PEPTIDIC BIOMARKERS IN URINE FOR THE DIAGNOSIS AND PROGNOSIS OF RENAL DISEASE ASSOCIATED WHIT DIABETES MELLITUS

PEPTIDIC BIOMARKERS IN URINE FOR THE DIAGNOSIS AND PROGNOSIS OF RENAL DISEASE ASSOCIATED WHIT DIABETES MELLITUS

1. INTRODUCTION

Renal disease associated to diabetes mellitus (DM) or diabetic nephropathy (DN) is the progressive and irreversible loss of renal function mainly characterized by microalbuminuria (Vassalotti, Centor et al., 2016). However, the presence of albumin in urine has a predictive value of only 43 % for DN (Becerra, Mata et al., 2012). Also, the estimation of renal damage in the early stages may be biased by the limitation of existing markers and quantification methods, which mainly detect the pathology in its later stages (Wahab, 2017). Therefore, it is desirable to identify organ-specific protein/peptide biomarkers for the diagnosis and evolution of this pathology, highlighting the need for targets of renal structural damage and prognosis of the patient.

Recently, a special interest in urinary peptides has been shown, and their search has being enhanced since they represent at least a 10-fold molar excess compared to protein content of urine (Ling, Mellins et al. 2010). Some studies indicate an increase in protein degradation (Osicka and Comper 1997), also trials of both diabetic human and mice have presented extensive degradation (~ 95%) of albumin to low molecular mass (~ 10 kD) peptides excreted in urine (Strong, Osicka et al. 2005). Furthermore, urinary peptide increasement in patients with renal damage has been clearly demonstrated (Wittke, Fliser et al. 2003). Also,  the concentration of excreted urinary peptides increased as a function of albuminuria values, suggesting damage in the protein degradation process (Osicka, Houlihan et al. 2000). Importantly, the same peptides has not been detected in blood  (Eppel, Pratt et al. 2002).

Recently, an international study Siwy et al., (2014); where nine DM research centers were included reported that (i) patients with DN presented a high abundance of peptides less than 10 kDa, (ii) identified peptides came from 17 different proteins and iii) the most abundant were alpha-1-antitrypsin and collagen-derived peptides, which has been strongly associated to the presence of functional alterations in glomerular filtration barrier or tubular reabsorption, that  could be early events in the establishment of DN.

In this sense, it is proposed to confirm the presence of the most relevant urinary peptides in DN and DM as well as the design of nanoparticles based on molecularly imprinted polymers (MIPs) that can dock to sensors.  The synthesis may be carried out by co-polymerization of the desired template (peptide of interest) with a functional monomer and stabilized by crosslinking agent. This interaction promotes the generation of a stereochemical complementary recognition site. In general, the application of these nanoparticles may be useful for the detection of potential peptide biomarkers of DN and DM which are present at a low concentration in urine samples and their identification may be limited to conventional methods.

2. RENAL DISEASE) AND ITS RELATIONSHIP WITH DIABETES MELLITUS

Renal disease associated to diabetes mellitus (DM) or diabetic nephropathy (DN) is the progressive (≥ 3 months) and irreversible loss of renal function characterized by i) structural or functional alterations determined by renal biopsy or imaging; ii) albuminuria with excretion rates greater than 250 mg / 24h (> 200 μg / min) or iii) lower glomerular filtration rate (GFR) of <60 mL / min / 1.73 m2 as well as an albumin-creatinine ratio of > 30 mg / G (> 3 mg / mmol) (Vassalotti, Centor et al., 2016). Epidemiological data suggest an incidence increasement of systemic diseases, progression to kidney disease and premature death in patients with DN. Globally, diabetes mellitus (DM) is the leading cause of DN (The National Center for Health Statistics, Centers for Disease Control and Prevention, 2015). In the United States alone, 1:10 people with DM and 1:20 with DN, of which at least 640,000 require dialysis as a treatment for end-stage renal failure, which translates into an annual cost of 50 billion dollars. On the other hand, there is a 90 %- 95 % probability that a patient with DM course with DM type 2. In Mexico, the prevalence reaches 14.4%, representing the second cause of death (Villalpando, De la Cruz et al., 2010, Geiss, Wang et al., 2014).

3. CURRENT RENAL FAILURE BIOMARKERS

The detection of microalbuminuria is the earliest clinical sign of renal damage and a predictor of cardiovascular disease in patients with DM. A 24-hour urine collection is the gold standard for the measurement of microalbuminuria (20 a 200 μg/min), but is well accepted as sieve shorter time collections as well as an isolated urine sample; usually the first morning urine (Gonzalez-Suarez et al,. 2013). This tool is based on albumin quantification present in urine by immunological methods and it is defined by levels of 30-300 mg / 24 hours of albumin in urine samples, as well as an albumin / creatinine ratio of > 30 μg / mg. However, it is recommended to calculate the estimated volume of globular filtration (VFGE), since, as altogether represent a greater accuracy and a significant improvement in the predictive capacity of the method (Chavan et al, 2011). The behavior of these signs across the different stages of pathology is shown in Fig. 1

However, estimation of renal damage in the early stages may be biased by the limitation of existing markers and quantification methods, mainly detecting the pathology in its later stages. Also, the presence of albumin in the urine is not a specific factor for ND since its presence is frequently related to inflammatory events, hypertension and exercise in non-diabetic patients with a 43 % predictive value for ND. There are also false positive reports, which evidence the poor accuracy of the method (Becerra, Mata et al., 2012). Therefore, it is desirable to identify organ-specific protein biomarkers for the diagnosis and evolution of this pathology, highlighting the need for identification of renal structural damage and prognosis markers.

Years after diagnosis[pic 1]

Figure 1. Progression of albumin excretion rate and volume of glomerular filtration with respect to time in patients with type 1 DM

4. PATOPHYSIOLOGY MECHANISM OF RENAL DISEASE

Hyperglycemia is the initiating event in DN, which causes structural and functional changes such as glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, and microalbuminuria, followed by the development of glomerular basement membrane thickening, accumulation of mesangial matrix, overt proteinuria, and finally glomerulosclerosis at end stage renal disease (Lim, 2014).

In this context, we will address the main physiological changes that comprehend this pathology, where, glomerular hyperfiltration corresponds to a detonating factor in the early stages of ND. This is a complex multifactorial hemodynamic phenomenon which included numerous humoral factors such as nitric oxide, prostaglandins, renin angiotensin aldosterone system, natriuretic atrial peptide, reactive oxygen species and other humoral and growth factor. Those, basically act either by potentiating vasodilation of the afferent arteriole or by vasoconstriction of the efferent arteriole, considered as primary vascular factors (Barber Fox and Barber Gutiérrez 2003). In this process, glomerular hemodynamics is being compromised due to shear stress and mechanical stain; releasing paracrine and autocrine cytokines and growing factors that produce structural changes. Some of the possible pivotal phenomena involved aim to an increasement of glomerular filtration rate by intact macula-densa mechanism that promotes sodium chloride reabsorption in proximal tubules or henle loop (Vinod 2012).

5. STRUCTURAL AND HISTOLOGICAL CHANGES DURING DN

The clinical manifestations of DN are strongly related to structural changes. Glomerular basement membrane thickness and mesangial expansion are the first noticeable changes related with glomerular lesion presence commonly, with nodular origin (Kimmelstiel-Wilson) leading to glomerular dysfunction (Tervaert, Mooyaart et al. 2010). Both mesangial expansion and glomerular basement membrane (GBM) and tubular basement membrane (TBM) thickening are a consequence of extracellular matrix (ECM) accumulation. due the increased deposition of the normal ECM local components of types IV and VI collagen, laminin and fibronectin due to their increased production, decreased degradation or both (Fioretto and Mauer 2007). Whereas the expression of heparin sulfate and the extent of sulfation decreases. In contrast to the mesangial matrix in which the α1 and α2 of type IV collagen are mainly expressed, the GBM contains α3, α4, and α5 chains. In DN, there is an up-regulation of α1 (IV) and α2 (IV) chains in mesangial cells, whereas α3 (IV) and α4 (IV) expression is increased in the GBM. Deposition of collagen type I and III in the mesangial area occurs late in glomerulosclerosis and is not an early event (Vinod 2012).

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