Vol 10 | Issue 1 | January-June 2024 | page: 13-29 | Swati P Panbude, Anita S Chalak, Prasad A Panbude, Rupesh Malla

https://doi.org/10.13107/jmt.2024.v10.i01.216

Author: Swati P Panbude [1], Anita S Chalak [2], Prasad A Panbude [3], Rupesh Malla [1]

[1] Department of Biochemistry, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra, India.
[2] Department of Biochemistry, K.E.M. Hospital, Mumbai, Maharashtra, India.
[3] Department of Anaesthesia, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra, India.

Address of Correspondence
Dr. Swati P Panbude,
Assistant Professor, Department of Biochemistry, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra, India.
E-mail: drswatibs@gmail.com

Abstract

Aim: To assess the risk of Renal microvascular involvement in patients with Type II Diabetes Mellitus.
Objectives: To study the usefulness of various biochemical parameters as marker of renal microvascular damage, Type II DM patients. To study the relationship of inflammatory marker (hs¬CRP) with renal microvascular damage, in all the three groups.
Material and Methods: In present study, 150 individuals were included & divided into 3 groups, depending on their HbA1C levels: Group I (n=50) – Normal individuals (HbA1C 8%), Parameters were measured by : 1) Plasma glucose – GODPOD method, 2) Glycated hemoglobin – Latex agglutination inhibition assay, 3) Urine Microalbumin – Immunoturbidemetric method, 4) Sr. Urea – Urease-Kinetic method, 5) Sr. Creatinine – Modified Jaffe’s kinetic method, 6) Sr. Uric acid – UricasePAP method, 7) eGFR – Cockcroft Gault equation and 8) hsCRP by Turbidimetric immunoassay.
Results: FPG, PMPG, HbA1C, Microalbumin, Sr. Urea, Sr. Creatinine, Sr. Uric acid & hsCRP in Group III was significantly increased, as compared to Group I and II. eGFR was significantly decreased in group III than Group I & II. There was poor positive correlation of hsCRP with Microalbumin & eGFR in Group II. There was poor negative correlation of hsCRP with Microalbumin & eGFR in Group III.
Conclusion: HbA1c is a good predictor of long term glycemic control of type II DM patients. Also, microalbumin is one of the best parameter to assess renal damage, since it directly predicts the poor glycemic status of type II DM patients. In addition, eGFR level is a sensitive marker to assess renal damage in type II DM, since its level falls markedly with the onset of diabetic nephropathy.
Keywords: Diabetes Mellitus, Microalbumin, Inflammatory marker, Diabetic nephropathy

Introduction
The term Diabetes Mellitus (DM) describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism, resulting from defects in insulin secretion, insulin action or both. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 [1].
The two broad categories of DM are designated type I and type II. Type I result due to beta cell destruction, usually leading to absolute insulin deficiency. Type II DM is a heterogeneous group of disorders characterized by variable degrees of insulin resistance, impaired insulin secretion, and increased glucose production. About one third of those affected, will eventually have progressive deterioration of renal function [2].
Diabetes results in both microvascular and macrovascular complications. Among the microvascular complications, diabetic kidney disease is one of the most serious, with significant impact on morbidity, mortality, and quality of life [3]. Diabetic nephropathy occurs in approximately one-third of all people with diabetes and is the leading cause of renal failure in developed and developing countries [4]. Death due to renal disease is 17 times more common in diabetics than in nondiabetics [5].
Diabetic nephropathy affects all the kidney cellular elements, that is, glomerular endothelia, mesangial cells, podocytes, and tubular epithelia [6]. It is characterized by excessive accumulation of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progresses to glomerulosclerosis and tubulointerstitial fibrosis [6-8].
The National Diabetes Data Group and World Health Organization have issued diagnostic criteria for DM based on the following premises:
(1) the spectrum of fasting plasma glucose (FPG) and the response to an oral glucose load varies among normal individuals,
(2) DM is defined as the level of glycemia at which diabete-specific complications occur rather than on deviations from a population-based mean.
Interventions effective in slowing progression from microalbuminuria to overt nephropathy include:
(1) near normalization of glycemia,
(2) strict blood pressure control,
(3) administration of ACE inhibitors or ARBs and
(4) treatment of dyslipidemia.
Improved glycemic control reduces the rate at which microalbuminuria appears and progresses in type I and type II DM. However, once overt nephropathy exists, it is unclear whether improved glycemic control will slow progression of renal disease. During the phase of declining renal function, insulin requirements may fall as the kidney is a site of insulin degradation. Furthermore, glucose-lowering medications (sulfonylureas and metformin) are contraindicated in advanced renal insufficiency. Different studies identified different markers for detection of diabetic nephropathy.
According to Shehnaz A Sheikh et al, screening for microalbuminuria (MAU) and glycosylated hemoglobin (HbA1c) test should be done in both newly and already diagnosed type II diabetic patients as an early marker of renal dysfunction and glycemic control [9].
Mohd. Idrees Khan et al, discovered that the early detection of MAU combined with glycemic control and improved lipid profile are fundamentals in prevention and control of diabetic complications [10]. Microalbuminuria also showed a significant correlation with HbA1c and duration of diabetes, thus serving as an invaluable tool in monitoring of glycemic status and screening for diabetic nephropathy [11].
There is an additive value of micro total protein estimation along with eGFR assessment in diagnosing incipient nephropathy and hence, increasing the chances of detecting renal damage at initial stages in type II diabetes mellitus patients [12]. New formulae for the calculation of eGFR corrected by the glycemic control indices were said to be better than the original eGFR, particularly in diabetic patients [13].
According to a survey done in year 2014, serum hs-CRP levels, independent of possible confounders, were associated with a subsequent risk of developing, not progressing, diabetic nephropathy in type II diabetic patients.14 In fact, serum hs-CRP may be useful for predicting the future risk of developing diabetic nephropathy [14].
In support one of the studies done in 2015, it was concluded that, the increase in serum hs-CRP value in type II diabetic patients increase the risk of diabetic nephropathy and thus increase the value of serum uric acid level [15]. Serum uric acid is also said to be a strong and independent risk factor for diabetes [16]. According to Suryawanshi K.S., et al, serum uric acid and urine microalbumin are not only early diagnostic markers for atherogenic cardiovascular disease and renal disease but also prognostic monitoring of the disease in type II diabetes mellitus patients [17].
Several studies were done in the past on identifying the various factors causing and not preventing, diabetic nephropathy. Subsequent studies showed that, microalbumin and eGFR are the markers of renal microvascular damage in type II diabetes mellitus [18]. These studies encouraged us to design the present study for early identification of at risk diabetic patients for diabetic nephropathy using screening tools like serum and urinary markers, that will help in preventing and/or postponing renal microvascular complications.
We, therefore, studied the usefulness of fasting and postmeal plasma glucose, glycosylated hemoglobin (HbA1c), microalbumin, serum urea, creatinine, uric acid, estimated glomerular filtration rate (eGFR) and highly sensitive C-reactive protein (hs-CRP), as a marker of renal microvascular damage in all the three groups i.e., normal, well & poorly controlled type II diabetes mellitus. We have also studied two important correlations of inflammatory marker with renal microvascular damage marker i.e., hs-CRP with microalbumin and hs-CRP with eGFR.
With this background, we aim to assess the risk of renal microvascular involvement in patients of type II diabetes mellitus.

Review Of Literature
Molnar M et. al. in 2000 [19], studied the prevalence of microalbuminuria (MA) and macroalbuminuria (MAA), their relationship with other diabetic complications and with some known cardiovascular risk factors in 200 in type II diabetic patients (100 females and 100 males). Sixty eight patients (33%) were normalbuminuric (NA), 55 (27.5%) had MA and 77 (38.5%) had MAA. There was no significant difference among these three groups in age, BMI or the time actually elapsed since the diabetes and hypertension were diagnosed. BMI was high in each group (28.8 ± 5.29, 28.0 ± 5.2 and 29.8 ± 4.6 kg/m2 mean ± SD). Sixty five percent of patients with NA, 77% of those with MA and 81% of patients with MAA had hypertension. MAA patients were more frequently smokers and former smokers, than MA and NA patients (56% vs 32% and 22%). Average GFR values (ml/min/1.73 m2) were 71.9 ± 26.8 in NA patients, 82.3 ± 36.8 in MA patients and 56.3 ± 32 in MAA patients. There was no significant correlation between the urinary albumin excretion (UAE) and glycemic control, serum cholesterol and serum HDL cholesterol. At the same time UAE showed a significant positive correlation with serum trigliceride (P < 0.01), serum uric acid (P < 0.01) and serum creatinine (P < 0.01) while a significant negative correlation was found with GFR (P < 0.01). Diabetic non-proliferative retinopathy (RP) was detected even in NA patients (27%) while 51% of MAA patients were without RP. Fifty six percent of NA patients, 57% of MA patients and 93% of MAA patients had macroangiopathy. They finally concluded that:
(1) renal function can be impaired even in type II diabetic pts with NA and MA,
(2) well-known cardiovascular risk factors seem to have a close relation with renal damage in type II diabetes,
(3) renal lesions in type II diabetic pts may be caused by diseases other than diabetes (e.g. arteriosclerosis, hypertension),
(4) unlike in type I diabetes, where the strict glycemic control is the main preventive factor of diabetic nephropathy, in type II diabetes, the control of hypertension, hyperlipidemia, obesity, hyperuricemia may have priority.
Garg, et. al. in 2002 [20], observed that a number of screening criteria, applied either at a single point in time or serially, can be used for the purpose of identifying individuals at risk of end-stage renal disease (ESRD). This study focused on two such criteria measured on a single occasion, proteinuria and renal insufficiency, and examined their prevalence in a sample representative of the adult U.S. non-institutionalized population. Such knowledge guides the utility of population screening to prevent ESRD. The prevalence of albuminuria (microalbuminuria and macroalbuminuria from a random urine albumin-to-creati- nine ratio) and renal insufficiency [GFR estimated from serum creatinine] was determined in different age categories in various adult screening groups in the cross-sectional ‘Third National Health and Nutrition Examination Survey (NHANES III)’. A total of 14,622 adult participants were included in the analysis. In the general population, 8.3% and 1.0% of participants demonstrated microalbuminuria and macroalbuminuria, respectively. To identify one case of albuminuria, one would need to screen three persons with diabetes mellitus, seven non-diabetic hypertensive persons, or six persons over the age of 60. When albuminuria and renal insufficiency were considered together, it was clear that these tests were identifying different segments of the population; 37% of participants with a GFR less than 30 mL/min/1.73m2 demonstrated no albuminuria. Non-albuminuric renal insufficiency was most evident in the ages of 60 to 79; 34% of diabetics and 63% of non-diabetic hypertensives with a GFR less than 30 mL/min/1.73m2 demonstrated no albuminuria. They concluded that, albuminuria is prevalent, and when considered together, screening tests of albuminuria and renal insufficiency measured on a single occasion identify different segments of the population. The prevalence of albuminuria and renal insufficiency in populations of interest should be considered, as this knowledge has implications for the effectiveness of screening.
Banerjee, et. al. in 2005 [21], studied the status of GFR estimation vis-a-vis other noninvasive modes of assessment of renal involvement in type II diabetes mellitus and assessment of the temporal profile of the prevalence of nephropathy with a cross sectional cohort. A total of 100 patients of type II diabetes mellitus were selected after screening and segregated into 3 groups according to duration of type II diabetes mellitus. Duration of <5 years constituted group A and had 31 patients, group B duration was between 5-15 years and had 40 patients, rest belonged to group C with duration >15 years. The parameters studied and compared were:
(1) various grades of albuminuria- normal, micro and macro by 24 hrs urinary albumin excretion rates (UAER-gm/24hr),
(2) sonologically detected renal size(normal, small, large) and morphology (loss or presence of corticomedullary differentiation,
(3) serum creatinine level (</> 1.4 mg/dl) and
(4) different levels (high, normal, low, very low) of GFR (ml/min) by Diethylene Triamine Tetra-acetic acid) DTPA renal scan.
There was high prevalence of nephropathy in all durations. Microalbuminuria had a high prevalence in patients of shorter duration (group A-74.2%). Albuminuria increased with duration but plateued off with longer duration (>15 yrs) (UAER- 0.0842 ± 0.083 vs. 0.906 ± 0.84 vs. 1.346 ± 1.28). Sonographic loss of corticomedullary differentiation and azotemia were late feature only and none had a contracted kidney. Only the parameter of GFR showed a graded and rather linear decrement with duration (132.57 ± 19.3 vs. 76.33 ± 20.8 vs. 40.08 ± 17.1). Hyperfiltration had a high prevalence in patients of early detection (61.3%) and was the earliest change noted before change in any other parameter. GFR shows wide variation in various grades of albuminuria, especially microalbuminuria and azotemia. A value in the normal range was uncommon (8%). They concluded that, GFR estimation is probably the most rational noninvasive mode of assessing the renal status in patients of type II diabetes mellitus, irrespective of the status of the other noninvasive methods as they express significant variation in inception and progression.
Parving, et. al. in 2006 [22], described the characteristics in a referred cohort of type II diabetic patients in the developing education on microalbuminuria for awareness of renal and cardiovascular risk in diabetes study evaluating the global prevalence and determinants of microalbuminuria (MA). A cross-sectional study evaluating 32,208 type II diabetic patients without known albuminuria from 33 countries was performed. Overall, 8057 patients were excluded, either because of prior known proteinuria or non-diabetic nephropathy (3670), or because of invalid urine collections (4387). One single random urinary albumin/creatinine ratio was obtained in 24,151 patients (75%). The overall global prevalence of normo, micro, and macroalbuminuria was 51%, 39% and 10%, respectively. The Asian and Hispanic patients had the highest prevalence of a raised urinary albumin/creatinine ratio (55%) and Caucasians the lowest (40.6), P<0.0001. HbA1c, systolic blood pressure, ethnicity, retinopathy, duration of diabetes, kidney function, body height and smoking were all independent risk factors of MA, P<0.0001. eGFR was below 60 ml/min/1.73m2 in 22% of the 11,573 patients with available data. Systolic BP below 130 mmHg was found in 33 and 43% had an HbA1c below 7%. The frequency of patients receiving aspirin was 32%, statins 29% and BP-lowering therapy 63%. A high prevalence globally of MA and reduced kidney function, both conditions associated with enhanced renal and cardiovascular risk, was detected in type II diabetic patients without prior known nephropathy. Early detection, monitoring of vascular complications and more aggressive multifactorial treatment aiming at renal and vascular protection are urgently needed.
Hanan M. Kamel, et. al. in the year 2008 [23], studied the assessment of the urinary α -1 microglobulin and its relation to microalbminuria as regarding early prediction of diabetic nephropathy and glycaemic control in diabetic patients. The study population included 60 subjects; they were classified into three groups: Group 1: 21 patients with type I diabetes mellitus under insulin therapy and dietary control; Group II: 21 patients with type 2 diabetes mellitus under oral hypoglycaemic therapy; Group III: 18 apparently healthy subjects. All cases and control groups were subjected to the following, complete history taking, complete physical examination, lab investigations included fasting, post prandial blood glucose, Glycated Hb (HbA1c), kidney function tests(blood urea, serum creatinine), lipid profile tests(total cholesterol, serum triglyceride(TG), high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, urine tests included creatinine urine, microalbuminuria, urinary α-1 microglobulin. They found that there was statistically significant increase in blood glucose level, serum urea level, TG, HDL-cholesterol in group I when comparing to group III (p-value=0.0001) also there was statistically significant increase in blood glucose level, all serum lipid profile in group II when comparing to group III (p-value=0.0001, but p-value=0.02 for serum total cholesterol, LDL-cholesterol).
Also there was statistically significant decrease in serum urea level (p-value=0.0001) and serum total cholesterol (p-value=0.05) when comparing group I to group II. There was statistically significant increase in microalbumin, α-1 microglobulin, HbA1c, microalbumin/creatinine ratio when comparing group I to group III (P-value= 0.05, 0.006, 0.02, <0.02 respectively). Also there was statistically significant increase in microalbumin, α-1 microglobulin, HbA1c, microalbumin/creatinine ratio and α-1 microglobulin/creatinine ratio, all with (P value = 0.0001) when comparing group II to group III but there was statistically significant decrease in microalbumin with (p value = 0.007), statistically significant increase in α-1 microglobulin (P value =0.01), statistically significant decrease in HbA1c with (p value= 0.009), statistically significant decrease in microalbumin/creatinine ratio with (p value = 0.01) when comparing group I to group II. In addition, in group I there was significant positive correlation between HbA1c and microalbuminuria (r value = 0.7, P =value 0.0001), the same in group II, also in group I there was significant positive correlation between α-1 microglobulin and HbA1c (r value 0.56, P valve 0.009),the same correlation in group II (r value 0.70 with p value 0.001) Also in group I and group II there was positive correlation between microalbumin/creatinine ratio and each of microalbumin, α-1 microglobulin, and HbA1c (r=o.6, p value=0.002), (r=0.66, p value=0.000), (r=0.40, p value=0.06) in group I respectively but in group II (r=0.73, p value=0.0001), (r=0.53, p value=0.01), (r=0.68, p value= 0.001) respectively, also this study showed significant correlation between α-1microglobulin/creatinine ratio and α-1microglobulin in group I only. They concluded that α-1 microglobulin, microalbumin are considered the best predicted markers for early glomerular and proximal tubular dysfunction predicting diabetic nephropathy.
Abbas Dehghan, et. al. in 2008 [24] study, investigated the association between serum uric acid level and risk of type II diabetes. The population for analysis consisted of 4,536 subjects free from diabetes at baseline. During a mean of 10.1 years of follow-up, 462 subjects developed diabetes. They observed that, the age- and sex-adjusted hazard ratios (HRs) (95% CIs) for diabetes were 1.30 (0.96 –1.76) for the second, 1.63 (1.21–2.19) for the third, and 2.83 (2.13–3.76) for the fourth quartile of serum uric acid, in comparison with the first quartile. After adjustment for BMI, waist circumference, systolic and diastolic blood pressure, and HDL cholesterol, the HRs decreased to 1.08 (0.78 –1.49), 1.12 (0.81–1.53), and 1.68 (1.22–2.30), respectively. They concluded that, the results of this population-based study suggest that serum uric acid is a strong and independent risk factor for diabetes.
Mohammad Afkhami-Ardekani, et. al. in 2009 [25], studied that, Type II diabetes is a common disorder recognized as a major health problem in Iran. Diabetes is a major source of morbidity, mortality and economic cost to society. Diabetic patients are at risk of experiencing macrovascular and microvascular complications of diabetes. The aim of this study was to assess the prevalence of type II diabetes complications and their contributing factors. This cross-sectional study was carried out on 1000 the type II diabetic patients referred to Yazd Diabetes Research Center. All diabetic patients underwent the specific tests for retinopathy, nephropathy, neuropathy, peripheral vascular diseases (PVD) and
cardiovascular diseases (CAD). Logistic regression analysis was used to find out strength of association of risk factors with a specific complication. In this study 1000 type II diabetic patients (457 male, 543 female) were studied. Nephropathy was diagnosed in 285 (28.5%), retinopathy in 519 (51.9%), CAD in 251 (25.1%), PVD in 143 (14.3%), CVA in 109 (10.9%) and foot ulcer in 84 patients (8.4%). They finally concluded that the most important contributing factors in diabetic complications were age, duration of diabetes, systolic and diastolic blood pressure, glycated hemoglobin and body mass index (BMI). So glycemic and blood pressure control can prevent diabetic complications or at least delay them.
According to a study by Shehnaz A Sheikh, et. al. in 2009 [10], diabetes is one of the most common endocrine disorders characterized by hyperglycemia. Diabetic nephropathy is a consequence of long standing diabetes. The prevalence of microalbuminuria predicts progression to diabetic nephropathy. The present study was conducted to determine the prevalence of microalbuminuria in relation to duration of diabetes, BMI, serum creatinine and HbA1c in an ethnic group of type II diabetes mellitus residing in Karachi. This cross-sectional descriptive study was carried out in a community diabetic centre, located at Garden East Karachi from july to december 2007. One hundred known type II diabetic patients with age 30–70 years were included in the study. Informed consent and a structured questionnaire of each patient were recorded. Fasting venous blood and morning urine sample was collected for analysis of creatinine, HbA1c and microalbuminuria respectively. Pearson correlation was applied to observe association of microalbuminuria with different parameters. All p-values <0.05 were considered as statistically significant. Microalbuminuria had a highly significant correlation with duration of diabetes, serum creatinine (p<0.001), HbA1c (p<0.05) and BMI (p<0.024). A strong correlation exists between age and serum creatinine (r=0.73). The present study found an early onset of microalbuminuria in the selected community which could be due to poor glycaemic control (high HbA1c >7%) or heredity factors. Screening for microalbuminuria and HbA1c test should be done in both newly and already diagnosed type II diabetic patients as an early marker of renal dysfunction and glycemic control.
Francisco Javier del Canizo Gomez, et. al. in 2011 [26], conducted a prospective study in patients with type II diabetes mellitus with no microvascular complications, analyzing the association between various baseline risk factors and development of microvascular complications at follow-up. A prospective, observational study in 376 patients with type II diabetes mellitus enrolled in 2004. The clinical end-point was urinary albumin excretion (UAE)>30 mg/24h and/or presence of retinopathy at follow-up in 2007. Baseline variables included age, gender, duration of type II diabetes mellitus, fasting plasma glucose, glycated hemoglobin (HbA1c), systolic and diastolic blood pressure, body weight, height, BMI, waist circumference, total cholesterol, TGs, HDL-C, LDL-C, hs-CRP, fibrinogen, UAE, creatinine, smoking status, exercise, alcohol consumption, use of hypoglycemic and lipid-lowering drugs, antihypertensive medications, and other data related to family history of diabetes and risk factors. Ninety-five subjects (25.2%) developed a microvascular complication at the end of the follow-up period. In logistic regression analyses, the main independent risk factors were UAE>12 mg/24h (odds ratio [OR]: 6.12; P = 0.000), hs-CRP> 3 mg/L (OR: 3.00; P = 0.004), and hypertension (OR: 2.43; P = 0.023). They found that, UAE levels higher than 12 mg/24 h, hs-CRP >3 mg/L, and presence of hypertension were all independent risk factors for development of microvascular complications in patients with type II diabetes mellitus studied.
Doyle M. Cummings et. al in another study of 2011 [27], studied that, reducing glycosylated hemoglobin (HbA1c) to near or less than 7% in patients with diabetes is associated with diminished microvascular complications, but this level is not consistently achieved. The purpose of this study was to examine the relationship between fluctuations in HbA1c and changes in eGFR and estimated stage of chronic kidney disease (CKD) in an academic primary care practice. They analyzed data from 791 diabetic primary care patients (25% white; 75% African American) enrolled between 1998 to 2002 and followed through 2008 (mean follow-up, 7.6-1.9 years). They calculated baseline and final follow-up eGFR using the modification of diet in renal disease equation. They examined the relationship between fluctuations in HbA1c and changes in eGFR and stage of CKD using multivariable linear and logistic regression models that controlled for demographic and clinical variables associated with CKD progression. From baseline to follow-up, mean eGFR in african americans declined to a greater extent and more rapidly than in whites. Age, mean systolic blood pressure, initial HbA1c, initial eGFR, and number of HbA1c values (all P <0.01) were significant predictors of change in eGFR. Among HbA1c fluctuation measures, the strongest predictor of change in eGFR was the proportion of HbA1c values >7% (P <0.02); however, this contributed little to explaining model variance. They finally found that traditional demographic and clinical risk factors remain significantly associated with changes in eGFR and that the pattern of variability in HbA1c is only modestly important in contributing to changes in eGFR among African-American and white diabetic patients in primary care.
Deepa.K, et. Al in 2011 [28], stated that, India as a developing country has more prevelance of diabetes and now has more people with type II diabetes (more than 50 million) than any other nation. Diabetes mellitus is a chronic metabolic disorder that can lead to cardiovascular, renal, neurologic and retinal complications. Type II diabetes has quickly become a global health problem due to rapidly increasing population growth, aging, urbanization and increasing prevalence of obesity and physical inactivity. A total of 40 diabetic patients of both sex aged between 35 to 75 years attending medicine OPD were included in the study. After obtaining informed consent from the study group 5 ml of fasting venous blood sample was collected. Plasma glucose was estimated by GOD – POD method. Estimation of plasma creatinine was done by the modified jaffe’s method. Serum urea was estimated by urease-berthelot’s method. There was significant increase in levels of serum urea, creatinine and FPG (p<0.001) in diabetic patients compared to healthy controls. On applying pearson’s correlation serum urea correlated positively with creatinine (p<0.001, r = 0.910) in cases and also in controls (p<0.001, r = 0.868). Blood urea and creatinine is widely accepted to assess the renal functions. Good control of blood glucose level is absolute requirement to prevent progressive renal impairment.
Nirmitha Dev, et al, in the year 2012 [29], discovered that, hs-CRP, a non specific inflammatory marker has been shown to be increased in metabolic syndrome a risk state for the development of cardiovascular disease and type II diabetes mellitus. Obesity is a predisposing condition to metabolic syndrome. Therefore, this study was intended to measure hs-CRP levels in obese females to assess their risk status. 55 healthy adult obese females with BMI >23 kg/m2 were taken as cases and 55 age matched healthy adult non-obese females with BMI <23 kg/m2 were taken as controls. Anthropometric measurements (waist circumference, hip circumference & waist to hip ratio) and biochemical estimations (blood glucose, hs-CRP and lipid profile) were carried out. There was significant increase in waist circumference, fasting blood sugar, total cholesterol, TG, VLDL, LDL levels and hs-CRP levels in obese females as compared to controls. Blood glucose levels & lipid profile were within the reference range in both obese and non obese females. hs-CRP did not show any correlation with blood glucose or lipid profile. hs-CRP behaved as an independent inflammatory marker in obesity. Therefore, hs-CRP might be an early novel marker of inflammation, for identifying the obese females who are at risk for obesity related co-morbidities.
Mohd. Idrees Khan, et al in 2012 [11], found that an Inflammatory marker hs-CRP may play role as predictor of inflammation in patients with type II diabetes. The aim of this study was to estimate hs-CRP levels and glycemic control status and to determine association with microalbuminuria. The relationship between inflammation and microalbuminuria complications in type II diabetes mellitus which has not yet been reported in North Indians. Forty two patients with microalbuminuria and twenty type II diabetes without microalbuminuria were enrolled. We analyzed serum concentrations of hs-CRP, serum lipid profile, HbA1c and urine microalbumin levels. HbA1C and hs-CRP were significantly higher in patients with microalbuminuria diabetic cases than without microalbuminuria (p<0.0001). Furthermore, hs-CRP was poorly correlated with urinary albumin excretion (p=0.002). This study concludes that inflammation is involved in the pathogenesis of microalbuminuria. The significance of these findings emphasizes the early detection of MAU combined with glycemic control and improved lipid profile which are fundamentals in prevention and control of diabetic complications.
Sudhindra Rao M, et al in 2012 [30], have reported that high serum levels of uric acid are strongly associated with prevalent health conditions such as obesity, insulin resistance, metabolic syndrome, essential hypertension and renal disease. This study aimed to investigate the level of serum uric acid in Type II diabetes mellitus, pre-diabetics and non diabetics (controls) in south Indian population. Uric acid level was measured by uricase-PAP methodology in patients with diabetes (n=71)/pre diabetes (n=12)/control groups (n=34). Using ANOVA test, uric acid levels in the above three groups were compared based on age, sex and other factors which can affect uric acid level. The mean serum uric acid level was lower in control group (3.84 mg/dl), rose in pre-diabetics (4.88 mg/dl) and again decreased in diabetics (3.78 mg/dl). P value comparing control and pre-diabetes was 0.009, p-value comparing pre-diabetes and diabetes was 0.003 and p-value comparing control and diabetes was 0.982 (p value <0.05 being significant).They found that, the serum uric acid level being higher in pre-diabetes than controls and lower in diabetes mellitus than pre-diabetes may serve as a potential inexpensive biomarker of deterioration of glucose metabolism.
Gurprit Grover, et. al. in another study conducted in year 2012 [31],, found that, diabetes affects more than 170 million people worldwide and the number will rise to 370 million people by 2030. About one third of those affected, will eventually have progressive deterioration of renal function. To estimate progression of renal disease among type II diabetic population, with serum creatinine, in the presence of covariates: fasting blood glucose, systolic BP, diastolic BP and LDL, duration of disease and age at which diabetes was diagnosed. Retrospective data collected from 132 patients, who were diagnosed as diabetic as per ADA standards with or without diabetic complications. Multiple linear regression (MLR) and logistic regression models were adopted to estimate and predict serum creatinine, a well-accepted marker for the progression of diabetic nephropathy. The fitted multiple linear regression models were found to be statistically significant, with p <0.001, fitted logistic models have 88.5% and 84.7% predictive power to assess the renal disease based on mean values of predictors and last record of predictors, respectively. It was concluded from the models, which were based on mean values of records, that high blood glucose and high blood pressure along with duration of diabetes are the main contributors for estimating serum creatinine and predicting diabetic nephropathy.
Sangeeta Kapoor, et. al. in 2014 [13], observed that, Diabetic nephropathy is a leading cause of end stage renal damage, characterized by decreased GFR and proteinuria in patients of Type II diabetes mellitus. In order to device a means to protect kidneys at an early stage, this study has examined micro total protein (MTP) in 24 hrs urine along with eGFR by modification of diet in renal disease (MDRD) and Cockroft-Gault (CG) prediction equations as predictor of early renal damage in type II diabetes mellitus. They examined the eGFR and MTP in 24 hrs urine sample as independent predictors of renal damage in type II diabetes mellitus patients and also to study the additive value of eGFR and MTP in diagnosing incipient diabetic nephropathy. Urinary 24 hrs proteinuria was assessed by pyrogallol red dye method and GFR estimated using MDRD and CG prediction equations. The mean ± standard deviation of MTP was compared between diabetic patients (1913.3 ± 2084.15 mg/24 hrs) and non-diabetic controls (189.5 ± 66.72 mg/24 hrs), found significant proteinuria in diabetic patients. The eGFR estimated by MDRD equation compared between diabetics (75.44 ± 30.85 mL/min/1.73 m2) and non-diabetic controls (103.52 ± 24.69 mL/min/1.73 m2) and eGFR by CG compared between diabetics (71.34 ± 32.63 mL/min) and non-diabetic controls (99.44 ± 25.37 mL/min) were found significantly decreased in diabetic patients. MTP correlated with eGFR estimated by both the equations (rMTP−MDRD = −0.544 and rMTP−CG = −0.452) and found to be significant at P <0.01 and <0.05, respectively. It has also been seen that MTP correlation with eGFR (MDRD) is better than MTP correlation with eGFR (CG).They finally concluded that, there is an additive value of MTP estimation along with eGFR assessment in diagnosing incipient nephropathy and hence, increasing the chances of detecting renal damage at initial stages in type II diabetes mellitus patients.
Geetha Bhaktha, et. al. in 2014 [32], found that, C-reactive protein is considered as one of the most sensitive markers of systemic inflammation. Studies have found that increase in the levels of C-reactive protein is associated with the vascular complications. Hence the author aimed in finding the correlation of hs-CRP with other risk factors like BMI, FBS and HbA1c in diabetic subjects who have still not developed any micro and macrovascular complications. 229 cases of type II diabetics and 205 healthy individuals were selected as per the criteria. BMI was calculated, FBS was estimated by glucose-oxidase-peroxidase method. hs-CRP was estimated by immunoturbidometric technique. The group was divided into low risk and high risk group as per their hs-CRP level. Correlation was seen with other factors like BMI, FBS and HbA1c. The level of hs-CRP was high in diabetic subjects when compared to normal individuals. Further when the diabetic subjects were divided into high risk and low risk groups, the difference between the groups were statistically significant. hs-CRP failed to show any correlation with BMI, FBS and HbA1c. Diabetes is considered as an inflammatory disease hence they observed an increase in the hs-CRP level in diabetes than in the normal. Since the vascular complication was totally absent hs-CRP failed to show any correlation with BMI, FBS and HbA1c.
Yasuaki Hayashino, et. al. in 2014 [15], assessed the prospective association between baseline serum hs-CRP concentration and the subsequent risk of development or progression of diabetic nephropathy. Longitudinal data was obtained from 2,518 patients with type II diabetes registered in a Japanese diabetes registry. To assess the independent correlations between serum baseline hs-CRP and either the development or progression of diabetic nephropathy 1 year later, the Cox proportional hazards model was used and adjusted for potential confounders. The mean patient age, BMI, and HbA1c level were 66.1 years, 24.6 kg⁄m2, and 7.5% (57.6 mmol/mol), respectively. Baseline serum hs-CRP levels were significantly associated with the urinary albumin-to-creatinine ratio at baseline (P <0.001). Multivariable adjusted hazard ratio for the development from normoalbuminuria to microalbuminuria was 1.31 (95% CI 0.80–2.17; P = 0.286), 1.55 (1.16–2.08; P = 0.003), and 1.57 (1.22–2.03; P = 0.001), respectively, for the second, third, and fourth quartiles of serum hs-CRP levels, showing a statistically significant linear trend across categories (P < 0.001). They did not observe a significant association between hs-CRP levels and the subsequent risk of diabetic nephropathy progression (P for trend = 0.575). They concluded that, serum hs-CRP levels, independent of possible confounders, were associated with a subsequent risk of developing, not progressing, diabetic nephropathy in type II diabetic patients. Serum hs-CRP may be useful for predicting the future risk of developing diabetic nephropathy.
Anwarullah, et. al. in another study of 2014 [33], discovered that, microalbuminuria is often the first sign of renal dysfunction (nephropathy) in diabetes mellitus. The current study was aimed at determining the microalbuminuria levels in type II diabetes and to correlate changes in microalbuminuria levels with the HbA1c levels in type II diabetic patients. The study was conducted at the Islamabad Diagnostic Centre, Islamabad, Pakistan. Patients with type II diabetes aged between 30-60 years were included in the study. Patients with systemic diseases like cardiovascular diseases, cerebrovascular diseases and urinary tract infection was excluded from the study. Fasting blood samples were used to analyze HbA1c levels for the estimation of diabetic control and subsequently random urine specimens to investigate microalbumin level of all the individuals under study. The study showed that microalbuminuria levels were linearly correlated to those of HbA1c levels. They found that, impaired glycemic control is associated with significant elevations in urinary microalbumin levels which suggest that the monitoring of microalbuminuria levels at the early stages of diabetes can avert and reduce the clinical and economic burden of auxiliary complications (nephropathy etc.) in the developing countries like Pakistan.
Ritika Kumar Tandon et. al. in 2014 [12], studied that, nephropathy is a common complication of diabetes mellitus that could lead to End Stage Kidney Disease (ESKD). Microalbuminuria is important as an ‘early marker’ of renal disease as it represents a time when renal biopsy shows no or minimal changes. HbA1c represents the average glucose concentration over the period of 2-3 months and is accepted as a useful index of mean blood glucose. The purpose of the study was to study the relationship between HbA1c and urinary microalbumin in patients of type II diabetes mellitus. A prospective study was conducted on 200 known diabetics. Detailed history was taken and thorough physical examination of all the patients was done followed by HbA1c estimation by Bio Rad D10 HPLC machine and microalbumin by Nyco Card Microalbumin test kit. 56.5% of the cases were males and 43.5% females. 43.5% cases were positive for microalbumin, of which 47% had duration of diabetes between 5 to 10 years (p<0.05). 35% cases had HbA1c in the range of 8.1-10% of which 67% had microalbuminuria (p<0.05) . Microalbuminuria showed a significant correlation with HbA1c and duration of diabetes, thus serving as an invaluable tool in monitoring of glycemic status and screening for diabetic nephropathy.
Sanjeev Kumar et. al. in the year 2014 [34], stated that, insulin resistance is characterized by a subnormal response to a given concentration of insulin and can be measured indirectly by a fasting insulin level. The prevalence of diabetes continues to grow worldwide, disease-related morbidity and mortality is emerging as major healthcare problems. Clearly, type II diabetes has a strong genetic component. Diabetic nephropathy is the leading cause of ESRD in US and a leading cause of diabetes mellitus related morbidity and mortality. Nephropathy complicates approximately 30% of type II diabetic patients. However no study has been performed that compared the HbA1c in type II diabetes mellitus with nephropathy to without nephropathy. Therefore aim of the study was to evaluate the glycosylated hemoglobin and their association with diabetic nephropathy in a western Uttar Pradesh. Venous blood was collected after 12 hours fasting into two test tubes; with no anticoagulant for serum creatinine, and with Ethylene Diamine Tetra Acetic Acid (EDTA) for HbA1c. They observed that Incidence of microalbuminuria increases with age as well as with increased duration of diabetes mellitus. Their study also evaluated relationship between diabetic retinopathy and nephropathy and found a significant correlation.
Thomas Vijatha et. al. in 2014 [35], studied the prevalence of DM and found that it has been increasing worldwide including India, both in rural and urban dwellers. Studies have shown prevalence rate of DM to be 2-4% in rural and 4-11% among urban dwellers. In parallel with increase in diabetes a dramatic increase in prevalence of diabetic nephropathy has been noted which is the single most common cause of ESRD. HbA1c is currently accepted as the most diagnostic and prognostic biomarker of glycemic control in subjects with diabetes. However, in diabetic patients with CKD, HbA1c may not be the most informative biomarker of glycemic index. Therefore objective of the study was to assess whether HbA1c is a reliable indicator of diabetic status in CKD patients in the advancing stage. 120 diabetic subjects with CKD, who were not on maintenance hemodialysis, were included in the study. They were divided into 4 groups depending on eGFR: Group I (n=30, eGFR ≥60ml/min/1.73m2), Group II (n=30, eGFR: 60-30ml/min/1.73m2), Group III (n=30, eGFR: 30-15ml/min/1.73m2), Group IV (n=30, eGFR <15ml/min/1.73m2). Their blood samples were used to measure glucose, HbA1c, serum creatinine and hemoglobin. eGFR was estimated using the four-variable MDRD formula using electronic abstraction. A significant correlation (p value of 0.00) was found between HbA1c and eGFR. Also significant change is seen in hemoglobin with change in eGFR. Significantly lower HbA1c values were seen in diabetic patients with advancing stage of CKD who were not on maintenance hemodialysis.
Akihiro Tsuda, et. al. in 2014 [14], found that, serum creatinine levels are lower in diabetic patients compared with their nondiabetic counterparts. Therefore, eGFR is higher in the former than in the latter group. Factors associated with overestimation of renal function in diabetic patients were examined and new formulae reflecting precise eGFR were created. Eighty subjects (age 56.5615.4years; 35 males[43.8%]; 40 patients with diabetes and 40 nondiabetic subjects) were enrolled. GFR was evaluated by inulin clearance (Cin). eGFR values were calculated based on serum creatinine and/or serum cystatin C levels. The factors related to the dissociation between eGFR and Cin in diabetic patients and the agreement among each of three eGFR and Cin were compared. Although Cin was not significantly different between the diabetic and nondiabetic subjects (P =0.2866), each of three eGFR measures from the diabetic patients was significantly higher than that of the nondiabetic subjects (P <0.01). There were significant and positive correlations between the ratio of each eGFR/Cin, HbA1c, and glycated albumin. The intraclass correlation coefficients in diabetic patients were weaker than those in the nondiabetic subjects and the intercepts of the regression lines between each eGFR measure and Cin in the diabetic patients were significantly higher than those of the nondiabetic subjects. New formulae for the calculation of eGFR corrected by the glycemic control indices were better than the original eGFR, particularly in diabetic patients. So, they concluded that, eGFR overestimates Cin as glycemic controls worsen. eGFR corrected by HbA1c is considered to be clinically useful and feasible.
Rohitash Kumar et. al. in 2014 [36], stated that, high values of renal function tests are associated with type II diabetes mellitus. But there are studies that found that the levels of creatinine and uric acid are low in cases of diabetes mellitus. Comparative studies related to serum renal function tests with eGFR and blood glucose in type II DM is less. Hence the study was undertaken to study the renal function test and its correlation with blood glucose and eGFR in type II DM. 25 freshly diagnosed cases of type II diabetes mellitus and 25 healthy controls were studied. It is found that mean serum urea levels were 25.80±6.75 mg/dl in controls and 34.08±9.62 in cases, which was statistically highly significant. Mean serum creatinine and uric acid values were also highly significant (p=0.0002) in cases, as compared to controls. Significant positive correlation was found between FBS (p<0.001) and PPBS (p<0.0001) with the eGFR and renal function tests in both cases and controls. Their study showed that, urea, creatinine and uric acid levels are towards higher reference limits in cases compared to controls.
Jay Prakash Sah, et. al. in the year 2015 [16], found that, hs-CRP is an α globulin produced by liver as a marker of inflammation. It may play a role as predictor of inflammation in diabetic nephropathic patients. The aim of present study was to estimate hs-CRP levels and blood uric acid and to determine association between them. The relationship between inflammation and blood uric acid level in type II diabetes has not yet been reported in Nepalese population. So a quantitative, analytical study were done by enrolling 89 type II diabetic patients conducted at Tertiary care Hospital. They analyzed serum concentrations of hs-CRP, serum uric acid, blood glucose and family history of the patients. In their study, they found the significant association between serum hs-CRP and serum uric acid level (P values=0.001). They also found the significant association between serum hs-CRP and blood glucose level (P values <0.01). Furthermore, serum hs-CRP was not correlated with family history of patients (P >0.599) and sex (P >0.08). They concluded that the increase in serum hs-CRP value in type II diabetic patients increase the risk of diabetic nephropathy and thus increase the value of serum uric acid level. And there is no correlation of both serum hs-CRP and uric acid level with the risk factors especially sex and family history of type II diabetes. The significance of these findings emphasizes to choose these association for early screening of diabetic nephropathy in type II diabetic patients to prevent from further complication.
Suryawanshi K.S, et. al. in 2015 [18], studied that, type II diabetes mellitus is one of the major cause of the mortality and morbidity in the world. Type II diabetes mellitus is a chronic disease characterized by relative deficiency of insulin, resulting in glucose intolerance. Their study was planned to understand more about hyperuricemia and microalbuminuria and its complications in type II diabetes mellitus patients. In present study, 565 patients of type II diabetes mellitus and age and sex matched controls were included. They found increased levels of serum uric acid and urine microalbumin in type II diabetic patients as compared to controls (p<0.001). They observed the positive correlation between serum uric acid and urine microalbumin (p<0.001). They finally concluded that, serum uric acid and urine microalbumin are not only early diagnostic markers for atherogenic cardiovascular disease and renal disease but also prognostic monitoring of the disease in type II diabetes mellitus patients.

Aim & objectives
Aim
To assess the risk of renal microvascular involvement in patients of type II diabetes mellitus.

Objectives
1. To study the usefulness of various biochemical parameters as marker of renal microvascular damage in patients of type II diabetes mellitus. 2. To study the relationship of inflammatory marker (hs-CRP) with renal microvascular damage, in all the three groups.

Material and methods
A prospective, observational study, “Assessing the Risk of Renal Microvascular Involvement in Patients of Type II Diabetes Mellitus” was carried out at Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi (Meghe), Wardha, during the period between January 2014 to August 2015. Permission from the college ethical committee was taken for the conduct of study. Informed written consent was obtained from each patient.
Source Population: The patients coming to the central clinical laboratory from different outpatient department (O.P.D.) of AVBRH were selected.
Study Population: Amongst the above, subjects coming particularly for plasma glucose estimation were selected.
Design of study: Prospective, analytical, case control study.

Inclusion criteria
1) Subjects coming for plasma glucose estimation
2) Age group >20 years
3) Patients willing to give informed consent.

Exclusion criteria
1) Type I diabetes mellitus
2) Known case of diabetic nephropathy
3) Known case of congestive heart failure
4) Patients having prostate disease or any infection.
Enrollment of patients: Informed consent of all patients for blood and urine investigations was taken. Name, age, sex, height and weight was noted. 5 ml of fasting venous blood and morning urine samples, were collected from the patients for assessment of parameters mentioned in table 1. Investigations like fasting and post meal plasma glucose, glycosylated hemoglobin, microalbumin serum urea, creatinine, uric acid, eGFR and hs-CRP were done.

Parameters:
Parameter Method
Plasma Glucose GOD-POD method
Glycated Haemoglobin Latex Agglutination Inhibition Assay
Microalbumin Immunoturbidimetric method
Serum Urea Urease Kinetic Method
Serum Creatinine Modified Jaffe’s Kinetic method
Serum Uric acid Uricase-PAP method
Egfr Cockcroft-Gault equation
hs-CRP Turbidimetric Immunoassay
method
Methods of Estimation
1. Plasma glucose estimation by god-pod method [37]: Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2).
Addition of the enzyme peroxidase and a chromogenic oxygen acceptor, such as o-dianisidine, results in the formation of a colored compound that is measured.

2. Glycated Haemoglobin by Latex Agglutination Inhibition Assay [38]: The agglutinator, a synthetic polymer containing multiple copies of the immunoreactive portion of HbA1C, binds the anti- HbA1C monoclonal antibody that is attached to latex beads. This agglutination produces light scattering, measured as an increase in absorbance. HbA1C in the patient’s sample competes for the antibody on the latex, inhibiting agglutination, thereby, decreasing light scattering.

3. Microalbumin estimation by Immunoturbidimetric method [33]: The multigent microalbumin immunoturbiditimetric that uses polyclonal antibodies against human albumin was used for the determination of urine microalbumin urea. The specimen was mixed with the reagents. Albumin in the specimen combined with the anti-human albumin antibody, in the reagent to yield an insoluble aggregate that causes increased turbidity in the solution. The degree of turbidity is proportional to the albumin in the specimen, which was measured optically.

4. Serum Urea by Urease Kinetic Method [39]: Method for the measurement of urea is based on preliminary hydrolysis of urea with Urease (urea with amidohydrolase), to generate ammonia which is then quantified.

5. Serum Creatinine by Modified Jaffe’s Kinetic method [40]: Creatinine reacts with picrate ion in alkaline medium to yield an Orange coloured complex (Creatinine picrate).

6. Uric acid estimation by Uricase-PAP Method [41]: Uricase oxidoreductase is used either as a single step or as the initial step to oxidize uric acid. Uricase acts on the uric acid to produce allantoin, hydrogen peroxide or carbon dioxide.

7. eGFR by Cockcroft-Gault equation [42]: Estimated GFR (eGFR; ml/min/1.73 m ) was calculated using the following CG(Cockcroft-Gault) formula:-
CG = [(140-age) x weight {x 0.85 if female}] / (72 x serum creatinine)

8. hs CRP by Turbidimetric Immunoassay (Latex) method [43]: The Latex method uses particle-enhanced technology. In this method, the specific antibodies coated to polystyrene particles formed a complex with CRP present in the measured study sample. The amount of scattered light was directly proportional to the size of the antigen-antibody complex and reflected the hs-CRP concentration present in the study sample.

Sample size: Total 150 subjects were studied and divided into three groups, depending upon their glycosylated hemoglobin level as follows:-

Group 1 (Normal subjects: 50 patients): HbA1c level <6%.
Group 2 (Well controlled diabetes: 50 patients): HbA1c level 6-8%.
Group 3 (Poorly controlled diabetes: 50 patients): HbA1c level >8%.
Statistical data: 1. Data was expressed as Mean ± SD. 2. For statistical analysis, SPSS Version 16 was done. 3. Anova test was applied for comparison between more than two groups & student ‘t’ test for comparison between two groups. 4. Pearson’s correlation was applied for correlating two parameters. 5. ‘p’ value of less than 0.05 was considered statistically significant.

Discussion
Diabetes mellitus (DM) comprises of a group of common metabolic disorders that share the phenotype of hyperglycemia. Several distinct types of DM exist and are caused by a complex interaction of genetics, environmental factors, and life-style choices. Depending on the etiology of the DM, factors contributing to hyperglycemia may include reduced insulin secretion, decreased glucose utilization, and increased glucose production. [44]
Type II DM is characterized by three pathophysiologic abnormalities: impaired insulin secretion, peripheral insulin resistance, and excessive hepatic glucose production. Obesity, particularly visceral or central (as evidenced by the hip-waist ratio), is very common in type II DM. Adipocytes secrete a number of biologic products (leptin, tumor necrosis factor-α, free fatty acids, resistin, and adiponectin) that modulate insulin secretion, insulin action, and body weight which may contribute to the insulin resistance.
In the early stages of the disorder, glucose tolerance remains normal, despite insulin resistance, because the pancreatic beta cells compensate by increasing insulin output. As insulin resistance and compensatory hyperinsulinemia progress, the pancreatic islets in certain individuals are unable to sustain the hyperinsulinemic state. Impaired glucose tolerance(IGT), characterized by elevations in postprandial glucose, then develops. A further decline in insulin secretion and an increase in hepatic glucose production lead to overt diabetes with fasting hyperglycemia. Ultimately, beta cell failure may ensue. Markers of inflammation such as IL-6 and C-reactive protein are often elevated in type II diabetes. [44]
DM can result into acute or chronic complications. Diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS) are acute complications of diabetes. Both disorders are associated with absolute or relative insulin deficiency, volume depletion and acid-base abnormalities. DKA and HHS exist along a continuum of hyperglycemia, with or without ketosis. Both disorders are associated with potentially serious complications if not promptly diagnosed and treated.
The chronic complications of DM affect many organ systems and are responsible for the majority of morbidity and mortality associated with the disease. Chronic complications can be divided into vascular and nonvascular complications of DM are further subdivided into microvascular and macrovascular complications. [44]
Diabetic nephropathy is the leading cause of ESRD in the United States and a leading cause of DM-related morbidity and mortality. Proteinuria in individuals with DM is associated with markedly reduced survival and increased risk of cardiovascular disease.
The pathogenesis of diabetic nephropathy is related to chronic hyperglycemia. The mechanisms by which chronic hyperglycemia leads to ESRD, though incompletely defined, involve the effects of soluble factors (growth factors, angiotensin II, endothelin, advanced glycated end products), hemodynamic alterations in the renal microcirculation (glomerular hyperfiltration or hyperperfusion, increased glomerular capillary pressure), and structural changes in the glomerulus (increased extracellular matrix, basement membrane thickening, mesangial expansion, fibrosis). Some of these effects may be mediated through angiotensin II receptors.
The natural history of diabetic nephropathy is characterized by a fairly predictable sequence of events that was initially defined for individuals with type I DM but appears to be similar in type II DM.
Glomerular hyperperfusion and renal hypertrophy occur in the first years after the onset of DM and cause an increase of the glomerular filtration rate (GFR).
During the first 5 years of DM, thickening of the glomerular basement membrane, glomerular hypertrophy and mesangial volume expansion occur as the GFR returns to normal.
After 5 to 10 years of type I DM, 40% of individuals begin to excrete small amounts of albumin in the urine. Microalbuminuria is defined as 30 to 300 mg/d in a 24-h collection or 30 to 300 μg/mg creatinine in a spot collection (preferred method). The appearance of microalbuminuria (incipient nephropathy) in type I DM is an important predictor of progression to overt proteinuria (>300 mg/d) or overt nephropathy. Blood pressure may rise slightly at this point but usually remains in the normal range. Once overt proteinuria is present, there is a steady decline in GFR, and 50% of individuals reach ESRD in 7 to 10 years. The early pathologic changes and albumin excretion abnormalities are reversible with normalization of plasma glucose. However, once overt nephropathy develops, the pathologic changes are likely irreversible.
The nephropathy that develops in type II DM differs from that of type I DM in the following respects: (1) microalbuminuria or overt nephropathy may be present when type II DM is diagnosed, reflecting its long asymptomatic period;(2) hypertension more commonly accompanies microalbuminuria or overt nephropathy in type II DM; and (3) microalbuminuria may be less predictive of diabetic nephropathy and progression to overt nephropathy in type II DM. Finally, it should be noted that albuminuria in type II DM may be secondary to factors
unrelated to DM, such as hypertension, congestive heart failure, prostate disease, or infection.
Type IV renal tubular acidosis (hyporeninemic hypoaldosteronism) also occurs in type I or II DM. These individuals develop a propensity to hyperkalemia, which may be exacerbated by medications [especially angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs)]. Patients with DM are predisposed to radio contrast-induced nephrotoxicity. [44]
The optimal therapy for diabetic nephropathy is prevention. As part of comprehensive diabetes care, microalbuminuria should be detected at an early stage when effective therapies can be instituted. The recommended strategy for detecting microalbuminuria is outlined below: [44]
Plasma glucose is a continuous variable, rising and falling about two-fold throughout the day in people without diabetes and up to some 10-folds in people with diabetes, given by Saudek CD, et al. [405 Rohitash Kumar, et al [36] showed a positive correlation existing between FPG and PMPG with eGFR, which may be due to the hyperfiltration of glomerulus during the early stage of diabetes, which at later stage is known to decrease eGFR with development of nephropathic changes. [2] In the study shown by Suryawanshi K.S., et al [18], FPG, PMPG, serum uric acid and urine microalbumin were markedly increased in diabetic patients as compared to healthy controls.
Our study shows that, FPG values are more in poorly controlled group(173.66 ± 52.73), as compared to normal(92.82 ± 8.43) and well controlled group(117.3 ± 22.67), which is statistically highly significant(<0.0001). Similarly, PMPG values are also found on a higher side in poorly controlled group(289.8 ± 83.58), compared to normal(136.82 ± 20.52) and well controlled group(185.72 ± 40.37), which is also statistically highly significant(<0.0001). As poor glycemic control increases UAE, rise in FPG and PMPG could be responsible for renal microvascular damage. Therefore, good control of plasma glucose level is absolute requirement to prevent progressive renal impairment.
It was reported by Shehnaz A Sheikh, et al [10] that, HbA1c can be used as a diagnostic test for Type II diabetes instead of relying only on FPG. The complications of both Type I and II diabetes do not develop or progress for 6–9 years when the average HbA1c level is kept at <7%, stated by Brownlee M, et al. [46] Marked increase in HbA1c level was found in diabetic patients with microalbuminuria, as compared to without microalbuminuria, which could be attributed to uncontrolled/ persistent higher blood sugar level as indicated by excessive glycosylation of hemoglobin. HbA1c is a good indicator of glycemic control in initial stages but, its reliability in advancing stage is questioned, as given by Thomas Vijatha, et al. [35]
In our study, HbA1c values are found to be more in poorly controlled group(10.32 ± 1.52), in comparison with normal(5.10 ± 0.42) and well controlled group(7.00 ± 0.52), which is statistically highly significant(<0.000`). Thus, tighter glycemic control will lead to greater reduction in the risk for microvascular complications. Also, substantial reductions in HbA1c level at an early stage have a long-lasting implications for reducing the risk of future microvascular complications.
Previous three studies by Dinneen SF, Borch-Johnsen K and Remuzzi G, et al established microalbumin as a powerful independent predictor of microvascular lesions, cardiovascular disease, cardiovascular mortality and kidney disease including endstage renal failure in patients with diabetes, hypertensive subjects and even in the general population. [47, 48, 2] H-H Parving, et al showed a clear association of higher levels of albuminuria with an increased frequency of renal insufficiency. [22] The frequency of microalbuminuria increased with the increase in duration of diabetes, was shown by Naz S, et al. [49]
Giunti S, et al [50] stated that, microalbuminuria is related to hyperglycemia and control of blood glucose level has been shown to prevent the development of nephropathy in Type I & II diabetes. A rapid decline in renal function can be predicted for patients having poor glycemic control and microalbuminuria, as given by Araki S and Wright J, et al in two separate studies. [51, 52] According to Derakhshan A, et al [53], minimizing microalbuminuria and having a tight glycemic control is an important treatment goal for patients with diabetes. Study by Shehnaz A Sheikh [10], et al concluded that, microalbuminuria and HbA1c testing should be done in both, newly diagnosed as well as already diagnosed Type II diabetic patients as an early marker of cardiovascular and renal risk factor.
A Study by Francisco Javier del Canizo Gomez, et al [26] concluded that UAE levels >12 mg/24hrs are the most significant independent risk factor for development of nephropathy and/or retinopathy in the type II DM patients studied. However, according to current guidelines, the UAE cut-off level for progression to nephropathy is 30 mg/24 hrs. [19] In addition, Hoefi eld RA, et al [54] found that, individuals with microalbuminuria also have an increased rate of decline of eGFR compared with people with normoalbuminuria.
The study by Anwarullah, et al [33] explicitly indicated that poor diabetic control is the leading cause of diabetic nephropathy as evidenced by elevated microalbuminuria. Many complications arise due to uncontrolled or poorly controlled diabetes mellitus amongst which the most destructive is diabetic nephropathy, found by Battisti WP, et al. [55] Chowta NK, et al [56] discovered that, the level of glycemic control also plays an important role in the transition from normoalbuminuria to microalbuminuria to macroalbuminuria. Jha P, et al [57] suggested that, to maximize prevention of microalbuminuria development, blood pressure should be maintained at less than 130/80 mm Hg, and HbA1c should be kept below 7%.
In our study, Microalbumin values are very high in poorly controlled group(164.18 ±41.95), in contrast with normal(15.21± 5.35) and well controlled group(81.55 ±26.82) and found to be statistically highly significant(<0.0001). Therefore, a screening criteria like microalbuminuria applied either at a single time or serially, should be used to identify individuals at high risk of ESRD. In this way, using screening tests of microalbuminuria to prevent multiple outcomes of interest such as ESRD, will improve the efficacy of screening, recognizing that the presence of microalbuminuria may have important predictive validity for future renal microvascular complications.
There is strong relationship of blood sugar level with urea level. An increase in urea level is seen when there is damage to the kidney or the kidney is not functioning properly. Increment of blood urea level with the increment of blood sugar level clearly indicates that the increase blood sugar level causes damage to the kidney. A research by Anjaneyulu, et al [58] has found that increase urea and serum creatinine in diabetic rats indicates progressive renal damage.
According to Deepa K, et al [28], the plasma urea and creatinine are estabished markers of GFR, though plasma creatinine is a more sensitive index of kideny function. Blood urea and creatinine is widely accepted to assess the renal functions. Another observations of Deepa K, et al were in accordance with various studies which showed raised plasma creatinine and urea levels in diabetic patients, which may indicate a pre-renal problem. [59, 60]
The measurement of serum creatinine concentration is widely used clinically as an index of renal function, given by Justesen TI, et al. [60] Adler AI, et al [61] stated that, the rate of rise in SrCr , a well-accepted marker for the progression of diabetic nephropathy, (creatinine value 1.4 to 3.0 mg/dl) is the indicator for impaired renal function.
Mitch WE and Schutte JE, et al [62, 63] in separate studies found that, serum creatinine and urea concentration change inversely with changes in GFR and therefore useful in gauging the degree of renal dysfunction. Also, microalbuminuria and serum creatinine increase significantly in Type II diabetes as reported in an earlier study by Justesen TI. [60] Some patients have a substantial decrease in glomerular filtration rate, while their serum creatinine concentration remains within the normal range and thus it is a poor screening test for mild kidney disease, observed by Hebert CJ, et al. [64]
In our study, serum urea values are found to be more in poorly controlled group (52.08 ± 9.96), as compared to normal (25.52 ± 6.26) and well controlled group (35.38 ± 10.07), which is statistically highly significant (<0.0001). Creatinine values are also found on a higher side in poorly controlled group (2.74 ± 0.83), compared to normal(1.06 ± 0.18) and well controlled group(1.58 ± 0.67) and which is also statistically highly significant(<0.0001). Serum urea & creatinine measurement is a convenient and inexpensive method of assessing renal function and a consistently elevated level indicates chronic kidney disease. Thereby, urea, not alone but alongwith creatinine must be assessed in type II diabetic patients, to prevent or postpone renal damage.
According to Abbas Dehghan, et al [24] one quarter of diabetic cases can be attributed to a high serum uric acid level. Recognition of high serum uric acid as a risk factor for diabetes has been a matter of debate for a few decades, since hyperuricemia has been presumed to be a consequence of insulin resistance rather than its precursor, concluded in a study by Butler R, et al. [65]
Hyperuricemia has been found to be associated with obesity and insulin resistance and consequently with type II diabetes mellitus, given by Idonije , et al. [66] Hyperuricemia indues endothelial dysfunction which results in nephropathy in type II DM patients [67] and study done by Tseng also says that, even mild hyperuricemia will results in kidney injury. [68] Fukui M and Chin-Hsiao Tseng, et al [69, 70], discovered separately that, the long duration of diabetes may cause hyperuricemia and microalbuminuria which in turn lead to micro and macrovascular complication. The uric acid and urine microalbumin are very good diagnostic markers for detection of kidney injury in initial stage of disease, stated by both CAI Xiao-ling and Saeed Behradmanesh. [71, 72]
We also found more uric acid levels in poorly controlled group(5.65 ± 0.50), compared to normal(4.22 ± 0.39) and well controlled group(4.9 ± 0.46) & which is also statistically highly significant(<0.0001). Increased levels of uric acid can be injurious to kidneys, therefore, uric acid levels should be estimated in patients with type II diabetes mellitus.
eGFR is the most rational noninvasive method of assessing the renal status in patients, according to two studies done by Gross JL and Mykkanen L, et al. [73, 74] The eGFR estimated from both the equations were found to be decreased in DM patients compared to non-diabetic controls, which show that eGFR decrease in diabetic patients more than non-diabetic controls. It has been suggested by Sangeeta Kapoor, et al that, using eGFR as a screening tool may also potentially predict and reduce the incidence of CKD, which is associated with increased risk of death.
Sangeeta Kapoor, et al [13] also observed that, micro total protein in 24 hrs urine sample and eGFR were examined as independent predictors for renal damage in Type II DM patients and their additive values have also been assessed to diagnose incipient diabetic nephropathy. Average GFR values determined by either the 24- hour creatinine clearance method or by the Cocroft-Gault formula were found to be lower not only in patients with macroalbuminuria but in normoalbuminuria and microalbuminuria as well. In order to avoid variations of protein concentration in urine, assessment of micro total protein in 24 hrs urine sample along with the eGFR by the prediction equations suggested by National Kidney Foundation, which are MDRD and CG equations must be advocated.
Another observation noted in a study by Rohitash Kumar, et al [36] was a positive correlation existing between FPG and PMPG with eGFR. On the other hand, Doyle M. Cummings, et al [27] used a novel approach to explore the association of patterns of HbA1c fluctuation with changes in eGFR and stage of CKD. Another finding by Molnar M, et al [19] told that, with the development of diabetic nephropathy, serum creatinine level starts to increase and GFR starts to fall.
Our study revealed eGFR values lower in poorly controlled group(40.13±3.83), in comparison with normal(98.63± 5.86) and well controlled group(78.45±5.04), which is statistically highly significant(<0.0001). We can thereby, come to the conclusion that, kidney functions are well-preserved in the normoalbuminuria stage of type II diabetes and the decrease in eGFR starts only during the transition from normoalbuminuria to microalbuminuria. eGFR estimation is one of the renal parameter which can provide a picture of the actual renal status of Type II DM patients at any duration irrespective of other parameters. Since, it is important to take steps to protect the kidneys before the problem advances, eGFR assessment must be done at an early stage of Type II DM in oreder to prevent overt diabetic nephropathy.
hs-CRP levels >3 mg/L were an independent risk factor for development not only of diabetic nephropathy, but also of diabetic retinopathy. As per observations done by Yasuaki Hayashino, et al [15], there is a temporal association between elevated levels of hs-CRP and the subsequent risk of developing, not progressing, diabetic nephropathy in a large registry of patients with diabetes, even after adjusting for possible confounders, including medication use, which may influence the natural course of renal function. Francisco Javier del Canizo Gomez, et al [26] suggested that, hs-CRP may be a marker of vascular disease, which indicates impaired self-regulation of glomerular pressure and/or dysfunction of glomerular endothelium. Both these factors may increase microalbuminuria.
Mohd. Idrees Khan, et al [11] made an attempt to test the hypothesis that systemic inflammation as indicated by hs-CRP and uncontrolled blood sugar level as indicated by HbA1C are associated with type II diabetic nephropathy which signify occurrence of significant microalbuminuria. Interestingly, hs-CRP level in microalbuminuria diabetic patients was many fold increased than without microalbuminuria. There is convincing evidence that type II diabetes mellitus presented with inflammatory component has been related to such diabetic complication as nephropathy. Inflammatory markers in early diabetic nephropathy in patients with type II diabetes are elevated and are independently associated with UAE. Therefore, screening of hs-CRP level in diabetics could be established as biomarker of vascular complications.
We found in our study that, hs-CRP values are high in poorly controlled group(7.12±0.57), in comparison with normal(1.96 ±0.52) and well controlled group(4.71±0.29) and it is statistically highly significant(<0.0001). It should be noted that, corrections of conditions like hyperglycemia may halt inflammation in type II diabetic patients and burden of ESRD. Hence, screening of hs-CRP must be considered under screening of type II DM, inflammatory marker like hs-CRP play a pivotal role in the development and progression of diabetic complications.
In our study, no correlations of hs-CRP with microalbumin(r value: -0.1372), as well as hs-CRP with eGFR (r value: -0.0898), was found in well controlled diabetic group, with no statistical significance of hs-CRP with microalbumin (p value: 0.3419) & eGFR (p value: 0.5350), respectively. Also, in our study, no correlations of hs-CRP with microalbumin (r value: 0.0435), as well as hs-CRP with eGFR (r value: 0.1280), was found in poorly controlled diabetic group, with no statistical significance of hs-CRP with microalbumin (p value: 0.7641) and eGFR (p value: 0.3758). Similar results were found in a study conducted by Mohd. Idrees Khan, et al, who established a poor correlation between UAE and inflammatory parameter in patients with type II DM at an early stage of diabetic nephropathy in north Indians.

Summary
Our study “Assessing the Risk of Renal Microvascular Involvement in Patients of Type II Diabetes Mellitus” is a prospective, analytical, case control study. We conducted our study at AVBRH, Sawangi (Meghe), Wardha. The subjects coming particularly for plasma glucose estimation, aged >20 years and those willing to give informed consent were included in the study. Patients of type I DM, known case of diabetic nephropathy, congestive heart failure and those with prostate disease or any infection, were excluded from the study.
Total 150 subjects were studied and divided into three groups (50 each), depending upon their HbA1c levels i.e. Group 1 (normal subjects): HbA1c <6%, Group 2 (well controlled diabetes): HbA1c =6-8% and Group 3 (poorly controlled diabetes): HbA1c >8%. List of investigations were done in all the patients by different methods: FPG and PMPG by GOD-POD method, HbA1c by latex agglutination inhibition assay, microalbumin by mmunoturbidemetric method, serum urea by urease kinetic method, serum creatinine by modified Jaffe’s kinetic method, serum uric acid by uricase-PAP method, eGFR by Cockcroft-Gault equation & hs-CRP by turbidimetric immunoassay method.
Amongst the above listed parameters, FPG and PMPG values were significantly raised in group III, compared to groups I and II. HbA1c also showed a significant increase in group III, than group II and normal in group I. In addition, urinary marker i.e. microalbumin was markedly elevated in group III, in contrast to groups I and II. Since, HbA1c is a better marker for assessing the glycemic status of the patient, its screening must be done on a routine basis in all the patients of type II DM, to prevent or postpone overt diabetic nephropathy.
Also, other serum markers like serum urea, creatinine and uric acid were found to be significantly increased in group III, than the other two groups. Another important urinary marker included in our study was eGFR, which was significantly decreased in group III and raised in I and II groups. It should be noted that, serum urea and creatinine are poor screening tools for assessing renal damage. Thus, eGFR estimation by Cockroft-Gault equation, should be done in all the type II DM patients, especially in the early stage of diabetic nephropathy.
hs-CRP, an inflammatory marker used in our study was observed to be on a higher side in group III, than groups I and II. Since, hs-CRP acts as a vascular component too, its assay must be considered, in order to prevent renal microvascular complications in patients of type II DM.

Conclusion
From this study it can be concluded that, the serum markers i.e. FPG, PMPG and HbA1c, are good predictors of renal microvascular damage in type II diabetes mellitus. Poor glycemic control directs to the development of diabetic nephropathy. Thus, a strict glycemic control, having a healthy lifestyle and maintaining standard body weight is especially important for diabetic patients and for those with a family history of diabetes.
There is a direct association of serum urea and creatinine with altered kidney function in type II DM. In addition, there could be an association of raised serum uric acid levels with the onset of diabetic nephropathy. Thereby, it is mandatory to include serum urea, creatinine and uric acid as a potential risk factors, especially in chronic diabetics with poor glycemic control.
Moreover, rise of both the urinary markers i.e. microalbumin and eGFR in type II diabetic patients have an important predictive value for future microvascular complications. Microalbuminuria & eGFR are considered as early predictors for renal dysfunction, either at glomerular or proximal tubular level. The screening for microalbuminuria and eGFR is a pre-requisite for preventing diabetic nephropathy. Therefore, microalbuminuria and eGFR testing should be done in both, newly diagnosed as well as already diagnosed type II diabetic patients.
In addition, raised level of hs-CRP in blood might not predict the progression, but the development of nephropathy in type II DM. Hence, screening of inflammatory marker like hs-CRP in diabetics could be established as biomarker of renal microvascular complications. Thereby, hs-CRP must be used as a powerful screening tool, in order to prevent and/or postpone renal microvascular damage in type II DM patients with poor glycemic control.

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