Transcription factors in the
pathogenesis of diabetic nephropathy
Amber Paratore Sanchez1,2 and Kumar Sharma1,2,*
Approximately a third of patients with diabetes develop diabetic kidney disease,and diabetes is the leading cause of end-stage renal disease in most developedcountries. Hyperglycaemia is known to activate genes that ultimately lead toextracellular matrix accumulation, the hallmark of diabetic nephropathy.Several transcription factors have been implicated in glucose-mediatedexpression of genes involved in diabetic nephropathy. This review focuses onthe transcription factors upstream stimulatory factors 1 and 2 (USF1 and 2),activator protein 1 (AP-1), nuclear factor (NF)-kB, cAMP-response-element-binding protein (CREB), nuclear factor of activated T cells (NFAT), andstimulating protein 1 (Sp1). In response to high glucose, several of thesetranscription factors regulate the gene encoding the profibrotic cytokinetransforming growth factor b, as well as genes for a range of other proteinsimplicated in inflammation and extracellular matrix turnover, includingthrombospondin 1, the chemokine CCL2, osteopontin, fibronectin, decorin,plasminogen activator inhibitor 1 and aldose reductase. Identifying themolecular mechanisms by which diabetic nephropathy occurs has importantclinical implications as therapies can then be tailored to target those at risk.Strategies to specifically target transcription factor activation and functionmay be employed to halt the progression of diabetic nephropathy.
Why do approximately a third of patients withdiabetes develop nephropathy irrespective ofglycaemic control? Hyperglycaemia is knownto play a critical role in the pathogenesis ofdiabetic nephropathy via activation of genesregulating extracellular matrix (ECM) synthesisand degradation. The accumulation of ECMaccounts for the prominent pathological
changes of diabetic nephropathy, as seen on thekidney biopsy in Figure 1. While ahyperglycaemic environment is necessary, itis not sufficient alone to cause diabeticnephropathy (Ref. 1). Marked ethnic variationand familial predisposition is also observed,suggesting a significant genetic component(Refs 2, 3). The pathogenesis of diabetic
1Division of Nephrology, University of California San Diego, La Jolla, CA 92093-0711, USA.
2Center for Renal Translational Medicine, University of California San Diego/VA Medical System,La Jolla, CA 92093-0711, USA.
*Corresponding author: Kumar Sharma, Center for Renal Translational Medicine, University ofCalifornia San Diego/VA Medical System, 9500 Gilman Drive, MC 0711, La Jolla, CA 92093-0711,USA. Tel: +1 858 822 0870; Fax: +1 858 822 7483; E-mail: [email protected]
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nephropathy likely results from a complexinteraction between both metabolic andhaemodynamic factors that depend on geneticpredeterminants within a specific environment(Ref. 4). The current proposed metabolicmechanisms mediating diabetic kidney diseaseinclude accumulation of advanced glycationendproducts in the kidney, oxidation ofrenal glycoproteins by reactive oxygen species(ROS), intracellular accumulation of sorbitolvia the reduction of glucose by aldosereductase, and activation of protein kinase C(PKC), the hexosamine biosynthetic pathway(HBP) and the mitogen-activated proteinkinase (MAPK) cascade – ultimatelyculminating in production of profibrotic growthfactors, such as transforming growth factor b1(TGF-b1) (Refs 4, 5).
This review focuses on a select group oftranscription factors that have been implicatedin the regulation of TGF-b1, as well as otherdownstream genes that are involved with theprogression of diabetic kidney disease (Fig. 2).Investigating transcription factors such as theupstream stimulatory factors (USF1 and 2),activator protein 1 (AP-1), cAMP-response-element-binding protein (CREB), nuclear factor(NF)-kB, nuclear factor of activated T cells(NFAT), and stimulating protein 1 (Sp1) may
help elucidate the translation of a high-glucosesignal into the production of mediators ofprogressive nephropathy.
Overview of transcription factoractivation and classification
Transcription factors are proteins that bind topromoter regions of genes and activate orsilence their transcription into mRNA (Ref. 6).In addition to availability of the DNA-bindingsite, activity of a transcription factor oftenrequires post-translational modification andbinding with nuclear-localisation proteins (Ref. 7).Thus, transient signals such as high glucose canbe converted into long-term changes in geneexpression by activating various signaltransduction pathways within the cell thatresult in transcription factor phosphorylationand glycosylation (Refs 8, 9, 10).
Most eukaryotic genes are packed intochromatin structures, comprising DNA andhistone proteins, that require chromatinremodelling to facilitate transcription (Ref. 11).Aberrant histone modifications may lead tochanges in chromatin structure, allowing fordysregulated gene transcription and diseaseprogression. Epigenetics is a term used todescribe heritable alterations in gene expressionby mechanisms other than changes in the
The pathological changes in the glomerulus with diabetic nephropathyExpert Reviews in Molecular Medicine © 2009 Cambridge University Press
a b c
Figure 1. The pathological changes in the glomerulus with diabetic nephropathy. (a) Light microscopy of ahuman glomerulus with diabetic nephropathy. Long arrows show areas of diffuse mesangial expansion. Shortarrow depicts arteriolar hyalinosis. (b) Electron microscopy of diabetic glomerulus. Long arrows depict diffusemesangial matrix expansion. Note that the mesangial matrix does not have clear borders. The short arrow pointsto a thickened glomerular basement membrane. (c) Electron microscopy of a normal glomerulus. The long arrowpoints to normal mesangial matrix. The short arrow shows the thickness of a normal glomerular basementmembrane.
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underlying DNA sequence (Ref. 12). Currentinterest in the epigenetics of diabeticcomplications has included evaluating theimpact of high glucose on histone lysineacetylation and methylation patterns in
vascular smooth muscle cells derived from type2 diabetic mice and in human monocytes(Refs 12, 13). These modifications can result in aheritable change in gene expression known ascellular or metabolic memory, which may
Metabolic pathways involved in the development of diabetic nephropathyExpert Reviews in Molecular Medicine © 2009 Cambridge University Press
Hyperglycaemia
PKCp38 MAPK
ERK
Hexosaminebiosynthetic
pathway
Polyolpathway
ROSproduction
Advancedglycation
endproducts
Activation of transcription factors:USF1/2, AP-1, CREB, NF- B, NFAT5, OREBP and Sp1
Increased expression of genes regulating extracellularmatrix production, inflammation and glomerulosclerosis:
TGFB1, SERPINE1, CCL2, THBS1, REN, SPP1, FN, ICAM1 and AKR1B1
Diabetic nephropathy
Figure 2. Metabolic pathways involved in the development of diabetic nephropathy. Hyperglycaemialeads to intracellular activation of the PKC, MAPK, HBP and polyol pathways, and also leads toaccumulation of ROS and production of advanced glycation endproducts. These pathways lead toactivation of the transcription factors USF1/2, AP-1, CREB, NF-kB, NFAT5, OREBP and Sp1. Thesetranscription factors have been demonstrated to increase the expression of genes that ultimatelyculminate in extracellular matrix accumulation, inflammation and glomerulosclerosis in the diabetic kidney.Abbreviations: AKR1B1, aldo-keto reductase family 1, member B1; AP-1, activator protein 1; CCL2,chemokine (C-C motif) ligand 2; CREB, cAMP-response-element-binding protein; ERK, extracellular-signal-regulated kinase; FN, fibronectin; HBP, hexosamine biosynthetic pathway; ICAM1, intercellularadhesion molecule 1; MAPK, mitogen-activated protein kinase; NF-kB, nuclear factor kB; OREBP,osmotic-response-element-binding protein; PKC, protein kinase C; REN, renin; ROS, reactive oxygenspecies; SERPINE1, serpin peptidase inhibitor, clade E, member 1; Sp1, stimulating protein 1; SPP1,secreted phosphoprotein 1; TGFB1, transforming growth factor b1; THBS1, thrombospondin 1; USF,upstream stimulatory factor.
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explain why patients with diabetes continue todevelop inflammation and vascularcomplications even after achieving glycaemiccontrol (Refs 11, 13). However, the emergingfield of epigenetics is not a major aspect of thisreview as we primarily focus on datademonstrating the role of specific transcriptionfactors in diabetic kidney disease.
Transcription factors can be classified by theirstructural motif or mechanistically, such asconstitutively active and regulatorytranscription factors (Refs 14, 15) (Fig. 3). Theconstitutively active transcription factorsfacilitate the expression of genes that are alwaystranscribed, such as structural proteins andcrucial metabolic enzymes, and include theubiquitously expressed transcription factor Sp1(Ref. 14). Regulatory transcription factors areeither developmental transcription factorsimportant in embryonic cell type differentiationor signal-dependent transcription factors, whichare largely inactive until exposed to theappropriate intra- or extracellular signal(Ref. 14). The signal-dependent transcriptionfactors can be further categorised into those thatare activated by internal stimuli (sterol-regulatory-element-binding proteins; SREBPs)and transcription factors activated byenvironmental stimuli. The transcription factorsthat are activated by environmental stimuli canbe further subcategorised into resident nuclearfactors (CREB, AP-1 and USF1/2), whichreside in the nucleus but must be activated toaffect transcription; latent cytoplasmic factors(NF-kB and NFAT proteins), which exist in thecytoplasm in an inactive form and thentranslocate to the nucleus upon activation; andthe steroid receptor superfamily (e.g. theperoxisome-proliferator-activated receptors andthe glucocorticoid receptor), which can reside inthe nucleus or cytoplasm and function astranscription factors once activated (Refs 6, 14)(Fig. 3).
The characteristics of the transcription factorsdiscussed in this review are summarised inTable 1.
Transcription factors involved indevelopment of diabetic nephropathy
Upstream stimulatory factors 1 and 2USF1 and USF2 are ubiquitously expressed andbelong to the Myc family of transcription factorsthat are characterised by a basic–helix–loop-
helix leucine-zipper motif (bHLH–zip)(Refs 16, 17). USF1 and USF2 are encodedby two separate genes but share the highlyconserved C-terminal domain that includesthe basic domain for DNA binding and theleucine zipper for dimerisation (Refs 10, 15, 16,18). Factors containing the basic bindingdomain must form dimers in order to bindDNA, and the USF proteins function primarilyas a USF1–USF2 heterodimer or a USF1homodimer (Refs 16, 19). The DNA-bindingbasic region recognises the E-box (CACGTG)or E-box motif (CANNTG), which for thepurposes of this review will be regarded asthe glucose-responsive element (GRE) andpart of the carbohydrate-response element(ChoRE), the latter consisting of two E-boxmotifs separated by 5 bps (Refs 20, 21, 22, 23).
In response to high glucose, activation of PKCappears to be a critical signalling pathwayaffecting USF1 levels, while USF2 appears to beregulated by several signalling pathwaysincluding p38 MAPK, ERK1/2 (extracellular-signal-regulated kinases 1/2) and HBP, as wellas PKC (Refs 22, 24, 25). USF1 and USF2 havebeen linked to a variety of glucose-regulatedgenes in mesangial cells, hepatocytes, epithelialcells and smooth muscle cells, and the relativeamounts of each protein vary among cell types(Refs 22, 26, 27). Mice lacking USF2 showgrowth delay and increased postnatal death,whereas USF1-knockout mice are phenotypicallynormal, possibly due to compensation fromUSF2 (Refs 26, 27). In streptozotocin-induceddiabetic rats, only USF1, and not USF2, isincreased in glomeruli after two weeks ofdiabetes (Ref. 22). However, overexpression ofUSF2 in transgenic mice leads to albuminuria innondiabetic mice by 6 months of age, which isfurther accelerated by inducing diabetes(Ref. 21). The role of USF1/2 in diabeticnephropathy likely involves transcriptionalupregulation of the genes for TGF-b1 (TGFB1),thrombospondin 1 (THBS1), and osteopontin(SPP1) in response to high glucose, althoughbinding sites have also been reported in thepromoters of the genes for plasminogenactivator inhibitor 1 (PAI-1; SERPINE1) andrenin (REN) (Refs 9, 21, 22, 24, 25, 28, 29).
Transforming growth factor b1The profibrotic cytokine TGF-b1 has beenimplicated in the development of diabetic
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Mechanistic classification of transcription factors activated in hyperglycaemic environmentsExpert Reviews in Molecular Medicine © 2009 Cambridge University Press
Resident nuclear factors Latent cytoplasmic factors
Sp1HIFNF1
Signal dependent Developmental
HNFs
SREBPs
PPARsGR
USF1/2CREBAP-1
NF-κBNFAT5OREBPSMADsChREBP
Constitutivelyactive
Conditionally active(regulatory)
Environmentalsignals
Steroid receptor proteins
Internal signals
Resident nuclear factors Steroid receptor proteins
Figure 3. Mechanistic classification of transcription factors activated in hyperglycaemic environments.This diagram shows a classification schema for transcription factors based on their functionalcharacteristics. Overall, transcription factors are either constitutively active or conditionally active. Includedin this table are transcription factors that are either activated in the diabetic state or important in glucosehomeostasis. This review focuses on signal-dependent transcription factors. Signals can arise fromthe intracellular environment, or from an environmental stimulus, such as glucose. Glucose has beenshown to activate transcription factors that reside in the nucleus as well as those that reside in thecytoplasm and translocate to the nucleus in order to affect gene transcription. The superfamily ofsteroid receptor proteins are transcription factors that reside in the cytoplasm or nucleus and havethe ability to directly bind DNA once activated. Abbreviations: AP-1, activator protein 1; ChREBP,carbohydrate-responsive-element-binding protein; CREB, cAMP-response-element-binding protein;GR, glucocorticoid receptor; HIF, hypoxia-inducible factor; HNFs, hepatocyte nuclear factors;NF1, nuclear factor 1; NFAT5, nuclear factor of activated T cells 5; NF-kB, nuclear factor kB; OREBP,osmotic-response-element-binding protein; PPARs, peroxisome-proliferator-activated receptors;Sp1, stimulating protein 1; SREBPs, sterol-regulatory-element-binding proteins; USF, upstreamstimulatory factor.
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kidney disease as a result of its role in excessECM accumulation in a high-glucoseenvironment (Ref. 22). The TGFB1 genecontains an E-box located within the murinepromoter at 2641 to 2636 and within thehuman promoter at 21013 to 21002 (Refs 9,22). Murine mesangial cells and porcinemesangial cells transfected with the humanTGFB1 promoter demonstrate increased USF1homodimer and USF1–USF2 heterodimerbinding activity to the TGFB1 GRE, althoughin murine mesangial cells there is preferentialbinding of USF1 (Refs 9, 22). USF2homodimers in vivo are scarce, and requireUSF1 as a heterodimer to regulate promoteractivity (Refs 22, 30). Overexpression of USF1or USF2 in transfected porcine mesangial cellsincreases the TGFB1 promoter activitytwofold, which is prevented by a mutation inthe GRE (Ref. 9). In murine mesangial cellsand aortic smooth muscle cells, USF1 proteinlevels increase with sequential elevations inthe glucose concentration, even within thehigh-normal range, while there have beenconflicting reports of high-glucose-enhancedUSF2 expression (Refs 20, 22, 29).Overexpression of USF1 or USF2 in humanembryonic kidney cells increases TGFB1promoter activity; however, only the USF1-transfected cells exhibited increased secretionof TGF-b1 protein (Ref. 22).
Renal TGF-b1 expression has been studiedunder fasting and carbohydrate refeedingconditions in USF1-knockout and USF2-knockout mice (Ref. 22). In mice lacking USF1,renal TGF-b1 mRNA is not stimulatedin response to high carbohydrate intake,whereas mice lacking USF2 have the samedegree of stimulation of renal TGF-b1 aswild-type mice (Ref. 22). USF2-expressingmesangial cells as well as the kidneys of USF2-transgenic mice display increased active TGF-blevels although in mesangial cells total TGF-blevels are not affected (Refs 21, 25). The data thusfar suggest that USF1 is necessary and sufficientfor stimulation of the renal TGFB1 gene in statesof high intake of carbohydrate (Ref. 22).
TGF-b2, along with TGF-b1, has beenimplicated in diabetic kidney disease; however,there are limited data on the regulation ofTGFB2 by high glucose. An E-box has beenidentified in the TGFB2 promoter that bindsUSF1/2, as well as a binding site for CREB, and
a mutation in either binding site results in a60–80% reduction in TGF-b2 expression(Ref. 31). No studies have yet been conductedevaluating glucose-induced binding of USF1/2or CREB to the TGFB2 promoter.
Thrombospondin 1Thrombospondin 1 (TSP1) converts latent TGF-b1 to the active form and therefore is animportant factor in the development ofdiabetic nephropathy (Refs 20, 25). USF1/2binds an E-box motif (CAGATG) in the humanTHBS1 promoter located at 2924 to 2919 thatregulates high-glucose-induced TSP1expression in mesangial cells (Ref. 25).Mutations in this GRE inhibit glucose-inducedUSF binding and THBS1 promoter activity(Ref. 25). USF2 overexpression in transfectedrat mesangial cells significantly augmentsTHBS1 promoter activity and TSP1 proteinlevels under normal glucose conditions (Ref. 25).USF2-transgenic mice with streptozotocin-induced diabetes exhibit increased TSP1compared with wild-type controls (Ref. 21). Therole of USF2 in regulation of TGF-b1 may bepredominantly via increased TSP1 expressionleading to active TGF-b formation.
OsteopontinOsteopontin is a bone matrix protein and pro-inflammatory cytokine produced in response tobiological stressors, and increased expressionstrongly correlates with albuminuria andglomerulosclerosis in human diabetic kidneys(Refs 29, 32, 33, 34). Osteopontin-knockout miceare protected from diabetes-inducedalbuminuria and mesangial expansion (Refs 32,33, 35). The promoter for the rat osteopontingene (SPP1; secreted phosphoprotein 1)contains a CCTCATGAC motif at 280 to 272relative to the initiation site, to which USFproteins and AP-1 bind as a complex (Ref. 29).In rat aortic vascular smooth muscle cells,transient expression of either USF1 or AP-1upregulates SPP1 promoter activity (Ref. 29).Glucose upregulates both AP-1 and USFbinding activities, and selectively upregulatesprotein levels of USF1 and the AP-1 componentFOS (Ref. 29).
Activator protein 1AP-1 belongs to the basic– leucine-zipper familyof transcription factors, which contain a basic
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region responsible for DNA binding and aleucine zipper that mediates dimer formation(Refs 10, 14, 15). The most common form ofAP-1 is a dimer of JUN and FOS that binds thesequences TGACTCT and TGTCTCA (Ref. 36).AP-1 is activated by high glucose, oxidativestress, angiotensin II, low-density lipoprotein(LDL) and oxidised LDL (Refs 36, 37, 38). PKCappears to be the key regulator for AP-1 viapost-translational phosphorylation of JUN andFOS, but the stress-activated kinases JNK and
p38 MAPK also regulate the transcriptionalactivity of AP-1 proteins (Refs 36, 37, 38, 39,40). In studies of vascular smooth musclecells, glucose-mediated AP-1 binding has beendemonstrated in the promoters for SPP1 andthe aldose reductase gene (AKR1B1), butfurther studies are needed in models ofdiabetic kidney disease (Refs 29, 41). Thefibronectin gene (FN) promoter contains an AP-1-binding site that displays increased angiotensin-II-induced transcriptional activation, but there
Table 1. Characteristics of transcription factors activated by high glucose andpotential gene targets in models of diabetic kidney diseasea
Transcriptionfactor
Structuralfamily
DNA bindingsequence
Gene targets indiabeticnephropathy
Refs
USF1 and USF2 Basic–helix–loop–helixleucine-zipper
E-box: CACGTGE-box motif:CANNTG
TGFB1, THBS1,SPP1, SERPINE1,REN, TGFB2
9, 16, 17, 18, 19,20, 21, 22, 23,24, 25, 26, 27,28, 29, 30, 31
AP-1 Basic– leucine-zipper
AP Box A and B:TGACTCT andTGTCTCA
TGFB1, SERPINE1,AKR1B1, FN, SPP1,ICAM1, RSOR
15, 29, 36, 37,39, 42, 43, 44,45, 49, 51, 52
CREB Basic– leucine-zipper
CRE: TGACGTCA FN, USF2, DCN,REN, TGFB2
15, 19, 20, 28,31, 53, 54, 56,57, 59
NF-kB Rel-homologyregion
kB site: GGG(A/G)(C/A/T)T(C/T)(C/T)CC
TGFB1, AKR1B1,CCL2, ICAM1, FN,PTGS2
42, 43, 46, 61,63, 64, 65, 66,67, 68, 69, 70,72, 77, 78, 84, 91
NFAT (NFAT5/OREBP)
Rel-homologyregion
ORE/TonE:TGGAAA(C/A/T)
AKR1B1, MIOX,RSOR
61, 62, 82, 92,93, 94
Sp1 Cys2His2 zinc-finger domain
GC-rich promoterregions:GGGGCGGGGC
TGFB1, SERPINE1,USF2, ADIPOQ,ICAM1, RSOR
8, 9, 37, 47, 48,91, 98, 99, 100
aThe first column in this table lists the transcription factors that have been associated with high-glucose-mediated activation in the kidney. The second column lists the structural class to which each transcription factorbelongs, and the characteristic DNA binding sequence is given in the third column. The fourth column lists thegenes or candidate genes that contain binding sequences within the promoter region for the associatedtranscription factor and display increased expression in hyperglycaemic conditions.Abbreviations: ADIPOQ, adiponectin; AKR1B1, aldo-keto reductase family 1, member B1; AP-1, activatorprotein 1; CCL2, chemokine (C-C motif) ligand 2; CRE, cAMP-response element; CREB, cAMP-response-element-binding protein; DCN, decorin; FN, fibronectin; ICAM1, intercellular adhesion molecule 1; MIOX, myo-inositol oxygenase; NFAT, nuclear factor of activated Tcells; NF-kB, nuclear factor kB; ORE, osmotic-responseelement; OREBP, osmotic-response-element-binding protein; PTGS2, prostaglandin-endoperoxide synthase 2;REN, renin; RSOR, renal-specific oxidoreductase; SERPINE1, serpin peptidase inhibitor, clade E, member 1;Sp1, stimulating protein 1; SPP1, secreted phosphoprotein 1; TGFB, transforming growth factor b; THBS1,thrombospondin 1; TonE, tonicity-response element; USF, upstream stimulatory factor.
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are limited data regarding glucose-inducedincreases in transcriptional activity in the kidney(Refs 42, 43, 44). In models of diabetic kidneydisease, glucose-mediated activation of AP-1 hasbeen demonstrated to increase expression ofTGF-b1 and PAI-1 (Refs 37, 45).
Transforming growth factor b1The human TGFB1 promoter region contains twobinding sequences for AP-1, designated AP-1 boxA (TGACTCT) and box B (TGTCTCA), whichmediate the upregulation of promoter activityvia a PKC-dependent pathway after exposureof cells to a high-glucose environment (Refs37, 38). Increased binding activity of AP-1 isobserved in peripheral blood mononuclearcells (PBMCs) from humans with diabeticnephropathy compared with control PBMCs(Ref. 46). Glucose markedly increases bindingactivity of nuclear AP-1 proteins JUND andFOS in mesangial cells to box B (Ref. 37). Amutation in AP-1 box A, box B or in both boxesprevents high-glucose-mediated increases inTGFB1 promoter activity, with the greatesteffect when both sites are mutated and lowesteffect with the AP-1 box B mutation (Ref. 37).Addition of the AP-1 inhibitor curcumin, ayellow pigment found in the spice turmeric,obliterates glucose- and oxidised-LDL-mediatedincreases in TGFB1 transcription (Refs 36, 37).In the murine model, the regulation of glucoseresponsiveness of the TGFB1 promoter cannotbe fully explained by AP-1 sites, as the glucose-responsive region of the mouse promoter waslocalised between 2835 and 2406 upstreamof the first transcriptional start site, a regionlacking AP-1-binding sites (Ref. 37).
Plasminogen activator inhibitor 1Elevated PAI-1 blood levels are observed indiabetes, and increased expression of its gene,SERPINE1 (serpin peptidase inhibitor clade E,member 1) appears to be involved in manyof the complications of diabetes includingglomerulosclerosis and tubulointerstitialfibrosis, presumably due to decreased plasmindegradation of ECM (Refs 45, 47, 48, 49, 50).PAI-1 deficiency has been shown to beprotective against diabetic nephropathy inexperimental animal models of both type 1 andtype 2 diabetes (Refs 45, 50). The promoter ofthe SERPINE1 gene contains four AP-1-bindingsites, as well as an oxidant-response element to
which AP-1 binds (Refs 49, 51). Glucoseupregulates SERPINE1 gene expression inAP-1-activated vascular smooth muscle cells viaactivation of MAPK and PKC (Ref. 52).Oxidative stress increases JUN mRNA,phosphorylated nuclear JUN, activation ofMAPK, and AP-1 DNA-binding activity, whichultimately results in increased SERPINE1transcription (Ref. 49). A mutation in the AP-1-binding site and use of the antioxidantglutathione abolishes the effect of oxidativestress on increased PAI-1 expression (Ref. 49).
cAMP-response-element-binding proteinCREB belongs to the basic–leucine-zipper familyof transcription factors and binds the 8 bppalindromic sequence TGACGTCA known asthe c-AMP-response element (CRE) (Refs 53,54). Tissue-specific regulation of CREB-targetedgenes depends on recruitment of a coactivatorknown as CREB-binding protein (CBP)(Ref. 55). CREB must be phosphorylated bykinases such as PKA, PKC and MAPK fordimerisation, binding of CBP, and increasedaffinity for the CRE (Refs 10, 14, 19, 54). Therole of CREB in the development of diabeticnephropathy includes transcriptionalupregulation of FN, USF2 and decorin (DCN)genes; although binding sites have beenidentified in the REN and TGFB2 genepromoters, there are limited data in regards toglucose-mediated activation in the kidney(Refs 20, 28, 53, 56).
FibronectinFibronectin is a major component of the ECM andis overexpressed in target organs of diabeticcomplications (Ref. 42). The 50 flanking regionof the human FN gene contains three functionalCREs that vary in sequence, to which activatedCREB binds leading to increased fibronectinmRNA expression (Refs 43, 54, 57). Treatmentof mesangial cells with high glucose increasesfibronectin synthesis, and phosphorylation ofCREB is mediated largely via HBP, althoughactivation via PKC and p38 MAPK pathwaysby high glucose has also been demonstrated(Refs 53, 57, 58). The enzyme 12/15-lipoxygenase (12/15-LO) has been implicated indiabetic nephropathy, as lipoxygenase productsinduce cellular hypertrophy and ECMdeposition (Ref. 58). Murine mesangial cellsderived from 12/15-LO-knockout mice display
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slower growth rates, reduced activation of MAPK,attenuated CREB and AP-1 DNA-bindingactivity, and lower amounts of fibronectinproduction (Ref. 58). A p38 MAPK inhibitor hasbeen shown to ameliorate CREB activation inhigh-glucose-stimulated mesangial cells (Ref. 53).
Upstream stimulatory factor 2High-glucose treatment in rat mesangial cellsincreases CREB binding to a CRE located at21740 to 21620 in the USF2 gene promoterand increases USF2 gene promoter activity(Ref. 20). Mutations in the CREB-binding site,and small interfering (si)RNA-mediated CREBknockdown, abolish glucose-induced USF2promoter activity and protein expression(Ref. 20). Activation of the HBP has been shownto increase USF2 mRNA, which may be due toO-glycosylation of other transcription factorssuch as Sp1, as a GC-rich region in the USF2promoter has also been reported (Ref. 9).
DecorinDecorin is a small proteoglycan component of theECM and inhibits active TGF-b, and appears to bea protective factor against the development ofdiabetic nephropathy (Refs 56, 59). Diabeticmice deficient in the DCN gene exhibitincreased glomerular active TGF-b and matrixaccumulation (Refs 59, 60). In advanceddiabetic nephropathy, decorin deposition isfound in fibrotic regions and is colocalised withdeposits of collagen type I (Ref. 60). Decorinmay modulate progression of TGF-b-mediatedfibrosis through formation of complexes ofdecorin, type I collagen and TGF-b (Ref. 60).Both high glucose and TGF-b upregulatedecorin in mesangial cells; however, a region inthe DCN promoter, known as the TGF-binhibitor element, has also been identified thatmediates downregulation of decorin expressionin response to TGF-b (Ref. 56). It appears thatDCN escapes the negative regulation of TGF-bvia increased CREB binding to a CRE-likesequence (TGACGTCA) in the promoter (Ref. 56).
Nuclear factor kBNF-kB belongs to the Rel family of transcriptionfactors (NF-kB, NFAT5, OREBP) which isdefined by the highly conserved Rel-homologyregion (RHR); the DNA-binding loop is in theRHR-N domain and dimerisation occurs at theRHR-C domain (Refs 61, 62, 63). The RHR-N
domain recognises the DNA consensussequence (kB site) GGGRNNYYCC, where R ispurine, Y is pyrimidine, and N is any base(Refs 64, 65, 66). The major form of NF-kB is thep50–p65 heterodimer, but it can also be ahomo- or heterodimer composed of variablesubunits: c-Rel, p65 (RelA), RelB, p50 and p52(Refs 63, 67, 68). NF-kB exists in the cytoplasmin an inactive state bound to the inhibitoryprotein IkB (Ref. 69). NF-kB is activated bya large variety of stimuli includinghyperglycaemia, oxidative stress or ROS,advanced glycation endproducts, elevated freefatty acids, and pro-inflammatory cytokines(Refs 46, 70, 71, 72, 73). High glucose inducesphosphorylation of IkB kinase (IKK), whichthen phosphorylates IkB leading to itsdegradation, allowing NF-kB to translocate tothe nucleus and induce expression of targetgenes (Ref. 41). High glucose increasesexpression of the p65 subunit of NF-kB viahistone methylation, and this effect persists upto six days in cultured aortic endothelial cells,despite a return to physiological glucose levels(Ref. 74). The PKC pathway has also beenshown to induce increased NF-kB bindingactivity and ROS generation in high-glucose-treated mesangial cells (Ref. 68).
High glucose leads to NF-kB activation inendothelial cells, retina, heart and kidneys ofdiabetic rats, with the most pronouncedactivation in the retina (Ref. 42). The activationof NF-kB in both human PBMCs and kidneybiopsies has been shown to correlate withseverity of proteinuria and degree of glycaemiccontrol in diabetic nephropathy (Refs 46, 65, 71,75, 76). NF-kB binds to the promoter regions ofseveral genes thought to be important in thepathogenesis of diabetic nephropathy: TGFB1,AKR1B1, CCL2 (encoding C-C motif chemokine2; also known as monocyte chemoattractantprotein, MCP-1) and ICAM1 (encodingintercellular adhesion molecule 1) (Refs 46, 67,68, 70, 77). Mutating the NF-kB-binding sites ofthe promoter of the cyclooxygenase 2 gene(PTGS2) in human mesangial cells cultured inhigh glucose abrogates PTGS2 promoteractivation as well as prostaglandin synthesis,which may be involved in the glomerularhyperfiltration observed in early diabetes(Ref. 78). Several potential NF-kB-binding siteshave also been identified in the FN promoter,and high-glucose-induced fibronectin mRNA
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synthesis in cultured endothelial cells is blockedby NF-kB inhibitors, but this relationship has notbeen investigated in mesangial cells or animalmodels of diabetic nephropathy (Refs 42, 43).
Transforming growth factor b1NF-kB appears to have an indirect regulatoryrole in glucose-induced TGF-b1 expression. Theregulatory protein SMAD7 is synthesised inresponse to TGF-b1, and inhibits TGF-b1signalling in a negative feedback loop (Ref. 79).The activity of the SMAD7 promoter isinhibited by NF-kB and enhanced by SMAD3(Ref. 67). The competitive interaction betweenthe SMAD proteins and NF-kB is mediated bythe transcriptional coactivator CBP (Ref. 67).NF-kB mediates disruption of the SMAD–CBPinteraction and may block SMAD activity;therefore, NF-kB may not directly affect theTGFB1 promoter itself but decrease theexpression of the inhibitory SMAD7 (Ref. 67).This is supported by models of downregulatedSMAD7 that show a progressive fibrosis inobstructive nephropathy (Ref. 79). Both urinaryTGF-b1 excretion and the TGF-b1 level inPBMCs significantly correlate with NF-kB andAP-1 DNA-binding activity in PBMCs ofpatients with diabetic nephropathy (Ref. 46).
Aldose reductaseThe AKR1B1 (aldo-keto reductase family 1,member B1) gene product, aldose reductase, isthe first and rate-limiting enzyme in the polyolpathway, and abnormal activation of thispathway in diabetes leads to osmotic andoxidative stress that results in chronic tissueinjury (Refs 41, 70, 80, 81, 82). Aldose reductasemRNA and protein levels are increased inpatients with diabetic microvascularcomplications compared with those withoutdiabetic complications, and it has been found tobe one of the TGF-b1-responsive genes (Refs 82,83). The AKR1B1 gene is activated by osmoticstress via osmotic-response elements (OREs)and is largely regulated by the transcriptionfactor NFAT5; however, NF-kB and AP-1 mayplay an important regulatory role as well(Refs 70, 80). NF-kB shares a binding site withNFAT5 at the ORE designated OREC, andhyperglycaemia-induced flux through thepolyol pathway leads to activation of NF-kB(Ref. 70). In patients with type 1 diabetesand nephropathy, PBMCs cultured under high-
glucose conditions have significantly increasedDNA-binding activities of NF-kB to the kBmotif of the AKR1B1 gene compared withpatients who have uncomplicated diabetes(Ref. 70). Levels of the aldose reductase proteinare also significantly increased in the PBMCs ofpatients with diabetic nephropathy culturedunder high-glucose conditions, compared withpatients who have uncomplicated type 1diabetes (Ref. 70). In vascular smooth musclecells cultured under high-glucose conditions,the presence of siRNA targeting NF-kB subunitp65 reduces transcription of aldose reductase(Ref. 41).
CCL2CCL2 is a potentially important mediator ofglomerular monocyte/macrophage infiltration,and urinary CCL2 levels correlate with thedegree of albuminuria in type 1 diabetes(Refs 72, 84, 85, 86, 87, 88, 89). The promoterregion of the human CCL2 gene has a bindingsite for NF-kB located between 22612 and22603, and in the mouse there are two bindingsites for NF-kB, at 22374 and 22348 (Refs 72,84). High glucose induces NF-kB activation inmurine mesangial cells, and leads to increasedNF-kB binding to the promoter region of CCL2,with resultant increased production of CCL2mRNA (Ref. 68). When human mesangial cellsare cultured in the presence of glycatedalbumin or high glucose, CCL2 gene and CCL2protein expression are upregulated, and thiseffect is abolished by use of PDTC, an NF-kBinhibitor (Ref. 89). In human monocytes, aorticendothelial cells, and vascular smooth musclecells, histone modifications have been shown tobe enriched at inflammatory gene promotersunder diabetic conditions, as well as regulateNF-kB activity at pro-inflammatory genes suchas CCL2, TNF-a (encoding tumour necrosisfactor a) and VCAM1 (encoding vascular celladhesion molecule 1) (Refs 74, 90).
Intercellular adhesion molecule 1During the process of glomerulosclerosis,adhesion of circulating monocytes tomesangial cells and endothelial cells is thoughtto be an early event, mediated by adhesionmolecules such as ICAM-1, VCAM-1 and E-selectin (Ref. 77). Cloning of the humanICAM1 gene revealed a sequence that closelyresembles the consensus sequence for NF-kB at
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position 2540, and there are binding sequencesfor AP-1 and Sp1 as well (Ref. 91). During theearly stages of diabetes, the expressionof ICAM-1 mRNA and protein is upregulatedand promotes recruitment of mononuclearcells in rat glomeruli through glomerularhyperfiltration (Ref. 77). In cultured murinemesangial cells, high glucose increasesmesangial cell proliferation and ICAM-1expression, but not VCAM-1 expression(Ref. 77). In human aortic endothelial cellstransiently exposed to high glucose, increasedexpression of ICAM-1 is preserved up to sixdays after return to normoglycaemia, possiblyvia histone modifications and increased NF-kBactivity (Ref. 74).
Nuclear factor of activated T cellsThe NFAT proteins belong to the RHR familyof transcription factors and include ORE-binding protein (OREBP) and NFAT5 (alsoknown as tonicity-response-element-bindingprotein; TonEBP) (Refs 62, 92). OREBP sharessignificant homology with NFAT5, but NFAT5is the largest of the Rel proteins and differsfrom OREBP at the N-terminus (Refs 61, 93).The DNA-binding domain of NFAT5 is thoughtto have evolved from NF-kB; however, the restof the protein differs considerably from otherRel transcription factors (Ref. 61). NFAT5 has atleast four spliced isoforms, which bind withvarying affinities to the ORE TGGAAA(C/A/T)(Refs 61, 82, 94). NFAT5 exists in the cytoplasmheavily phosphorylated, but is activated byhyperosmolar stress in which an influx ofcalcium activates calcineurin (a type 2bphosphatase), which dephosphorylates NFAT5and allows for nuclear translocation (Refs 14,92, 93). NFAT proteins have a potential role inthe pathogenesis of diabetic nephropathy viaglucose-mediated activation of the geneAKR1B1 (Ref. 82). NFAT proteins also regulateexpression of myo-inositol oxygenase (MIOX),which is an enzyme important in cellularadaptation to a hypertonic environment, andrenal-specific oxidoreductase (RSOR) (Refs 92,93, 94). Expression of RSOR is remarkablyincreased in parallel to the degree ofhyperglycaemia in mouse models of diabeticnephropathy and during embryonictubulogenesis, and exposure to high glucosehas a differential effect on renal RSORexpression (Refs 94, 95, 96, 97). Further
investigation is warranted of the role of NFATproteins in glucose-mediated MIOX and RSORexpression in models of diabetic kidney disease.
Aldose reductaseThe expression of AKR1B1 is tightly regulated byNFAT5 binding the three OREs (OREA, OREBand OREC), which are also known as thetonicity-response element (TonE) (Refs 70, 80,82). High glucose increases the binding activityof NFAT5 to the OREs of AKR1B1, and thisincrease is significantly higher in patients withtype 1 diabetes and nephropathy than in thosewithout diabetic complications (Ref. 70). InPBMCs and human mesangial cells culturedunder normal conditions, NFAT5 binding toOREA is the highest in patients withuncomplicated type 1 diabetes (Ref. 82). PBMCsand human mesangial cells from those withdiabetic nephropathy cultured in high-glucoseconditions display increased DNA-bindingactivity of NFAT5 to all three OREs, althoughOREB and OREC binding is greater in diabeticnephropathy than controls (Ref. 82). Thus, OREBand OREC may have a greater role incontributing to stress conditions than OREA.Silencing NFAT5 with siRNA reduces high-glucose-stimulated increases in AKR1B1transcription (Ref. 82).
Stimulating protein 1Sp1 belongs to the two-cysteine–two-histidinezinc-finger (Cys2His2 zinc-finger) family ofubiquitous transcription factors that are mainlyinvolved in basal promoter activity and cellcycle regulation (Refs 47, 98). Sp1 exists in avariety of isoforms that bind with varyingaffinities to GC-rich promoters at sequencesdesignated as Sp1 sites (50-GGGGCGGGGC-30)in order to regulate the expression of a widevariety of genes, including many ECM genes(Ref. 98). Flux through the HBP leads to O-linked glycosylation of Sp1, which is crucial forSp1 transcriptional activation (Refs 46, 48). Thisprocess is dynamic, reversible and responsiveto extracellular stimuli (Ref. 48). Sp1 oftencooperates with other transcription factors orproteins such as AP-1 and SMADs in order toregulate transcription (Ref. 47). Sp1 is activatedby glucose and has been implicated in diabeticnephropathy via the upregulation of TGFB1 andSERPINE1 (Refs 37, 47, 48). Binding sites forSp1 have been identified in the promoter regions
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of the genes ADIPOQ (encoding adiponectin),USF2, ICAM1 and RSOR, although the role ofSp1 in glucose-mediated gene expression in thekidney has not been defined (Refs 9, 91, 94, 99).
TGF-b1Sp1 has a high-affinity GC-box binding site on theTGFB1 promoter, and thus has been considered tobe one of the transcription factors involved in thedevelopment of diabetic nephropathy (Refs 37,100). However, in human studies, binding activityof Sp1 does not correlate with urine or PBMClevels of TGF-b1 or with 24 h urinary albuminexcretion in patients with or without diabeticnephropathy, unlike NF-kB and AP-1 (Ref. 46). Inporcine mesangial cells transfected with thehuman TGFB1 promoter, a mutation in the Sp1-binding site decreases the activity of the TGFB1promoter in both normo- and hyperglycaemicmedia, likely due to Sp1-mediated changes onbasal activity as hyperglycaemia still significantlyincreases promoter activity (Ref. 37). Additionally,introduction of mithramycin, an inhibitor of Sp1,does not abolish the high-glucose effect on theTGFB1 promoter (Ref. 37). Mutation of thebinding site for Sp1 in the human TGFB1promoter also does not change angiotensin-II-mediated increases in TGFB1 promoter activity(Ref. 38). Although activation of Sp1 by glucosehas been demonstrated, it does not appear to havea significant effect on glucose-induced TGFB1expression.
PAI-1The human SERPINE1 promoter has two Sp1-binding sites located at 285 to 263 that arerequired for glucose-induced activation inmesangial cells (Refs 47, 48). Disruption at thetwo putative Sp1-binding sites blocks inductionof SERPINE1 promoter activity by glucose- orglucosamine-induced HBP flux (Ref. 47). O-linked glycosylation is crucial for Sp1transcriptional activation, SERPINE1 promoteractivation, and high-glucose-induced SERPINE1gene expression (Ref. 48). Suppression of O-linked glycosylation in mesangial cells preventshigh-glucose-induced increases in PAI-1 mRNAlevels and SERPINE1 promoter activity (Ref. 48).
Clinical implications and applicationsIdentifying the molecular mechanisms by whichdiabetic nephropathy occurs has importanttherapeutic implications, as drug therapies can
then be tailored to target those at risk. Single-nucleotide polymorphisms (SNPs) in the genesencoding certain transcription factors have beenlinked to development of diabetic nephropathy,perhaps by altering the way these transcriptionfactors interact with target genes underhigh-glucose conditions. Recently, a SNP in theSp1-binding site of the ADIPOQ promoterwas found to be associated with diabeticnephropathy among patients with type 1diabetes (Ref. 99). Polymorphisms in the USF1gene have been linked to incident type 2diabetes in some populations, as well asthe metabolic syndrome, which is associatedwith modest fluctuations in blood glucoselevels (Refs 101, 102, 103). Additionally,polymorphisms in the promoter regions ofAKR1B1, CCL2 and SERPINE1 are associatedwith diabetic nephropathy in certainpopulations (Refs 104, 105, 106).
Interfering with transcription factor binding toa promoter region has shown a reduction inprogression of diabetic nephropathy in variousmodels of diabetes, which may have importanttherapeutic implications in the treatment ofpatients with uncontrolled diabetes. Severalstrategies have been employed to interfere withupregulation of target genes by blockingthe action of transcription factors. Syntheticdouble-stranded oligodeoxynucleotides (ODNs)containing a consensus sequence for a specifictranscription factor have been developed thatdownregulate expression of the targeted gene(Ref. 107). Both an AP-1 and Sp1 decoy ODNhave been shown to diminish TGF-b1 and PAI-1 expression, as well as synthesis of other ECMproteins in mesangial cell cultures and animalmodels of diabetes (Refs 51, 98, 100, 107).Pyrrole-imidazole polyamides are smallsynthetic nuclease-resistant compounds thatrecognise and bind specific nucleotidesequences in the minor groove of double-helicalDNA with high affinity and block binding ofspecific proteins, and can be used to targettranscription-factor-binding elements (Ref. 108).In vivo studies using a polyamide targeting theAP-1-binding site of the rat TGFB1 promoterresult in decreased expression of TGF-b1,connective tissue growth factor, collagen typeIa1 and fibronectin mRNA, resulting in areduction of urinary protein and albumin inrats fed a high-salt diet (Ref. 108). Use ofpolyamides and ODNs can be particularly
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helpful as regulatory sequences that bind specifictranscription factors can be targeted while stillmaintaining the baseline expression of thecandidate gene (Ref. 108).
Another therapeutic approach is to inhibitpathways of transcription factor activation. Useof the PKC inhibitor calphostin C reducesNF-kB-mediated increases in CCL2 and ICAM-1 expression in cultured mesangial cells(Refs 68, 77). Diabetic rats treated for threemonths with a p38 MAPK inhibitor displayreduced CREB activation, reduced fibronectinexpression, and decreased apoptosis inglomeruli and mesangial cells (Ref. 53). 1,25-dihydroxyvitamin D3 stabilises the inhibitoryprotein IkB, thereby reducing nucleartranslocation of NF-kB, and blocks high-glucose-induced CCL2 expression in mesangialcells and may reduce hyperglycaemia-inducedrenal injury (Ref. 84). Aldose reductaseinhibitors and curcumin have both been shownto prevent hyperglycaemia-induced NF-kBactivation via suppression of IKK activation,thereby preventing degradation of IkB (Refs 41,60, 70, 109). Curcumin also inhibits AP-1 viadirect interaction with the AP-1-binding motif,and has been shown to ameliorate renal diseasein diabetic rats and block high-glucose-inducedfibronectin mRNA accumulation in culturedendothelial cells (Refs 43, 60, 109). Treatmentwith the antioxidant a-lipoic acid decreasesNF-kB binding activity in PBMCs of patientswith diabetic nephropathy, and the antioxidantspyrrolidine dithiocarbamate (PDTC), N-acteylcysteine (NAC) and trolox abolish NF-kB-mediated increases in CCL2 protein secretion inmurine mesangial cells cultured in high glucose(Refs 68, 75). Pirfenidone, an antifibrotic, is apromising agent being studied in diabeticnephropathy that also likely acts via reducedactivation of NF-kB (Refs 110, 111).
Recent interest in the prevention and treatmentof diabetic nephropathy has also centred onhistone modifications, which are believed to bea core process behind the phenomenon ofmetabolic memory. Resveratrol, a polyphenolfound in red wine, administered tosteptozotocin-induced diabetic rats decreasesplasma creatinine, lowers blood pressure, andprevents activation of p53 anddephosphorylation of histone H3, possiblypreventing cell death (Ref. 112). Resveratrol hasalso been demonstrated to lower albuminuria
and markers of cardiovascular disease in ageingmice fed a high-calorie diet (Ref. 113).Curcumin treatment in diabetic rats also affectspost-translational modifications of histone H3,expression of MAPK p38, and HSP27 (heatshock protein 27) and appears to protect againstdiabetic nephropathy (Ref. 114). Histonedeacetylase inhibitors are currently beinginvestigated as novel agents in the treatment ofdiabetic nephropathy (Ref. 115).
Research in progress and outstandingresearch questions
In the quest for personalised medicine, theidentification of transcription factors that areactivated in response to a hyperglycaemicenvironment may lead to more effectivestrategies for identifying and treating patientsat risk for diabetic nephropathy. Further studiesto delineate clearly the role of eachtranscription factor individually or inco-operation with other transcription factorswill allow for a systems biology understandingof the pathogenesis of diabetic nephropathy.These studies will hopefully lead to targetedtherapies based on transcription factor biologyto arrest nephropathy in the early stages ofdevelopment.
Acknowledgements and fundingThe authors acknowledge the NIDDK for grantsR01DK053867, R01DK63017 and U01DK076133,and the American Diabetes Association. Wethank Dr Noel Weidner (University ofCalifornia San Diego Medical Center, CA, USA)for providing the electron microscopy image ofa normal glomerulus, and the anonymous peerreviewers for their constructive comments onthis article.
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Further reading, resources and contacts
ReferencesBrivanlou, A.H. and Darnell, J.E., Jr (2002) Signal transduction and the control of gene expression. Science 295,
813-818This review article covers the classification of positively acting transcription factors in the human genome,
focusing on transcription factors that receive signals at the cell surface.
Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813-820This review describes the metabolic mechanisms behind hyperglycaemia-induced diabetic complications,
including flux through the polyol pathway, formation of advanced glycation endproducts, activation ofprotein kinase C, and flux through the hexosamine biosynthetic pathway.
(continued on next page)
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Further reading, resources and contacts (continued)
Zhu, Y., Usui, H.K. and Sharma, K. (2007) Regulation of transforming growth factor beta in diabetic nephropathy:implications for treatment. Seminars in Nephrology 27, 153-160
This review discusses the central role of TGF-b1 in diabetic nephropathy, detailing the pathways by whichglucose leads to its upregulation, the transcription factors involved, and rationale behind anti-TGF-btherapies.
Zhu, Y. et al. (2005) Role of upstream stimulatory factors in regulation of renal transforming growth factor-beta1.Diabetes 54, 1976-1984
This paper presents data that USF1 is stimulated by modest increases in glucose concentration in murinemesangial cells and USF2-knockout mice with fasting–refeeding, where USF1 binds to the TGFB1promoter thereby increasing TGF-b1 expression.
WebsiteThe Animal Models of Diabetic Complications Consortium website is a useful resource for investigators in
this field:
http://www.amdcc.org
Features associated with this article
FiguresFigure 1. The pathological changes in the glomerulus with diabetic nephropathy.Figure 2. Metabolic pathways involved in the development of diabetic nephropathy.Figure 3. Mechanistic classification of transcription factors activated in hyperglycaemic environments.
TableTable 1. Characteristics of transcription factors activated by high glucose and potential gene targets in models
of diabetic kidney disease.
Citation details for this article
Amber Paratore Sanchez and Kumar Sharma (2009) Transcription factors in the pathogenesis ofdiabetic nephropathy. Expert Rev. Mol. Med. Vol. 11, e13, April 2009, doi:10.1017/S1462399409001057
expert reviewshttp://www.expertreviews.org/ in molecular medicine
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&2009 Cambridge University Press
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