*3.3. Analysis of Survival and MACE Occurrence*

Based on obtained data, specifically, renalase levels and correlations between renalase and occurrence of MACE and all-cause death in the CKD group, we first divided the CKD group into two subgroups with RNLS levels higher and lower than 25 μg/mL.

MACE occurred in 20 patients with CKD: 11 with elevated renalase levels (>25 μg/mL) and 9 with lower renalase levels (<25 μg/mL). A Mantel–Cox test *p <* 0.01 showed a significant difference in MACE occurrence between analyzed groups, the odds of which were significantly higher in the group with elevated renalase levels, as shown in Figure 3.

**Figure 3.** Kaplan Meier curve for MACE in groups with low and elevated renalase levels.

There was no difference in the occurrence of death between subgroups divided basing on the 25 μg/mL threshold.

When a higher threshold was assumed, and patients with CKD were divided into groups with levels of RNLS higher or lower than 30 μg/mL, not only did the higher probability of MACE remain significant, but a higher probability of all-cause death was also observed (but without statistical significance in log-rank test: *p =* 0.09), as presented in Figure 4.

In the HD group, in which a significant correlation between RNSL levels and all-cause death was observed, a Kaplan–Meier analysis of survival was not possible as the deaths were observed only in patients with renalase concentration higher than 30 μg/mL. At the same time, there was no significant difference in MACE occurrence in this subgroup.

**Figure 4.** Kaplan–Meier curve for all-cause death in groups with low and high renalase levels.

#### **4. Discussion**

CKD significantly contributes globally to both morbidity and mortality. Exact numbers are unknown, but screening programs in high-income countries have shown that more than 10% of adults have markers of kidney disease. The global burden of CKD in 2017 was estimated at 6975 million cases. Even though primary causes of CKD vary in different populations, the most common causes are hypertension and diabetes [5]. In Poland, an estimated 4.2 million people have CKD, and about 6500 yearly develop ESRD and require RRT [6]. The lives of patients afflicted with CKD, whether they deal with earlystage disease or end-stage renal disease (ESRD), are associated with high morbidity and increased healthcare utilization [7].

Patients with CKD have an established, higher CVD-related morbidity and mortality, and multiple analyses of studies have shown that the presence of CKD is independently associated with cardiovascular events, especially in groups with preexisting known risk factors [8–10]. The exponential increase in risk for cardiovascular mortality risk at low eGFR was shown in a large meta-analysis comprising ten cohorts with 266,975 patients. Hazard ratios at eGFRs of 60, 45 and 15 mL/min per 1.73 m<sup>2</sup> were 1.03, 1.38 and 3.11, respectively, compared to an eGFR of 95, after adjustment for albuminuria and cardiovascular risk factors [11]. A total of 7.6% of all CVD deaths (1.4 million globally in 2017) could be attributed to impaired kidney function [12].

CVD burden is also qualitatively different in CKD patients than those without renal pathology. Aside from atherosclerosis-related diseases (coronary artery disease, ischemic stroke and peripheral artery disease), other cardiac events become more and more prevalent as kidney function declines. Non-atherosclerotic CVD, arrhythmias, sudden cardiac deaths, arterial calcifications, valve calcifications and haemorrhagic strokes may be caused by uraemia, CKD-MBD, LVH or be dialysis-related events [13]. A growing trend of nonischemic CVD, to which CKD undoubtedly contributes, has been observed in developed countries [14].

Standard risk factors and assessment tools underperform in CKD patients, as their predictive risk is much lower than observed CVD events [15]. Moreover, we do not have particularly well-designed or widely used recalibration tools, which should at least account for eGFR and albuminuria [10]. Standard and widespread CVD risk assessment tools name CKD as an additional factor to be taken into consideration but do not clearly stratify the eventual risk. CVD risk equations can be of great importance to both patients and clinicians during decision-making. Even the classic symptoms of acute myocardial infarction are reported far less often by CKD patients than people without kidney pathology [13]. Riskaware and evidence-based medical interventions are needed as CKD prevalence continues to grow. Early action can delay disease progression and mitigate the fact that patients with ESRD consume a disproportionate share of health care resources.

Renalase has been linked to CKD and CVD in numerous studies. In a seminal paper describing renalase discovery, Xu et al. postulated a link to the cardiovascular system [16]; unfortunately, the enzymatic function of catecholamine degradation primarily proposed in the study, directly linking renalase to CVD, was disputed [17–21] and eventually refuted [22]. Different enzymatic activity was observed and described; renalase was found to be an oxidase/isomerase, using molecular oxygen to convert α-NAD(P)H into β-NAD(P)+, with hydrogen peroxide as a reaction byproduct [23], which also turned out to be incorrect [24]. Eventually, renalase has come to be described as an isomerase catalyzing rapid oxidation of 6DHNAD(P) and 2DHNAD(P) (which are inhibitory to metabolism dehydrogenases) to their active form—β-NAD(P)+ [22].

Additionally, since 2014, renalase's other function has been observed—that of a cytokine. It was found that a part of renalase protein – RP 220 (described previously [25], containing amino acids 220–239) as well as its variations RP-224 (amino acid 224–233) and RP-H220 (histidine-tagged RP 2020) – improved HK-2 cell line survival when treated with cisplatin, independently of amine oxidase activity by activation of the AKT and MAPK signaling pathways [26]. A receptor for renalase was found a year later. In 2015, PMCA4b (plasma membrane ATPase) was discovered to bind with renalase and activate the aforementioned signaling pathways [27], thus rekindling the search for the causative interplay of renalase and CVD, as PMCA4b is connected to both cardiac hypertrophy [28] and hypertension [29]. Aside from catalytic and cytokine mechanisms, yet another metabolic role came to light through further research in 2019: data collected in models of liver injury indicated that renalase might activate SIRT1 by elevating NAD+ levels [30].

Renalase affects redox balance in all functions and is beneficial in states of ischemia and/or reperfusion injury.

Its role in kidney disease has been shown in animal model interventional studies. Contrast-induced nephropathy in rat models was mitigated by supplying exogenous renalase by reducing oxidative stress, among other influences [31]. Renalase has also been found to reduce cisplatin-induced acute kidney injury by decreasing mitochondrial fission and reactive oxygen production [30].

Heart disease is also linked to redox state [32]. As such, studies have shown associations between higher renalase levels and unstable angina pectoris [33], lower ejection fraction in heart failure patients [34,35] and coronary microvascular dysfunction diagnosed with Rb-82 PET/CT imaging [36]. When performed in patients with stable angina, Percutaneous Coronary Intervention decreased serum renalase levels within a few days [37]. Extensive reviews of other observed relationships can be found elsewhere [38–40].

Even though we do not fully understand renalase's mechanism of action, as paradigm shifts seem to occur every few years, renalase displays a wide range of correlations and effects in CVD and both chronic kidney disease and acute kidney injury, which have been found in observational and interventional studies.

Other studies have also tried to assess renalase levels as a predictive factor. A study by Gluba-Brzózka et al. [41] positively correlated renalase with worsening CKD stages, as did markers (both biochemical and functional) of cardiovascular risk, but no observational follow-up was mentioned. Chang et al. [42] conducted a study in which patients (all male) were observed up to 10 years. A statistically significant correlation was found between renalase levels at baseline and both 1- and 5-year all-cause mortality. No correlation was found at 10 years. Using data from a Korean K-STAR cohort, Na et al. [43] observed higher all-cause mortality but not more frequent MACE in CKD patients with higher renalase levels. Another group studied patients recruited during scheduled PCI. In the group characterized by higher renalase levels, more endpoints (comprising myocardial infarction, stroke or death) were recorded during follow-up (median follow-up time lasted 4.1 years) [37]. In yet another recent study by Cerqueira et al., 40 pre-dialysis CKD patients were followed up for a mean period of 65 months. RNLS was associated with CKD progression, hospitalizations and all-cause mortality, but not with MACE occurrence [44]. Our study results are in line with these findings. Renalase levels found in studies mentioned in this paragraph are summarized in Table 6.

**Table 6.** Renalase levels in various groups in studies by other authors.


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