Diabetic Nephropathy & Diabetic Mellitus

Diabetes mellitus (DM) is a major epidemic that affects over 347 million people worldwide (Danaei et al. 2011). The World Health Organization predicts that the incidence of DM will rapidly increase with the number doubling by 2030 (Alwan et al. 2011; World Health Organization 2014). There are two major forms of DM, type 1 and type 2, which are clinically characterized by hyperglycaemia due to insulin insufficiency (Mathis et al. 2001). Type 1 diabetes (T1D), an autoimmune response resultant of genetic and environmental factors in which pancreatic β-cells within the islets of Langerhans are destroyed, comprises of approximately 10% of the total diabetic population but accounts for 40% of diabetes treatment costs. This is due to the generally early onset (usually before the age of 30 years) (Keenan et al. 2010). Type 2 diabetes (T2D) accounts for the majority of diabetes cases with a much larger environmental influence where lifestyle (reduced exercise), diet (high dietary sugar and fat intake) and obesity are strong factors. T2D is characterized by an increase in insulin resistance in many tissues, including skeletal muscle, live and adipose tissue, and a decline in pancreatic islet secretory function leading to hyperglycaemia (Kahn et al. 1993).

A large proportion of people with DM will go on to develop additional complications. As well acute metabolic complications, such as diabetic ketoacidosis from hyperglycaemia and coma as the result of hypoglycaemia (usually due to faulty or incorrect insulin treatment), there is a range of hugely damaging longterm vascular complications – macrovascular (damage to large blood vessels and arteries) and microvascular (damage to small blood vessels) (Forbes & Cooper 2013). Macrovascular complications include the acceleration of cardiovascular disease increasing the risks of myocardial infarction and cerebrovascular disease manifesting as strokes. Microvascular complications include diseases of the eye (retinopathy), brain and nervous system (neuropathy), and kidney (nephropathy) (Forbes & Cooper 2013).

Diabetic Nephropathy is a microvascular disease. It afflicts approximately 25-40% of patients with long-standing diabetes and is the leading cause of end stage renal disease (ESRD). DN affects kidney function and adds additional risk of cardiovascular disease and mortality (Byrne et al. 2010; Collins et al. 2010; Collins et al. 2012). The characterization of DN is by the increased presence of albumin detected in the urine with a decline in glomerular filtration rate (GFR), which indicates the progressive loss of kidney function. Over the course of decades, microalbuminuria can develop to macroalbuminuria (Mogensen & Christensen 1984; Viberti et al. 1982; Parving et al. 1982). The progression from microalbuminuria to macroalbuminuria is, however, not an assumed outlook (Forsblom et al. 1992; Steinke et al. 2005) and there have been studies showing regression to normoalbuminuria (Hovind et al. 2004; Perkins et al. 2003). Current therapies for DN target system blood pressure and intraglomerular hypertension – Angiotensin Converting Enzyme (ACE) inhibitors (Lewis et al. 1993) and Angiotensin II (ANG II) receptor antagonists (Brenner et al. 2001) are considered the first line of defence. Early diagnosis of DN is preferred but critically DN is not clinically detectable until significant kidney damage has developed, necessitating the need to identify early-stage biomarkers (Brennan et al. 2013).

There are many factors that lead to the development of Diabetic Nephropathy and its progression to terminal kidney failure. Chronic elevated blood glucose (hyperglycaemia) and glomerular hypertension combine to cause renal inflammation, glomerulosclerosis and tubulointerstitial fibrosis. Additionally, interplay between many factors, high glucose, Advanced Glycation End-products (AGEs), ANG II, Connective Tissue Growth Factor (CTGF) and TGF-β in the kidney contribute to the damage of many renal cell types, such as expansion of mesangial cell matrix, loss of podocytes, apoptosis of epithelial tubule cells, dedifferentiation, recruitment of macrophages and fibroblast activation (Brennan et al. 2013; Brownlee 2005; Marrero et al. 2005; Sharma & Ziyadeh 1995; Yan et al. 2008; McMahon et al. 2002; Borgeson & Godson 2010). Furthermore, key modulators of DN pathogenesis are oxidative stress, protein kinase C (PKC), activation of Receptor of AGEs (RAGE) and the nuclear factor – κB family of transcription factors (Brennan et al. 2013).

Even though it is still relatively unclear as to why some diabetics are susceptible to Diabetic Nephropathy, the most effective method of risk minimization is the optimal control of blood glucose levels (Turner et al. 1998). Prolonged exposure to hyperglycaemia contributes to the initiation of DN by renal cellular hypertrophy and hyperfiltration, followed by progressive albuminuria and a decline in kidney function as measured by GFR. Approximately 10-15 years after the onset of DM, the onset of microalbuminuria (urinary albumin excretion rate of 30-300 mg/24hr) is detected which progressively deteriorates to macroalbuminuria (urinary albumin excretion of over 300 mg/24hr). Outlined by Brennan et al. (2013), hyperglycaemia is thought to play a role in several mechanisms that contribute to DN pathology, including glucose flux through the hexosamine and polyol pathways, oxidative stress and overproduction of AGEs. A unifying hypothesis was proposed by Brownlee to explain the link between hyperglycaemia and vascular damage, which is centered around the induction of stress caused by reactive oxygen species (ROS). In his hypothesis, Brownlee proposes that the increase in upstream glycolytic intermediates is due to production of mitochondrial superoxide limiting the flux of glucose through the glycolytic pathway (Brownlee 2005). This leads to increased activation of protein kinase C (PKC) isoforms, increased production of AGEs and accelerated glucose flux through the polyol and hexosamine pathways (Brownlee 2005; Brennan et al. 2013). In recent years, it has been observed that the microRNA, miR-377, is also induced by high glucose in mouse and human mesangial cells, and drives increased fibronectin expression (Wang et al. 2008).

The three main focuses for Diabetic Nephropathy research are currently unravelling and identifying genetic mechanisms that dictate whether a diabetic will progress to DN, development of effective clinical biomarkers to aid early diagnosis and development of new therapeutic interventions that arrest or even reverse damage. Many genome-wide association studies (GWAS) have been performed and have identified signals that reside in novel genes with little known about their function making likely of the implication of novel pathways playing an important role in progression of DN (Sandholm et al. 2012). It is also likely that mutations spread across the genome may have protective or causal effects in the development and progression of DN. Larger genetic studies across multiple populations will strengthen the statistical significance of future studies. The need for clinical biomarkers is certainly required to necessitate accurate and early diagnosis of Diabetic Nephropathy. Much effort is being exerted to find renal biomarkers of higher sensitivity and specificity. The development of such biomarkers may also provide better understanding of the complex pathophysiological processes of DN and several approaches are being attempted to identify markers of renal fibrosis, inflammation and oxidative stress (Jha et al. 2014). This multifactorial approach is also being employed in current small molecular drug design efforts. Strategies to control risk factors include the development of RAS blockades, dipepitidyl peptidase 4 (DPP-4) inhibitors and mineralocorticoid receptor (MR) antagonists (DCCT/EDIC Research Group 2011; Hattori 2011; Groop et al. 2013; Meyers et al. 2010; Piotrowski 2012). Further, drug discovery efforts are being trialed for multiple targets of oxidative stress, inflammation and fibrosis (Lv et al. 2014). BMP and their antagonists are also emerging as key targets for development as pharmacological agents for the treatment of bone repair and tissue fibrosis (Ali & Brazil 2014).

The BMP antagonist, Gremlin-1, is associated with DN (Diabetic Nephropathy) progression. This association between Gremlin-1 expression and diabetic nephropathy was identified in experimental mice models of DN and human renal biopsy specimens from patients with DN where Gremlin-1 was found to be overexpressed (Dolan et al. 2003; Dolan et al. 2005). Importantly, in normal adult human tissue, the level of Gremlin-1 was not detected and as disease progression increased there was a correlated increase in detected Gremlin-1 expression, with maximal expression in areas of tubulointerstitial fibrosis – a marker of ESRD.

The promoter region of grem-1 was analysed and revealed several transcription factor binding sites in common with other genes implicated in renal fibrosis, including those of the Notch pathway. This hints at the reactivation of developmental processes in Diabetic Nephropathy as a maladapted repair process (Walsh et al. 2008; Niranjan et al. 2008; Walsh et al. 2010). Allelic depletion of grem-1 in T1D mice was also shown to attenuate several markers of kidney damage (Roxburgh et al. 2009) and grem-1 knockout mice die shortly after birth due to kidney and lung defects (Khokha et al. 2003).

Recently it has been observed that Gremlin-1 may have its own intrinsic signalling capability (treatment of mouse mesangial cells with high glucose or conditioned medium containing Gremlin-1 increased the expression of TGF-β1, CTGF and collagen type IV proteins associated with diabetes-induced damage to the glomerulus) (Huang et al. 2013) and may also interact with several non-BMP binding partners, such as Slit proteins, where Gremlin-1 acts as a negative regulator of monocyte chemotaxis (B. Chen et al. 2004). This new data stresses the critical and delicate balance of BMP and TGF-β signalling in many cells. Upsetting this balance, such as with the upregulation of Gremlin-1, can contribute to the development and progression of human disease (D. Brazil et al. 2015).

The onset of DN perturbs the finely balanced and extensive BMP and TGF-β signalling pathways which ultimately leads to tubulointerstitial fibrosis manifesting with loss of kidney function and organ failure in ESRD. As stated above, the level of Gremlin-1 expression is commensurate to disease progression. Mice lacking one copy of the Grem-1 gene (grem-1+/-) also lack early markers of kidney damage (Roxburgh et al. 2009). Zhang and colleagues have also shown the therapeutic potential of in vivo delivery of an si-RNA plasmid targeting Gremlin-1 restores BMP-7 levels (Zhang et al. 2010). Therefore as a strategy, sequestering Gremlin-1 maybe the most effective means of protecting against diabetic nephropathy.

BMP-7 is one of the most prominent members of the BMP family to be involved in renal development and disease. BMP-7 is also known to be expressed primarily in kidney tubules and glomeruli. Loss of endogenous BMP-7 in occurs in diabetic rats, which is associated with profibrotic activity (McMahon et al. 2000; Morrissey et al. 2002). It has been shown that TGF-β decreases BMP-7 expression in cultured tubule cells. In Diabetic Nephropathy, the rise in TGF-β levels maybe causally linked to the loss of BMP-7. Additionally, CTGF has also been linked with the inhibition of BMP-7 signalling in DN, leading to ECM thickening and albuminuria (Nguyen et al. 2008).

Furthermore, when recombinant human (rh) BMP-7 is exogenously administered leads to a pro-resolution outcome with a partial return of renal function (Morrissey et al. 2002; McMahon et al. 2000). However, BMP-7 was shown not to attenuate TGF-β1 mediated epithelial-tomesenchymal transition (EMT) in human proximal tubule epithelial cells but a protective effect was observed in mouse renal tubular epithelial cells (Dudas et al. 2009). Furthermore, BMP-7 was also shown not to have protective effects against fibrosis or reverse EMT in a mouse model of lung or skin fibrosis (Murray et al. 2008). These results suggest that rhBMP-7 may not be suitable as a therapeutic for use in human disease but the potential for BMP-7 based anti-fibrotic therapeutics is still strong (Ali & Brazil 2014).