**1. Introduction**

Renalase (RNLS) is a small flavoprotein produced mainly by the kidney. However, the latest investigations show that RNLS might be an "organolase", as the *RNLS* gene is expressed in many other cells and tissues, including the nervous system, endocrinal and digestive tract organs, lungs or heart in humans and some other mammals [1]. RNLS shows both intracellular and extracellular activity. Intracellular RNLS acts as an enzyme that, in the presence of a FAD cofactor, oxidizes 2- and 6- DHNAD(P) to β-NAD(P)+, its biologically active form. This action prevents toxicity resulting from the inhibition of many β-NAD(P)+ dependent enzymes and reactions. Additionally, extracellular renalase and RP-200 and RP-220 peptides, which are fragments of the protein, activate some signaling pathways, including Akt and MAP kinases, therefore promoting cell survival. This activity is mediated by the binding of renalase to its recently discovered receptor–plasma membrane Ca2+-ATPase-4b (PMCA4b) [2].

Despite reported discrepancies in observed serum renalase levels in humans, most analyses indicate that serum RNLS levels significantly increase in people with chronic kidney disease (CKD). As in the case of many other markers, this relationship would demonstrate the usefulness of the RNLS concentration assessment in the diagnosis and prognosis of kidney diseases and accompanying disorders. Seeing that CVD is the most significant contributor to mortality of CKD patients, we sought to answer whether RNLS could be a predictive factor for CVD in CKD.

## **2. Materials and Methods**

#### *2.1. Study Design*

One hundred twenty people (aged 40–79) were enrolled into the study. We divided 90 CKD patients into subgroups, each consisting of 30 participants: CKD stage III (CKD III), CKD stage IV (CKD IV), CKD stage 5D who were hemodialyzed (HD) and 30 adults (15 women and 15 men) with no signs of chronic kidney disease (control). Both groups were signed up for the study during treatment at a Nephrology Ward and its Admissions Department, Outpatient Clinic and Dialysis Centre.

#### *2.2. Material*

To obtain blood serum, blood was drawn into test tubes (S-Monovette, Sarstedt, Germany) and left for 30 min at room temperature. The tubes were then centrifuged (10 min, 1000× *g*, room temperature). The obtained material was then transferred to separate tubes and frozen at −80 ◦C until renalase levels were measured. eGFR, HDL, LDL and TG levels were assessed in an analytical laboratory as soon as possible after sample collection.

#### *2.3. Methods*

eGFR was calculated using the CKD-EPI Equation (1), where Scr is serum creatinine (mg/dL), κ is 0.7 for females and 0.9 for males, α is −0.329 for females and −0.411 for males, min indicates the minimum of Scr/κ or 1 and max indicates the maximum of Scr/κ or 1) [3]. Renalase levels were measured in blood serum using an ELISA kit (Cloud-Clone Corp, Houston, TX, USA). Biochemical parameters in serum were evaluated using routine laboratory methods.

$$cGFR = 141 \times \min\left(\frac{Scr}{\kappa}, 1\right)^{\alpha} \times \max\left(\frac{Scr}{\kappa}, 1\right)^{-1.209} \times 0.993^{\text{age}} \times 1.018 \left[\text{if female}\right] \times 1.159 \left[\text{if black}\right] \tag{1}$$

Cardiovascular risk (CV) was calculated using the ASCVD (atherosclerotic cardiovascular risk) algorithm, which includes: age, sex, race, systolic and diastolic blood pressure, total cholesterol and HDL/LDL fractions, diabetes, smoking and hypertension [4]. Participants with acute kidney injury or a history of kidney transplantation were excluded from the study. All patients have given written informed consent.

Follow-up was administered by means of a questionnaire during an outpatient clinic visit, dialysis treatment or a telephone call. Mean follow-up time was 17.3 months. Two patients were lost to follow-up—one patient in control and one in the CKD IV group. In the follow-up, we asked about the first major adverse cardiovascular event (MACE). We defined MACE as an occurrence of either ischemic stroke, acute coronary syndrome, hospitalization due to heart failure or death attributed to a cardiac event. We also looked for all-cause mortality and a need to start renal replacement therapy (RRT).

#### *2.4. Statistical Analysis*

Obtained data were evaluated using Statistica 13.0 software (StatSoft, Tulsa OK, USA). Since almost all parameters had distributions different than normal, non-parametric tests were used. Differences between two groups (control and CKD) were evaluated using the Mann–Whitney U test. Differences between analyzed parameters in subgroups (control, CKD III, CKD IV, HD) were assessed using Kruskal–Wallis ANOVA with post-hoc Dunn's test. Correlations were evaluated using Spearman's Rank Correlation Coefficient. The analysis of the occurrence of MACE and overall survival was performed using the Kaplan– Meier method. The significance level was set at α = 0.05.
