Activities of Fetuin-A protein in type-2 diabetic nephropathy patients

Abstract

2 ABSTRACT Background: Diabetic nephropathy (DN) is a serious complication of type 1 and type 2 diabetes mellitus (T2DM). It is also known as diabetic kidney disease (DKD). It is characterized by albuminuria and decreased eGFR. Excess blood sugar leads to damage of endothelial cells, renal glomerular cells along with central and peripheral nervous system involvement.. C-reactive protein (CRP) is a systemic inflammation marker that has been revealed to be correlated with DN development. Based on albuminuria and eGFR, diabetic nephropathy is divided into 5 grades/stages by KDIGO (Kidney Disease Improving Global Outcomes) guidelines. It is a terminal disease requiring early diagnostic markers to protect renal function and individual life. Many studies have shown CRP levels increased in DN cases, showing its relation to proportional rise as the disease progress. Traditional biomarkers like serum creatinine, eGFR, albuminuria are used routinely. They have limitations in detecting early renal changes as they are influenced by age, sex, and muscle mass. Fetuin-A is an acute phase protein, inhibits receptor tyrosine kinase leading to insulin insensitivity. The Fetuin-A gene (Thr256Ser) plays a important role in Diabetes and its complications. Free fatty acids have a role in glucose intolerance resulting in diabetes which is reported by many authors. Studies have demonstrated high serum Fetuin-A levels and free fatty acids along with the elevated urinary Fetuin-A levels as early predictor of diabetic nephropathy in comparison to the usage of the traditional biomarkers like creatinine, albuminuria. Therefore, study was done to determine how Fetuin-A and its gene relate to the severity of various diabetic nephropathy stages. Aim: To estimate serum Fetuin-A levels and assess the severity of diabetic nephropathy3 Objectives: This study was under taken to estimate and compare the glycemic, renal parameters, CRP and lipid profile in controls and different stages DN cases. To estimate and compare the serum Fetuin-A, free fatty acid and urinary Fetuin-A levels in controls and various stages of diabetic nephropathy. To study the polymorphism of Fetuin-A gene in diabetic nephropathy cases. To find the best cut-off value by ROC curve analysis of these parameters for early diagnosis of diabetic nephropathy and prevent the further complications. Methods: This is a hospital based comparative study, done at medicine department, HSK hospital, in Bagalkot, Karnataka. Approval for the study was taken from institution ethical committee. Consent was taken by study participants. Confidentiality of the participants was maintained as per Helsinki declaration. 40 healthy controls and 40 type 2 diabetic nephropathy cases in each first 3 stages and 19 cases in 4th stage of diabetic nephropathy were selected between the age group of 35-65 yrs based on KDIGO guidelines for nephropathy. Serum FBS, PPBS levels were estimated by spectrophotometric method, HbA1c by HPLC, fasting insulin by CLIA, HOMA-IR by formula. Serum urea, creatinine, uric acid, urine microalbumin were estimated by spectrophotometric method, eGFR by MDRD equation. Serum TC,TGL, HDL-C, and VLDL-C were estimated by spectrophotometric method LDL-C by Friedwald formula. Serum CRP is estimated by latex turbidometric method. Serum and urinary Fetuin-A, Serum free fatty acids were estimated by ELISA method. Fetuin-A gene polymorphism study was done. Statistical analysis was done using SPSS software version 19.stages of DN progressed. Serum CRP also showed significant increase in all the stages of diabetic nephropathy compared to controls (p<0.001). Serum lipid profile showed increased levels in all the stages of DN compared to controls except the HDL-C which decreased as the stages of DN progressed (p<0.001). Serum Fetuin-A level was significantly increased in first 2 stages (p=0.003) and decreased in 3rd and 4th stages of diabetic nephropathy indicating the urinary loss of this protein. Urinary Fetuin-A was significantly increased (p=0.001) in all the stages of diabetic nephropathy. There was significant gene polymorphism noted with change in the frequency of G allele (G>C) which denotes the alteration of serum Fetuin-A levels in diabetic nephropathy cases. Serum free fatty acids also significantly increased in all the stages of diabetic nephropathy (p=0.000). Best cut-off value of serum Fetuin-A, Urinary Fetuin-A and serum free fatty acids were 48.21ng/ml, 41.28ng/ml and 395.4mmol/L respectively and area under the curve 0.802, 0.947 and 0.979. Conclusion: Serum Fetuin-A levels were significantly increased in first two stages of diabetic nephropathy but decreased in 3rd and 4th stages, whereas urinary Fetuin-A levels increased in all the stages compared to controls. There was significant positive correlation of serum and urinary Fetuin-A levels with parameters like urea, creatinine, microalbuminuria, and CRP and lipid profile observed in cases. eGFR was decreased in all the stages of diabetic nephropathy cases compared to controls, showed negative correlation with serum Fetuin-A, FFA and urinary Fetuin-A levels.Gene polymorphism showed missense mutation for the Fetuin-A gene with the 100% frequency of G allele which can be one of the factor affecting the concentration of Fetuin-A in diabetic nephropathy. Cut-off values of serum and urinary Fetuin-A, serum free fatty acids can beused in predicting the severity of diabetic nephropathy compared to other routine biomarkers of DN