Correlation of microalbuminuria with glycemic status in diabetic nephropathy

Abstract               Diabetes mellitus is the most common endocrine disorder and has become one of the most challenging health problems of the 21st century. Diabetic patients have increased risk of developing both microvascular and macrovascular complications. Kidney damage caused by diabetic nephropathy, one of the microvascular complications is the leading cause of mortality in diabetic patients. Microalbuminuria is the earliest manifestation of diabetic nephropathy and is a predictor of progressive renal damage. Hence the study has been designed to evaluate microalbuminuria and glycated hemoglobin levels (HbA1c) and to assess the correlation of microalbuminuria with glycemic control and markers of renal dysfunction (urea and creatinine) and hypertension in diabetic nephropathy patients. Study involved 100 participants of which 50 were age and sex matched healthy controls and 50 were diagnosed to have diabetic nephropathy. Detailed medical history including duration of diabetes was taken. Blood samples were drawn and analyzed for fasting blood sugar (FBS), postprandial blood sugar (PPBS), blood urea, serum creatinine and glycated hemoglobin levels and an early morning urine sample was collected for microalbuminuria levels. Blood pressure both systolic and diastolic was recorded in cases and controls. Statistical analysis was done using Student ‘t’ test. Pearson correlation was done to establish the relation between the study variables. Statistically significant increase in levels of FBS, PPBS, blood urea, serum creatinine, glycated hemoglobin, systolic and diastolic blood pressure and urinary microalbumin was observed in cases compared to controls (p<0.001). A significant large positive correlation was found between urinary microalbumin levels and glycemic status in diabetic nephropathy patients.

Key Word: Diabetes mellitus, Diabetic nephropathy, Glycated hemoglobin, Microalbuminuria.

INTRODUCTION

Diabetes mellitus is the most common endocrine disorder and is characterized by metabolic abnormalities due to relative or absolute deficiency of insulin or insulin resistance resulting in hyperglycemia and is associated with development of both microvascular and macrovascular complications. Diabetes mellitus is not a single disease entity rather a group of metabolic disorders sharing the common underlying feature of hyperglycemia¹. Globally, it is estimated that 415 million people had diabetes in 2015 which is expected to reach 642 million by 2040. This global pandemic principally involves type 2 diabetes mellitus². Diabetes mellitus is associated with microvascular complications including retinopathy, neuropathy and nephropathy. It also increases the risk of stroke, myocardial infarction and peripheral vascular diseases. Diabetic nephropathy is one of the major debilitating microvascular complications of diabetes that leads to end stage renal disease. Diabetic nephropathy also called as Kimmelsteil Wilson syndrome is characterized by a progressive increase in the excretion of protein particularly albumin, an early and continuing rise in blood pressure and a late decline in glomerular filtration rate (GFR) resulting in end stage renal disease³. Hyperglycemia through increased mitochondrial production of reactive oxygen species (ROS) mediate the adverse effects through various metabolic pathways like increased activity of polyol pathway, increased intracellular advanced glycation end product (AGEs), increased flux through hexosamine pathway and activation of protein kinase C^4,5. The earliest functional abnormality in diabetic kidney disease is renal hypertrophy associated with raised GFR related to poor glycemic control and appears soon after diagnosis. When kidney gets damaged, the afferent arteriole gets dialated to a greater extent than the efferent glomerular arteriole which damages the glomerular capillaries and increases the intraglomerular filtration pressure leading to increased shearing forces which contribute to mesangial cell hypertrophy and increased secretion of extracellular mesangial matrix material resulting in glomerular sclerosi⁶. The initial structural lesion in the glomerulus is thickening of the glomerular basement membrane and other associated changes result in the disruption of protein cross linkages leading to progressive leakage of large molecules (particularly protein) into urine resulting in proteinuria⁶. Microalbuminuria is defined as urinary excretion of 30-300 mg/day of albumin. Microalbuminuria is not a different form or fraction of albumin but just a very small amount of albumin. Albumin is often the first protein to excrete in the urine after the kidney is damaged. Detection of microalbuminuria is the most important screening tool for early phase of diabetic renal disease called incipient diabetic nephropathy. Urine is negative for standard dipsticks at this stage and must be screened by the use of sensitive tests. If protein is detectable on a standard dipstick suggestive of macroalbuminuria >300 mg/day. Glycated hemoglobin is a standard measure of severity of diabetes mellitus and gives an idea about long term glycemic control. Early diagnosis and treatment delays or prevents the onset of diabetic nephropathy and progression of diabetic kidney disease. Hence the study was designed to estimate urinary microalbumin levels and glycated hemoglobin levels and to know about the correlation of microalbuminuria with glycated hemoglobin in diabetic nephropathy patients.

MATERIALS AND METHODS

Clinically diagnosed 50 diabetic nephropathy cases attending medicine department in Adichunchanagiri hospital and research centre, B G Nagar, Mandya were included in the study. They were tested for urinary albumin by albustix method and albustix negative patients were included. Age and sex matched 50 healthy individuals were taken as control group. The study was approved by ethical and research committee of the institution. Informed consent was taken from both cases and healthy controls. Patients suffering from acute and chronic inflammatory conditions, other metabolic conditions like ketoacidosis, cerebrovascular accidents, preeclamptic patients, pre-existing chronic kidney disease, chronic glomerulonephritis, nephrotic syndrome and urinary albumin positive subjects were excluded from the study. Blood sample was drawn and urine sample was collected from both diabetic nephropathy patients and controls. 2 ml of blood in both fasting and postprandial state was collected with an anticoagulant sodium fluoride for the estimation of blood glucose (Glucose oxidase method). 2 ml of blood was taken without anticoagulant and allowed to clot and serum was separated and used for the estimation of blood urea (Glutamate dehydrogenase (GLDH) Urease method) and serum creatinine (Jaffe’s method), Whole blood was collected in heparin coated tube for the estimation of glycated hemoglobin (Affinity Chromatography). Early morning urine sample was collected under aseptic precautions for the estimation of microalbuminuria (Immunoturbidimetry). Systolic and diastolic blood pressure was recorded in both cases and controls. By using Student ‘t’ test statistical analysis was done and statistical significance was compared between case and controls. The statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc 9.0.1, Systat 12.0 and R environment ver.2.11.1 were used for the analysis of the data and Pearson correlation was performed to establish the relation between the study variables. Results were represented as mean ±SD. Statistical significance and difference from control and test values were evaluated by student t test.

OBSERVATIONS AND RESULTS

Clinically diagnosed 50 diabetic nephropathy cases and 50 healthy controls were included in the study to evaluate FBS, PPBS, blood urea, serum creatinine, glycated hemoglobin levels, systolic and diastolic blood pressure and urinary microalbumin levels. There was an increase in FBS, PPBS, blood urea, serum creatinine, glycated hemoglobin, systolic and diastolic blood pressure and urinary microalbumin levels in cases as compared to controls which was statistically highly significant (p<0.001) as shown in the table 1. As shown in the table 2, Correlation study shows a significant positive correlation between urinary microalbumin levels with glycated hemoglobin and FBS, PPBS, blood urea, serum creatinine, systolic and diastolic blood pressure in cases which indicates as urinary microalbumin level increases, glycated hemoglobin and other parameter also increases.
DISCUSSION

Diabetes is associated with a variety of metabolic abnormalities principle among them is hyperglycemia. The metabolic dysregulation associated with diabetes mellitus causes secondary pathophysiologic damages in multiple organ system leading to a tremendous burden in the individual with diabetes and on the health care system. Several factors are involved in the genesis of diabetic nephropathy include glomerular hyperfiltration with increased intra glomerular pressure which antedates the development of nephropathy and contribute to the diabetes associated renal injury⁷. Between 20 and 40% of diabetic patients ultimately develop diabetic nephropathy⁸. In our study the mean values of FBS and PPBS were 90.86±13.29 and 119.72±10.57 in controls and 187.60±58.14 and 295.22±81.87 in diabetic nephropathy patients which is statistically highly significant with p<0.001. Hyperglycemia dependent metabolic abnormalities play a role in the development of nephropathy by various biochemical pathways including AGEs, polyols and activation of protein kinase C. Measurement of blood urea is used to evaluate renal function, to determine nitrogen balance and to aid in the diagnosis of renal disease. The mean blood urea values were 23.79 ±4.44 and 61.63±12.37 in controls and in diabetic nephropathy cases which is statistically highly significant with p<0.001. Measurement of serum creatinine is done to determine kidney function, severity of kidney damage and to monitor the progression of kidney disease. The mean serum creatinine levels were 1.06±0.20 and 2.83±0.68 in controls and in cases respectively which is statistically highly significant with p<0.001. Our study is in accordance with several studies where increased serum creatinine levels were noted in diabetic nephropathy cases compared to controls^9,10. Glycated hemoglobin is effective in monitoring long term control of blood glucose level in patients with diabetes mellitus. The complication of diabetes depends not only by the duration of diabetes mellitus but also by mean average level of chronic glycemia as measured by glycated hemoglobin level¹¹. Determination of HbA1c is not for the diagnosis of diabetes mellitus but only for monitoring the response to treatment. The mean values of HbA1c were 5.50±0.45 in controls and 11.17±1.63 in cases. This increase in HbA1c levels in diabetic nephropathy cases is statistically highly significant with p<0.001. Our study is in accordance with other studies where several authors Shahid SM, Mahaboob T⁹ Shivananda Nayak B and Geetha Bhanktha¹⁰ and Chen JW, Gall MA¹² found that HbA1c levels were increased in diabetic patients with and without diabetic nephropathy compared to controls ^9,10,12. Hypertension is very common in diabetic patients. It is a cause and also a result of diabetic kidney disease. Mean systolic blood pressure values were 116.00±6.32 in controls and 155.36±14.78 in cases and mean diastolic blood pressure values were 76.92±4.56 in controls and 92.88±4.92 in cases which is statistically highly significant. Our study is in consistent with several studies^12,13. Microalbuminuria is a marker of microvascular damage that has already occurred. Microalbuminuria arises from the increased passage of albumin through the glomerular filtration barrier which results from ultrastructural changes rather than alterations in glomerular pressure or filtration rate alone¹⁴. The mean values of urinary microalbumin were 10.35±2.77 and 153.38±69.64 in controls and in diabetic nephropathy cases which is statistically highly significant with p<0.001 and correlated well with the diagnosis. Our study is in accordance with several studies. In a study done by Chen JW, Gall MA and Melidonis A, Tournis S it was shown that urinary microalbumin levels were higher in diabetic nephropathy patients compared to controls^12,13. Shivananda Nayak B and Geetha Bhaktha demonstrated that urinary microalbumin levels were significantly increased in diabetic nephropathy patients compared to healthy controls¹⁰. Correlation between microalbuminuria with glycated hemoglobin and other study parameters: In a correlation study there is a significantly large positive correlation between microalbuminuria and glycated hemoglobin (r=0.608)is noted in diabetic nephropathy cases indicating that risk of microalbuminuria increases with poor glycemic control. This correlation is distorted when cases were compared with controls as controls showed a small negative correlation between microalbuminuria and glycated hemoglobin (r= -0.100). There is a small positive correlation between urinary microalbumin levels and FBS (r= 0.217)) and moderate positive correlation between urinary microalbumin levels and PPBS (r= 0.321) in diabetic nephropathy cases which indicates hyperglycemia has an important role in renal damage. Correlation study revealed a moderate positive correlation between urinary microalbumin levels and both blood urea (r= 0.375) and serum creatinine levels (r= 0.389) indicating that as urinary microalbumin level increases, blood urea and serum creatinine levels also increases. There is a moderate positive correlation between urinary microalbumin levels and systolic BP in cases (r= 0.413) and a small positive correlation between urinary microalbumin levels and diastolic BP in cases (r= 0.276) compared to controls where a small negative correlation is observed in systolic BP (r= -0.270) and diastolic BP (r= -0.290). Our study has highlighted the relationship of microalbuminuria with HbA1c levels where significant large positive correlation was found which is in accordance with other studies where Jorgensen et al and Mayurasakorn K et al have shown that microalbuminuric state is reversed and onset of complications will be delayed by tight glycemic control^15,16,17. Gupta M, Singh JP [18], Baig et al¹⁹ and Raman S, Dai H²⁰ in their study concluded that poor glycemic control (high HbA1c level) is significantly correlated with high level of microalbumin suggesting that higher value of HbA1c in diabetes patient is strongly associated with microabuminuria.

Table 1: Comparison of study variables in two group studied

Table 2: Pearson correlation of Microalbumin (mg/l) and other study variables in cases and controls

Significant figures

+Suggestive significance (P value: 0.05<P<0.10); *Moderately significant (P value:0.01<P £ 0.05); **Strongly significant (P value: P£0.01)

SUMMARY AND CONCLUSIONS

Diabetic nephropathy is the most common cause of end stage renal disease. Hyperglycemia is a common etiological factor in diabetic patients with nephropathy. Onset of microalbuminuria is the first practical evidence of renal involvement in diabetes. Once microalbuminuria is present progression cannot be prevented but only be delayed. Every diabetic patient must be screened for microalbuminuria atleast once in 6 months irrespective of age and duration of diabetes. Poor glycemic status play an important role in microvascular complications. Early stages of damage can be prevented by vigorous control of hypertension, strict glycemic control and protein restriction. Hence the study was done to estimate microalbuminuria and glycated hemoglobin levels and to assess the correlation of microalbuminuria which is an early marker of renal damage with glycated hemoglobin and markers of renal dysfunction (urea and creatinine) and hypertension in diabetic nephropathy patients. Statistically significant increase in FBS, PPBS, blood urea, serum creatinine, glycated hemoglobin, systolic and diastolic blood pressure and urinary microalbumin levels were noted in cases as compared to controls. From the correlation study a large positive correlation was observed between urinary microalbumin levels with glycemic status (blood sugar and HbA_1c) in diabetic nephropathy patients which indicates that the risk of microalbuminuria increases with poor glycemic control and elevated urinary microalbumin levels were strongly related to the presence of microvascular complications like diabetic nephropathy.

REFERENCES

• Kumar R, Abbas A, Fausto N, Aster JC, editors.: Robbins and Cotron “Pathologic basis of disease” 8^th Ed. Saunders Elsevier Publishing Division; 2010; 934-935, 1131-1146.
• Ralston SH, Penman ID, Strachan MWJ, Hobson RP: “Davidson’s principles and practice of medicine” 23^rd ed. Elsevier publishing division; 2018: 719-762.
• Saleh Ben Hamed SR, Pavkovic P, Metelko Z: Microalbuminuria and Diabetes mellitus. Diabetologia Croatica. 2002; 31(4):209-221.
• Kronenberg HM, Melmed S, Kenneth S, Polonsky, Larsen PR, editors: Williams textbook of endocrinology. The complications of diabetes mellitus. 11^th ed. Saunders Elsevier publishing Division; 2008: 1417-1482.
• Kowrluru RA, Chan P.:Oxidative stress and diabetic retinopathy. Experimental diabetes research Hindawi publishing corporation volume 2007: article ID 43603.
• Kumar P, Clark M: Clinical medicine. Diabetes mellitus and other disorders of metabolism. 9th ed. Saunders Elsevier; 2009:1241-1276.
• Shahid SM, Shaikh R, Nawab SN, Qader SA, Azhar A, Mahaboob T Serum Nitric Oxide and Sialic acid: Possible Biochemical Markers for Progression of Diabetic Nephropathy. World Academy of Science, Engineering and Technology 73 2011:404-406.
• Dronavalli S, Duka I, Bakris GL: The pathogenesis of diabetic nephropathy Nature Clinical Practice Endocrinology and Metabolism.2008 July; 8(4):444-452.
• Shahid SM, Mahaboob T: Clinical correlation between frequent risk factors of diabetic nephropathy and serum sialic acid. Int J Diabetes and Metabolism 2006; 14:138-142.
• Shivananda Nayak B, Geetha Bhaktha: Relationship between sialic acid and metabolic variables in Indian type 2 diabetic patients. Lipids in Health and Disease. 2005 Aug; 10; 4:15.
• Burtis CA, Ashwood ER, Burns DE: Teitz textbook of clinical chemistry and molecular diagnosis. 4th ed. Elseveir, a division of Reed Elsevier India Private Ltd; 2006.
• Chen JW, Gall MA, Yokoyama H, Jensen JS, Deckert M, Parving HH: Raised serum sialic acid concentration in NIDDM patients with and without nephropathy. Diabetes Care 1996 February; 19(2):130-134.
• Melidonis A, Tournis S, Hraklioti S, Hraklianou S: Serum sialic acid concentration and diabetic nephropathy in type 2 diabetes mellitus. Diabetologia Croatica 1998 Decemb; 29;27(4).
• Satchell SC, Tooke JE: What is the mechanism of microalbuminuria in diabetes; a role for the glomerular endothelium? Diabetologia 2008 May; 51(5):714-725.
• Jorgensen K, Brinchmann-Hansen O, Hanssen KF, Ganes T, Kierulf P, Smeland E et al: Effect of near normoglycaemia for two years on progression of early diabetic retinopathy, nephropathy, and neuropathy. The Oslo Study. Br Med J 1986; 293:1195-1199.
• Mayurasakorn K, Somthip N, Caengow S, Chulkarat N, Wanichsuwan M: Glycemic control and microvascular complications among type 2 diabetes at primary care units. J Med Assoc Thai. 2009; 92:1094-101.
• Krishnamurthy U, Halyal SS, Jayaprakash Murthy DS. Serum sialic acid and microalbuminuria in non insulin dependent diabetes mellitus. Biomedical Research 2011;22(1):31-34.
• Gupta M, Singh JP: Correlation of microalbuminuria with glycosylated haemoglobin in patients of diabetes having nephropathy. Int J Adv Med 2017; 4:805-8.
• Baig JA, Asif N, Sarfaraz A, Alam JM: Correlation of microalbuminuria with glycosylated hemoglobin (HbA1c) and duration of type 2 diabetes mellitus (T2DM) in male and female patients. Middle-East J Sci Res. 2016; 24(9):2900-3.
• Raman S, Dai H, DeLurgio SA, Williams DD, Lind M, Patton SR, et al: High hemoglobin A1c variability is associated with early risk of microalbuminuria in children with T1D. Pediatr Diabetes. 2016; 17(6):398-406.