Urine klotho is a potential early biomarker for acute kidney injury and associated with poor renal outcome after cardiac surgery

Acute kidney injury (AKI) remains a highly prevalent and serious complication of cardiac surgery. AKI develops in up to 40% of patients who undergo cardiac surgery [1, 2]. Despite decades of research, the mortality of cardiac surgery-associated AKI (CSA-AKI) remains high, even for those patients whose renal function has completely recovered [3]. A major reason for the disappointing outcome is the scarcity of early biomarkers. Current diagnosis of AKI is made based on the changes of serum creatinine (SCr) and urine output, which lack sensitivity and reliability [4]. First, SCr reflects the loss of glomerular filtration function rather than the renal tubular lesions. Second, SCr is vulnerable to several nonrenal factors such as sex, muscle mass, diet and hemodynamic alterations.

Klotho is a multifunctional protein that includes transmembrane and soluble forms [5]. The latter is almost entirely derived from membrane-bound Klotho shedding and has been found in serum, cerebrospinal fluid and urine [6‐9]. Soluble Klotho functions as a hormone and plays a role of anti-oxidative stress [10], anti-apoptosis [11], and anti-fibrosis [12]. The renal tubular epithelial cells are the principal cells that contribute to Klotho synthesis and excretion [13, 14]. Klotho regulates transporters and ion channels through autocrine or paracrine to the urinary luminal side [9, 14]. Klotho in the urine can be derived from plasma and interstitium through transcytosis, and can also originate from the tubule through secretion rather than filtered across the glomerular barrier [9, 14].

To date, few studies have focused on the urine Klotho in prediction of AKI. In the present study, we firstly assessed the diagnostic and prognostic performance of urine Klotho in patients undergoing cardiac surgery.

To our knowledge, we are the first to examine the time course of uKlotho among adults undergoing cardiac surgery. We found that the elevated first postoperative uKlotho may be an early predicator for the occurrence of CSA-AKI. Further, urine Klotho in patients with AKI stage 2 and 3 was higher than that in patients with AKI stage 1 at the first postoperative time. Urine Klotho in AKI patients with incomplete recovery of renal function was significantly higher than that in patients with complete recovery at the first postoperative time.

AKI following cardiac surgery is a world health issue. Serum creatinine and urine output remains the gold standard for clinical diagnosis of AKI, although both were believed as unspecific markers for kidney injury. Novel biomarkers including NGAL, KIM-1 and interleukin-18 (IL-18) were evaluated for early diagnosis of AKI. Unlike Klotho that mainly expressed in kidney tubule, NGAL and IL-18 are nonspecific for kidney and are expressed in variety of tissues [4, 21]. Moreover, large, prospective, multicenter trails failed to show troponin-like diagnostic performance of plasma NGAL, urine NGAL, urine Kim-1, and urine IL-18 for AKI detecting with AUCs of less than 0.77 [22, 23]. Thus, efforts to validate potential markers are needed.

In animal experiments, Klotho deficiency in kidney tissues has been observed in both acute and chronic kidney injuries [24, 25]. More recently, a small study with 35 patients observed reduced serum Klotho in adults who developed AKI after cardiac surgery [26]. However, no significant difference of serum Klotho was found between patients with and without AKI on 1 day postoperatively and thereafter. Moreover, their study did not evaluate the change of urine Klotho and did not comment on the severity or renal outcome of AKI.

We firstly showed that uKlotho quickly increased as early as transferred to the ICU in patients who developed AKI later. Urine Klotho kept significantly higher in AKI patients than non-AKI patients until day 3 after surgery. The elevation of uKlotho occurred earlier than that of uNGAL demonstrated in the present cohorts and our previous data [27]. However, there was no difference in preoperative levels of uKlotho between patients with and without AKI. The early performance of urine Klotho may allow earlier detection of AKI and thus increase the success of therapeutic interventions.

To date, only two studies had examined uKlotho levels in patients or rodents with AKI. Isidro and his colleagues [28] examined the levels of uKlotho at 12 h post-cardiac surgery and showed a similar elevation of that in AKI patients (n = 15) compared with the healthy volunteers (n = 10). When comparing to the non-AKI (n = 15) group, the uKlotho levels in AKI patients increased but without significance. However, our results conflict with the reports from Hu et al. [24]. They found that urine Klotho levels in 17 AKI patients were significantly lower than that in 14 healthy controls by using immunoblotting assay. There are several reasons for the conflicted results. First, Hu did not describe the methods of pretreatment and the collecting time of urine samples. Klotho is unstable in urine and any additional freeze-thaw cycle decreases Klotho concentrations [29]. In the present study, urine samples were frozen at − 80 °C and were thawed for the first time for measurement of Klotho. Furthermore, uKlotho in patients with AKI was comparable with that in patients without AKI on 7 days post-surgery. Thus, a delayed collecting time may lead to different results. Second, the different causes of AKI may also lead to the contrary results. In the study of Hu et al., the causes of AKI are heterogeneous including sepsis, hypertension, CKD, nephrontoxin, pre-renal and others. Different measuring assays and fewer patients may also contribute the difference. Further researches with large sample size, multi-center and extensive time course are needed to clarify the change pattern of uKlotho.

Histologically, proximal tubular epithelial cells lose their brush border membrane as well as the Klotho protein expressed in the apical brush border, which may result in an acute increase of uKlotho at early AKI. With the progression of AKI, renal tubular epithelial cells were necrotic and exfoliated, accompanied with shedding of Klotho protein. This may contribute to the continuous elevation of uKlotho during AKI. We have previously showed similar phenomenon in the mouse model of AKI induced by renal ischemic-reperfusion injury [30]. Under these circumstances, the increase of uKlotho may indicate the severity of tubule injury. In support of this, we found that uKlotho was strongly associated with the occurrence of CSA-AKI and was significantly higher in AKI stage 2 and 3 than in stage 1.

Our study has several limitations. First, The present study is a single center study with a relatively small number of patients and short follow-up. Second, we did not compare the performance of predicting AKI between Klotho and other biomarkers besides NGAL. Third, the time points of uNGAL detection are insufficient. Additionally, the use of creatinine as a reference standard for biomarker assessment is imperfect for the known insensitivity and non-specificity.

In conclusion, our study implies that first postoperative uKlotho may be an early predictor for the occurrence of AKI among patients undergoing cardiac surgery. The first postoperative uKlotho may be associated with the severity and prognosis of AKI. This may help to early identify at-risk patients before the progression to overt kidney injury and implement early interventions.