them, 100 patients presented STEMI and 55 patients had USTEMI.Te demographic, clinical presentation, laboratory tests and therapies were listed in Table 1.Compared with the non-AKI group, patients with AKI contained more women and with higher incidence of CKD.
In cardiac manifestation, the AKI group had lower systolic pressure, faster heart rate, higher Killip class, more STEMI, more extensive anterior myocardial infarction, severer cardiac function, more cardiac complications including diferent types of arrhythmias and acute heart failure.In terms of laboratory tests, we found that it was consistent in renal function of both groups at admission, but during hospitalization AKI group developed a deterioration in renal function.Moreover, AKI group's myocardial necrosis was more serious, tending to TNI and CK-MB were obviously higher.And larger doses of diuretics, less use of angiotensinconverting enzyme inhibitors/angiotensin receptor blocker (ACEI/ARB), more device-assisted treatment were found in AKI groups.

Discussion
In this study, we aim to examine α-Klotho's early diagnostic value in diagnosis of AKI after AMI, as well as to predict efect of CKD and readmission.Te main fndings of our study can be summarized as follows: (1) α-Klotho is a superior biomarker than serum creatinine for AKI in AMI patients with eGFR ≥60 ml/min * 1.73 m 2 , (2) Higher α-Klotho levels at admission indicates greater morbidity of CKD, and (3) with numerous studies fnding α-Klotho decreased in AKI rodent models, our team frstly reported that α-Klotho increased signifcantly in AMI induced AKI patients, which deserves further research.
Development of AKI following AMI is regarded as an important complication in patients with AMI due to its enhancive in-hospital and long-term mortality and subsequent risk of CKD/ESRD.Previous observational studies showed the related risk factors of AKI after AMI, including use of hypertension, diabetes, CKD, hemodynamic instability, cardiogenic shock, decreased cardiac output, contrasts, and medicines, which was mostly consistent with the clinical data of our study [1,2,[19][20][21].In this study, the incidence of AKI was 17.4%, and an approximate quarter of patients with AKI died in hospital.It worth noting that even we enrolled patients with eGFR ≥60 ml/min * 1.73 m 2 , there still has an increased prevalence of CKD in AKI   International Journal of Clinical Practice group.Furthermore, AKI group tend to have more female, cardiac dysfunction and complications, less use of ACEI/ ARB, more use of loop diuretics, and supportive therapies.And, logistic regression model indicates extensive anterior myocardial infarction, Killip classifcation ≥2 grade, α-Klotho ≥516.9 pg/mL, eGFR (decrease per 10 ml/min- * 1.73 m 2 ), Hgb, and nonuse of ACEI/ARB are the risk factors of AKI after AMI.
According to AKI's defnition in 2012, it takes at least 48 hours to observe the dynamic variation of serum creatinine for AKI's diagnosis.For the acknowledgement of early detection of AKI may allow better therapy and potentially avoid its undesirable prognosis, we compared the precision and discriminative ability of serum creatinine and α-Klotho for AKI prediction after AMI at admission.Te results indicated that α-Klotho may be a more sensitive and reliable biomarker for AKI.Compared with serum creatinine, α-Klotho was signifcantly elevated in the AKI group of AMI on admission, when AKI had not developed.And, at this moment, there was no statistical diference in serum creatinine between the two groups.In addition, the comparison of other conventional risk factors included hemoglobin, blood pressure, eGFR, BNP, and LVEF of the predictive value for AKI after AMI were conducted.We found that, compared with traditional renal and other indicators, the factors represented cardiac function (SBP, LVEF, and BNP) might have better predictive value, with AUROC greater than 0.7.However, it was still not as good as α-Klotho.Te cut-of value of α-Klotho was 516.9 pg/mL (sensitivity, 0.778; specifcity, 0.694; Youden index, 0.472); and AUROC was 0.792.
Emerging evidence has revealed that α-Klotho defciency may be an early event in AKI rodent models, and a pathogenic factor that exacerbates acute kidney damage and contributes to long-term consequences [6][7][8][9].Studies in human and animal models have shown low levels of serum α-Klotho in CKD and ESRD, and treatment with α-Klotho could improve kidney damage in rodent models [6,7].Currently, few studies have been focused on α-Klotho expressions in human AKIs.Seibert et al. [22] have reported that serum α-Klotho was increased in AKI patients and progressively decreased in patients with CKD stage 1 to 5, and in this study, the etiology of AKI includes infection, parenchymal renal disease, acute tubular necrosis, prerenal kidney failure, cardiorenal syndrome (CRS), and other renal disease.Another research on renal α-Klotho expression determined by immunohistochemical staining showed that renal α-Klotho expression decreased signifcantly according to the severity of AKI, and that low expression was associated with a poor short-term outcome [23], which suggests that Klotho expression is opposite in circulation and kidney when AKI happens, manifested as high circulating Klotho and low organ Klotho expression.
AMI induced AKI belongs to type I CRS.Jerin et al. [24] found that α-Klotho levels of AKI group increased in few hours after cardiac surgery and decreased in 48 hours postoperatively.Qian et al. [25] discovered higher urine Klotho levels in AKI groups after cardiac surgery, which was associated with poor short-term renal prognosis.Tese results were consistent with our study on AMI related AKI.
Te possible reasons for the opposite results in animal and human researches could be that animal models of AKI were established by drugs or ischemia-reperfusion injury, resulting in recoverable kidney damage, while human AKIs might be relieved properly.And, there were various mechanisms of animal AKI models, which might explain the reason for the opposite clinical fndings in the present study.Terefore, we speculate that the elevated level of α-Klotho might be a negative feedback phenomenon of the body, suggesting that α-Klotho might be a protective factor indirectly.And, in an AMI rodent model, α-Klotho was found to inhibit the expression of proinfammatory cytokines in the peri-infarct regions and signifcantly attenuate the apoptosis and production of intracellular reactive oxygen species by myocardial ischemia/reperfusion injury [26].Further studies are needed on whether Klotho could be considered as a selfprotection biomarker during AKI after AMI.Dynamic changes of α-Klotho profles might be conducive to the potential hypothesis.
Te study also has its limitations.Firstly, the enrolled sample capacity was relatively small, and all patients were from a single center.Secondly, α-Klotho was only detected on admission, although it is more predictable for AKI, but we did not monitor the creatinine and α-Klotho to observe continuous changes.Tirdly, the biomarker needs to be proven in a general population without exclusion criteria, including who died in 48 h since it could not diagnose AKI according to current defnition.Since Klotho is defnitely declined in chronic renal failure patients, we excluded this part of the population in the study design stage and it is necessary to explore the dynamic evolutions of Klotho levels in AMI patients complicated with CKD 4-5 stages when AKI occurs.
In summary, the study provides sound evidence that Klotho could be a better biomarker for AKI in AMI patients with eGFR >60 ml/min * 1.73 m 2 .Elevated α-Klotho levels are related to the development of AKI during hospitalization and suggest a higher prevalence of CKD after discharge.On contrary to rodent experiments, whether the increased expression of Klotho is a self-protective factor secreted by AKI after AMI, still remains further study.

Table 1 :
Baseline characteristics in AMI patients with or without AKI.

Table 3 :
Te areas under the receiver operating characteristic curves for serum levels of α-Klotho and other conventional risk factors to predict AKI after AMI.

Table 4 :
Te binary logistic regression model for predicting AKI in AMI patients.