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Abstract

Cardiovascular disease (CVD) remains one of the most common causes of morbidity and mortality in patients with chronic renal disease. It has been recently postulated that the loss or reduced levels of renalase in patients with chronic renal disease are, at least in part, responsible for elevated plasma catecholamine levels, which leads to increased CVD. Therefore, the aim of the present study was to evaluate whether renalase administration might serve as a therapeutic drug, decreasing the severity of CVD in 5/6 nephrectomized (Nx) rats. The current study was conducted on 30 male Wistar albino rats divided into the following groups: group I: sham-operated rats that received phosphate-buffered saline (PBS) subcutaneously (s.c.) for 4 weeks following sham operation, group II: rats in which 5/6 Nx was done and then the rats received PBS daily s.c. for 4 weeks following 5/6 Nx, and group III: rats in which 5/6 Nx was done and then the rats received recombinant renalase daily s.c. for 4 weeks following 5/6 Nx. 5/6 nephrectomy resulted in a significant increase in mean arterial pressure, left ventricular (LV)/body weight ratio, LV hydroxyproline concentration, plasma creatinine, blood urea nitrogen (BUN), and noradrenaline (NA) levels as well as significant decrease in LV papillary muscle developed tension in group II compared with the sham-operated group I. Administration of renalase to group III resulted in significant amelioration of all studied parameters with the exception of plasma creatinine and BUN which were not significantly different from nontreated group II. The results of the current study identify renalase as a new therapeutic modality that might modulate cardiac function and systemic blood pressure in renalase-deficient states like chronic renal disease.

Keywords

cardiovascular disease left ventricular hypertrophy hypertrophy renal

Introduction

The incidence of chronic kidney disease (CKD) and end-stage renal disease (ESRD) has increased worldwide.¹ Cardiovascular disease (CVD) remains one of the most common causes of morbidity and mortality in this patient group.² A number of causative conditions for CVD in these patients with renal disease have been identified, including traditional risk factors such as hypertension and hyperlipidemia. Additional risk factors such as increased oxidative stress and arterial calcification also appear to play a key role in the development of CVD in the patients with CKD.³

Another factor contributing to CVD in ESRD is activation of the renin–angiotensin–aldosterone system (RAAS). Thus, in the past, research into therapeutic strategies focused mainly on interventions targeting volume control and RAAS which contributed to slowing down the progression of renal disease.⁴

However, more recently several additional factors have been implicated in the pathogenesis of hypertension in renal disease, such as increased sympathetic nervous system activity.⁵ An elaborate identification and understanding of such factors is the key to successful treatment.

The recent identification of renalase, a novel flavin adenine dinucleotide-dependent amine oxidase that is secreted into the blood by the kidney, metabolizes circulating catecholamines and is deficient in CKD, provides a plausible theory for the increased catecholamines levels observed in CKD.⁶ Renalase circulates in the blood and has recently been discovered to modulate cardiac function and systemic blood pressure.⁷ It has been found that renalase is virtually undetectable in patients with ESRD.⁸

Thus, it can be postulated that the loss or reduced levels of renalase in patients with ESRD are, at least in part, responsible for elevated plasma catecholamine levels, which leads to increased CVD.

Previous studies have documented that the rats subjected to removal of ≈85% of kidney tissue (5/6 nephrectomy [Nx]) develop progressive CKD and many of the associated abnormalities, including increased sympathetic activation, hypertension, and left ventricular (LV) hypertrophy. The 5/6 Nx rat is considered an excellent animal model of CKD, and it has been reported that these rats develop severe blood renalase deficiency 2 to 3 weeks after surgery.⁶

Therefore, the aim of the present study was to evaluate whether renalase administration might serve as a therapeutic drug, decreasing the severity of CVD in 5/6 Nx rats.

Materials and Methods

The current study was conducted on 30 male Wistar albino rats weighing 200 to 250 g. Rats were fed a standard diet and acclimatized for 2 weeks before the experiments. The animal protocol was reviewed and approved by the Ethical Committee of Faculty of Medicine, Alexandria University. Rats were randomly divided into the following groups (each of 10 rats):

Group I: sham-operated rats that received phosphate-buffered saline (PBS) in a dose of 0.5 mg/kg body weight (BW) daily subcutaneously (s.c.) for 4 weeks following sham operation.

Group II: rats in which 5/6 Nx was done, according to the method described below, and then the rats received PBS in a dose of 0.5 mg/kg BW daily s.c. for 4 weeks following 5/6 Nx.

Group III: rats in which 5/6 Nx was done and then the rats received recombinant renalase (Novus Biologicals, Littleton, Colorado) in 0.5 mL PBS in a dose of 0.5 mg/kg BW⁹ daily s.c. for 4 weeks following 5/6 Nx. The dose of renalase was chosen based on the previous studies demonstrating the effectiveness of this dose in reducing significantly elevated blood pressure in rats.

5/6 Nephrectomy Procedure

The procedure was carried out according to the method of Ghosh et al.¹⁰ Briefly, animals were anesthetized with pentobarbitone in a dose of 40 mg/kg BW. A left flank incision was made and the left kidney was exposed. The renal artery was temporarily occluded, and the upper and lower thirds of the kidney were ligated and excised. Bleeding was controlled by compression, until it stopped. Thus, one third of the left kidney remained. The muscle and skin incisions were sutured with polypropylene suture. The animals were returned to the cages to recover. One week later, a right flank incision was made, the renal vessels and ureter were tied, and the right kidney was excised. Animals were returned to the cages to recover.

Biochemical Study of Blood

At the end of the experimental period, blood samples were collected from the retro-orbital venous plexus of the rat, in tubes containing EDTA. Tubes were immediately centrifuged at 3000g for 8 minutes for the separation of plasma that was stored at −20°C for the determination of plasma NA¹¹ (by enzyme-linked immunosorbent assay using CatCombi ELIZA-DLD Diagnostika, Hamburg, Germany), creatinine,¹² and blood urea nitrogen (BUN)¹³ (by colorimetric determination using kits supplied by Sigma Chemical Co, St Louis, Missouri).

Mean Arterial Blood Pressure Study

Rats were anesthetized with an intraperitoneal injection of pentobarbital in a dose of 40 mg/kg BW and were subjected to femoral artery catheterization for recording the mean arterial blood pressure (MABP) using a blood pressure transducer (Letica, PanLab, Spain), connecting the catheterized femoral artery with a mercury manometer.

Myocardial Structure Study

Rats were then killed by exsanguination. The beating heart was excised from the chest cavity and atria were excised, and the ventricles separated into LV muscle mass. The ratio of LV and BW (LV/BW) was calculated for each rat as a marker of cardiac hypertrophy.¹⁴

Mechanical Study of the Papillary Muscles

Contractility was studied in papillary muscle preparations that enable the assessment of the contractile status regardless of loading conditions, chamber geometry, and neurohormonal disorders. The left ventricle was opened from the septum to expose the papillary muscles that were isolated for determining the LV papillary muscle developed tension (DT). The LV papillary muscles were dissected free in a bath containing oxygenated modified Krebs bicarbonate buffer (mmol/L): NaCl 120, KCl 5.9, dextrose 5.5, NaHCO₃ 25, NaH₂PO₄ 1.2, MgCl₂ 1.2, and CaCl₂ 2.5. The ends of the muscle were grasped with spring clips, and the muscle was suspended vertically from an isometric force transducer (PanLab, Letica, Spain) in a tissue bath containing the Krebs-Henseleit solution. Field stimulation at 0.5 Hz was delivered through a pair of platinum wire electrodes placed parallel to the muscle. The muscle was allowed to stabilize for 1 to 2 hours, with a resting tension of approximately 1 g/mm² and attached to an isometric force transducer for determination of isometric force (DT). After the recordings were concluded, muscle length at L _max was measured and the muscle was weighed. The cross-sectional area ([SA], mm²) was calculated (mass/[length × density]), with the assumption of a density of 1.06 g/cm³. Developed tension of the papillary muscles was normalized for SA.¹⁵

Biochemical Study of Cardiac Tissue

The heart was then sampled for the determination of LV hydroxyproline (LV HPO) concentration as a measure of fibrosis.¹⁶ After the left ventricles had been dried for 24 hours, the specimens were hydrolyzed in 6N hydrogen chloride solution at 120°C. After resolution in a buffer at pH 7.0, p-dimethylaminobenzaldehyde (Ehrlich reagent) was added to form a complex with HPO. The concentration of HPO was measured by spectrophotometric analysis at a wavelength of 560 nm.

Statistical Analysis

Data were fed to the microcomputer program Statistical Package for Social Science SPSS version 16.0 (IBM, Armonk, New York). Results were expressed as a mean ± standard error. Tabulation and analysis of data were done using analysis of variance. Least significant difference was used to identify the significance of pairwise comparison of mean values among the groups. Statistically significant differences were assumed at P value less than or equal to .05.¹⁷

Results

Mortality rate: 3 rats died in group II and 1 rat died in group III, whereas no rat died in group I.

Body weight gain: a significant decrease in BW gain between 5/6 Nx and sham-operated groups was detected, whereas renalase-treated 5/6 Nx group did not show a significant difference in BW gain compared with sham-operated rats (data not shown).

Results of MABP, LV/BW, and DT of papillary muscles:

5/6 Nx resulted in a significant increase in MABP in group II (157.21 ± 8.45 mm Hg) compared with the sham-operated group I (100.98 ± 5.53 mm Hg). The LV/BW ratio was significantly increased in group II (8.58 ± 0.76 mg/g) compared with group I (4.68 ± 0.67 mg/g). Renalase-treated 5/6 Nx group showed a significant decrease in MABP level (131.45 ± 7.95) and in LV/BW ratio (6.34 ± 0.52) versus nontreated 5/6 Nx group. The isolated papillary muscles in group II exhibited a significant decrease in DT compared with the papillary muscle from normal sham-operated rats (2.87 ± 0.32 vs 4.59 ± 0.41 g/mm²). In renalase-treated groups, there was a significant increase in DT compared with group II (3.52 ± 0.37 vs 2.87 ± 0.32 g/mm²; Table 1).

Biochemical results: 5/6 Nx resulted in a significant increase in LV HPO concentration in group II (7.56 ± 0.39 mg/g wet tissue weight) compared with the sham-operated group I (4.37 ± 0.26 mg/g wet tissue weight). Plasma creatinine, BUN, and NA levels were significantly increased in 5/6 Nx compared with the sham-operated group I (2.98 ± 0.06 mg/dL, 85.67 ± 7.87 mg/dL, and 865.60 ± 25.18 pg/mL vs 0.24 ± 0.07 mg/dL, 27.25 ± 3.35 mg/dL, and 464.55 ± 11.37 pg/mL, respectively). Renalase-treated 5/6 Nx group showed a significant decrease in plasma NA level and LV HPO concentration (663.14 ± 25.43 pg/mL and 6.03 ± 0.59 mg/g wet tissue weight, respectively) versus nontreated 5/6 Nx group. However, renalase treatment did not result in significant change in plasma creatinine or in BUN (2.80 ± 0.07 mg/dL and 80.45 ± 8.21 mg/dL, respectively) compared with nontreated 5/6 Nx group (Table 2).

Table 1.

The MABP, LV/BW Ratio, and DT of Isolated LV Papillary Muscles (Mean ± SE) 4 Weeks Post-Renalase Administration in Rats

Groups	MABP (mm Hg)	LV/BW (mg/g)	DT (g/mm²)
Sham-operated (group I) n = 10	100.98 ± 5.53	4.68 ± 0.67	4.59 ± 0.41
5/6 Nx (group II) n = 7	157.21 ± 8.45^a	8.58 ± 0.76^a	2.87 ± 0.32^a
5/6 Nx and renalase (group III) n = 9	131.45 ± 7.95^a, ^b	6.34 ± 0.52^a,b	3.52 ± 0.37^a,b
F value	68.09^c	59.18^c	39.85^c
P	<.001	<.001	<.001

Abbreviations: SE, standard error; MABP, mean arterial blood pressure; LV, left ventricular; LV/BW, left ventricular and body weight; DT, developed tension; n, number of rats in each group.

^aValues are significant compared with sham-operated group (I).

^bValues are significant compared with 5/6 Nx group (II).

^cValues are statistically significant.

Table 2.

Plasma Creatinine, BUN and Noradrenaline Levels as well as LV HPO Concentration (Mean ± SE) 4 Weeks Post-Renalase Administration in Rats

Group	Plasma Creatinine (mg/dL)	Plasma BUN (mg/dL)	Plasma Noradrenaline (pg/mL)	LV HPO (mg/g wet tissue weight)
Sham-operated (group I) n = 10	0.24 ± 0.07	27.25 ± 3.35	464.55 ± 11.37	4.37 ± 0.26
5/6 Nx (group II) n = 7	2.98 ± 0.06^a	85.67 ± 7.87^a	865.60 ± 25.18^a	7.56 ± 0.39^a
5/6 Nx and renalase (group III) n = 9	2.80 ± 0.07^a	80.45 ± 8.21^a	663.14 ± 25.43^a,b	6.03 ± 0.59^a,b
F value	68.09^c	59.18^c	39.85^c	58.34^c
P	<.001	<.001	<.001	<.001

Abbreviations: SE, standard error; n, number of rats in each group; BUN, blood urea nitrogen; LV HPO, left ventricular hydroxyproline.

^aValues are significant compared with sham-operated group (I).

^bValues are significant compared with 5/6 Nx group (II).

^cValues are statistically significant.

Discussion

Renalase is a recently discovered amine oxidase that is secreted into the blood mainly by the kidney. It has been also detected but at lower level in the heart, skeletal muscle, and small intestine. Circulating renalase is inactive at baseline and is rapidly activated by catecholamines. Once activated renalase, in turn has been found to metabolize catecholamines. The kidney releases this protein into the bloodstream to regulate blood pressure.¹⁸ It has been recently reported that renalase levels are found to be low in patient with CKD and in partially nephrectomized rats¹⁹ indicating that the kidneys are the major contributor to circulating renalase.

Downregulation or knockout of renalase is associated with increased catecholamine levels, increased blood pressure, and higher susceptibility to ischemic myocardial damage.¹⁸ The results of the present study have shown that administration of renalase to 5/6 Nx rats resulted in a significant decrease in MABP compared with 5/6 Nx rats that were not injected with renalase; however, the values were still higher than those of the sham-operated group. The ability of renalase to decrease MABP could be explained by its ability to metabolize increased catecholamines as evidenced by a significant decrease in plasma NA levels. These findings are consistent with the results of Xu et al⁶ who found that renalase-treated animals showed a reduction in the blood pressure which was associated with decreased concentration of circulating catecholamines.

A number of studies have shown that plasma catecholamine levels are elevated in patients with ESRD^20,21 contributing to the pathogenesis of hypertension. Catecholamines are mainly metabolized by the enzymes l-monoamine oxidase and catechol-o-methyl transferase which are located intracellularly, so they degrade catecholamines after they have been taken up by the cells or before release from the nerve terminal. Unlike the classical amine oxidases, renalase has been postulated to metabolize circulating catecholamines.²² Thus, renalase deficiency in 5/6 Nx rats contributes to the increased concentration of catecholamines by a reduction in their degradation.⁶

A decrease in LV papillary muscle DT, an increase in LV/BW ratio, denoting cardiac hypertrophy, as well as an increase in LV HPO concentration and in plasma NA level have been demonstrated in the current study. Cardiac hypertrophy is frequently encountered in patients with CKD and is related to multiple factors including excess sympathetic activity.²³ Recently, a correlation was reported between plasma NA and LV mass in patients on hemodialysis.⁸ Sustained sympathetic activation can also lead to deterioration of cardiac function, as evidenced by a significant decrease in LV papillary muscle DT, through a variety of mechanisms including stimulation of cardiomyocyte apoptosis,²⁴ direct toxicity to cardiomyocytes,²⁵ and induction of ventricular dysrhythmias.²⁶

Modification of adrenergic activity by renalase could test for the hypothesis that high plasma norepinephrine is involved in cardiac hypertrophy and cardiovascular complications in patients with CKD. Supporting this is a recent study reporting that a functional missense polymorphism in renalase (Glu37Asp) is associated with cardiac hypertrophy.²⁷ In the present study, HW/BW ratio, LV HPO concentration, and plasma NA level of renalase-treated 5/6 Nx group were significantly lower than nontreated group but still higher than the control group. In addition, LV papillary muscle DT in renalase-treated group was significantly higher than nontreated group.

These findings indicate that renalase-injected rats had a lesser degree of cardiac hypertrophy and cardiac dysfunction than untreated rats and that renalase modifies the adrenergic activity. Thus, the amelioration of studied cardiac parameters in renalase-treated group could be explained by renalase-induced decrease in norepinephrine level as norepinephrine is reported to promote myocardial cell hypertrophy in vitro.²⁸ This is assumed based on the fact that not all agents that lower blood pressure reverse LV hypertrophy. Those that do include adrenergic blocking agents, calcium blockers, and angiotensin-converting enzyme inhibitors, whereas diuretics and smooth muscle vasodilators on their own are less effective, probably because they do not interfere with sympathetic reflexes and so lead to tachycardia and increase in cardiac output.²⁹

However, renalase had a partial effect in reduction of cardiac hypertrophy in the present study, which points to the multifactorial etiology of this event in patients with CKD. Increased level of norepinephrine may be the only factor responsible for cardiac hypertrophy in these patients. It has been pointed out that arterial rigidity is perhaps another important factor triggering LV hypertrophy in patients with CKD, which is due to high calcium phosphate production and endothelial dysfunction.³⁰

The results of the current study identify renalase as a new therapeutic modality that might modulate cardiac function and systemic blood pressure in renalase-deficient states like CKD. Recombinant forms of renalase may eventually serve to attenuate the CVD risk seen in patients with ESRD, among whom renalase levels are abnormally low.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Cardioprotective Effect of Renalase in 5/6 Nephrectomized Rats

Abstract

Keywords

Introduction

Materials and Methods

5/6 Nephrectomy Procedure

Biochemical Study of Blood

Mean Arterial Blood Pressure Study

Myocardial Structure Study

Mechanical Study of the Papillary Muscles

Biochemical Study of Cardiac Tissue

Statistical Analysis

Results

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References