Review Article

Accepted Article Nephroprotective Effects of Metformin in Diabetic Nephropathy†

Sreenithya Ravindran, Vinitha Kuruvilla, Kerry Wilbur and Shankar Munusamy*

College of Pharmacy, Qatar University, PO Box 2713, Doha, Qatar

*Address for Correspondence:

Shankar Munusamy, MS (Pharm), PhD
College of Pharmacy
Qatar University
PO Box 2713, Doha, Qatar
Tel: (+974) 4403-5592
Fax: (+974) 4403-5551
Email: shankar.munusamy@qu.edu.qa

Running Head: Metformin and its nephroprotective effects

Keywords: Metformin; Diabetic Nephropathy; Chronic Kidney Disease; AMPK; ER Stress;

Autophagy

Contract grant sponsor: Qatar National Research Fund (a member of The Qatar Foundation), Doha,
Qatar; Contract grant number: NPRP 7-1189-3-304

†
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/jcp.25598]

Received 9 September 2016; Accepted 12 September 2016

Journal of Cellular Physiology
This article is protected by copyright. All rights reserved
DOI 10.1002/jcp.25598

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 Abstract
Accepted Article Metformin, a well-known anti-diabetic agent, is very effective in lowering blood glucose in
patients with type 2 diabetes with minimal side-effects. Metformin is also being recommended in the
treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via
activation of AMP-activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric
conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and
activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic
reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), oxidative stress and hypoxia. As
these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin
therapy in patients with type 2 diabetes would extend beyond its anti-hyperglycemic effects. However,
since metformin is eliminated unchanged through the kidneys and some studies have shown the
incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was
contraindicated in patients with renal disease until recently. With more studies indicating the relatively
low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US
FDA has now approved metformin to be administered in patients with established renal disease based
on their renal function. The purpose of this review is to highlight the various mechanisms by which
metformin protects renal cells that have lost its functionality in a diabetic or non-diabetic setting and to
enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with
diabetic nephropathy and other non-diabetic forms of chronic kidney disease. This article is protected by
copyright. All rights reserved

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 Introduction
Accepted Article The high prevalence and ongoing increasing incidence of diabetes mellitus is placing more
individuals at risk for diabetes-related complications and associated morbidity and mortality.
According to the International Diabetes Federation, more than 415 million adults suffer from diabetes
worldwide and the diabetes population is predicted to rise to 552 million by 2030 (Diabetes Atlas, 7th
Edition). Fortunately, research continues to offer enhanced understanding of the pathophysiology and
outcomes of this disease, which can help people manage diabetes better with proper diet, physical
activity, and medication.

Diabetic nephropathy (DN) is one of the serious complications of diabetes mellitus and
accounts for 40% of all new end-stage renal disease cases in the United States. DN is characterized by
progressive albuminuria followed by a gradual decline in glomerular filtration rate (GFR) leading to
kidney failure accompanied by podocyte loss, progressive glomerulosclerosis and ultimately to
progressive tubulointerstitial fibrosis (Lieberthal and Levine, 2009). Hence, DN can be categorized into
three different stages (Figure 1). In stage I of DN, there will be no signs of microalbuminuria and the
blood pressure will be normal in patients. The plasma flow and GFR increases leading to a reduced
serum creatinine level. The stage II of DN is characterized by stabilized serum creatinine levels, the
absence of proteinuria, and normal GFR except few morphological changes that include increased
thickness of basement membrane and mesangial expansion. Currently, there are no clinical markers to
identify DN at early stages. DN is most evident at stage III where patients are diagnosed with
microalbuminuria, hypertension and a declining GFR with the appearance of glomerulosclerosis
(Appel, 2013).

Metformin, a biguanide derivative, is the first-line of oral therapy in patients with type 2
diabetes mellitus (T2DM). Metformin decreases hepatic gluconeogenesis with less risk of causing
hypoglycemia, decreases intestinal absorption of glucose and increases insulin sensitivity in peripheral
tissues (Sena et al., 2010). Furthermore, metformin decreases the levels of LDL cholesterol and
triglycerides, and promotes weight loss in patients with obesity (Bailey, 2008; Bodmer et al., 2008)

Clinical trial evidence supports metformin’s ability to reduce the incidence of macrovascular
complications such as myocardial infarction, angina, sudden death, stroke and peripheral vascular
disease in patients with T2DM-diabetes (Kim et al., 2015). The UK Prospective Diabetes Study
(UKPDS) Group demonstrated that metformin reduces the risks for any diabetes-related endpoint in
diabetes patients by 32%, diabetes-related death by 42% and all-cause mortality by 36% when
compared to treatment with sulfonylurea or insulin (Group, 1998). A meta-analysis by Salpeter et al.
aggregating results from 31 trials with 4,570 participants followed for 8,267 patient-years showed
metformin treatment reduced the incidence of new-onset of diabetes by 40% in patients at risk for
diabetes. This study suggests that metformin is capable of preventing the onset of diabetes in at-risk
patients. (Salpeter et al., 2008).

Recent reports support the use of metformin even in patients with renal disease based on the
estimated glomerular filtration rates (eGFRs), which is a better measure of renal function than the

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 serum creatinine level. An eGFR of 30 to 45 mL/min is equivalent to a serum creatinine level of about
2 mg/dL and this could be considered as a cut-off to evaluate the use of metformin in patients with
Accepted Article
established kidney disease (Lipska et al., 2011). Based on these reports, the US FDA was requested to
relax and reframe the contradiction of use of metformin in terms of eGFR measure (Flory and
Hennessy, 2015). In April 2016, the US FDA has conceded to revise the guidelines; the current
recommendations allow metformin and metformin-containing medications to be administered to
patients to lower glucose levels but with precautions and based on eGFR levels as shown in Table 1.

Before this report from US FDA, metformin was contraindicated in a large population of T2DM
patients mainly due to concerns over metformin-associated lactic acidosis (MALA). Metformin is
eliminated unchanged via kidney and patients with existing renal dysfunction or insufficiency will have
reduced glomerular filtration rate (GFR) thus, increasing the risk of lactic acidosis. Metformin
enhances anaerobic metabolism by reducing pyruvate dehydrogenase activity and transport of reducing
agents by mitochondria (Ncomanzi et al., 2014). The anaerobic metabolism along with reduced insulin
in the body impairs the ability of cells to shift to aerobic metabolism and results in an increased
production of lactic acid (Ncomanzi et al., 2014). But evidence supports the fact that lactic acidosis is
not being induced by metformin per se. In a meta-analysis by Salpeter et al. on T2DM-diabetic
patients, there were no cases of fatal or nonfatal lactic acidosis in 36,893 patient-years in the metformin
group, or in 30,109 patient-years in the non-metformin group in 194 cases considered (Salpeter et al.,
2003). The Comparative Outcome Study on Metformin Intervention vs Convention (COSMIC)
approach study conducted by Bristol-Myers Squibb on about 9,000 patients out of which 7,227 patients
received metformin concluded that there was no incidence of lactic acidosis (Cryer et al., 2005).
Therefore, metformin can be administered according to the regulations listed by the US FDA without
being concerned about developing lactic acidosis.

Pharmacodynamics and Pharmacokinetics of Metformin

It has been widely accepted by researchers that metformin mainly works by suppressing hepatic
gluconeogenesis through phosphorylation of AMP-activated kinase (AMPK) in hepatocytes. AMPK
acts as a fuel sensor and a master regulator of energy homeostasis in many organs and tissues by
inhibiting anabolic pathways and stimulating catabolic pathways (Stephenne et al., 2011). Although the
specific pathway(s) responsible for phosphorylation of AMPK is not yet clear, multiple studies suggest
that the molecular component LKB1 or STK11 (upstream kinase of AMPK) plays an important role.
Shaw et al. showed that deletion of LKB1 in hepatocytes of adult mice significantly reduced AMPK
activity indicating the importance of LKB1 in AMPK signaling (Shaw et al., 2005). Studies have
reported that the primary target of metformin is the mitochondrial respiratory complex 1 (Stephenne et
al., 2011). Metformin is transported into hepatocytes by organic cation transporter 1 (OCT1), which in
turn inhibits respiratory complex 1 resulting in decreased ATP synthesis and a rise in the cellular AMP:
ATP ratio. This decrease in cellular energy status, in turn, activates LKB1/AMPK (Stephenne et al.,
2011). Activation of AMPK leads to inhibition of hepatic gluconeogenesis, increased glucose uptake in
the skeletal muscle and stimulation of free fatty acid oxidation in the muscle and adipose tissue
(Stephenne et al., 2011; Viollet et al., 2006). Evidence from a study by Shu et al. supports that OCT1 is
the primary transporter of metformin in hepatocytes as OCT1 deficient mice treated with metformin

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 showed significant reductions in hepatic gluconeogenesis. The study also demonstrated that individuals
with genetic variations in OCT1 gene are unresponsive to metformin therapy, reconfirming that OCT1
Accepted Article
is the key player in metformin’s therapeutic action on the liver (Shu et al., 2007).

Additionally, activated AMPK phosphorylates cAMP response element binding protein

(CREB)-regulated transcription coactivator 2 (CRTC2 or TORC2) and inhibits the expression of
Glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK), the enzymes involved in
gluconeogenesis, thereby inhibiting gluconeogenesis. Impairment in LKB1/AMPK signaling will result
in non-phosphorylated CRTC2, which translocates to the nucleus and binds to phosphorylated CREB.
This results in the expression of proliferator-activated receptor-γ coactivator-1α (PGC-1α) and its
gluconeogenic target genes, PEPCK and G6Pase and leads to increased gluconeogenesis(Shaw et al.,
2005; Viollet et al., 2006; Yee et al., 2012).

In peripheral tissues such as skeletal muscle, metformin improves insulin sensitivity by various
mechanisms including increased insulin receptor tyrosine kinase activity, glycogen synthesis,
recruitment and activation of GLUT4 glucose transporters. In adipocytes, metformin promotes the
reduction of free fatty acids and inhibits lipolysis. This may indirectly improve insulin sensitivity
through reduced lipotoxicity (Giannarelli et al., 2003; Stephenne et al., 2011).

Metformin is mostly absorbed in the small intestine and has an oral bioavailability of 50 to
60%. Metformin does not appear to bind to any hepatic or plasma proteins (Graham et al., 2011).
About 90% of the absorbed fraction is excreted unchanged in the urine by filtration and active tubular
secretion. The mechanism of transport of metformin into the intestinal cell is still not elucidated. A
study by Zhou et al. shows that intestinal absorption of metformin is primarily mediated by the plasma
membrane monoamine transporter (PMAT), which is mainly expressed on the luminal side of the
enterocytes (Zhou et al., 2007). Metformin regulates glucose uptake by regulating sodium/glucose
symporter SGLT1 on the luminal side of the intestine and the glucose uniporter GLUT2 on the blood
side through rapid activation of AMPK (Sakar et al., 2010).

Mechanisms underlying the nephroprotective effects of metformin

The beneficial effects of the use of metformin in patients with DN outweigh its potential risks.
In this review, we will be shedding more light on the major signaling pathways that appear to be
modulated by metformin. These include: AMPK/mTOR pathway, Endoplasmic Reticulum (ER) Stress,
Epithelial-to-Mesenchymal Transition (EMT), Autophagy, Oxidative Stress and Advanced Glycation
End Products, Hypoxia Inducible Factor (HIF) and Lipotoxicity.

AMPK/mTOR pathway
The mammalian target of Rapamycin (mTOR) is a serine-threonine kinase that plays a major
role in cell proliferation and survival. Numerous studies have demonstrated that hyperglycemia triggers
the activation phosphoinositide-3 kinase (PI3K) and AKT pathways, which subsequently lead to the
activation of mTOR. Activated mTOR induces the synthesis of matrix proteins associated with
basement membrane thickening and mesangial matrix accumulation (Estacio and Schrier, 2001). In

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 addition, mTOR enhances renal fibrosis through fibroblast proliferation, epithelial-to-mesenchymal
transition (EMT) and the expression of pro-fibrotic cytokines such as transforming growth factor-β1
Accepted Article
and connective tissue growth factor (Kume et al., 2014; Lieberthal and Levine, 2009). This leads to loss
of functional nephrons in the kidney. mTOR-dependent infiltration of macrophages and production of
pro-inflammatory cytokines, such as MCP-1, has been shown to exacerbate the inflammatory process
associated with DN (Kume et al., 2014).

Many reports also demonstrate that mTOR contributes to renal tubular cell apoptosis and
damage in diabetes (Sakaguchi et al., 2006; Velagapudi et al., 2011). Inhibition of mTOR by rapamycin
treatment in animal models has shown amelioration of the above events (Sakaguchi et al., 2006;
Velagapudi et al., 2011). In addition, mTOR signaling pathway plays an important role in the
regulation of autophagy, a cellular defense mechanism, in the kidney. mTOR activation suppresses
autophagy initiation in kidney cells thus, leading to apoptosis and necrosis in kidney cells causing
irreversible damage (Xiao et al., 2014). Thus, it is evident that mTOR plays a central role in the
development of the major features of DN, although additional mechanisms are likely to be involved.
Thus, inhibition of mTOR would prevent the pathophysiological and structural alterations in the DN
(Lieberthal and Levine, 2009).

AMP-activated protein kinase (AMPK) is a fuel sensor present in cells and is mainly involved
in cellular metabolisms such as regulation of fatty acid oxidation and ATP synthesis. AMPK consists of
an alpha (α), a beta (β), and a gamma (γ) subunit and is activated by the upstream kinases such as
calcium-calmodulin-dependent protein kinase kinase (CaMKK) and serine/threonine kinase 11 (LKB1)
via phosphorylation of threonine residue 172 (Kume et al., 2012; Long and Zierath, 2006). A fall in
ATP levels leads to activation of AMPK and subsequent phosphorylation of its multiple substrates, in
an effort to enhance catabolism and suppress anabolic energy consumption. For instance, glucose
deprivation leads to an increase in AMP:ATP ratio and subsequently leads to activation of AMPK and
inhibition of mTOR by phosphorylation (Lieberthal and Levine, 2009; Long and Zierath, 2006).
Conversely, when there is an energy surplus, AMPK activation is reduced and mTOR is activated,
which in turn stimulates protein synthesis, cell growth, and storage.

Multiple studies support the fact that high glucose levels inhibit AMPK signaling in both
glomerular and tubular compartments of the kidney. Moreover, the restoration of AMPK activity using
AMPK activators such as metformin and 5-aminoimidazole-4-carboxamide-1-riboside (AICAR) has
been shown to attenuate renal injury induced by hyperglycemia (Lee et al., 2013; Lee et al., 2007;
Takiyama et al., 2011). A study by Lee et al. revealed the role of AMPK inactivation and mTOR
activation in glomerular hypertrophy induced by high glucose levels in vitro and in vivo. The study also
highlighted that the effects of metformin to inhibit renal hypertrophy is independent to that of its effects
on hyperglycemia (Lee et al., 2007). Similarly, a study by Kim et al. documented low levels of
phosphorylated AMPK in renal podocytes of diabetic mice. Consistent with other studies, metformin-
mediated restoration of AMPK pathway in renal podocytes rendered protection from diabetes-induced
injury (Kim et al., 2012).

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 Decreases in AMPK signaling is also demonstrated in renal tubular cells exposed to high
glucose milieu, and the reinstitution of AMPK with metformin has been shown to prevent
Accepted Article
hyperglycemia-induced tubular injury (Takiyama et al., 2011). In a report by Sokolovska et al., AMPK
activation via metformin treatment in diabetic mice has lowered blood glucose levels and normalized
the GLUT1 expression in diabetic kidney (Sokolovska et al., 2010). Thus, it is clear that in conditions
of hyperglycemia, the AMPK is inhibited and the mTOR pathway is activated, resulting in hypertrophy
and other structural alterations leading to DN (Figure 2).

A similar phenomenon is observed with the progression of CKD in non-diabetic settings. In a

study by Zhang et al., metformin treatment restored the expression of phosphorylated AMPK protein
and improved metabolic function of the kidney in the high fat diet in C57BL/6J mice (Zhang et al.,
2014). In another study by Lu et al., activation of AMPK pathway via metformin in mouse renal
fibroblasts inhibited the activation of TGF-β-induced production of collagen, which suggests that
activation of AMPK would attenuate renal interstitial fibrosis in chronic kidney disease (Lu et al.,
2015).

Furthermore, the nephroprotective effect of metformin is also demonstrated in acute kidney

injury. A study in a rat model of unilateral renal ischemia-reperfusion by Taheri et al. demonstrated
that activation of AMPK using metformin prevents hypoxia and protects the renal cells from functional
and structural damage induced by ischemia-reperfusion injury via inhibition of mitochondrial
respiratory complex I (Taheri et al., 2012).

The interplay between mTOR and AMPK is also documented in other kidney diseases. Liu et
al. demonstrated that metformin inhibits cell proliferation in renal carcinoma cells in vitro and in vivo
by down-regulating cyclin-D1 (a protein involved in cell cycle progression) and inducing G0/G1 cell
cycle arrest. The study also showed that metformin upregulates AMPK activity and inhibits
mammalian target of rapamycin (mTOR) pathway (Liu et al., 2013). Therefore, activation of AMPK
could be associated with cell cycle arrest leading to phosphorylation and inactivation of mTOR that
will further lead to downregulation of cell metabolism, cell growth, cell proliferation and survival.
Another study by Takiar et al. evaluated the effect of metformin in autosomal dominant polycystic
kidney disease (ADPKD). ADPKD is an inherited systemic disorder affecting the kidneys and mainly
involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride
channel of the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in
the fluid secretion and mTOR is involved in the proliferation of cyst epithelial cells and both are
negatively regulated by AMPK. The study found that metformin induces AMPK resulting in the
inhibition of both CFTR and mTOR, thereby ameliorating cyst fluid secretion and epithelial cell
proliferation (Takiar et al., 2011).

Altogether, these reports suggest that restoration of AMPK signaling and/or inhibition of
mTOR pathway with metformin treatment may serve as viable therapeutic strategies to preserve renal
function in patients with DN and other forms of CKD.

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 Endoplasmic Reticulum Stress
Accepted Article The endoplasmic reticulum (ER) is the central site for protein folding and post-translational
modifications and transport of several proteins. The main functions of ER are: 1) synthesis, folding,
and maturation of proteins; 2) Ca2+ storage and 3) lipid biosynthesis. The process of protein folding is
controlled by molecular chaperones such as glucose-regulated protein 78/immunoglobulin binding
protein (GRP78/BiP) and calreticulin (a Ca2+-dependent chaperone). Disruption of the ER protein
folding machinery during pathological conditions results in a buildup of misfolded and potentially toxic
proteins that give rise to ER stress. The homeostatic response of the ER to stress comprise the unfolded
protein response (UPR) (Cameron, 2013). The UPR consists of intricate signaling systems that are
based on three stress sensors.

1) Protein kinase R-like endoplasmic reticulum kinase (PERK): PERK is usually bound to
the ER chaperone GRP78/BiP. The accumulation of unfolded proteins within ER leads to dissociation
and activation of PERK. Activated PERK phosphorylates eukaryotic initiation factor 2α (eIF-2α), and
ultimately results in general inhibition of protein translation and synthesis (Cameron, 2013).

2) Inositol-requiring enzyme-1 (IRE-1): Dissociation of GRP78/BiP from IRE-1 facilitates

splicing of the transcription factor X-box-binding protein 1 (XBP-1) mRNA, which leads to induction
of ER chaperones such as GRP78 and a pro-apoptotic molecule CAAT/enhancer-binding protein
homologous protein (CHOP). This ultimately contributes to ER-dependent degradative processes
including cell death if ER stress is severe and prolonged (Cameron, 2013).

3) Activating transcription factor 6 (ATF-6): Upon dissociation from GRP78/BiP, ATF6

translocates to the Golgi apparatus where the proteases process ATF6 to an amino-terminal fragment
that is taken up to the nucleus. Together IRE-1 and ATF6 pathways stimulate the expression of
chaperones and other enzymes involved in protein folding and alleviate ER stress (Cameron, 2013).

In DN, the abnormal levels of glucose serve as the principal stimulus for initiation of ER stress
in renal cells. Nevertheless, analyzing the UPR response in kidney cells is quite challenging owing to
the heterogeneity of the renal architecture and the complexity of the ER stress pathways. It is still
unclear whether activation of one the UPR sensors is enough, or all the three sensors need to be
activated to evoke a full-fledged ER stress. However, studies in experimental models of DN (Liu et al.,
2008; Wu et al., 2010) and in human kidney biopsies from patients with DN (Lindenmeyer et al., 2008)
have consistently documented the presence of ER stress in diabetic kidneys.

Studies show that both the PERK/eIF2α and IRE1/JNK pathways are activated in DN (Liu et
al., 2008; Wu et al., 2010). One possible mechanism by which hyperglycemia and proteinuria (a
diagnostic and prognostic marker of DN) initiate ER stress in renal cells is via generation of reactive
oxygen species (ROS). Increased ROS generation necessitates increased synthesis of antioxidants and
membrane proteins in the kidney to combat oxidative stress and repair oxidative membrane damage.
This increased protein synthesis may overwhelm the ER and result in ER stress (Lindenmeyer et al.,
2008). Other mechanisms such as the activation of growth factor receptors may also contribute to the

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 development of ER stress in DN. Studies by Zhang et al., showed that inhibition of renal epidermal
growth factor receptors (EGFRs) attenuates DN by reducing ER stress, and activating AMPK and
Accepted Article
autophagy signaling in renal cells (Zhang et al., 2014). Thus, maintaining the ER’s ability for handling
misfolded proteins in renal cells is considered as a new therapeutic target for protecting the kidney
from hyperglycemia-induced cellular injury and apoptosis.

Emerging evidence indicate the protective role of metformin against ER stress induced by
multiple factors in the kidney (Kim et al., 2015; Lee et al., 2012; Theriault et al., 2011). In a study by
Lee et al., albumin-induced ER stress in human proximal tubular cells was shown to be mediated
through the reactive oxygen species-Src Kinase-mTOR pathway. The study also indicated that
metformin reduces ER stress and caspase 3-dependent apoptosis induced by albumin and thereby
decreases tubulointerstitial injury in proximal tubular cells (Lee et al., 2012). In a recent study by Kim
et al., metformin treatment was shown to attenuate ER stress and fibrosis induced in renal cells by
various noxious stimuli including high glucose (Kim et al., 2015). It was shown that metformin
abrogated ER stress via suppression of GRP78 induction and induction of antioxidant protein heme
oxygenase 1. Interestingly, both studies also indicate that the ability of metformin to alleviate ER stress
is mediated via AMPK activation, as chemical inhibition and genetic silencing of AMPK have blocked
the protective effects of metformin. Contrary to these findings, a study by Thériault et al., indicate that
the metformin-mediated attenuation of UPR response occurs independently of the AMPK pathway in
renal tubular epithelial cells (Theriault et al., 2011). Nonetheless, it is clear that metformin has the
potential to curb ER stress - with or without activation of AMPK - in renal cells (Figure 3).

Epithelial-to-Mesenchymal Transition (EMT)

Renal fibrosis is a collective product of hyperfiltration, persistent proteinuria, cellular
inflammation, and the local release of morphogenic cytokines that alter the structure–function
relationships among nephrons (Eddy and Neilson, 2006). Tubular EMT is a highly structured process in
which tubular epithelial cells undergo a phenotypic change into fibroblasts. These fibroblasts then
migrate through the tubular basement membrane and into the interstitium. The process of tubular EMT
is proposed to comprise of four key steps (Liu, 2004).
(1) Loss of epithelial cell adhesion
(2) De novo -smooth muscle actin expression and actin reorganization;
(3) Disruption of the tubular basement membrane
(4) Enhanced cell migration and invasion of the interstitium

In kidneys, epithelial cells are polarized with their basal surface attached to a basement
membrane and their apical side facing the lumen of a tubular structure. They interact with adjacent
epithelial cells through adherens junctions, desmosomes, and tight junctions laterally. Mesenchymal
cells have two key characteristics that are important in embryonic development; they can migrate
through an extracellular matrix and subsequently differentiate into divergent cell types of the
mesenchymal cell lineage like fibroblasts (Fragiadaki and Mason, 2011). In DN, the conversion of
mesenchymal cells into myofibroblasts is driven by factors released from surrounding kidney cells (in
response to hyperglycemia, proteinuria and/or hypoxia), and from inflammatory and immune cells,

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 which infiltrate the interstitium in renal disease. These factors include a diverse range of proteins such
as the growth factors – TGF-β1 and connective tissue growth factor (CTGF), cytokines - IL-1 and
Accepted Article
oncostatin M, angiotensin II, proteases, plasminogen activator inhibitor-1, and advanced glycation end
products (AGE) (Fragiadaki and Mason, 2011; Thiery et al., 2009)

Hyperglycemia is one of the primary factors that stimulates EMT in the renal cells of diabetes
patients, thus leading to DN. In a recent study by Zhang et al., exposure of rat renal proximal tubular
cells to high glucose was sufficient to induce EMT, evidenced by a decrease in epithelial markers - E-
Cadherin and cytokeratin19, and an increase in mesenchymal markers - α-SMA and vimentin (Zhang et
al., 2015). However, a study by Xu et al., indicate that lipid (triglyceride and fatty acid) accumulation
occurs ahead of EMT in DN (Xu et al., 2014). Thus, lipotoxicity caused by high glucose levels prior to
EMT, if inhibited, could be a potential therapeutic target in the treatment of DN. In addition, the
advanced glycosylation end products (AGE) formed under hyperglycemic conditions are shown to
activate the receptor of advanced glycation end products (RAGE) and promote the expression of
growth factors such as TGF-β1 and CTGF in renal proximal tubular cells and result in renal fibrosis
(Cheng et al., 2015).

Studies in non-diabetic forms of kidney diseases implicate the involvement of additional factors
such as oxidative stress and epigenetic modifications in the pathogenesis of EMT (Rhyu et al., 2005;
Rodriguez-Romo et al., 2015). Using an experimental model of TGF-β-induced EMT in renal tubular
cells, Rhyu et al. demonstrated that TGF- induces reactive oxygen species (ROS) generation in renal
cells. Scavenging of ROS with antioxidants prevented TGF- mediated activation of MAPK pathway,
which plays a central role in the induction of EMT (Rhyu et al., 2005). In a recent study by Rodriguez
et al., the team reported that epigenetic modifications such as chromatin compaction, DNA
methylation, and histone acetylation/deacetylation increase the production of proinflammatory and
profibrotic cytokines such as monocyte chemoattractant protein-1 (MCP-1), complement protein 3 (C3)
and TGF-β, which play a major role in induction of EMT and fibrosis in the kidney (Rodriguez-Romo
et al., 2015). Together, suppression of ROS generation and/or epigenetic modification would inhibit the
transition of renal epithelial cells into mesenchymal cells and prevent renal fibrosis.

The anti-fibrotic effects of metformin have been well-documented in both diabetic and non-
diabetic models of CKD. Cufi et al. studied the effect of metformin on MDCK cells against TGF-β,
which is a major driving force in EMT. Their study concluded that metformin attenuates the activity of
TGF-β by reducing the accumulation of mesenchymal marker vimentin in MDCK cells (Cufi et al.,
2010). A study by Lee et al. further demonstrated that metformin inhibits EMT induced by diverse
stimuli including TGF-β and high glucose. Interestingly, the anti-fibrotic effects of metformin were
diminished in the presence of chemical inhibitors and small interference RNA of AMPK, indicating
that metformin exerts its nephroprotective effects through AMPK dependent pathway (Lee et al.,
2013). These findings suggest that metformin could prevent renal cells from undergoing fibrosis, which
plays an important role in the progression of DN and other forms of CKD (Figure 4).

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 Autophagy
Accepted Article Autophagy is a well-coordinated, multi-step cellular process of degradation of protein
aggregates and damaged organelles by lysosomes. In this process, the cytosolic portions containing
damaged cellular components are taken up by autophagosomes, which subsequently fuse with
lysosomes to form autolysosomes, the ultimate autophagy apparatus that mediates lysosomal
degradation to maintain cell survival and cellular metabolism (Inoki, 2014; Liu et al., 2012; Liu et al.,
2014). This process occurs in a sequence of 3 major steps as follows (Ding and Choi, 2015; Ravikumar
et al., 2010).

1. Initiation mechanisms that regulate the formation of pre-autophagosomal structure (PAS):

mTOR-Atg13-ULK1 complex and Beclin-Vps34 complex. [Atg - autophagy-related gene, ULK -
Unc 51-like kinase, VPS - Vacuolar protein sorting]
2. Elongation mechanisms for PAS (Ubiquitin-like conjugation systems): [Atg5-Atg12
conjugation and LC3-phosphatidyl ethanolamine (PE) conjugation]. The conversion of cytosolic
truncated form (LC3I) into its autophagosomal membrane-associated phosphatidylethanolamine-
conjugated form (LC3II) indicates autophagosome formation. [LC3- microtubule-associated protein
1 light chain 3].
3. Maturation step (Fusion): UVRAG interacts with Class C VPS proteins and activates Rab7 and
this promotes the fusion of autophagosomes with lysosomes. [UVRAG- UV-radiation associated
gene].

mTOR is one of the most important nutrient-sensing pathways within cells. mTORC1 and
mTORC2 complexes of mTOR negatively regulate autophagy by inhibiting the initiation of formation
of PAS via direct phosphorylation of ULK1 (Kim et al., 2011; Lee et al., 2010). Under the conditions
of nutrient starvation, ER stress or hypoxia, autophagy is activated and acts as a protective mechanism
for renal tubular cells (Ding and Choi, 2015; Inoki, 2014). For example, in a study conducted by Xiao
et al., inhibition of mTOR with rapamycin restored autophagy and inhibited apoptosis in the podocytes
of diabetic mice and ultimately delayed the progression of DN (Xiao et al., 2014).

Unlike mTOR, activated AMPK has been shown to positively regulate autophagy (Ding and
Choi, 2015). For instance, activation of AMPK (and consequent inhibition of mTOR) by metformin has
been shown to induce autophagy via phosphorylation of key autophagy proteins such as ULK1, VPS34
and Beclin 1 (Kim et al., 2011). A study conducted by Kim et al. revealed how phosphorylation of
ULK1 at different serine residues by mTOR and AMPK modulates ULK1 activity and consequently
autophagy. Glucose-starvation induced AMPK activation has been shown to phosphorylate ULK1 at
Ser-317 and Ser-777 and induce autophagy. Whereas conditions that favor high mTOR activity such as
nutrient excess caused phosphorylation of ULK1 at Ser-757 and subsequently hindered the interaction
of ULK1 with AMPK and prevented autophagy induction. A similar modulation by mTOR and AMPK
on ULK1 and autophagy activation has been demonstrated in albumin-induced renal cell injury – an in
vitro model of non-diabetic CKD (Lee et al., 2011). Furthermore, the effects of activated AMPK on
autophagy induction was also shown in experimentally-induced renal ischemia-reperfusion injury, a
model of acute kidney injury (Decleves et al., 2014). These studies provide compelling evidence that

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 autophagy induction via AMPK activation is one of the major mechanisms by which metformin exert
its nephroprotective effects (Figure 5).
Accepted Article
Oxidative Stress and Advanced Glycation End Products
Numerous studies suggest that increased production of mitochondrial ROS from mitochondrial
electron transport chain plays an important role in the development of DN (Brownlee, 2005; Giacco
and Brownlee, 2010). Increased levels of mitochondrial ROS overwhelm the cellular antioxidant
machinery and causes renal damage by four major mechanisms such as induction of the polyol
pathway, the hexosamine pathway, increased production of advanced glycosylation end products
(AGE) and activation of protein kinase C (PKC) (Brownlee, 2005; Giacco and Brownlee, 2010). A
study by Brownlee et al. found that blocking increased ROS production or interfering with ROS
signaling attenuates the activity of all the above pathways (Brownlee, 2005). Recently, a study by
Wang et al. concluded that even in non-diabetic rats, acute hyperglycemia - induced by glucose
infusion - can trigger oxidative stress and inflammation in kidney cells (Wang et al., 2015).

Under hyperglycemic conditions, glucose combines with amino groups in proteins, nucleic
acids and lipids in circulation through an irreversible, non-enzymatic process to form AGEs (Wolf,
2004). Studies have shown that formation of AGEs are increased in diabetes patients and doubled in
diabetes patients with ESRD compared to diabetes patients with no renal disease. This accumulation of
AGEs in patients with DN is due to enhanced formation of AGEs and decreased renal clearance
(Makita et al., 1991).

AGEs exerts intracellular effects via its receptor, RAGE in renal cells leading to the activation
of NF-κB. This would increase the generation of ROS leading to oxidative stress. AGEs induce the
expression of growth factors and cytokines such as TGF-β and connective tissue growth factor (CTGF).
AGEs also stimulate the production of extracellular matrix proteins and interact with the renin-
angiotensin system (Forbes et al., 2003). Thus, AGEs and its receptor RAGE play an important role in
the development of tubulointerstitial damage in DN.

Metformin treatment was shown to inhibit the deleterious effects of AGEs by down-regulating
the expression of RAGE and subsequently suppress ROS production. A study by Ishibashi et al.
revealed that metformin inhibits the generation of hyperglycemia-induced ROS and other
profibrotic/proinflammatory factors such as TGF-β, intercellular adhesion molecule-1 and monocyte
chemoattractant protein-1 in the proximal tubule, all of which are activated through AGE-RAGE
interaction. This study also indicated that the antioxidant effects of metformin are mediated through
AMPK activation (Ishibashi et al., 2012).

Moreover, incubation of renal podocytes with metformin has been shown to decrease ROS
generation via suppression of NADPH oxidase, a pro-oxidant enzyme system that generates superoxide
anions (Piwkowska et al., 2010). This antioxidant effect of metformin is also shown to be AMPK
dependent. In a study by Kim et al., metformin prevented podocyte loss by decreasing oxidative injury

This article is protected by copyright. All rights reserved 12

 to podocytes in a rat model of type-2 diabetes (Kim et al., 2012). Similarly, treatment with metformin
in experimental models of type 1 diabetes has also shown to ameliorate oxidative stress in the diabetic
Accepted Article
kidney (Alhaider et al., 2011). An even more convincing evidence for the antioxidant effects of
metformin was demonstrated by Chakraborty et al. in T2DM patients. In validation to the findings from
preclinical studies, the metformin-treated group demonstrated reduced levels of AGE and advanced
oxidation products, and improved antioxidant status as compared to the placebo-treated group
(Chakraborty et al., 2011b).

Studies in models of acute kidney injury (AKI) also indicate that metformin exerts antioxidant
effects on the kidney. A study by Morales et al. revealed that metformin prevents acute renal failure
induced by gentamycin in rats. The improvements in the in vivo markers of AKI observed with
metformin treatment is accompanied by decreases in the levels of mitochondrial ROS and lipid
peroxidation and an increase in antioxidants levels (Morales et al., 2010). Another study by Sahu et al.,
tested the nephroprotective effects of metformin against cisplatin-induced AKI in rats. Although
metformin did not prevent the renal damage induced by cisplatin, it was shown to significantly reduce
the levels of ROS and increase the antioxidant pool in the kidney (Sahu et al., 2013). Thus, it is clear
that metformin inhibits ROS generation via multiple pathways and the resultant reduction in oxidative
stress contributes to its nephroprotective effects in DN (Figure 6).

Hypoxia Induced Factor (HIF)

Hypoxia occurs when oxygen delivery is reduced and/or when oxygen consumption is increased
secondary to increased respiratory coupling. Chronic hypoxia, induced by early hemodynamic and
metabolic changes in the glomerulus, has been proposed as an important factor in the development of
tubulointerstitial fibrosis leading to DN. Accumulating evidences show that chronic hypoxia via the
activation of hypoxia-inducible factor-1α (HIF-1α) accelerates the progression of DN (Gu et al., 2013;
Takiyama et al., 2011).

HIF-1α plays an important role in wound healing via expression of multiple angiogenic growth
factors, recruitment of progenitor cells and cell motility (Botusan et al., 2008). At low oxygen levels,
HIF-1α acts as the key regulator in many cells by activating several pathways to adapt and survive
hypoxic conditions (Nangaku et al., 2013). Active HIF-1 is a heterodimer composed of HIF-1β and
HIF-1α subunits. HIF-1β is stable under hypoxic and normoxic conditions, whereas HIF-1α is highly
induced by hypoxia. Under normoxic conditions, HIF-1α is rapidly degraded by ubiquitin-proteasome
pathway due to hydroxylation of its proline residues by prolyl hydroxylases. In its hydroxylated form,
HIF-1α binds to the tumor suppressor protein, von Hippel-Lindau (VHL), which is part of an E3
ubiquitin ligase complex and subjected to proteasomal degradation. Under hypoxia, proline residues on
HIF-1α are no longer hydroxylated and HIF-1α is stabilized against degradation. Accumulated
unmodified HIF-1α in the cytosol translocates to the nucleus where it heterodimerizes with HIF-1β and
up-regulates the transcription of target genes (Botusan et al., 2008; Nangaku et al., 2013).

Although activation and upregulation of HIF-1α have been shown to be associated with
protective effects in acute ischemic injury, prolonged HIF-1α activation is considered pathogenic in

This article is protected by copyright. All rights reserved 13

 CKD (Nangaku et al., 2013). This is because HIF-1α has the ability to stimulate collagen accumulation
by activating fibrogenic factors such as plasminogen activator inhibitor (PAI) and tissue inhibitor of
Accepted Article
metalloproteinase (TIMP). Apart from hypoxia, angiotensin II also stimulates HIF-1α accumulation
(Wang et al., 2011). Studies indicate that angiotensin II inhibits prolyl-hydroxylases through
stimulation of hydrogen peroxide production and leads to accumulation of HIF-1α. This results in
accumulation of collagen in renal cells and leads to renal fibrosis (Wang et al., 2011). Furthermore,
silencing of HIF-1 in renal cells using short hairpin RNA has been demonstrate to attenuate
overexpression of collagen and -smooth muscle actin and prevent glomerulosclerosis in a rat model of
CKD. Hence, inhibition of HIF would serve as an effective therapeutic strategy to preserve renal
structure and function in chronic renal disease states such as DN (Figure 7).

Similar to the findings from various models of CKD, a study by Takiyama et al. in a genetic
model of type-2 diabetes showed increased expression of HIF-1α in conjunction with
glomerulosclerosis and tubulointerstitial injuries in the kidney (Takiyama et al., 2011). Metformin
treatment significantly decreased expression of HIF-1α and ameliorated tubular injury compared to
insulin-treated rats; however, in an AMPK-independent manner (Takiyama et al., 2011). The study
further indicated that metformin inhibits HIF-1α levels through inhibition of mitochondrial respiratory
chain complex I, thereby inhibiting oxygen consumption and ATP production. The resultant increased
intracellular oxygen redistributes to prolyl hydroxylase, leading to proteasomal degradation of HIF-1α.
Thus, the beneficial effects of metformin in ameliorating this progression of DN are partly attributed to
its ability to inhibit HIF-1 expression in renal cells under diabetic milieu.

Lipotoxicity
Accumulation of lipid deposits can also cause renal injury leading to DN. The underlying
mechanism that promotes accumulation of lipids in diabetic kidneys in not fully understood. Studies
implicate that sterol regulatory element binding protein-1 (SREBP-1), a transcription factor that
regulates fatty acids and triglycerides, plays a critical role in lipid deposition into the kidney.
Specifically, an in vitro study by Jun et al. indicated that hyperglycemia can induce the expression of
SREBP-1 in human kidney cells. Increased SREBP-1 expression can therefore lead to lipid
accumulation in renal cells by up-regulating the accumulation of extracellular matrix cells and
expression of fatty acid synthase (FAS) by inducing TGF-β1 (Jun et al., 2009). An in vivo study by
Wang et al. not only confirmed that DN is associated with accumulation of triglycerides and lipids in
the kidney, but also demonstrated that this renal lipotoxicity can be ameliorated by metformin therapy.
The study indicated that metformin reduces fat content by down-regulating the expression of SREBP-1,
FAS and acetyl-CoA carboxylase or ACC (an enzyme involved in the biosynthesis of fatty acids)
(Wang et al., 2006) as shown in Figure 8.

Conclusion
The therapeutic use of metformin in DN and other non-diabetic kidney diseases was restricted
by the US FDA due to the risk of patients developing lactic acidosis on its administration.
Nevertheless, multiple studies have shown that incidence of lactic acidosis is very rare and is often
triggered by other underlying conditions. Hence, the US FDA has recently revised their guidelines and

This article is protected by copyright. All rights reserved 14

 approved the use of metformin in patients with underlying kidney disease based on their eGFR levels.
This marks a new beginning for metformin as the healthcare professionals could use metformin or
Accepted Article
metformin-containing medicines and to effectively assess the renoprotective effects of metformin to
hinder the progression of kidney disease.

This review highlights that metformin not only activates AMPK signaling but also modulates
other signaling pathways described earlier, thus protecting renal cells from damage-induced by high
glucose. For instance, activation of mTOR leads to inhibition of AMPK, thus leading to cell
proliferation and nephropathy. Metformin has been shown to effectively activate AMPK in renal cells
(Lee et al., 2007) but does it directly inhibit mTOR pathway independent of AMPK activation
(Kalender et al., 2010) is an area to be researched on. As mTOR signaling is activated by nutrients such
as glucose and amino acids, and growth factors, it opens the door for investigation into other pathways
that are regulated similarly such as PIP3/AKT pathway. Moreover, when AKT is activated by PIP3, it
leads to activation of mTORC1. So, more research needs to be done to understand how the activation
of AMPK by metformin inhibits pathways such as mTOR and PIP3/AKT, and protects the renal cells
from cell damage induced by high glucose levels.

Studies have also shown that metformin prevents ER stress induced by chemicals such as
tunicamycin and thapsigargin and other non-chemical inducers such as TGF-β, ANG II, aldosterone
and high glucose (Lee et al., 2013; Theriault et al., 2011). Interestingly, majority of the evidence
suggest that this reduction in ER stress is due to activation of AMPK by metformin (Hou et al., 2010;
Kim et al., 2015; Lee et al., 2012). This raises a question that how AMPK activation by metformin
changes the expression of other UPR markers. It is still unclear as to when the cells divert from UPR
and undergo ER stress as the three pathways in UPR have their own temporal dynamics and functions.
Hence, concluding on metformin’s effects on UPR specifically may be difficult as all the pathways are
highly intertwined with each other. However, if metformin can reduce the vicious UPR response in
renal cells that are growing in an abnormal physiological milieu, then it would prevent the cells from
marching towards ER stress and promote cell survival.

As mentioned earlier, EMT culminates in tubulointerstitial fibrosis during the onset of DN.
Studies have shown that metformin possess anti-fibrotic properties as it decreases the induction of
EMT induced by cytokines and growth factors such as TGF-β by activating AMPK (Cufi et al., 2010;
Lee et al., 2013; Lu et al., 2015). Metformin could also reduce EMT by reducing renal lipotoxicity as
studies suggest that lipid accumulation occurs in early stages of DN before the onset of EMT (Xu et al.,
2014).

During DN, mTORC1 is activated in renal cells and this leads to inhibition of autophagy, a
defense mechanism within cells. Since metformin inhibits mTORC1 and activates AMPK in renal
cells, it could also be used as means to induce autophagy in the kidney and to restore the cell repair
machinery. Thus, metformin therapy would help the kidney to remove damaged cells and protect the
healthy cells from further cell injury in case of DN and CKD.

This article is protected by copyright. All rights reserved 15

 It is also well-established that hyperglycemic conditions lead to overproduction of ROS
(Brownlee, 2005; Giacco and Brownlee, 2010). Thus, studies demonstrating the ROS scavenging
Accepted Article
effects of metformin (Alhaider et al., 2011; Chakraborty et al., 2011a; Ishibashi et al., 2012; Kim et al.,
2012) reinforce the therapeutic potential of metformin as a nephroprotective drug and could be of
greater benefit in the treatment of DN and CKD.

Chronic hypoxia, which leads to induction of HIF-1, is regarded as an important event in

pathogenesis of DN and CKD. This makes HIF pathway as a viable target to protect renal cells from
cell damage. Metformin has shown to reduce HIF-1 expression by reducing ATP levels and renal
oxygen consumption (Takiyama et al., 2011). The study further revealed that metformin was able to
suppress HIF-1α expression even when the AMPKα unit was knocked out. Other AMPK activators and
inhibitors did not affect the HIF pathway. This suggests that metformin-mediated reduction in HIF does
not require the activation of AMPK, and metformin protects renal cells from hypoxia conditions by
mechanisms independent of AMPK signaling cascade.

Lipid accumulation in renal cells is another factor that contributes to renal injury in diabetes
patients (Jun et al., 2009). Metformin is effective in reducing fat content in kidneys by reducing the
expression of SREBP-1, FAS and acetyl-CoA carboxylase, all of which involved in lipid accumulation
(Wang et al., 2006).

In summary, metformin, apart from its use as an anti-diabetic drug, possess nephroprotective
properties owing to its pleiotropic effects (as shown in Table 2) on multiple signaling pathways (Figure
9), which warrants additional studies to be conducted to employ it as a nephroprotectant to treat
patients with DN and non-diabetic kidney diseases.

This article is protected by copyright. All rights reserved 16

 Acknowledgements
Accepted Article This review was made possible by a NPRP award [NPRP 7-1189-3-304] from the Qatar
National Research Fund (a member of The Qatar Foundation). The statements made herein are solely
the responsibility of the author[s]. The authors have no conflict of interest, financial or otherwise, to
disclose.

This article is protected by copyright. All rights reserved 17

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 Table 1: Revised Guidelines (2015) from the US FDA on the use of metformin in patients with
kidney disease
Accepted Article
Estimated Glomerular Filtration Recommendations on the Use of Metformin in
Rate (eGFR) Patients with Reduced Renal Function
Recommended with continuous risk assessment; should
Above 45 mL/min/1.73 m2
be stopped when eGFR falls below 30 mL/min/1.73 m2
Not recommended but can be continued with risk
Between 30 to 45 mL/min/1.73 m2
assessment of therapy and reduction of 50% of the dose
Below 30 mL/min/1.73 m2 Not recommended

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 Table 2: Effect of Metformin on the major signaling pathways implicated in DN and other non-
diabetic renal disease states
Accepted Article
Pathways/Biomarkers Effect of metformin on biomarker References
involved
ER stress  Reduces GRP78 expression in mouse (Cameron, 2013; Kim
 GRP78 model of unilateral ureteral et al., 2015; Wu et al.,
 CHOP obstruction and attenuates renal 2010)
 p-eIF2α fibrosis
 Inhibits UPR in renal tubular cells by
reducing the expression of pro-
apoptotic marker CHOP
 Suppresses the expression of p-eIF2α
in human proximal tubular (HK-2)
cells
Epithelial to Mesenchymal  Regulates levels of E-Cadherin (Cheng et al., 2015;
Transition (EMT) expression and inhibits accumulation Maezawa et al., 2015;
 E-Cadherin of vimentin in canine kidney cells Xu et al., 2014)
 α-SMA (MDCK) cells by decreasing the
 Desmin effect of TGF-β-induced inflammation
 Vimentin in renal cells and attenuating renal
fibrosis and CKD
 Inhibits the upregulation of α-SMA
and downregulates E-Cadherin in
human proximal tubular (HK-2) cells
in which EMT is induced by factors
like TGF-β, ANG-II, aldosterone, high
glucose and urinary albumin
Autophagy  Upregulates LC3II protein in renal (Ding and Choi, 2015;
 LC3I/II cells during DN and AKI Kim et al., 2011; Li et
 P-ULK1  Phosphorylation of ULK-1 was al., 2016)
stimulated
Oxidative Stress and  Inhibits reactive oxygen species
Advanced Glycation End generation in rat kidneys induced by (Alhaider et al., 2011;
Products (AGE) streptozotocin-induced diabetes. Ishibashi et al., 2012;
 GSTα  Regulates the levels of GSTα, NQO1 Verzola et al., 2004;
 NQO1 and CAT gene expression Yamagishi and
 CAT  Inhibits the generation of AGEs Matsui, 2010)
Hypoxia Inducible Factor  Reduces HIF-1α expression by (Han et al., 2013;
(HIF) reducing ATP synthesis in DN and Takiyama et al., 2011;
 HIF-1α attenuates chronic renal hypoxia Viollet et al., 2012)

This article is protected by copyright. All rights reserved 25

 Figure Legends
Accepted Article
Figure 1: Stages in the progression of diabetic nephropathy (DN). Initial stages of DN show minimal
changes in albumin excretion, creatinine levels and blood pressure. Kidney function tests may show
changes in the GFR depending on the extent of glycemic control by the patient. Later stages of DN are
associated with extreme changes in morphology of kidney cells, reduction in GFR, high blood pressure
and abnormal increase in urinary albumin excretion.

Figure 2: Effect of metformin on AMPK and mTOR pathway in diabetic nephropathy. Hyperglycemia
leads to uncontrolled cell proliferation and hypertrophy of kidney cells due to high levels of mTOR
activity and inactivation of AMPK. Metformin treatment stimulates AMPK phosphorylation and
subsequently reduces the expression of mTOR. This augments autophagy and prevents the renal cells
from undergoing fibrosis, hypertrophy, EMT and apoptosis. Upward and downward arrows indicate
stimulation and inhibition of the parameter respectively by metformin. PI3K: Phosphatidylinositol-3-
kinase, AKT: Protein kinase B, mTOR: Mammalian target of Rapamycin, AMPK: Adenosine
monophosphate- activated protein kinase.

Figure 3: Effect of metformin on endoplasmic reticulum (ER) stress in diabetic nephropathy. ER stress
is induced under hyperglycemic conditions through accumulation of proteins, ROS generation and
activation of mTOR in the renal cells. Activation of AMPK by metformin protects the renal cells from
ER stress via inhibition of sustained unfolded protein response, ROS generation and mTOR mediated
signaling. GRP78: Upward and downward arrows indicate stimulation and inhibition of the parameter
respectively by metformin. Glucose-regulated protein 78, CHOP: CAAT/enhancer-binding protein
homologous protein, p-eIf2α: phosphorylated Eukaryotic Initiation Factor-2, ROS: reactive oxygen
species.

Figure 4: Effect of metformin on epithelial to mesenchymal transition (EMT) in diabetic nephropathy.

The driving force of EMT under hyperglycemic conditions is the activation of a growth factor, TGF-β.
Phosphorylation of AMPK by metformin inhibits activation of TGF-β and attenuates EMT and thereby,
protects the renal cells from undergoing fibrosis and injury. Upward and downward arrows indicate
stimulation and inhibition of the parameter respectively by metformin. TGF-β: Transforming Growth
Factor- beta, E-Cadherin: Epithelial Cadherin, α-SMA: alpha Smooth Muscle Actin.

Figure 5: Effect of metformin on autophagy in diabetic nephropathy. Autophagy is inhibited in

diabetic kidney due to inactivation of AMPK and/or activation of mTOR. Metformin induced
activation of AMPK stimulates phosphorylation of autophagy proteins such as ULK-1, VPS34 and
Beclin 1 and up-regulation of LC3II for autophagosome formation. Upward and downward arrows
indicate stimulation and inhibition of the parameter respectively by metformin. ULK-1: Unc-51-like
kinase 1, VPS34: Vacuolar protein sorting 34, LC3- microtubule associated light chain 3.

Figure 6: Effect of metformin on oxidative stress in diabetic nephropathy. Under hyperglycemic

conditions, glucose combines with proteins, nucleic acids and lipids to form AGEs. AGE via their
receptors, lead to activation of NF-κB and ROS generation, which causes oxidative stress in the renal
cells. Metformin attenuates oxidative stress in renal cells by multiple pathways such as suppression of

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 NADPH Oxidase, ROS generation in mitochondria, oxidative lipid degradation and increase in anti-
oxidant levels. Upward and downward arrows indicate stimulation and inhibition of the parameter
Accepted Article
respectively by metformin. AGE: Advanced Glycation End-products, NADPH: Reduced Nicotinamide
Adenine Dinucleotide Phosphate, AMPK-PGC-1α: AMP-activated protein kinase- peroxisome
proliferator-activated receptor gamma coactivator 1-alpha, ROS: Reactive Oxygen Species, NF-κB:
Nuclear factor kappa B, TGF-β: Transforming growth factor-beta.

Figure 7: Effect of metformin on hypoxia-inducible factor (HIF) in diabetic nephropathy. Hypoxic

conditions and elevated levels of angiotensin II in DN causes severe kidney damage, hypertension and
accumulation of collagen. Metformin inhibits mitochondrial respiratory chain I and redistributes the
oxygen within the cells. This reduces the oxygen consumption and ATP levels, which in turn promotes
the degradation of HIF-1α by proteasomes and prevents the hypoxia-induced renal cell injury.
Downward arrows indicate inhibition of the parameter by metformin. HIF-1α: Hypoxia-inducible
factor-1 alpha, ATP: Adenosine triphosphate.

Figure 8: Effect of metformin on lipotoxicity in diabetic nephropathy. Hyperglycemic conditions

promote accumulation of lipid deposition and matrix cells in renal cells via enhanced expression of
FAS and activation of TGF-β. Metformin reduces lipotoxicity by down-regulating the expression of
SREBP-1, a transcription factor that plays a vital role in lipid deposits in hyperglycemic kidneys.
Downward arrows indicate inhibition of the parameter by metformin. SREBP-1: Sterol regulatory
element binding protein-1, FAS: Fatty acid synthase, TGF-β: Transforming growth factor-beta.

Figure 9: Summary of the effects of metformin on various signaling pathways in diabetic nephropathy.
Metformin protects renal cells under hyperglycemic and hypoxic conditions through mechanisms
dependent and independent of phosphorylation of AMPK. Upward and downward arrows indicate
stimulation and inhibition of the parameter respectively by metformin.

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