Prediction of Renal Injury Risk by Expressions of KIM-1 and NGAL in Type 2 Diabetic Nephropathy

Background: About 10 to 40% Type 2 diabetes (T2DM) and 30% Type 1 diabetes (T1DM) suffer from kidney failure increases huge financial burden for care for patients [1]. Patients with uncontrolled diabetes prone to end-stage renal disease (ESRD) which required kidney transplantation, haemodialysis or peritoneal dialysis which adds psychological & financial burden. Early kidney injury can be prevented by evaluating gene expression (KIM1, NGAL) in T2DM with microalbuminuria. Methodology: This study includes 241 subjects (118 male, 123 women, and age ranges 30-70 years, distributed in two groups; 30-45 years and 45-70 years) were included after screening for T2DM by measurement of blood glucose in fasting, post-prandial, glycosylated haemoglobin and micro albumin in urine. Subjects were randomised after written consent; subject examined by physician and enrolled as per inclusion/exclusion criteria. Categorization of subjects in three study groups were done on the basis of T2DM duration 3-5 years, Glycosylated haemoglobin level (HbA1c) ≥ 7.0% with fasting blood glucose ≥ 126 mg/dl) and micro albuminuria in study group. Equal numbers of age and sex matched healthy volunteers enrolled in control group. Blood samples were processed for other renal parameters &rt-PCR to check expressions of KIM1 and NGAL. Results: In study groups all renal parameters are within normal range except albumin creatinine ratio (p<0.012) & e-GFR (p<0.000). Other parameters showed marginal significance within and between the groups. KIM-1 and NGAL showed high degree of significance (p<0.000). Conclusion: Biochemical renal parameters are not enough to identify risk of DN even in microalbuminuria. Early detection of gene expressions of KIM1 and NGAL may help to evaluate the status of kidney functions which help to prevent morbidity & mortality of kidney due to diabetic nephropathy. Extensive study in large population size was recommended.


INTRODUCTION
Recent estimates by the National Institutes of Health indicate that diabetes represents the single largest cause of ESRD [1]. Today it has become the single most common cause of (ESRD) in the entire world. T2DM subject undergoing dialysis consumes significantly more financial resources than those with non-diabetic ESRD. In addition, T2DM patients do poorly on dialysis and have an excess mortality. Clinical management and therapeutic intervention in stage of DN has major role in prevention and progression of DN to ESRD. DN is typically defined by microalbuminuria, i.e., renal albumin excretion more than 300 mg in 24 hrs collection (urine dipstick positive) and abnormal renal function (increase serum creatinine or GFR). Clinically nephropathy with T2DM was characterized by progressive increase in proteinuria and decline in GFR. There are several biomarkers in use to detect kidney damage. Conventional biomarkers for kidney damage include glomerular filtration rate (GFR), plasma creatinine, blood urea nitrogen (BUN), urinary micro-albumin excretion rate and several urine qualities such as proteinuria and hematuria as well as liver function test, glycated hemoglobin, RBC indices and urine routine. However these routinely used parameters to evaluate kidney function are nonspecific and insensitive, hence new specific markers are being developed [2].
Poorly sensitive methods are failed to recognized early detection of DN, these patients has greater chances of ESRD. Use of novel biomarkers has potential to detect early DN progression. Several novel biomarkers of kidney injury have been shown to increase in the urine & plasma of individuals with diabetes at early stage [3]. DN [5]. Other putative risk factors are GFR, smoking, dyslipidemia, proteinuria levels, and dietary factors, the main potentially modifiable DN initiation [6,7]. A higher proportion of individuals with T2DM are found to have microalbuminuria and overt nephropathy shortly after the diagnosis of their diabetes, since diabetes is actually present for many years before the diagnosis was made and also due to the fact that presence of albuminuria may be less specific for the presence of DN [8]. Without specific interventions, 20-40% of T2DM patients with microalbuminuria progress to overt nephropathy, but after 20 years onset of overt nephropathy, only 20% will have progressed to ESRD. Once the GFR begins to fall, it differs from one individual to another, but overall, they may not be substantially different between T1DM patients and T2DM Patients [9]. The prediction of microalbuminuria is an indication for screening for possible vascular disease and aggressive intervention to reduce all cardiovascular risk factors (e.g., reducing LDL cholesterol, antihypertensive therapy, cessation of smoking, institution of exercise, etc.). Additionally, there is some preliminary indication that suggests lowering of cholesterol also lowers the level of proteinuria [10].
Routinely used investigations for kidney injury are serum creatinine & blood urea. These are conventional biomarkers have several disadvantages. Serum levels of creatinine may only change after about 50-60% of the kidney function has been lost. Altered levels of serum creatinine take time to establish & make it impossible to detect acute kidney injury early.
There is utmost need to know highly sensitive biomarkers for the early detection of. Inadequate tools are failed to recognize early detection of DN at an early stage. So it was postulated that KIM1 and NGAL may help to identify the risk of DN.
Kidney Injury Molecule (KIM1) is a type-1 transmembrane glycoprotein not detected in normal kidney, tissue or urine but it appears at high levels after ischemic or toxic kidney injury [11]. (KIM-1) is a promising biomarker for kidney damage. It is structurally related to the immunoglobulin gene family [12].
There are many reasons to consider that KIM-1 may be released into the circulation after kidney proximal tubule injury. In injury, tubular cell polarity is lost, such that KIM-1 may be released directly into the interstitium. Further, increased trans-epithelial permeability after tubular injury leads to leak of tubular contents into the circulation [13]. Also, altered micro vascular permeability is an important contributor to the pathophysiology of kidney injury [14].
NGAL is a member of the lipocalin family of proteins and is expressed only at very low levels in several human tissues, including kidney [15]. Therefore NGAL protein has been extensively studied in different models of acute kidney injury (AKI). In numerous studies, the increase of NGAL in urine and serum has been a good predictor of a onset of AKI, especially anticipating the increase in serum creatinine [16][17][18][19][20]. Literature survey revealed that NGAL could be a potential marker of CKD [21,22].
Human NGAL was originally identified as a novel protein isolated from secondary granules of human neutrophils [23,24]. NGAL mRNA is normally expressed in a variety of adult human tissues, including bone marrow, uterus, prostate, salivary gland, stomach, colon, trachea, lung, liver and kidney [25].

MATERIALS AND METHODS
Present research conducted at Department of Biochemistry, Dr D. Y. Patil University, Navi Mumbai. Patients referred to Diabetic clinic OPD were recruited in this study. The enrolled patients were randomly selected & distributed into three different groups; subjects of T2DM between ages 30-45 years; subjects of T2DM between 45-70 years and healthy volunteers (Non-diabetic) between 30-70 years.T2DM of diabetes duration between 3-5 years, HbA1c ≥ 7.0%, pre-prandial blood glucose (FBS-126 mg/dl), post-prandial glucose (PPBS-200 mg/dl) and microalbuminuria (30-300 mg/dl) were included in this study. Subjects satisfying above criteria but suffering with chronic conditions were excluded from the study. Other renal parameters (blood urea, serum creatinine, urine creatinine calcium and uric acid was measured and e-GFR, albumincreatinine ratio were calculated from previously collected serum & urine samples. 3 ml whole collected for gene expression separately. All biochemical renal parameters were measured by Dade Dimension dry chemistry auto-analyser (Roche Diagnostics), isolation and amplification of m-RNA was performed by "One Step Prime Script RT-PCR (Perfect Real Time)" designed by using Taq Man® probe. KIM-1 and NGAL expressions were measured from buffy coat, a concentrated leukocyte suspension of whole blood. Procedure for preparing buffy coat from whole blood; whole blood was collected in a blood collection tube containing anticoagulant EDTA with aseptic precautions. Added 1 part phosphate buffered saline (PBS) + 2% FBS (Fetal bovine serum) to 1 part fresh whole blood. Reaction mixture centrifuged at room temperature and 200 x g for 10 minutes. Concentrated leukocyte band called buffy coat was removed carefully. Contamination in the buffy coat can be minimized by avoiding RBC lees band with minimum plasma.

RESULT
Screening parameters used for diagnosis of microalbuminuria & non-microalbuminuria showed significant P value. Results are expressed as means ± SD and standard error. Statistical calculations were performed using the R software package. Means in control and type 2 diabetic groups were compared.
Statistical analysis (descriptive and post hoc) of Tables 1 & 2 showed significant difference in P value of HbA1c and urine creatinine. Though GFR is considered as gold standard parameter for renal assessment but in early stage of DN it has less clinical usefulness. Observation of e-GFR in this study was supported by Nielsen SE, et al. study [26].
In this study it was found significant P-value (p <0.000) in all study groups. High degree of significance was found in both KIM-1 and NGAL (Table 3) despite all routine renal parameters reported within reference interval. KIM1 and NGAL m-RNA expressions were measured from a plot (Figs. 1 & 2) which is mentioned in Table 3.

DISCUSSION
KIM1 is a type 1 trans-membrane protein which is exclusively and abundantly expressed in damaged kidney cells. The ectodomain of KIM-1 is shed into urine and easily detectable. According to Waanders F, et al. [27] KIM-1 expressions are measurable within a day after the onset of kidney damage. It is a marker for acute and chronic kidney disease. It was observed that blood KIM1 expressions were unregulated in this study. Similar results were demonstrated by Markus Alter, et al. [28] in experimental diabetic nephropathy study, which shows proximal tubular injury in rodent. In contrast, blood KIM-1 was reported raised shortly after proximal tubular injury [29]. Another study

CONCLUSION
It was concluded that early detection of renal injury in T2DM patients with routine biochemical parameters create dilemma. Outcome of this study evaluated with gene expressions (KIM-1 and NGAL), may help in prediction of risk of DN. Early measurement of KIM-1 and NGAL may prevent ESRD. Further extensive research on large number of subjects with population diversity has been recommended.

CONSENT
All authors declare that 'written informed consent was obtained from patients for publication of outcome of this study' copy of written consent may retrieve from us, if required.