Advanced glycation end products mediated cellular and molecular events in the pathology of diabetic nephropathy

Introduction

Proteins and polypeptides are routinely subjected to several post-translational modifications during their lifetime, which includes phosphorylation, glycosylation, deamination, ubiquitination and sumoylation. These post-translational modifications may facilitate proteins to achieve proper folding and sorting into distinct cellular compartments, elicit intracellular signaling and confer stability during stress conditions and degradation and regulation of their intracellular levels. Proteins are also susceptible to post-translational modifications that could alter their structure, function, and half-life during normal aging and pathological conditions such as diabetes. One such post-translational modification is non-enzymatic glycation (NEG, also called non-enzymatic glycosylation). NEG of client proteins ensue crosslinking and aggregation that may adversely affect the structural and functional properties of proteins. In addition to affecting the physicochemical properties of the afflicted proteins, NEG also induces aberrant cellular signaling, activation of transcription factors and alter gene expression profiles. The final products that accumulate owing to NEG are termed Advanced Glycation End Products (AGEs). Although NEG occurs at a lower rate over a lifetime, it occurs more rapidly in clinical conditions such as diabetes and is implicated in the pathophysiology of several complications of diabetes and other age-related disorders such as cataract, renal failure, cardiovascular complications, and Alzheimer’s disease. Tissue AGEs correlate very tightly with early kidney, eye, and nerve disease in patients with diabetes mellitus (DM). The focus of this review is to provide insights of chemical reactions of NEG and AGE formation, details about predominant client proteins affected by NEG, fate of AGEs, details about the interaction between AGEs and their receptors and down-stream signaling cascades. Further, we discuss the mechanism of AGE-mediated cellular and molecular events that promote pathological events during diabetic nephropathy (DN). Possible therapeutic and dietary interventions to prevent AGE formation and to cleave AGE crosslinks will also be discussed.

Chemical reactions of non-enzymatic glycation (NEG)

In 1912, the French chemist Louis-Camille Maillard reported for the first time the formation of brown colored substances during the enzyme-free reaction between reducing sugars and glycine (1). The complex inter-related reactions between the free amino group and reducing sugars are known as the Maillard reaction, which is largely considered as a browning reaction that takes place in the processing of foods and beverages (2). The significance of the Maillard reaction was dormant for a very long time till browning reactions were identified in vivo by Monnier and Cerami in 1981 (3). The formation of fluorescent yellow pigments and crosslinks upon incubation of eye lens proteins with reducing sugars was reported, similar to those reported in aging and cataractous lenses (3). The classical reaction of NEG occurs through the covalent bonding of reducing sugars (e.g. glucose or fructose) with free amino groups of proteins at N-terminal amino acid residues and/or ϵ-amino groups of lysine and arginine. NEG preferably occurs at amino groups that are either close (~5 Å) to an imidazole residue or a part of lysine doublet (4). The initial reaction between the reactive carbonyl group and a free amino group of a protein results in the formation of a labile Schiff’s base (Figure 1A). The initial Schiff’s base (aldimine) undergoes spontaneous rearrangements to a relatively stable ketoamine (1-amino-1-deoxy-2-ketose), which is known as an Amadori product, after Mario Amadori, an Italian scientist, who attempted to characterize the products of a condensation reaction between glucose and p-phenetidine (5). Both the Schiff’s base and Amadori product can undergo further reactions with accessible lysine/arginine residues of the client proteins and form AGEs (6), (7). The rate at which the Schiff’s base forms takes hours to days, whereas it may take days to weeks and weeks to years to progress to Amadori products and the formation of AGEs, respectively (8). The term AGEs was coined by Cerami to refer to fluorescent pigments arising from the degradation of fructosamine, which can crosslink proteins (9). The term AGEs is used to describe any protein-bound adduct detected after the formation of the initial Schiff’s base/Amadori product, and that could be considered as the final product of Maillard reaction. AGEs may result from a one-step conversion from the Amadori product, as it happens in the case of carboxymethyllysine (CML), or a series of complex reactions leading to a crosslink such as pentosidine and glyoxal-lysine dimer (GOLD) (Figure 2A). In addition, the oxidative breakdown of Amadori products can lead to the formation of reactive carbonyls such as glyoxal, methyl-glyoxal, and 3-deoxy-glucosone (3DG). These carbonyl compounds can, in turn, react with free amino groups of proteins to form intermediate glycation products. A series of reactions including dehydration, successive β-eliminations, and condensation reactions of both Amadori products and intermediate glycation products eventually result in the formation of irreversible inter- or intra-protein crosslinking AGEs that could persist for the lifetime of the modified substrate. John Hodge summarized the complex network of NEG reactions that take place during food processing and storage in his seminal review (10). The formation of AGEs from autoxidation of Amadori products is known as ‘Hodge pathway’ and cleavage of dicarbonyl compounds from Schiff’s base refers to ‘Namiki pathway’ (Figure 1B). Alternatively, the formation of dicarbonyls from autoxidation of glucose, ribose, fructose, and glyceraldehyde is known as ‘Wolff pathway’ (11).

Figure 1:

Mechanism and pathways of formation of advanced glycation end products.

(A) Classical reactions in the formation of advanced glycation endproducts (AGEs). The initial nonenzymatic interaction between the highly reactive carbonyl group of glucose with a free amino group on proteins creates a reversible Schiff’s base, which spontaneously undergoes rearrangement itself into a partially reversible Amadori product. Amadori products rearrange to form heterogeneous AGEs including CML, pentosidine, FFI, MOLD and GOLD. (B) A complex network of NEG reactions. Highly reactive carbonyl intermediates (glyoxal, methylglyoxal and 3-deoxyglucosone) can be formed from the auto-oxidation of monosaccharides (e.g. glucose: Wolff pathway) or from Schiff’s base (Namiki pathway) or from Amadori products (Hodge pathway). The highly reactive intermediates that are formed by these three pathways can react with free amino groups to form diverse AGEs. The possible sites that could be targeted to inhibit NEG and to prevent AGEs mediated complications are indicated in numbers [1, 2, 3, 4].

Figure 2:

Schematic representation of predominant AGEs and receptors for AGEs (RAGE).

(A) AGEs can exist as protein adducts (CML) or AGEs can crosslink proteins (GOLD and pentosidine). (B) Structure of full-length RAGE and soluble RAGE (sRAGE). sRAGE derived via proteolytic cleavage of full-length RAGE by ADAMS 10, known as cleaved (cRAGE) or via alternative splicing of RAGE mRNA lacking the cytoplasmic tail and transmembrane domains known as endogenous secretory RAGE(esRAGE). Full-length RAGE, upon interaction with a ligand (for example, CML), elicit intracellular signaling, whereas sRAGE fails to transduce intracellular signaling and forms a natural defense mechanism to combat AGEs.

Advanced glycation end products

The AGEs comprise a large number of heterogeneous chemical structures and a majority of them have a crosslinking propensity between proteins, which alter their structure and function. AGEs are relatively insoluble, yellow-brown in color and a majority of them exhibit a fluorescence phenomenon. CML, pentosidine, argypyrimidine, pyrraline, imidazolone, 2-(2-furoyl)-4(5)-furanyl-1H-imidazole (FFI), glyoxal-lysine dimer (GOLD), methylglyoxal-lysine dimer (MOLD), 3-deoxyglucosone-lysine dimer (DOLD), carboxyethyl-lysine, fructose-lysine, methylglyoxal-derived hydroimidazolones are well-known AGEs (Table 1). Both biochemical and immuno-histochemical studies suggested that CML modifications of proteins are predominant AGEs that accumulate in the majority of tissues from diabetic subjects (12), (13). CML is nonfluorescent protein adduct and it can be formed by the oxidative degradation of Amadori products or the direct reaction of glyoxal with lysine. Quantification of CML levels is considered as an index of AGE content in food, and CML is the major epitope of the commonly used polyclonal antibodies employed to detect AGEs. On the other hand, pentosidine is a fluorescent glycoxidation product that forms protein-protein crosslinks. It is composed of an arginine and a lysine residue crosslinked to a pentose (14), (15). Carbonyl compounds (glyoxal, methylglyoxal, and 3-DG) derive from both oxidative degradation or auto-oxidation of Amadori products and are highly reactive molecules leading to protein crosslinks (16). AGEs such as carboxymethyl-hydroxy-lysine, carboxyethyl-lysine, fructose-lysine, methylglyoxal-derived hydroimidazolones, and pyrraline form non-fluorescent protein adducts, while GOLD and MOLD form nonfluorescent protein crosslinks.

Among commonly available sugars, glucose exhibits the slowest glycation rate, while intracellular sugars such as fructose and glucose-6-phosphate form AGEs at a much faster rate and form heterogeneous crosslinking products (17). Reactive intermediates produced from degraded AGEs can bind and form ‘second’-generation AGEs. Intermediate AGEs such as glyoxals, glycolaldehydes, and hydroxyl aldehydes can be formed during oxidation of both carbohydrates and fatty acids. The increase in glycoxidation and lipoxidation in diabetes contribute to the accelerated accumulation of carbonyl compounds in addition to NEG, and such a condition is generally referred to as carbonyl stress. The intermediates arising as a result of carbonyl stress also react with free amino groups of proteins to form AGEs. Therefore, apart from the direct action of reducing sugars with free amino groups, AGEs can also be formed via distinct oxidative reactions. Formation of AGEs is not only restricted to proteins, but lipoproteins and nucleic acids are also susceptible. It is noteworthy that AGEs can also be exogenously ingested through food, particularly from baked and processed food (18), (19). AGEs in cooked food remain active in the circulation after oral uptake. Although AGEs accumulate endogenously at an accelerated rate during diabetes, AGEs are also formed at lower rates during normal metabolic processes. However, they can sequester by the natural anti-AGE cellular defense system of the host. Environmental factors such as diet, smoking, and consumption of alcohol influence the rate of AGE accumulation.

Tissue targets of AGE modification

Free AGEs and protein-bound AGEs are found in several sites including plasma, vasculature, eye lens, retina and various renal compartments including mesangium, glomerulus, and basement membrane. Both extracellular and intracellular proteins are susceptible to modification with AGEs. In general, proteins and polypeptides have a limited life span and are periodically degraded and resynthesized. Exceptions to this are proteins that are stable throughout the lifetime of the individual, such as crystallins in the eye lens. Proteins with a relatively long biological half-life are expected to be more susceptible to modification with AGEs. Extracellular matrix (ECM) proteins such as collagen, laminin, elastin, and plasma proteins including hemoglobin and albumin,with a relatively short half-life, are also prone to NEG and crosslinking. Nevertheless, proteins in E. coli cultured under normal growth were shown to undergo NEG (20).

The accumulation of AGEs in eye lens crystallins contribute to the age-related incidence of brown color, fluorescence and decreased solubility of lens crystallins, thus contributing to senile cataract. α-crystallin, a predominant soluble eye lens protein, upon glycation with either reducing sugars such as glucose, fructose, glucose-6-phosphate or sugar derivatives such as methylglyoxal undergo crosslinking and form a range of high-molecular-weight aggregates (17), (21). Glycated α-crystallin displayed reduced chaperoning activity compared to unmodified α-crystallin to prevent thermal or stress-induced aggregation of co-lenticular proteins and substrate proteins. Reduced chaperoning activity of glycated α-crystallin is implicated in the development of diabetic cataract and impaired vision (21), (22).

Collagen and hemoglobin are extensively studied proteins for modification with glycation. Glycation results in decreased solubility and flexibility of collagen and is resistant to degradation by proteases. Glycation with glucose abolishes the positive charge of lysine and the formation of CML imparts a negative charge on lysine. Perturbations in charge profile of collagen monomers affect their aggregation into fibers and normal architecture of basement membrane (23), (24). In addition to affecting inter-monomeric interactions, glycation of collagen alters its interaction with other components of the ECM such as proteoglycans, vitronectin, and laminin. In the case of glycated hemoglobin (HbA_1c), NEG occurs between glucose and the free amino group at N-terminal valine of the β-chain. HbA_1c is a measure to predict the extent and duration of glycemic control. Unlike ECM proteins, HbA_1c is more prone to proteolytic degradation compared with native hemoglobin. The life span of erythrocytes decreases by ~7 days for every 1% increase in HbA_1c levels. Free radicals arising as a consequence of HbA_1c degradation manifest in a plethora of events such as (a) increased fragility and decreased life span of erythrocytes, (b) erythrocyte aggregation, and (c) increased viscosity of blood and reduced blood flow.

Other than hemoglobin, plasma proteins including low-denisty lipoproteins (LDL) and immunoglobulins also undergo glycation. NEG alters LDL clearance and increases susceptibility to oxidative modifications. Macrophages have more affinity for uptake of glycated LDL over normal LDL via scavenger receptors and these events ultimately manifest in foam cell formation, which is a characteristic of atherosclerotic plaques (25). AGEs were also shown to alter the endothelial surface from an anti-coagulant nature to a pro-coagulant nature by reducing thrombomodulin expression and the concomitant induction of tissue factor expression (26). Besides these popular targets of NEG, several other proteins are also vulnerable to NEG and AGE accumulation. Glycated laminin has decreased affinity to polymerize and is unable to bind collagen and heparin sulfate proteoglycans, thus affecting the integrity of matrix interactions (27). NEG-mediated crosslinking of structural proteins and the resultant accumulation of AGEs contribute to the long-term complications of diabetes such as cataract, retinopathy, neuropathy, nephropathy, amyloidosis, and Alzheimer’s disease. Histones, which play an important role in chromatin organization and epigenetic regulation of gene expression are vulnerable to NEG as evidenced by the accumulation of CML, pentosidine, imidazolone, and FFI (28). Among several histones, H1 and H2B are extensively subjected to glycation and all of the identified glycated residues were lysine (28). Glycation of histones results in structural alterations, which are speculated to alter the integrity of chromatin structure and function. Carboxyethyl guanine is reported to contribute to mutations and DNA transpositions (29). Glycosylation of immunoglobulins (IgG, IgA, and IgM) was noticed in both type 1 and type 2 DM patients with DN (30). Glycated IgG undergoes structural perturbations, compromised complement fixation, and reduced interaction with complementary antigens (31). Extensive NEG is reported during autoimmune disorders (32). AGEs formed on IgG become targets for circulating antibodies in American Indians with rheumatoid arthritis (32). Anti-IgG-AGE antibodies recognize and bind to glycated IgG, thus preventing the clearance of glycated antibodies by AGE receptors. Other plasma proteins such as albumin, complement C3, fibrinogen, transferrin, haptoglobin, and anti-trypsin were also found to be the targets of NEG. Although proteins with the longest biological half-life are more prone for glycation, glycability of client proteins depends on intrinsic chemical characteristics including charge and localization of target lysine residues and the availability of glycating agents. In general, basic proteins show a greater glycation rate than acidic proteins despite shorter half-life (33).

AGEs and diabetic nephropathy

Both clinical and experimental studies in type 1 and type 2 diabetes strongly suggest AGE involvement in the pathogenesis of diabetic complications including DN. DN is the most common microvascular chronic complication and develops in 20%–40% of type 1 and type 2 diabetic patients. DN accounts for approximately one-third of all new cases of end-stage renal disease (ESRD). Glomerular capillaries that are affected during DM ensue a progressive decline in glomerular filtration rate, accompanied by several stages of proteinuria such as microalbuminuria, macroalbuminuria and overt proteinuria, and culminate in ESRD warranting renal replacement. Progression to ESRD is further enhanced by hyperglycemia and hypertension, which are other features of type 2 diabetes. Renal disease in diabetic patients is characterized by both hemodynamic (hyperfiltration) and structural abnormalities (glomerulosclerosis, interstitial fibrosis). Prominent glomerular changes include thickening of basement membranes, mesangial expansion and hypertrophy and loss of podocytes. Thickening of tubular basement membranes, tubular atrophy, interstitial fibrosis, and arteriosclerosis are prominent changes in the tubulointerstitial compartment during DN. The critical role of AGEs in the pathogenesis of renal damage has been evidenced by a study in nondiabetic rats wherein administration of AGEs induced proteinuria and degenerative changes that were observed in DN (44).

In type 1 diabetic patients, advance from normal renal function to ESRD have significantly increased serum levels of fluorescent non-CML AGEs (45). In contrast, another study revealed that CML in type 1 diabetic patients correlates with the severity of nephropathy (46). In type 2 diabetic patients, there is a significant increase in CML- and hydroimidazolone-AGEs (47). CML-human serum protein levels were higher in those patients with proteinuria and increase in circulating AGE peptides correlated with the severity of renal impairment (41). The severity of DN correlates to the extent of AGE formation and RAGE expression in glomerular and tubulointerstitial compartments. The course of DN is further characterized by decreased glomerular filtration rate (GFR). AGEs induce asymmetric dimethylarginine (ADMA) in renal tubular cell production, which is an endogenous inhibitor of nitric oxide (NO) synthase. Prevention of renal NO production by AGEs is implicated in the decreased GFR (48).

AGEs alter the metabolism of ECM components and manifest in DN

Accumulation of ECM in the mesangium, tubular interstitium, and glomerular basement membrane (GBM) are hallmark features of DN. AGEs deregulate the balance between synthesis and degradation of ECM components, particularly collagen. Collagen is one of the extensively studied proteins for modification with glycation. AGE formation ensues intra and intermolecular crosslinks in collagen that lead to significant structural changes, including altered packing density and surface charge, together manifested in reduced solubility (15), (49). Glycated collagen compromises its self-assembly and interaction with sub-ECM components. Impaired intermolecular interactions within the collagen fibers and with other components of basement membranes ensue decreased elasticity of arterial vessel walls and capillaries in the glomeruli. Apart from affecting the metabolism of collagen, AGEs alter the interactions among ECM components. Besides, intra-ECM interactions, cell-matrix interactions may also be disrupted by glycation, contributing to impaired cell growth, altered cellular adhesion, and loss of the epithelial phenotype. Modification of various ECM proteins with AGEs impairs their degradation by matrix metalloproteinases, contributing to thickening of the basement membrane and mesangial expansion (50).

The ECM of podocytes and renal capillaries are negatively charged and are able to prevent loss of serum albumin into the glomerular filtrate owing to charge-charge repulsion. However, glycation and accumulation of AGEs in the ECM components of renal capillaries alter the electrostatic interactions that ensue vascular permeability to albumin. AGEs accumulated in ECM components could trap plasma proteins, lipoproteins, and immunoglobulins and affect the homeostasis of normal renal function and aggravate glomerulosclerosis (50). Glycated collagen from the basement membrane increases aggregation propensity of platelets (51). Adverse effects of AGE accumulation on ECM components are also evidenced by studies that employed AGE breakers. Cleavage of AGE-induced crosslinks by ALT-711 and N-phenacylthiazolium bromide improved the solubility of collagen and reduced matrix accumulation in the kidney (52). AGEs stimulate the expression of transforming growth factor β (TGF-β) in podocytes, tubular cells, and mesangial cells. TGF-β is a pro-fibrotic factor that augments the production of type IV collagen, laminin, and fibronectin. AGEs also induce platelet-derived growth factor, which is also pro-fibrotic. Besides hyperglycemia, glycoxidation and lipoxidation products also contribute to AGEs, including CML, pentosidine, and malondialdehyde-lysine. Altogether, AGEs directly and indirectly alter the metabolism and expression of ECM components that manifest in an advanced nephropathy phenotype (Figure 3).

Figure 3:

AGE modification of ECM proteins results in remodeling and thickening of the basement membrane.

Stimulation of RAGE by AGEs in addition to increased production of reactive oxygen species resultesin the activation of various signaling events and the secretion of inflammatory cytokines and growth factors and culminates in several histological and hemodynamic changes implicated in the pathology of DN. Interaction of AGEs with AGE-R2 leads to adverse effects on cells such as inflammation, cell activation and altered metabolic effects, whereas the interaction of AGEs with AGE-R1/R3 results in cellular uptake and degradation of AGE-modified client proteins.

AGEs induce renal inflammation and fibrosis

Infiltrating macrophages also compounds to disproportionate synthesis and altered metabolism of ECM components during DN. AGEs stimulate monocyte chemoattract protein-1 (MCP-1) expression in mesangial cells, which is associated with monocyte/macrophage infiltration into the mesangium (53), (54). Plasma MCP-1 levels were associated with the rate of albumin excretion. Urinary MCP-1/creatinine ratios in diabetic patients with microalbuminuria were much higher than those in healthy controls (55). Selective targeting of MCP-1 significantly improved fibrosis and albuminuria in streptozotocin-induced DN (56), suggesting that AGE accumulation in the glomeruli could be implicated in inflammatory and fibrogenic reactions. Another prominent feature of DN is tubulointerstitial injury, which correlates with a decline in renal function. Activation of RAGE induces the expression of TGF-β and other cytokines that are suggested to mediate the epithelial-myofibroblast transdifferentiation of renal tubular cells (57). Exposure to AGEs induced dose-dependent epithelial-myofibroblast transdifferentiation of tubular epithelial cells as determined by morphological changes, alpha-smooth-muscle actin expression, and loss of epithelial cadherin (E-cadherin) expression. Epithelial-myofibroblast transdifferentiation was apparent in the proximal tubules of diabetic rats and a kidney biopsy from a diabetic patient (57).

AGEs induce podocytopathy and mesangiopathy

Glomerular podocytes offer a size-selective filtration barrier that restricts the entry of plasma proteins from the circulation into the urine. Studies in both diabetic subjects and animal models of DN revealed that proteinuria is associated with decreased density of podocytes (58). Renal biopsies from patients with DN suggested that AGE accumulation was predominant in the GBM and its accumulation involves up-regulation of RAGE in podocytes (59). In a recent study, we demonstrated that CML activates RAGE expression in podocytes and activates NF-κB signaling, which in turn induces the expression of zinc finger E-box binding homeobox 2 (ZEB2) (60). ZEB2 is a transcription factor that orchestrates epithelial-mesenchymal transition (EMT) by promoting a ‘cadherin switch’, which involves suppression of E-cadherin (epithelial marker) and activation of N-cadherin (a mesenchymal marker). EMT of podocytes is implicated in their detachment from GBM, thus decreasing podocyte count in the glomeruli of diabetic rats. On the other hand, exposure of podocytes to CML is associated with loss of placental cadherin (P-cadherin), a key protein localized to the podocyte slit-diaphragm (60). Slit-diaphragm is the size-selective filtration barrier that regulates podocyte perm-selectivity and impedes the loss of high-molecular-weight molecules into the urine. Both CML-induced EMT and loss of P-cadherin expression could be linked to albuminuria in diabetic conditions (Figure 4). It was shown that an activated AGE-RAGE axis ensues TGF-β-induced EMT in normal rat kidney epithelial cells. Mesangial cells play a critical role in maintaining the integrity of glomerular capillary tufts, whereas AGEs-induced apoptosis of these cells may contribute to glomerular hyperfiltration, an early manifestation of renal dysfunction during DN.

Figure 4:

The mechanism by which CML induces podocyte injury and proteinuria.

CML, via RAGE, induces NF-κB activation, which in turn stimulates ZEB2 expression. Elevated ZEB2 expression orchestrates podocyte damage by two different ways: (1) ZEB2 transcriptionally suppresses E-cadherin expression by which podocytes may undergo epithelial-mesenchymal transition and detach from the basement membrane, which manifests in decreased podocyte count per glomeruli. (2) ZEB2 also suppresses P-cadherin expression, which is localized to slit-diaphragm (SD) protein. Reduced P-cadherin expression ensues in compromised function of SD. CML also suppresses nephrin, another key protein of SD by an unknown mechanism. Together, the loss of E- and P-cadherin and nephrin manifest in decreased podocyte count and proteinuria.

AGEs and the renin-angiotensin system

Renin-angiotensin system (RAS) comprises renin, angiotensinogen, angiotensin I, angiotensin-converting enzyme (ACE), angiotensin II and their receptors. The kidneys synthesize all the components of RAS and the predominant function of RAS is to regulate blood pressure and fluid balance. Activation of the local RAS occurs in the proximal tubular epithelial cells, mesangial cells, and podocytes during DN (61). Renal angiotensin II activity is augmented during the early stages of DN. Angiotensin II is a potent vasoconstrictor, providing a possible explanation for increased renal filtration in the early stages of DN. Inhibitors of ACE, which converts angiotensin I to angiotensin II, decrease renal hyperfiltration and prevent glomerular injury. Angiotensin II triggers hypertrophy of both mesangial and tubular epithelial cells (62), (63). Angiotensin II elicits its action via angiotensin II type 1 receptor (AT1R), whereas AGEs stimulate AT1R expression. Therefore, AGEs are implicated in enhanced angiotensin II activity and a series of adverse events and degenerative changes associated with DN. It was also reported that RAS induces MCP-1 and TGF-β (62), (63), (64), (65). MCP-1 promotes macrophage recruitment, thus ensuing extracellular matrix accumulation and renal fibrosis. It was also reported that angiotensin II induces ROS production, which may contribute to podocyte autophagy (66). Our earlier study revealed that accumulation of glomerular CML is associated with elevated expression of MCP-1 and proteinuria in diabetic rats (67).

Anti-glycation strategies

The normalization of blood glucose levels in diabetic patients is challenging. Intensive treatment strategies to reduce hyperglycemia in diabetic patients are associated with severe hypoglycemia and increased mortality risk. Although strict control of blood glucose levels inhibits the progression of albuminuria levels, it may not offer significant differences in the outcome of clinical kidney disease. Therefore, to prevent diabetic renal complications, additional therapeutic options are required than that prescribed for normalization of blood glucose. The steady-state abundance of AGEs reflects not only on the status of glycemia, but also on the balance of oral intake, endogenous formation and their degradation. A natural protective mechanism against deleterious post-translational modifications including glycation is protein turnover (68). Proteins with shorter half-lives are prone to rapid degradation and are minimally affected with glycation. Another natural AGE clearing system includes sRAGE, which binds with AGEs and facilitates their degradation. Considering the adverse events and pathologies associated with AGE accumulation, several natural and synthetic compounds have been screened for their anti-AGE effects (Table 2). Anti-glycation compounds are broadly classified as 1) anti-AGEs that prevent AGE formation, 2) AGE breakers that break preformed AGEs and Amadori products, 3) RAGE inhibitors, 4) redox regulators, and 5) neutraceuticals that serve as anti-glycating agents.

Dietary recommendations

In addition to the AGEs that are formed in the body, AGEs are also obtained from various dietary sources. Dietary AGEs are highly present in heat-processed foods and are known to contribute to increased oxidative stress and inflammation. The role of dietary AGEs in human health and disease was ignored as it was assumed that they are poorly absorbed. Nevertheless, it was demonstrated that a significant proportion of ingested AGEs are absorbed from food (96). Consumption of AGE-rich diets is associated with the incidence of kidney disease, whereas dietary restriction of AGEs improved kidney dysfunction (97). Dietary AGEs correlate with circulating AGEs (CML and methylglyoxal) and markers of oxidative stress in healthy subjects (98). Increasing evidence suggests that restriction of dietary AGEs help in combating chronic kidney disease. Several dietary sources rich in antioxidants prevent glycation and accumulation of AGEs. Feeding green tea extract prevented AGE-related collagen crosslinking and formation of fluorescent AGEs in C57BL/6 mice (99). Epigallocatechin-3-gallate revealed a promising in vitro anti-glycation potential by acting as a reactive carbonyl scavenger and antagonized AGE-induced pro-inflammatory changes (100). Foods high in carbohydrates have the lowest amount of AGEs (19). Processed foods high in protein and fat, such as meat, cheese, and egg yolk are rich sources of AGEs. A diet heavy in AGEs results in proportional elevations in serum AGE levels in diabetic patients. On the other hand, restriction of AGEs in the diet results in a 30%–40% decrease of serum AGE levels in healthy individuals (18), (101). In diabetic subjects, a diet with high AGE content induced an elevated expression of inflammatory mediators including C-reactive protein, NF-κB, and VCAM-1 compared with a regular diet (102), (103). The excretion of AGEs is suppressed in DN patients compared to healthy controls. The renal clearance of dietary AGEs is impaired in diabetic subjects with renal insufficiency, whereas dietary restriction of AGEs in patients with renal failure improved tissue injury. Therefore, the dietary intake of AGEs might represent an additional risk for AGE toxicity.

Many spices and herbs including curcumin, ginger, cinnamon, and cloves were shown to inhibit glycation in vitro, and their anti-glycation potential is associated with their polyphenolic content (100), (104). Naturally occurring flavonoids, such as lutein, quercetin, and rutin were shown to inhibit AGE formation. It was found that curcumin administration combat oxidative stress and inflammation, thus ameliorating DN (105), (106), (107). Feeding of cinnamon to diabetic rats prevented CML accumulation in the glomeruli and improved AGE-mediated loss of expression of key podocyte proteins nephrin and podocin. Procyanidin-B2, an active component of cinnamon also prevented accumulation of CML in the glomerular region (67). It is interesting to note that cinnamon and procyanidin-B2 supplementation to diabetic rats exhibited systemic anti-glycation potential as evidenced by a reduction in HbA_1c levels and glycation-prone RBC-IgG crosslinks (67). Together, by preventing AGEs in blood and in the kidney, cinnamon and procyanidin-B2 improved proteinuria in diabetic rats. Feeding of ellagic acid (EA), a polyphenol present in fruits and vegetables, to diabetic rats prevented the glycation-mediated RBC-IgG crosslinks and HbA_1c accumulation (108). EA ameliorated the accumulation of CML in the kidney and prevented proteinuria in experimental diabetic rats (108). Plant-derived foods like cereals, legumes, vegetables, and fruits that are rich in carbohydrates contain few AGEs unless they are prepared with added fats and exposed to dry-heat cooking methods. By avoiding fatty meats, full-fat dairy products and solid fats dietary, the intake of AGEs can be restricted (19). Dry-heat cooking methods such as broiling, roasting, grilling, baking, sautéing, pan-frying, and deep fat frying propagate and accelerate new AGE formation. AGE formation during cooking can be significantly reduced by cooking food at low temperatures, cooking for short durations, using moist heat methods and by using acidic ingredients like lemon juice.

Conclusion

Considering the magnitude of diabetes incidence, the burden of diabetes on human health and the importance of renal function, it is important to have potent treatment strategies to prevent DN. The dearth of affordable and effective renal care for subjects with chronic kidney disease is also contributing to the global healthcare burden. Proteinuria is also a strong predictor of other diseases, including cardiovascular and cerebrovascular complications. Regulation of glucose homeostasis has been a major treatment option for diabetics. The accumulated evidence argues for the critical role for AGEs in the pathophysiology of DN as evidenced by the involvement of AGEs in hypertrophy, thickening of the GBM and EMT of podocytes (Figure 5). Therefore, evaluation of the efficacy and safety of anti-AGE compounds including AGE blockers/crosslink breakers, ACE inhibitors, sRAGE and inhibitors of AT1R is the choice of preventive medicine for combating DN. Successful accomplishment of anti-AGE therapy could be a part of a regimen for subjects with diabetic renal complications. Discovering and developing a novel drug target against AGE-mediated renal pathology will be an effective therapeutic intervention against DN. It is evident that both dietary AGEs and local AGEs contribute to tissue injury and impaired renal function. A regimen besides regulating blood glucose to the normal range, antioxidants and anti-AGE compounds in the diet protects from AGE-mediated complications and improve renal function, particularly in diabetics.

Figure 5:

Illustration of the comprehensive effect of AGEs on the glomerular architecture.

AGEs induce thickening of the glomerular basement membrane, hypertrophy of podocytes and podocyte epithelial-mesenchymal transition. These events account for the altered structure of the glomerular filtration barrier and reduced podocyte count that ultimately manifest in proteinuria.