Diabetes induces the onset and progression of renal injury through causing hemodynamic dysregulation along with abnormal morphological and functional nephron changes. The most important event that precedes renal injury is an increase in permeability of plasma proteins such as albumin through a damaged glomerular filtration barrier resulting in excessive urinary albumin excretion (UAE). Moreover, once enhanced UAE begins, it may advance renal injury from progression of abnormal renal hemodynamics, increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix accumulation, and glomerulosclerosis to eventual end-stage renal damage. Interestingly, all these pathological changes are predominantly driven by diabetes-induced reactive oxygen species (ROS) and abnormal downstream signaling molecules. In diabetic kidney, NADPH oxidase (enzymatic) and mitochondrial electron transport chain (nonenzymatic) are the prominent sources of ROS, which are believed to cause the onset of albuminuria followed by progression to renal damage through podocyte depletion. Chronic hyperglycemia and consequent ROS production can trigger abnormal signaling pathways involving diverse signaling mediators such as transcription factors, inflammatory cytokines, chemokines, and vasoactive substances. Persistently, increased expression and activation of these signaling molecules contribute to the irreversible functional and structural changes in the kidney resulting in critically decreased glomerular filtration rate leading to eventual renal failure.
Diabetes is a group of chronic metabolic diseases marked by high plasma glucose levels (usually fasting plasma glucose (FPG) is ≥126 mg/dL) resulting from defects in insulin secretion or insulin action or both. The chronic hyperglycemia of diabetes induces several pathophysiological complications including cardiovascular abnormalities to renal failure. According to the American Diabetes Association [
The prevalence and incidence of diabetes and diabetic kidney diseases have alarmingly increased during recent decades. According to a 2014 national diabetes statistics report, 29.1 million United States citizens have diabetes which is 9.3% of the U.S. population. Every year, 1.4 million Americans are diagnosed with diabetes [
In line with the increasing incidence of diabetes, cases of chronic kidney disease (CKD) or end-stage renal damage (ESRD) have been growing significantly, since CKD is directly related to diabetes and/or hypertension. Approximately 1 of 3 adults with diabetes and 1 of 5 adults with high blood pressure have CKD. According to the 2014 National Chronic Kidney Disease Fact Sheet, more than 20 million adults (10% of all adults) have CKD in the United States. CKD is more prevalent in older people and most common among adults older than 70 years of age. It has also been observed that diabetes and hypertension are the leading causes of ESRD. In 2011, diabetes and hypertension were identified as the primary cause for 7 of 10 new United States cases of ESRD [
Diabetes-mediated chronic hyperglycemia evokes the onset and progression of renal injury because of its role in causing hemodynamic dysregulation along with abnormal morphological and functional nephron changes. The most important event that precedes renal injury is an increase in permeability of plasma proteins such as albumin through a damaged glomerular filtration barrier. This results in excessive urinary albumin excretion (UAE) through the nephron. Excess albumin excretion into urine is used as a prominent marker for diabetic kidney disease. Increased albumin leakage results from the impaired integrity of the glomerular filtration barrier (GFB), which is primarily responsible for retention of all plasma proteins. It is noted that GFB consists of three layers, where the visceral epithelial cells (podocytes) layer is highly vulnerable to ROS because of its nonproliferative nature even in response to injury [
Reactive oxygen species promote renal injury which eventually develops into chronic kidney disease. Diabetes-mediated ROS could be generated in both enzymatic and nonenzymatic pathways. Among many, NADPH oxidase (Nox) (enzymatic) and mitochondrial electron transport chain (mETC) (nonenzymatic) pathways are the prominent sources of ROS generation in the diabetic kidney and play a critical role in promoting pathophysiological events in kidney disease. In addition to NADPH oxidase (Nox) and mETC, other sources of ROS such as advanced glycation end products (AGEs) and uncoupled nitric oxide synthase (NOS) have been discussed in the current manuscript.
Hyperglycemia-induced ROS, particularly of Nox and mETC origin, are believed to cause the onset of albuminuria followed by progression of renal damage through podocyte depletion. ROS play a significant role in the onset of microalbuminuria through damaging the integrity of all the layers of the GFB. Once microalbuminuria occurs, ROS along with increased protein levels in the tubular ultrafiltrate can activate diverse aberrant signaling pathways to facilitate renal damage from the progressive stage to eventual end-stage renal damage (ESRD). Increased activation and/or production of various signaling mediators such as transcription factors, inflammatory agents, growth factors, cytokines, chemokines, and vasoactive molecules produce deleterious structural and functional glomerular alternations. These abnormal signaling cascades advance renal injury from progression of abnormal renal hemodynamics, increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix accumulation, interstitial fibrosis, and glomerulosclerosis to eventual end-stage renal damage. Though, at the outset, hyperglycemia-induced renal damage exhibits moderate structural and functional glomerular changes, such as hyperfiltration, untreated kidney develops most abnormal structural (Kimmelstiel-Wilson syndrome, nodular form of mesangial matrix) and functional (critically decreased filtration rate, <15–29 mL/min/1.73 m2) condition that warrants kidney dialysis. Albeit the role of glomerulus in progressive renal damage is substantial, tubular segment is not less important at all. Renal tubules rather increasingly contribute to the development of advanced stage of kidney damage which is beyond the scope of this review.
Both kidneys receive about 22 percent of cardiac output that is equal to 1100 mL of blood in an adult. Blood flow into the glomerulus of the kidney is controlled by the afferent and efferent arterioles. The afferent arteriole drives the blood into the glomerulus, whereas efferent arteriole helps the blood flow out of the glomerulus into peritubular capillaries. Both of these arterioles can contribute to the filtration process by either facilitating blood flow to the glomerulus (via afferent arteriolar vasodilation) or increasing intraglomerular pressure (via efferent arteriolar vasoconstriction). Moreover, other physical factors play an important role in maintaining the net intraglomerular filtration pressure. Three critical pressures govern the filtration through the glomerular capillaries. They are (1) hydrostatic pressure inside the glomerular capillaries, also known as glomerular hydrostatic pressure (
The glomerular filtration barrier (GFB) is recognized as a highly specialized ultrafiltration device that is capable of filtering large volumes of plasma fluids with a high permeability to water and small and midsized solutes in plasma, while efficiently retaining relatively larger macromolecules within the circulation. The barrier is composed of three layers: the innermost fenestrated vascular endothelium, the glomerular basement membrane, and the outermost podocyte layer (also called the glomerular visceral epithelial cells) [
The endothelial layer is composed of unusually flattened endothelial cells with a height around the capillary loops of approximately 50–150 nm. Remarkably, endothelial cell bodies are completely perforated by open holes or fenestrae which constitute 20–50% of the entire endothelial surface. The fenestrae are usually round having a diameter of 40–100 nm which is similar in size to that found by Bearer et al. [
This negatively charged layer can selectively restrict access of negatively charged plasma proteins such as albumin to the endothelial cell membrane leading to limited filtration of albumin. This is manifested by a recent study, where the investigators infused hyaluronidase (ESL degrading enzyme) solution into right jugular vein of mice for 4 weeks and found significant decrease in ESL thickness resulting in increased albumin filtration [
Glomerular basement membrane is a gel-like layer interwoven between endothelium and epithelial layer. Electron microscopic study demonstrates that GBM is composed of inner, middle, and outer sublayers designated as lamina rara externa, lamina densa, and lamina rara interna, respectively [
The podocytes are terminally differentiated visceral epithelial cells covering the outer surface of the glomerular capillaries and maintain the integrity of the kidney filter. They consist of a voluminous cell body, primary processes (arm-like projections coming from the cell body), and foot processes (numerous slender feet projected from primary processes). The cell body faces urinary space and gives rise to primary processes. Both processes are enriched in microtubules and intermediate filaments such as desmin and vimentin. The primary processes further elongate toward the capillary to make secondary/foot processes that contain an actin-based cytoskeleton. Besides actin, foot processes (FP) also contain other contractile proteins such as myosin,
On the other hand, slit diaphragm plays a remarkable role in filtration by providing charge and size selective barrier to the macromolecules because of its architectural nature (physical sieve having pore size of ~3.8 nm, the same diameter of an albumin molecule [
Mesangial cells are smooth muscle-like pericytes located in the intercapillary regions of the glomerulus. Though the mesangial cells are not an integrated structural part of the glomerular capillary barrier in the kidney filter, their contribution to the fluid filtration cannot be underestimated. They, along with the capillary barrier, form a coordinated biochemical unit and control the filtration rate as they have the capacities of regulating filtration surface area, intraglomerular blood volume and filtration pressure, and hormone as well as growth factor secretion. Contracting (e.g., Ang II and vasopressin) and relaxing (i.e., ANP and NO) hormones secreted by GMC can control blood flow to the capillary loops via preferential constriction and dilation of efferent and afferent arterioles, respectively, thus maintaining constant glomerular filtration rate (GFR) [
The term reactive oxygen species (ROS) can be defined as highly reactive oxygen-centered chemical species containing one or two unpaired electrons, where an unpaired electron is one that exists in an atomic or molecular orbital alone. The unpaired electron containing chemical species can also be called “free radicals.” In medical literature, the term “ROS” is used as a “collective term” to include both radicals and nonradicals, the latter being devoid of unpaired electron. So, ROS are classified into two categories: (1) oxygen-centered radicals and (2) oxygen-centered nonradicals. Oxygen-centered radicals include superoxide anion (
High glucose-induced ROS can be generated by both enzymatic and nonenzymatic pathways. The enzymatic pathways include nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), uncoupling of nitric oxide synthase (NOS), cytochrome P-450 (CYTP450), cyclooxygenase (COX), lipoxygenase (LOX), xanthine oxidase, and myeloperoxidase (MPO). Conversely, the nonenzymatic pathways include mitochondrial electron transport chain (mETC) deficiencies, advanced glycation end products (AGEs), glucose autooxidation, transition-metal catalyzed Fenton reactions, and polyol (sorbitol) pathway [
NADPH oxidase is one of the principal sources of ROS production in hyperglycemic conditions of different organs including the kidney. NADPH oxidase is a respiratory burst enzyme that was initially identified in phagocytes in 1933. The enzyme is responsible for production of millimolar amounts of superoxide using cytosolic NADPH as substrate, and the superoxide or its downstream metabolite H2O2 can kill microorganisms in burst-dependent manner of phagocytes. Since its early detection in phagocytes, a growing body of scientific studies identified and cloned five major subunits constituting the enzyme, NADPH oxidase. They are membrane-bound flavocytochrome b558 forming subunits such as
Mitochondria are another potential source of ROS production in diabetic condition. However, there is a controversy as to which source of NADPH oxidase and mitochondria is predominantly contributing to ROS generation in diabetic condition, since some scientists identify the first [
Any dysregulation in the coordinated transfer of the electrons by the enzyme complexes results in the leakage of electrons. The leaked electrons in turn reduce O2 to form superoxide (
Complex I generates superoxide (
More ROS production occurs when antimycin is used. Because antimycin stabilizes the ubisemiquinone at ubiquinol binding site
On the other hand, ROS generation by complex II should not be underestimated, albeit it is considered to have limited role in ROS release. Complex II appears to produce ROS in a condition of high succinate concentration and membrane potential (
In diabetic milieu, certain factors such as excess reducing equivalents NADH/FADH2 [
AGEs are a group of heterogeneous compounds produced from the nonenzymatic reaction of reducing sugars with the amino groups of proteins, lipids, and nucleic acids. Their generation involves few steps. The first step is “Maillard reaction” which involves the attachment of the carbonyl group (aldehyde or ketone) of reducing sugars with nucleophilic lysine or N-terminal amino groups of a variety of proteins, lipids, and nucleic acids to form Schiff base. In second step, the Schiff bases undergo reorganization to form more stable ketoamines called Amadori products. Amadori products are highly reactive intermediates that include
AGEs evoke diverse physiological and pathological effects through interaction with their receptors called receptor for AGEs (RAGE). RAGE is multiligand member of immunoglobulin superfamily, usually located on the cell surface of different cells such as macrophages, adipocytes, endothelial cells, vascular endothelial muscle cells, podocytes, and mesangial cells [
In addition to many receptor-dependent signaling effects of AGEs, ROS generation resulting from AGE-RAGE-mediated activation of NADPH oxidase and mitochondrial ETC is also prominent. For example, evidence from a study showed that interaction of AGE with its receptor RAGE in INS-1 cells caused early phase of ROS production from mitochondria. Moreover, this AGE-mediated early mitochondrial ROS in turn induce NADPH oxidase to produce more ROS, supporting the notion that AGE-RAGE interaction stimulates ROS generation via mitochondrial or NADPH oxidase pathway [
Uncoupling nitric oxide synthase (NOS) is also another important enzymatic source for superoxide generation. Three quite distinct isoforms of NOS have been identified with their different location, regulation, catalytic properties, and inhibitor sensitivity. They are (1) nNOS isoform which is first (also predominantly) found in the neuronal tissue, (2) iNOS isoform which is induced in diverse cells and tissues, and (3) eNOS isoform which is first identified and usually located in vascular endothelial cells. Of these, nNOS and eNOS isoforms are constitutively expressed, whereas iNOS is inducible [
Sources of ROS generation and their impact on cellular components and signaling pathways.
All isoforms of NOS can generate superoxide in absence of L-arginine and/or cofactor tetrahydrobiopterin. For example, saphenous veins and internal mammary arteries collected from diabetic patients showed significantly elevated levels of superoxide production especially in the endothelium as demonstrated by fluorescent microtopography. In addition, either denudation of endothelium or inhibition of NOS by N-methyl-l-arginine in diabetic mammary arteries significantly reduced superoxide production suggesting the involvement of eNOS as the mediator of superoxide generation which is reversed in presence of sepiapterin, a BH4 precursor [
As per above discussion, it is clear that superoxide may produce in both physiological and pathological conditions. Once produced, superoxide is immediately neutralized by cellular superoxide dismutase (SOD) to limit its damaging effects on cellular components. Three isozymes of SOD are found in the cell. They are Cu, Zn-SOD, Mn-SOD, and EC-SOD. Among these, Cu, Zn-SOD is believed to be located in cytosol, whereas Mn-SOD and EC-SOD are thought to be localized in mitochondrial matrix and on the outer surface of cell membranes, respectively [
Hyperglycemia-induced onset of renal injury is marked by microalbuminuria, glomerular hemodynamic abnormalities, increased kidney and glomerular size, and hyperfiltration. Once these conditions are set in, diverse pathological events are induced due to aberrant signaling cascades with the progress of time. Impaired signaling functions cause a variety of structural and functional changes ranging from increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix deposition, glomerulosclerosis, overt proteinuria, and decreased glomerular function and filtration rate to eventual end-stage renal damage. Since diabetic renal injury advances through different stages of structural and functional changes in the glomerulus, we will discuss ROS-mediated renal damage in three steps: onset of injury, progression of injury, and end-stage renal damage.
There is an established notion that passage of macromolecules including albumin is highly restricted through normal glomerular capillary wall. However, increased ROS levels in diabetic milieu cause aberration in signaling pathways of different glomerular capillary layers leading to their structural or functional abnormalities which compromise on the glomerular ability of retention of macromolecules. This in turn results in increased leakage of proteins. As all the barriers constitute the capillary wall, it is assumed that each layer’s damage by ROS might have a contributory role in protein leakage. Moreover, each layer is likely to communicate with other layer(s) through release of different mediators for the development and the maintenance of functional as well as structural integrity of the glomerular filtration barrier as a composite layer. For example, endothelial layer can communicate with podocytes through secretion of cytokines and growth factors and vice versa [
Earlier we have discussed microalbuminuria. Here we will focus on how microalbuminuria and hyperfiltration occur at the early phase of renal injury due to ROS-mediated effects inflicted on different glomerular filtration barriers.
From the previous discussion, we have already known that luminal surface of the endothelium is covered by a layer of glycocalyx and endothelial cell coat forming endothelial surface layer (ESL). The glycocalyx is a dynamic hydrated layer largely composed of proteoglycans and glycoproteins of which proteoglycans such as glycosaminoglycans (GAGs) are enriched in heparan sulphate (HS) which gives anionic charge characteristics to the ESL. Interestingly, endothelial glycocalyx can be a major site of action of ROS and different proinflammatory cytokines, which causes degradation of GAGs leading to decreased anionic charges and increased permeability to macromolecules [
Glomerular endothelial cells have also been reported to increase the expression of dysfunctional endothelial nitric oxide synthase (eNOS) due to increased monomeric isoforms instead of dimeric in hyperglycemic condition. Either eNOS impairment or its deficiency results in increased superoxide generation as opposed to NO and the superoxide in turn can scavenge NO decreasing its bioavailability. Attenuation of NO levels impairs endothelium-dependent capillary relaxation as well as vasodilation by enhancing formation of vasoconstrictors and alters renal autoregulation which in combination results in increased glomerular intracapillary pressure and filtration rate (hyperfiltration) which is an early sign of diabetic renal injury [
Like endothelium, glomerular basement membrane is also considered to have charge- and size-selective properties because of its anionic heparan sulfate (HS) side chains attached to proteoglycan core proteins (e.g., agrin and perlecan) and extracellular matrix (ECM) network, respectively. It has been found that the heparan sulfate component of GBM can be depolymerized from its core proteoglycan proteins by the action of ROS, whereas uses of ROS scavengers inhibited degradation of HS [
Podocytes, also known as visceral epithelial cells, are the most restrictive barrier to macromolecules, since podocytes form slit diaphragm with very smaller pores by interdigitating neighboring foot processes that ultimately provide efficient size-based permselectivity. However, its charge selectivity for plasma proteins should not be overlooked as apical membrane domain of podocytes contains anionic surface proteins such as podocalyxin (rich in sialic acid) [
Hyperglycemia-induced ROS can trigger early loss of podocytes by inducing a variety of pathological events which include apoptosis, detachment of podocytes, foot process effacement, reorganization of cytoskeleton, and dysregulation of any single or a group of podocyte proteins. Due to impaired DNA synthesis and hypertrophy podocyte proliferation may be reduced during cell division [
Reactive oxygen species induce dysregulation of different redox signaling cascades in the podocytes causing their apoptosis or detachment. In doing so, high glucose or ROS can upregulate and activate diverse proinflammatory cytokines and transcription factors, proapoptotic molecules, and growth factors. Recently, using type 1 and type 2 diabetic models of mice, Susztak et al. [
Major signaling pathways for induction of apoptosis and hypertrophy of podocyte and mesangial cells.
On the other hand, in nondiabetic in vivo and in vitro studies treated with puromycin aminonucleoside (PAN), loss of nephrin and podocin expression has been observed in line with increased foot process effacement and cytoskeletal actin reorganization of podocytes. Actin reorganization that is accompanied by loss of synaptopodin may induce FPE. These pathological modulations are found to be caused by an underlying mechanism of ROS generation and subsequent activation of p38-MAPK pathway. Triptolide has showed restoration of nephrin and podocin levels with remarkable improvement in cytoskeleton and foot processes by reducing ROS levels and p38-MAPK activation and ultimately decreased proteinuria [
Moreover, diabetes is characterized not only by oxidative stress levels but also by other complications including insulin resistance, reduced adiponectin, and increased inflammatory mediators which are highly common in obese subjects. Thus, obese patients with diabetes are more susceptible to renal injury. This notion is supported by a recent study where the investigators using normoglycemic Zucker-fatty rats showed that some podocyte proteins such as nephrin, podocin, podocalyxin, and CD2AP are remarkably downregulated which can be attributed to increased oxidative and inflammatory mediators [
All the hyperglycemia-induced ROS-mediated pathological events discussed above are adequate to initiate protein leakage across structurally and functionally impaired glomerular barrier resulting in primary urine with excessive proteins. Though increased leakage of protein through glomerulus is the initiating point for microalbuminuria, other factors such as inability of renal tubule to increase protein reabsorption corresponding to increased protein levels in the tubular filtrate and decreased capacity of tubular reabsorption of proteins due to increased injury to the tubular cells can be held responsible for eventual increased urinary excretion of protein or albumin leading to microalbuminuria which, with progress of time, results in macroalbuminuria with advanced pathological changes in the kidney.
Podocytes are terminally differentiated cells with a limited proliferative capacity. Therefore, the fate of a podocyte depends on its ability to cope with the stress. Fortunately, podocytes exhibit a high level of autophagy even under nonstress conditions, suggesting that podocytes need to keep cellular homeostasis under basal conditions [
Evidently, autophagy plays an important renoprotective role by mainly maintaining homeostasis of podocytes in diabetic nephropathy. It has been manifested by podocyte-specific expression of autophagy related proteins such as Beclin-1, Atg5–Atg12, and LC3 (rat microtubule-associated protein 1 light chain 3) which results in increased basal level of autophagy in podocytes [
The mechanism underlying diabetes-induced impairment of podocyte autophagy is still ambiguous. However, in podocytes of diabetic mice and patients, mTORC1 (mammalian target of rapamycin complex 1) is highly activated and may be involved in the mechanisms of diabetes-induced autophagy inhibition in podocytes [
Mature podocytes reduce expression of Ki-67, a proliferation marker, cyclin A, and cyclin B1, while CKIs and cyclin D1 are intensively increased. Cyclins and CDKs can be modulated in human and experimental podocyte injury. For example, in the cellular type of human FSGS (focal segmental glomerulosclerosis), studies have found absent p27, p57, and cyclin D1 expression and increased cyclin E, cyclin A, cyclin B1, CDK2, and p21 [
Podocyte hypertrophy is a characteristic of diabetic nephropathy. It occurs in different diabetic animal models due to increased expression of CKIs. For example, Zucker diabetic rats and db/db mice, both models of type 2 diabetes, or type 1 models, induced by streptozotocin administration, increase the expression of p27 and p21 resulting in podocyte’s cell cycle arrest in response to injury induced DNA damage and this in turn causes glomerular hypertrophy and development of progressive renal failure [
Increased glomerular filtration rate (GFR) or hyperfiltration also marks the early sign of diabetic renal injury and may play a major role in the pathogenesis of diabetic nephropathy. Glomerular hyperfiltration occurs due to increased dilation of afferent arterioles leading to increased blood flow to the glomeruli. This afferent arteriolar dilation can be attributed to increased prostaglandin E2 synthesis, impaired responsiveness to vasoconstrictors (i.e., thromboxane and norepinephrine), elevated levels of atrial natriuretic peptide (ANP), and hyperglycemia-mediated inactivation of tubuloglomerular feedback (TGF) [
Though microalbuminuria may be initiating step for glomerular damage, progression of damage actually is achieved through activation of diverse pathological pathways. We have already discussed some of the signaling molecules that evoke some structural and functional damage to the filtration barrier to increase glomerular permeability. Now we will have a holistic view on some more signaling mediators in greater detail which are responsible for advanced pathological damage to the glomerulus if initial symptoms are not corrected. Of note, signaling mediators can be activated in any part of the glomerulus in response to high glucose, AGEs, and/or ROS. However, their activation in any glomerular cell type may affect surrounding cells as the whole glomerulus acts as a coordinated unit to regulate its functions.
PKC is also an important signaling molecule playing central role in glomerular injury. In high glucose ambience, PKC is activated by diacylglycerol (DAG), a signaling molecule increasingly produced from an intermediate product of glycolytic pathway such as glyceraldehyde-3-phosphate (G3P) which is abundantly produced from high glucose flux into glycolytic pathway. Interestingly, under high glucose conditions, PKC can also be activated by higher concentrations of ROS, perhaps through tyrosine phosphorylation or DAG synthesis. Moreover, PKC-
Major signaling pathways for induction of ECM accumulation following mesangial expansion, increased GBM, glomerulosclerosis, and fibrosis. This results in subsequent end-stage renal damage.
AP-1 is another redox-regulated transcription factor involved in transcription of various inflammatory genes in response to activation by diverse stimuli. ROS can activate AP-1 through phosphorylation of upstream MAPKs such as ERK and p38 kinases as shown by a study [
HIF is a heterodimeric transcription factor that is composed of two subunits, an oxygen sensitive HIF-
Other transcription factors including CREB (c-AMP-response-element-binding protein), NFAT (nuclear factor of activated T cells), and Sp1 (stimulating protein 1) are also activated in hyperglycemic milieu. These transcription factors can also regulate genes related to inflammation and ECM turnover [
Cytokines are small, nonstructural proteins with low molecular weights having autocrine, paracrine, and juxtacrine effects and very complex activities. They can act as regulators of host response to infection, immune response, trauma, and inflammation with their both pro- and anti-inflammatory role based on the type of cell, the time of action, and cellular environment. There are a lot of proinflammatory cytokines that can be activated in response to high glucose or oxidative stress. These include IL-1, IL-6, IL-18, and TNF-
IL-1
IL-6 has also been reported to be significantly high in type 2 diabetic patients with nephropathy (DN) in comparison to DM patients without DN. Analysis of kidney biopsies in patients with type 2 DN evidenced increased expression of IL-6 in cells infiltrating mesangium, interstitium, and tubules. Moreover, there is a positive relationship between mesangial expansion (glomerulopathy) and expression of IL-6 mRNA in both mesangial cells and podocytes, implying an important role of IL-6 in influencing extracellular matrix dynamics at mesangial and podocyte levels [
IL-18 is potent inflammatory cytokine that is involved in different functions, such as induction of interferon-
TNF-
Growth factors are activated by different effectors to induce secretion of matrix forming proteins to increase mesangial expansion as well as GBM thickness and express many cellular entities to promote cellular hypertrophy, apoptosis, and foot process effacement. Major GFs that play critical role in the pathogenesis of renal injury include TGF-
TGF-
In addition to its apoptotic role, TGF-
VEGF being expressed predominantly by podocytes and in some cases by mesangial cells in the kidney can induce angiogenesis and vascular permeability. VEGF elicits its action by interacting with its receptor located on the endothelium and mesangial cells [
At initial stage, though VEGF increases filtration rate accompanied by microalbuminuria via enhanced neoangiogenesis, its subsequent reduction resulting from increased podocyte loss during progressive period of the disease eventually diminishes GFR [
Though many studies exhibited the salutary effects of anti-VEGF agents to treat diabetic nephropathy, some other studies have shown potential complications associated with anti-VEGF treatment. Studies have found that administration of anti-VEGF neutralizing antibodies can significantly decrease hyperfiltration, albuminuria, and glomerular hypertrophy [
In contrast to these renoprotective effects, many investigations found deleterious effects associated with anti-VEGF therapy for neoplastic diseases. These deleterious effects may include but are not limited to proteinuria, hypertension, and thrombotic microangiopathy [
CTGF is an important downstream mediator of TGF-
PDGF is a cytokine that is involved in mediating and modulating many biological processes which occurred during renal injury. PDGF mediates its diverse effects, including proliferation, differentiation, extracellular matrix accumulation, tissue permeability, pro- as well as anti-inflammatory mediators, and migration of mesenchymal cells. It evokes its actions by interacting with its receptor, PDGFR, which can be expressed on mesenchymal, mesangial, and glomerular endothelial cells. PDGF is also important for physiological angiogenesis by the recruitment of perivascular cells, for example, pericytes, and it regulates vascular tone and platelet aggregation. PDGF binding with its receptor can trigger many signaling pathways, for example, Ras-MAPK, JAK/STAT, PLC-
Adhesion molecules such as ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) play crucial role in infiltration of immune cells to endothelium, mesangium, and GBM. Invasion of immune cells (leukocytes) follows few steps: cell tethering, selectin-mediated rolling of cell on the endothelium, chemokine-dependent integrin activation and leukocyte adhesion, and finally transmigration of leukocytes across the endothelium. Interestingly, these processes can be advanced by the help of any adhesion molecules mentioned above to initiate immune response in local tissues [
ICAM-1 is a cell surface glycoprotein belonging to Ig superfamily and binds to
VCAM-1, a member of Ig superfamily, is also a cell surface protein expressed on endothelial cells and some leukocytes such as macrophages and helps in their adhesion. It has been reported to be overexpressed on endothelial cells and infiltrating leukocytes in renal interstitium in diabetic animal models. In type 2 diabetes, serum level of VCAM-1 is likely to be increased and it positively correlates with albuminuria [
Chemokines are small cytokines that are secreted by cells/leukocytes to induce recruitment of leukocytes to nearby host cells. They are induced and activated by primary proinflammatory mediators, for example, IL-1 and TNF-
This is a potent chemokine belonging to CC chemokine family that is also recognized as chemokine (C-C motif) ligand 2 (CCL2). MCP-1 plays a key role in migration of monocytes, T cells, and macrophages to the diabetic kidney. In diabetic nephropathy, MCP-1 can be excessively produced by both inflammatory and renal resident cells which in turn induce progressive glomerular and tubule-interstitial injury by increasing macrophage infiltration. Its increased expression in type 2 diabetes is confirmed by its elevated urinary excretion accompanied with progressive tubulointerstitial damage [
In addition, macrophage inflammatory protein-1
These are circulating substances that regulate vascular tone and systemic as well as local blood pressure. Among many, angiotensin II and endothelin have been reported to be increased in response to high glucose, ROS, and AGEs in diabetic kidney and impair structural and functional integrity of the glomerulus.
Ang II not only increases vasoconstriction of glomerular capillary followed by intraglomerular pressure but also elicits more progressive pathological change to the glomerulus and renal tubules. Increasing evidence of experimental and clinical studies has shown injurious effects of Ang II during progressive kidney injury that ranges from vascular and mesangial cell proliferation, hypertrophy, podocyte apoptosis, and increased synthesis of matrix forming proteins to eventual glomerular and tubular sclerosis by induction of profibrotic mediators, namely, TGF-
An example of damage inflicted by Ang II is matrix protein synthesis in mesangial cells. Kagami et al. [
On the other hand, endothelin-1 is a potent vasoconstrictor that is highly produced in diabetic kidney. In addition to its vasoconstriction effect, endothelin-1 can induce proteinuria, proinflammatory mediators, ECM accumulation, and infiltration of macrophages in kidney of streptozotocin-induced diabetic rats [
At the outset of diabetes, though renal injury is triggered by ROS-mediated loss of podocyte to a certain threshold level following microalbuminuria, major structural and functional changes occur in progressive stage which are induced by activation of diverse mediators and their signaling pathways. Major progressive pathological changes that have already been discussed include increased mesangial expansion, ECM deposition, hypertrophy and proliferation of mesangial cells, increased apoptosis of podocytes beyond threshold level, increased GBM thickening resulting from matrix forming protein deposition and expression of TIMPs, glomerular sclerosis that may have a nodular appearance (classic Kimmelstiel-Wilson nodules), inflammatory cell infiltration, and tubulointerstitial fibrosis (Figure
Moreover, denuded GBM which has already been left by increased podocytes depletion is no longer able to resist glomerular hydrostatic pressure allowing the GBM to be stretched to come in contact with the parietal cells of Bowman’s capsule resulting in synechiae formation through capillary tuft adhesion to Bowman’s capsule (adhesion of capillary basement membrane with Bowman’s capsule). This tuft adhesion further degenerates the remaining podocytes located at the flanks of an adhesion leading to more podocyte loss that invokes excessive protein leakage that is termed “overt proteinuria” (macroalbuminuria) [
Progressively increased tubular protein load in tubular filtrate appears to keep the renal tubule under continuous challenge that results from its sustained exposure to diverse bioactive molecules including proteins. It is assumed that excessive proteins in the tubular infiltrate may elicit proinflammatory and profibrotic effects that directly contribute to chronic tubulointerstitial damage. This is initiated through the interaction of filtered proteins with proximal tubular cells, which excrete increased chemokines (e.g., MCP-1, RANTES, and complement component 3), profibrotic molecules (e.g., TGF-
As a result, at a point, kidney mass greatly reduces resulting in gradual decrease in glomerular blood flow and filtration rate. A limited number of nephrons also receive higher workload necessitating higher filtration pressure which can further weaken the attachment of podocytes to GBM. These are also complicated by increased mesangial expansion that reduces the filtration surface, thereby significantly reducing filtration rate and increasing intraglomerular pressure. Reduction of GFR can be used to represent severity of renal injury. For example, in the patient without kidney disease, GFR usually remains >90 mL/min/1.73 m2. However, GFR reduces to the range between 59 and 30 mL/min/1.73 m2 during moderate renal failure which further comes down to 15–29 mL/min/1.73 m2 in patients with severe renal failure. Moreover, GFR having <15 mL/min/1.73 m2 indicates end-stage renal damage requiring either dialysis or kidney transplantation [
Chronic hyperglycemia is one of the most important risk factors for progressive renal damage. Patients having diabetes are more likely to develop microalbuminuria (proteinuria) that is used as a marker for abnormal renal function. High glucose plays pivotal role in causing abnormal renal function through stimulation of ROS generation. Increasing body of evidence shows that ROS is elevated in diabetic milieu both in vivo and in vitro. ROS are considered as important second messengers for different signaling pathways which maintain necessary biochemical interactions for the functions and survival of the tissues. However, accumulation of ROS resulting from their imbalanced generation and neutralization promotes diverse aberrant signaling pathways. Abnormal signaling in the kidney causes functional and structural changes of the glomerulus which is the center for renal damage. Though being generated from many sources, ROS originated from mitochondria and NADPH oxidase are thought to cause the onset of albuminuria followed by progression of renal damage through podocyte depletion.
It is assumed that all the components of glomerular filtration barrier remain under persistent strain in oxidative stress environment. But many studies have attributed initial renal damage to highly sensitive podocytes (visceral epithelial layer) that undergo apoptosis, foot process effacement, and detachment in high glucose-induced ROS environment. Accumulation of ROS in hyperglycemic ambience activates proapoptotic signaling pathways through upregulation and activation of p38-MAPK and caspase-3 which are downstream mediators of TGF-
Increased albuminuria from the compromised functions of glomerular filtration barrier sets the platform for excessive activation of diverse signaling molecules. Among many, we have discussed transcription factors, inflammatory agents, growth factors, cytokines, chemokines, and vasoactive molecules in this paper in detail. Dysregulation of these abnormal signaling molecules advances the renal injury from progression of abnormal renal hemodynamics, increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix accumulation, interstitial fibrosis, and glomerulosclerosis to eventual end-stage renal damage. Lack of pharmacological intervention during progression of abnormal functional and histological change of the glomerulus may evoke irreversible end-stage renal damage which is marked by invasion of excess immune cells, classic Kimmelstiel-Wilson nodule and critically decreased glomerular filtration rate (<15 mL/min/1.73 m2) (Figure
Comparison between normal and diabetic glomeruli with regard to pathological events which occurred during onset and progression of diabetes. I, parietal epithelial cells; II, Bowman’s capsule; III, primary urine majorly containing water, urea, electrolytes, glucose, and so forth; IV, podocyte; V, glomerular basement membrane (GBM); VI, endothelial cells; VII, glycocalyx layer; VIII, mesangial cells; IX, extracellular matrix (ECM) proteins.
The authors declare that there are no competing interests.