View of Association of Serum Renalase with Insulin Sensitivity Indices in ESRD Patients

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Association of Serum Renalase with Insulin Sensitivity Indices in ESRD Patients

R.Sudha^1*, C.K.Vijayasamundeeswari², S.Bharanidharan³

1- Associate Professor, Department of Biochemistry, Vinayaka Mission’s Kirupananda Variyar Medical College and Hospitals, Vinayaka Missions Research Foundation (Deemed to be University), Salem, Tamilnadu, India - 636308.

2 – Professor, Department of Biochemistry, Vinayaka Mission’s Kirupananda Variyar Medical College and Hospitals, Vinayaka Missions Research Foundation (Deemed to be University), Salem, Tamilnadu, India - 636308.

3 – Professor, Department of Paediatrics, Annapoorana Medical College and Hospitals, Salem, Tamilnadu, India - 636308.

1* Corresponding author Email: [email protected]

ABSTRACT

Introduction and Aim: The most common cause of chronic kidney disease (CKD) in India is diabetic nephropathy or in combination with hypertensive nephropathy leading to the most advanced stage, end-stage renal disease (ESRD). Insulin resistance (IR) is highly prevalent in chronic hemodialysis (CHD) patients and is associated with poor cardiovascular outcomes. Renalase, a recently discovered monoamine oxidase secreted by the kidney metabolises circulating catecholamines and is likely to regulate blood pressure. Recent data have shown increased serum renalase levels in type 2 diabetic patients with renal dysfunction. So, the study was designed to compare serum Renalase levels between healthy controls, ESRD patients with and without diabetes and to assess the relationship of serum renalase with insulin sensitivity indices in ESRD patients.

Methodology: The study was conducted on 120 subjects comprising of three groups. Group 1 includes 40 healthy controls, group 2, 40 ESRD patients without Diabetes Mellitus and Group 3, 40 ESRD patients with Diabetes Mellitus(DM). All the study subjects were analysed for fasting blood glucose, renalase, urea, creatinine, lipid profile, HbA1c, and Insulin. HOMA IR, QUICKI and eGFR were calculated. Appropriate statistical analysis was done.

Results: The mean serum renalase levels of group 1, group 2 and group 3 were 43.20 ± 22.39 ng/ml, 67.56 ± 34.18 ng/ml and 87.77 ± 38.16 ng/ml respectively which were significantly different from each other(p- value<0.05). In ESRD patients with Diabetes, serum renalase had a significant positive correlation with Fasting blood glucose, HbA1c, Urea, Creatinine, and HOMA IR (r=0.34, 0.41, 0.25, 0.53, 0.34 respectively, p- value<0.05). Serum Renalase had a significant negative correlation with HDL-Cholesterol, QUICKI and eGFR (r= -0.26, -0.30, -0.26) (p-value<0.05).

Conclusion: Serum renalase levels were significantly increased in ESRD patients with DM and had a significant association with fasting blood glucose, HbA1c, HOMA IR, QUICKI, urea, creatinine and eGFR in ESRD patients with Diabetes..

Keywords:

Serum Renalase, End stage Renal Disease, Diabetes Mellitus, Fasting blood glucose, HOMA IR, QUICKI

1. Introduction

End stage renal disease is a growing public health problem globally because of its huge social, financial and individual burden. It is well established that diabetic nephropathy is now the leading cause and or probably with hypertensive nephropathy are the most common causes of ESRD.

ESRD patients are known to be insulin resistant (1,2) and have multiple cardiovascular risk factors, advanced atherosclerosis and an increased risk of cardiovascular mortality. (3-5) Many prospective studies have shown that the presence of diabetes is associated with an increased risk of ESRD of both diabetic and non-diabetic origin. (6-8) A number of methods are widely used in epidemiologic studies and in clinical setting to quantify insulin resistance in general population and ESRD population. The hyperinsulinemic euglycemic glucose clamp (HEGC) technique is the gold standard for measuring insulin sensitivity (IS). (9) More practical and popular methods are homeostatic model assessment of insulin resistance (HOMA-IR) and quantitative insulin

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sensitivity check index (QUICKI). Studies have emphasized the importance of, unravelling the precise molecular mechanisms responsible for the pathogenesis of CKD-related insulin resistance and identification of novel therapeutic targets aimed at reducing renal and perhaps cardiovascular damage in this condition. (10)

Increase in the level of catecholamines have been reported in cardiovascular diseases. Renalase is a recently identified monoamine oxidase enzyme which is involved in the degradation of catecholamines and also in the regulation of blood pressure. (11, 12) Recent studies have indicated that circulating renalase might be a predictor of mortality and adverse renal outcomes in patients with CKD (13,14) and support the measurement of renalase in patients with USAP and MetS for a more specific risk assessment. (15) The current study was performed to determine whether renalase was associated with insulin sensitivity indices in ESRD population.

2. Methodology:

A cross sectional study was conducted on 120 subjects comprising of three groups. Group 1 consists of 40 healthy controls, group 2, 40 ESRD patients without Diabetes Mellitus and Group 3, 40 ESRD patients with Diabetes Mellitus. All the study subjects were age and gender matched.

The inclusion criteria for group 2 and 3 were, age more than 18 years and patients on maintenance hemodialysis (3 times weekly) for more than a year. Patients with acute infections, known cardiac diseases, hepatic diseases, endocrinopathies were excluded. Group 1 included apparently healthy individuals over 18 years, not under any medication. Institutional ethical clearance was obtained before the study and informed consent was obtained from all the study subjects. For all the study subjects, details of age, sex, symptoms and aetiology of ESRD, history of medication and duration of hemodialysis were noted. Measurements of systolic and diastolic blood pressure were recorded for all the subjects.

Fasting blood samples were collected for all the study subjects immediately before the HD procedure and transferred to appropriate containers for further analysis. All the samples were analysed for glucose, urea, creatinine and lipid profile using a fully automated analyser, Erba EM200. HbA1c was estimated by Immunoturbidimetry method and Insulin by ELISA. LDL-C was calculated by the Friedwald formula. Homeostatic model assessment (HOMA-IR) and Quantitative insulin sensitivity check index (QUICKI), were used for assessing β-cell function and insulin resistance (IR) from fasting glucose and insulin concentrations. eGFR was calculated using the MDRD formula.

The Insulin sensitivity indices were measured by using the following formulae. Homeostasis model assessment (HOMA-IR)(16), insulin (µU/ml) X glucose (mg/dl)/405 and Quantitative insulin sensitivity check index (QUICKI)(17), 1/(log glucose [mg/dl] + log insulin [µU/ml]). The reference values for insulin resistance in the general population (7,16) are HOMA, 2.6 and QUICKI, 0.33. (16) Serum renalase was measured by ELISA method (Wuhan, China) and presented as ng/ml. The sensitivity of the renalase assay was <0.469ng/ml and the intra and inter assay coefficients were <8% and <10% respectively.

Statistical analysis: SPSS software was used for all the statistical analysis. The results were presented as Mean ± Standard deviation. Student’s t-test was applied to estimate the differences between the groups. Pearson’s correlation analysis was used to test the association of renalase with other parameters in group 2 and 3. A p-value of less than 0.05 was considered as significant.

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3. Results:

The data of 80 hemodialysis patients and 40 healthy controls were analysed. Of 120 study subjects, 45% were women and 55 % were men. The mean age of controls, ESRD patients with and without diabetes were 52.7±13.32yrs, 54.03±13.43yrs and 57.35±12.64yrs. General and biochemical characteristics of the study subjects are given in Table 1. Table 2 depicts the glycaemic status and lipid profile of the study subjects. The groups did not differ in terms of age and gender.

Correlation of serum renalase levels with other parameters in Group 2 and Group 3 subjects are depicted in Table 3 and Table 4 respectively.

Table 1. General and Biochemical characteristics of study subjects Variable Group 1

(M ± SD)

Group 2 (M ± SD)

Group 3 (M ± SD)

p-value

Number(n) 40 40 40 -

Age (yrs) 52.7±13.32 54.03±13.43 57.35±12.64 0.26 SBP (mm/Hg) 123.63±9.41 159.25±17.45^# 160.50±17.09^# 0.75 DBP (mm/Hg) 82±4.63 90.50±8.15^# 93±9.15^# 0.20 Dialysis

Duration(M)

- 27.15±17.72 25.95±15.92 0.75

Renalase (ng/ml) 43.20 ± 22.39 67.56±34.18^#$ 87.76±38.16^#$ 0.04 Urea (mg/dl) 17.38±3.67 98.03±20.65^# 91.39±27.63^# 0.21 Creatinine (mg/dl) 0.85±0.10 7.10±2.61^# 7.46±2.45^# 0.52 eGFR 89.19±11.02 9.22±3.14^# 8.18±4.10^# 0.21

# - When compared to Group 1; $ - When compared to Group 2

Table 2. Glycaemic status and Lipid profile of study subjects

Parameter Group 1

(M±SD)

Group 2 (M±SD) Group 3 (M±SD) P-value

FBG(mg/dl) 97.96±13.93 94.03±11.63 169.50±40.76^#$ 0.000

HbA1c 4.4±0.5 5.13±0.41 7.84±1.33^#$ 0.000

Insulin(μU/ml) - 8.70±9.59 17.11±8.40^$ 0.000

HOMA IR - 1.98±2.04 7.55±4.50^$ 0.000

QUICKI - 0.37±0.04 0.31±0.03^$ 0.000

Total

cholesterol(mg/dl)

181.94±30.09 221.15±42.17^#$ 196.75±29.57^#$ 0.004

Triglycerides (mg/dl) 151.46±72.82 150.43±32.87 142.95±25.26 0.26 HDL(mg/dl) 45.28±6.49 46.55±4.65 46.55±9.88 1 LDL(mg/dl) 106.25±27.10 144.52±45.52 121.61±25.42^$ 0.007 VLDL(mg/dl) 24.53±4.25 30.09±0.57 28.59±5.05 0.26

# - When compared to Group 1; $ - When compared to Group 2

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Table 3. Correlation of Renalase with other parameters of Group 2 Variable r-value p-value

SBP 0.12 > 0.05

DBP 0.09 > 0.05

Urea 0.66 < 0.05*

Creatinine 0.26 < 0.05*

eGFR -0.23 < 0.05*

FBG 0.52 < 0.05*

HbA1c 0.25 < 0.05*

Insulin -0.25 < 0.05*

HOMA IR -0.22 > 0.05

QUICKI 0.02 > 0.05

Total Cholesterol -0.06 > 0.05 Triacylglycerol 0.43 < 0.05*

HDL 0.24 > 0.05

LDL -0.14 > 0.05

VLDL 0.43 < 0.05*

* Statistically significant

Table 4. Correlation of Renalase with other parameters of Group 3 Variable r-value p-value

SBP 0.22 > 0.05

DBP 0.54 < 0.05*

Urea 0.25 < 0.05*

Creatinine 0.53 < 0.05*

eGFR -0.25 < 0.05*

FBG 0.34 < 0.05*

HbA1c 0.41 < 0.05*

Insulin -0.15 > 0.05

HOMA IR 0.34 < 0.05*

QUICKI -0.30 < 0.05*

Total Cholesterol -0.03 > 0.05 Triacylglycerol 0.13 > 0.05

HDL -0.26 < 0.05*

LDL 0.04 > 0.05

VLDL 0.13 > 0.05

* Statistically significant

4. Discussion:

Insulin resistance(IR) is common and an early metabolic alteration in hemodialysis (HD) patients and use of indices like HOMA-IR and QUICKI may allow better detection of alterations in insulin sensitivity in HD patients. IR is also an independent predictor of cardiovascular mortality in ESRD [18] and is increasingly recognized as a non-traditional risk factor. (19, 20) Renalase is a novel protein likely to be involved in the regulation of blood pressure. The present study

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investigated the association of renalase with insulin sensitivity indices in ESRD patients with and without DM.

The results of the study showed that serum renalase levels in ESRD patients were significantly increased when compared to healthy controls. Recent studies have demonstrated that renalase levels are increased in CKD patients and few studies have related it to declining renal function.

(21-24) Elevated circulating renalase levels in dialysis patients may be related to kidney function and activation of the sympathetic nervous system detected in these patients. (11,25) Serum renalase levels were significantly elevated in group 3 patients when compared to group 2 patients.

Concentration of renalase in DM patients was similar to the range reported by Serwin NM et al in a study on 49 diabetic Nephropathy patients. (26)

In ESRD patients without diabetes, Serum renalase had a significant positive correlation with urea, creatinine, fasting blood glucose, HbA1c, triacylglycerol & VLDL and a significant negative correlation with eGFR and Insulin. In ESRD patients with DM, serum renalase had a significant positive correlation with diastolic blood pressure, urea, creatinine, fasting blood glucose, HbA1c & HOMA IR and a significant negative correlation with eGFR, QUICKI &

HDL. These findings confirm the presence of strong association of renalase with renal function and also the glycemic status of the ESRD patients.

Prevalence of IR in dialysis patients is 67% as compared to 31.6% in Sit et al(27) and 19.4% in Atwlin et al.(28) Even though Euglycemic clamp technique is the more reliable indicator of IR in HD patients, because of the laboriousness of the technique, most epidemiological and clinical studies are based on HOMA IR.(10) But Oterdoom and associates have shown that all investigated insulin resistance indices in their study were valid estimates of IR in the long term stable renal transplant population.(29) Association of renalase with IR has been suggested by the positive correlation with FBG, HbA1c, HOMA IR and negative correlation with QUICKI in Group 3 patients. These results suggest the association of renalase with DM which may be in its occurrence or the course of the disease as we could find the association in ESRD patients without Diabetes also. The findings need to be confirmed by longitudinal studies in a large population.

Wang et al have shown that the levels of serum renalase and R/C ratio of T2DM patients were significantly higher than those of healthy subjects and appeared correlated with changes in blood pressure, glomerular filtration rate and IR.(30) Eman W Gober and Hoda A El Attar have shown that serum levels of renalase are increased in type 2 diabetic patients with renal dysfunction and renalase levels may be increased to compensate for the increase in dopamine level.(31)

Despite many recent studies on the renalase concentration and its association with diseases, Renalase is still a very enigmatic molecule, with attributed variety of properties, including anti- inflammatory, cytokine-like activity, and enzymatic activity involved in the regulation of catecholamine levels in blood, and therefore blood pressure.(26) Hence, addressing the assessment and treatment of IR and CVD risks along with renalase in clinical practice could help with improving the hemodialysis outcomes.

5. Conclusion:

Serum renalase levels were elevated in ESRD patients especially in patients with DM. Serum renalase had an association with renal function parameters like urea, creatinine and eGFR and also was associated with insulin resistance indices like HOMA IR and QUICKI. Renalase might possibly play a role in insulin resistance which is multifactorial, thereby targeting renalase can be useful in the reduction of cardiovascular damage in ESRD patients.

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