Fatty Liver and Chronic KidneyDisease: Novel MechanisticInsights and TherapeuticOpportunitiesDiabetes Care 2016;39:1830–1845 | DOI: 10.2337/dc15-1182

Chronic kidney disease (CKD) is a risk factor for end-stage renal disease (ESRD) andcardiovascular disease (CVD). ESRD or CVD develop in a substantial proportion ofpatients with CKD receiving standard-of-care therapy, and mortality in CKD remainsunchanged. These data suggest that key pathogenetic mechanisms underlying CKDprogression go unaffected by current treatments. Growing evidence suggests thatnonalcoholic fatty liver disease (NAFLD) and CKD share common pathogeneticmechanisms and potential therapeutic targets. Common nutritional conditionspredisposing to both NAFLD and CKD include excessive fructose intake and vitamin Ddeficiency. Modulation of nuclear transcription factors regulating key pathways oflipid metabolism, inflammation, and fibrosis, including peroxisome proliferator–activated receptors and farnesoid X receptor, is advancing to stage III clinical devel-opment. The relevance of epigenetic regulation in the pathogenesis of NAFLD andCKD is also emerging, and modulation of microRNA21 is a promising therapeutictarget. Although single antioxidant supplementation has yielded variable results,modulation of key effectors of redox regulation and molecular sensors of intracel-lular energy, nutrient, or oxygen status show promising preclinical results. Otheremerging therapeutic approaches target key mediators of inflammation, such aschemokines; fibrogenesis, such as galectin-3; or gut dysfunction through gut micro-biota manipulation and incretin-based therapies. Furthermore, NAFLD per se affectsCKD through lipoprotein metabolism and hepatokine secretion, and conversely,targeting the renal tubule by sodium–glucose cotransporter 2 inhibitors can improveboth CKD andNAFLD. Implications for the treatment ofNAFLD and CKD are discussedin light of this new therapeutic armamentarium.

EPIDEMIOLOGICAL EVIDENCE LINKING NAFLD AND CKD

Chronic kidney disease (CKD) affects up to 8% of the world’s adult population, withits prevalence increasing in an aging population beset by lifestyle-associated dis-eases such as obesity, the metabolic syndrome, diabetes, hypertension, and smok-ing (1). CKD may progress to end-stage renal disease (ESRD) and is an importantcardiovascular disease (CVD) risk factor. Importantly, most patients with CKD dieas a result of CVD before renal replacement therapy is initiated (1).There is potential for improving recognitionand treatmentofCKD. In theThirdNational

Health and Nutrition Survey, awareness among patients with stage 3 CKD was,8% (1).Furthermore, ESRD or CVD develop in a substantial proportion of patients with CKDreceiving standard-of-care therapy, and all-cause mortality remains unchanged in theCKD population (2). These data suggest that key pathogenetic mechanisms underlying

1Humanitas Gradenigo Hospital, University ofTurin, Turin, Italy2Department of Medical Sciences, San GiovanniBattista Hospital, University of Turin, Turin, Italy3Department of Nephrology, Western & RoyalMelbourne Hospitals, Melbourne, VIC, Australia

Corresponding author: Giovanni Musso, giovanni_musso@yahoo.it.

Received 3 June 2015 and accepted 10 July 2016.

© 2016 by the American Diabetes Association.Readers may use this article as long as the workis properly cited, the use is educational and notfor profit, and the work is not altered. More infor-mation is available at http://www.diabetesjournals.org/content/license.

Giovanni Musso,1 Maurizio Cassader,2

Solomon Cohney,3 Franco De Michieli,2

Silvia Pinach,2 Francesca Saba,2 and

Roberto Gambino2

1830 Diabetes Care Volume 39, October 2016

REV

IEW

renal disease progression are unaffectedby current treatment and prompt thesearch for easily identifiable risk factorsand novel pharmacological targets.The presence and severity of nonalco-

holic fatty liver disease (NAFLD) hasbeen related to the incidence and stageof CKD (3) independently of traditionalCKD risk factors; conversely, the pres-ence of CKD increases overall mortalityin patients with NAFLD compared withthe general population (4). Further sup-porting a pathogenic link between NAFLDand CKD, nonalcoholic steatohepatitis(NASH)–related cirrhosis carries a higherrisk of renal failure than other etiologiesof cirrhosis, is an increasing indication forsimultaneous liver-kidney transplanta-tion, and is an independent risk factorfor kidney graft loss and CVD (5,6). Collec-tively, these data suggest that commonpathogenic mechanisms underlie bothliver and kidney injury and could be tar-geted to retard the progression of bothNAFLD and CKD.

POTENTIAL PATHOGENICMECHANISMS CONTRIBUTING TONAFLD TO CKD AND THERAPEUTICIMPLICATIONS

Nutritional Factors in NAFLD and CKD:Fructose and Vitamin D IntakeDietary intake of fructose, the main con-stituent of sugar sweeteners, increasedtwofold over thepast decade (7). Fructosemay contribute to liver and kidney injurythrough several mechanisms, includinguric acid overproduction, and consis-tently, uric acid–lowering agents haveimproved fructose-induced experimen-tal NAFLD and CKD (8,9) (Table 1). Onthis basis, the impact of xanthine oxidaseinhibitors on CKD progression is beingevaluated in the trials CKD-FIX (ControlledTrial of Kidney Disease Progression Fromthe Inhibitionof XanthineOxidase; clinicaltrial reg. no. ACTRN12611000791932, www.anzctr.org.au) and FEATHER (FebuxostatVersus PlaceboRandomizedControlled TrialRegarding Reduced Renal Function in Pa-tients With Hyperuricemia Complicated

by Chronic Kidney Disease Stage 3; clin-ical trial reg. no. UMIN000008343, www.umin.ac.jp/ctr) (Table 2).

Vitamin D attracted considerable in-terest because of its pleiotropic func-tions, with roles in regulation of cellproliferation, differentiation, immunity,inflammation, fibrogenesis, and metab-olism (Table 1), concurrent with an un-suspected high prevalence of vitamin Ddeficiency, approaching 25% of the gen-eral adult population (37). Vitamin Ddeficiency has been linked to the path-ogenesis and severity of NAFLD and CKDby observational and experimental data(38–40) (Table 1). However, the few trialsof vitamin D supplementation yieldedmixed results, and the benefit of vitaminD supplementation remains uncertain(41). NAFLD and CKD are characterizedby vitamin D resistance, which is partlydetermined by impaired hepatic 25hydroxylation and increased renal tubular25 hydroxyvitamin D loss, and may re-quire higher-dose supplementation of

Table 1—Nutritional factors involved in liver and renal disease in NAFLD and CKD

Biological effect

Dietary fructoseCellular effectsHypothalamus Weight gain↑ Dopaminergic tone→↑ appetite and calorie intake

Hepatocyte, skeletal myocyte Ectopic fat deposition↓ FFA oxidation and REE

Adipocyte Insulin resistanceAdipose tissue dysfunction→↓ adiponectin secretion

Liver and kidney cells (uric acid mediated) InflammationNLRP3 inflammasome activation→↑ IL-1 secretion→↑ macrophage accumulation↓ AMPK activity→ ↓ FFA oxidation FibrosisSREBP-1c activation→↑ de novo lipogenesisNOX activation→ ROS generation→ podocyte loss, tubule cell EMT, endothelial injury Albuminuria

Pancreatic b-cell↓ Postprandial insulin response Hyperglycemia

Vitamin D deficiencyCellular effectsSkeletal myocyte↓ IRS-1 activity→↓ insulin signaling Insulin resistance

Hepatocyte, Kupffer cell↑ TLR-2/4/9 expression→↑ sensitivity to LPS and FFA-induced inflammatory cytokines ↑ ROS production→↑

hepatocyte apoptosis, ↑ inflammatory cell recruitment Ectopic fat depositionHepatic stellate cellVDR downregulation→ ↑ TGF-b secretion and SMAD2 activation→↑ fibrogenesis Inflammation

Glomerular podocyte↑ p38- and ERK-mediated apoptosis→↑ podocyte injury→ glomerular barrier disruption↑ RAS activation→ glomerulosclerosis Hepatic fibrosis

Renal tubule cell and interstitial macrophage↑ SREBP-1c/SREBP-2→↑ toxic cholesterol and FFA lipid accumulation↓ FXR/PPAR-a activation→↑ toxic cholesterol and FFA lipid accumulation Proteinuria↑ NF-kB pathway activation→↑ inflammation and macrophage accumulation

ERK, extracellular signal–regulated kinase; IL, interleukin; IRS-1, insulin receptor substrate 1; NLRP3, NOD-like receptor family, pyrin domaincontaining 3; NOX, NADPH oxidase; RAS, renin-angiotensin system; REE, resting energy expenditure; TLR, Toll-like receptor; VDR, vitamin D receptor.

care.diabetesjournals.org Musso and Associates 1831

Table

2—Mech

anismsofactionanddeve

lopmentalstagesofdru

gstarg

etingboth

NAFL

DandCKD

Developmen

talstage

Mechanism

ofaction

Molecule

NAFLD

CKD

Xanthineoxidaseinhibitors

Allopurinol,febuxostat

Preclin

ical

IIa CKD

-FIX

(clin

icaltrialreg.no.ACTR

N12

6110007919

32)

FEATH

ER(clin

icaltrialreg.no.UMIN00

0008343)

Vitam

inDsupplemen

tation

Natural(ergocalciferol,cholecalciferol,calcitriol),

IIaIIa

SyntheticVDRagonists(doxercalciferol,paricalcitol)

Clinicaltrialreg.no.NCT0209

8317

Clinicaltrialreg.nos.NCT00893451an

dNCT0162

3024

PPAR-d

agonists

GW0742

dPreclin

ical(50)

GW610742

dPreclin

ical(51)

PPAR-a/d

agonists

GFT50

5IIb

GOLD

EN-505(10)

d

PPAR-a/g

agonists

Saroglitazar

IIa(clinicaltrialreg.no.CTR

I/2010/091

/000108)

d

Aleglitazar

dIIb

(54)

SREB

P-2

antago

nists

Naturalantioxidan

ts(m

yricetin)

dPreclin

ical(13)

FXRagonists

Obeticholic

acid

IIaFLINT(17)

Preclin

ical(56,57)

miRNA21antago

nists

Antago

mir-21

Preclin

ical(59)

Preclin

ical(60)

AMPKactivators

Natural:curcumin

berberine,

monascinan

kaflavin

Preclin

ical(19)

Preclin

ical(20)

Synthetic:oltipraz

IIa(clinicaltrialreg.no.NCT0137

3554)

d

HIF-1ainhibitors

YC-1

Preclin

ical(65)

d

DualmTO

RC1/2inhibitor

Rap

amycin

Preclin

ical(24)

Preclin

ical(25)

ASK1inhibitor

GS-49

97IIa

(clinicaltrialreg.no.NCT0246

6516)

IIa(clinicaltrialreg.no.NCT0217

7786)

Nrf2activators

Oltipraz

IIa

Bardoxolonemethyl

Clinicaltrialreg.no.NCT0137

3554

Clinicaltrialreg.no.NCT0231

6821

CCR2receptorantagonist

CCX14

0-B

dIIa

(82)

CCR2/5receptorantago

nist

Cen

icriviroc

IIa:C

ENTA

UR(clin

icaltrialreg.no.N

CT02217475)

BMS-813160

IIa

PF-0463481

7d

Clinicaltrialreg.nos.NCT01752985an

dNCT0171

2061

Galectin-3

inhibitors

GM-CT-01

Preclin

ical(84)

d

GR-M

D-02

PhaseI(clinicaltrialreg.no.NCT0189

9859)

d

GCS-10

0d

IIa(clinicaltrialreg.no.NCT0184

3790)

Incretin-based

therap

ies

Incretin

mim

etics

Liraglutide,

exen

atide

IIaLEAN(92)

IIa(93,94)

DPP

-4inhibitors

Saxagliptin,lin

agliptin

Preclin

ical(90)

IIa(M

ARLINA-T2D

,CARMELINA)

Gutmicrobiota

man

ipulation

Preb

iotics,p

robiotics,synbiotics

IIa(106)

IIa(clinicaltrialreg.no.ACTR

N126130

00493741

)

CETPinhibitors

Fc-CETP6

Preclin

ical(112)

d

Inhibitors

ofsyndecan

-1shed

ding

Sphingo

sine-1-phosphate

dPreclin

ical(119

)

FGF21analogs

PEG-FGF21,recombinantFG

F21,anti-FGFR

1mAb

IIa(123)

d

SGLT2inhibitors

Rem

ogliflozinetab

onate,

luseogliflozin,ipragliflozin

IIa(131,132

)d

Dap

agliflozin,can

agliflozin,e

mpagliflozin

dIIa

(127,CRED

ENCEtrial)

FGFR

,FGF21receptor;mAb,m

onoclonalantibodies;PEG,p

egylated

;VDR,vitamin

Dreceptor;YC

-1,1

-ben

zyl-3-(substitutedaryl)-5-methylfuro[3,2-c]pyrazole.

1832 NAFLD and Chronic Kidney Disease Diabetes Care Volume 39, October 2016

vitamin D, calcitriol, or vitamin D receptoragonists (e.g., paricalcitol) to be overcome(42,43). The effects of vitamin D supple-mentation inCKDandNASHarebeing eval-uated in several clinical trials (clinical trialreg. nos. NCT00893451, NCT01623024,and NCT02098317, www.clinicaltrials.gov).

Reversing Ectopic Fat Deposition byTargeting Nuclear TranscriptionFactors in NAFLD and CKDNAFLD and CKD are characterized by ec-topic toxic lipid accumulation, which is de-termined by an extensive derangement inhepatic and renal lipid metabolism and

triggers lipoperoxidative stress, cell apo-ptosis, inflammation, and fibrosis (44–46)(Fig. 1). Underlying these abnormalitiesis an extensive deregulation of nucleartranscription factors that regulate lipidmetabolism, inflammation, and fibrogen-esis, including peroxisome proliferator–activated receptor (PPAR)-a, PPAR-d,and PPAR-g; SREBP-2; and farnesoid Xreceptor (FXR),which represent an attrac-tive target for the treatment of NAFLDand CKD (47,48) (Fig. 1).

On the basis of the finding that PPAR-aand PPAR-d are downregulated in NAFLDand CKD (47,49), potent, selective PPAR-a,

PPAR-d, and dual PPAR-a/d agonists(K-877, GW501516, MBX-8025 and GFT505,respectively) were evaluated in thesetwo conditions, with encouraging resultsin preclinical models (49–51). Some ofthese compounds advanced to the clini-cal stage of development, and GFT505, adual PPAR-a/d agonist, improved steato-hepatitis, fibrosis, and the glycolipid profilein the recently completed GOLDEN-505trial (10) (Table 2).

The PPAR-g agonists thiazolidine-diones comprise another pharmacolog-ical class that has significantly improvedNASH and albuminuria in clinical trials(11,12), but their clinical use was lim-ited by their side effects. These draw-backs prompted the development ofnew compounds, including dual PPAR-a/gagonists, which maintained the thera-peutic effectiveness of PPAR-g agonistsbut were devoid of their unwantedeffects (Fig. 1). Saroglitazar, a potentPPAR-a/g agonist, did not induce weightgain, peripheral edema, or other adverseevents after 1 year (52) and improvedmarkers of NAFLD in patients with dia-betes (53), whereas aleglitazar slowedestimated glomerular filtration rate(eGFR) decline in diabetic nephropathy(54) (Table 2). A small, phase IIa trial ofpatients with biopsy-proven NASH hasbeen completed, but the results arenot yet available (clinical trial reg. no.CTRI/2010/091/000108, www.ctri.nic.in). Larger and longer randomized clini-cal trials (RCTs) are needed to evaluatelong-term clinical safety and effective-ness of these compounds in patientswith and without diabetes.

Among various lipotoxic species accu-mulating in NAFLD and CKD, free choles-terol is believed to play a key pathogenicrole in liver and renal injury (13,55). Ec-topic cholesterol accumulation is drivenby an inappropriate upregulation oftranscription factor SREBP-2, with conse-quently increased cholesterol synthesis,influx, and retention and reduced cho-lesterol excretion by liver and renal cells(13,55) (Fig. 1). Such pervasive deregula-tion in all steps of cholesterol metabo-lism may diminish the effectiveness ofavailable cholesterol-lowering drugsthat target single steps in cholesterolmetabolism (14). Therefore, modulationof SREBP-2 activity represents an attrac-tive therapeutic tool. Although selectiveSREBP-2 antagonists are under develop-ment, several natural antioxidants, such

Figure 1—The role of nuclear transcription factors in the pathogenesis of NAFLD and CKD. Eachnuclear transcription factor is reported in the text box at the center of the scheme and is markedwith a superscript number. The various molecular pathways affected by each nuclear transcrip-tion factor are shown in the boxes surrounding the central box, with the superscript numberreferring to the corresponding nuclear transcription factor affecting the pathway. eNOS, endo-thelial nitric oxide synthase; LPL, lipoprotein lipase; NLRP3, NOD-like receptor family, pyrin domaincontaining 3; NOS, nitric oxide synthase; NOX, NADPH oxidase; PAI-1, plasminogen activator in-hibitor 1.

care.diabetesjournals.org Musso and Associates 1833

asmyricetin, repress SREBP-2 expressionand ameliorate cholesterol-induced in-flammation and fibrosis in experimentalmodels (13) (Table 2).FXR is a nuclear transcription factor

with prominent insulin sensitizing, antilipo-genic, anti-inflammatory, and antifibroticproperties; furthermore, its activationimproves endothelial function (16,56,57)(Fig. 1). FXR expression is downregulatedin the liver and kidney of patients withNAFLD and CKD, respectively, and is in-versely related to disease severity. Onthis basis, potent semisynthetic bileacid FXR agonists have been developed(Table 2). Obeticholic acid (or INT-747), asemisynthetic chenodeoxycholic acidderivative, improved liver histology in thephase IIa, multicenter, randomized FXRLigand NASH Treatment (FLINT) trial andin ameliorated renal histology and protein-uria in nutritional models of CKD (16,57).Several issues remain, however, includingthe effectiveness of FXR agonists in sub-jects without diabetes and the impact ofHDL cholesterol reduction on long-termCVD risk.

Epigenetic Regulation in NASHand CKDmicroRNAs (miRNAs) are small (;22 basepairs) endogenous noncoding RNAs thatregulate gene expression of at least 60%ofprotein coding genes.miRNAs recognizemRNA targets through sequence comple-mentarity between themiRNAandbindingsites in the 39 untranslated regions of thetarget mRNAs or through interactionwith RNA-binding proteins.miRNAs regulate geneexpression in one

of two ways, depending on the degree ofcomplementarity between themiRNA andits target. miRNAs that bind to mRNAtargets with imperfect complementarityblock target gene expression throughtranslational silencing while miRNAsbinding their mRNA targets with perfectcomplementarity enhance target geneexpression (15,58). To date, .2,500miRNAshavebeen identified in the humangenome, and each can regulate severalhundred target genes involved in diversedevelopmental and cellular processes, in-cluding cellular metabolism proliferation,differentiation, and apoptosis. SeveralmiRNAs have been found to be dysregu-lated in NAFLD and CKD (15,58). On thisbasis, approaches inhibitingoverexpressedmiRNAs by antisense oligonucleotides orrestoring the expression of downregulated

miRNAs by synthetic miRNA mimics havebeen attempted. miRNA21 in particular isan attractive target because it regulateskey metabolic, proinflammatory, and pro-fibrogenic pathways and its hepatic andrenal overexpression in NASH and CKDleads to PPAR-a downregulation, SREBP-2upregulation, mitochondrial dysfunction,and profibrogenic hepatic stellate cell acti-vation and proximal tubular cell epithelial-to-mesenchymal transition (EMT) (59,60).Consistently, in experimental models ofNASH and CKD, anti-miRNA21 antisense ol-igonucleotides induced weight loss, nor-malized metabolic dysregulation, andimproved hepatic and renal inflammationand fibrosis, effects at least partlymediatedby PPAR-a upregulation (59,60). Despitethese premises, several issues remain,including the stability and selective deliv-ery of the pharmacological modulators tothe target organs and long-term safety ofthis approach because miRNAs also

regulate cell proliferation and cell cycleprogression in diverse tissues and long-term consequences of its modulation ontumor onset and progression are unclear(15,59).

ROLE OF CELLULAR ENERGY,OXYGEN, AND NUTRIENT SENSORS

In mammals, cellular metabolism is finelyorchestrated by molecular sensors of en-ergy, nutrient, and oxygen status to adaptto changing substrate availability. Thedysregulation of some of these sensors,including AMPK, hypoxia-inducible factor(HIF)-1a, and mammalian target of rapa-mycin (mTOR), has been implicated in thepathogenesis of NAFLD and CKD andcould be targeted for the treatment ofNAFLD and CKD. AMPK is a ubiquitouskinase that preserves cell survival undercalorie restriction or high energy de-mand (18). In response to cellular ATPdepletion or to an increase in AMP/ATP

Figure 2—Mechanisms connecting cellular sensors AMPK (A) and HIF-1a and mTORC1 (B) in thepathogenesis of NAFLD and CKD. HIF-1a and mTORC1 are reported in the text box at the centerof the scheme and are marked with a superscript number. The various molecular pathwaysaffected by each sensor are shown in the boxes surrounding the central box, with the superscriptnumber referring to the corresponding cellular sensor affecting the pathway. eNOS, endothelialnitric oxide synthase; LOXL, lysyl oxidase-like; NOX, NADPH oxidase; PAI-1, plasminogen activa-tor inhibitor 1; PGC-1a, peroxisome proliferator–activated receptor g coactivator 1a.

1834 NAFLD and Chronic Kidney Disease Diabetes Care Volume 39, October 2016

ratio, AMPK activation enhances sub-strate oxidation and has antioxidant,anti-inflammatory, and antifibrotic ef-fects (18,20,61) (Fig. 2A). On this basis,several natural AMPK activators were as-sessed in preclinical models of NASH andCKD, with encouraging results (18,20,61),and the synthetic AMPK activator oltiprazis being evaluated in patients with non-cirrhotic NAFLD (clinical trials reg. no.NCT01373554, www.clinicaltrials.gov)(Table 2).HIF-1a is a ubiquitous oxygen-sensitive

protein that regulates transcription ofgenes involved in metabolic adaptation,energy conservation, angiogenesis, andcell survival in response to cellular hyp-oxia and nonhypoxic stimuli like choles-terol overload (21). Inappropriate HIF-1aactivation following such stimuli aschronic intermittent hypoxia and choles-terol overload has been found to be in-volved in NASH pathogenesis (22,62),whereas chronic hypoxia has been impli-cated in renal injury in early diabetic andobesity-related CKD (63,64) (Fig. 2B). Onthis basis, small-molecule synthetic HIF-1a inhibitors, including YC-1, have beendeveloped and show potent antifibrotic

properties in experimental models ofNASH (65) (Table 2). Several issues re-main, however. In some studies, HIF-1aactivation protected against kidney in-jury (66), suggesting that HIF-1a maynot be the sole, or the best, therapeutictarget for reversing cellular effects ofhypoxia in CKD.

mTOR is a serine/threonine kinasethat in response to changes in cellularnutrient levels, growth factors like insulinand IGF and other stressors associateswith companion proteins to form twodistinct signaling molecular complexes,mTOR complex 1 (mTORC1) and mTORC2(23). mTORC1 has been more extensivelystudied and found to promote cellularanabolism by stimulating synthesis ofprotein, lipid, and nucleotides and block-ing catabolic processes. InappropriatemTORC1 activation in NAFLD and CKDinhibits autophagy (23,67) and promotesinsulin resistance, ectopic lipid accumu-lation, lipotoxicity, and proinflammatorymonocyte recruitment in the liver andkidney (Fig. 2B) (24,25). Consistently,mTORC1 inhibition has reversedmetabolicabnormalities and attenuated lipid accu-mulation, inflammation, and fibrosis in

diverse models of NASH and CKD (68,69)and may represent a therapeutic optionfor NAFLD and CKD (70) (Table 2).

TARGETING REDOX REGULATIONIN THE PATHOGENESIS OF NAFLDAND CKD

Although increased oxidative stress isbelieved to play a central role in NASHand CKD progression, single antioxidantsupplementation strategies have yieldedvariable results (11), and other ap-proaches targeting common effectorsof redox regulation, like apoptosis signal-regulating kinase 1 (ASK1) and nuclearerythroid 2–related factor 2 (Nrf2), arebeing investigated. ASK1 is a serine/threonine kinase belonging to themitogen-activated protein kinase (MAPK)kinase kinase family, which is activatedin response to stresses like reactive oxy-gen species (ROS), tumor necrosis factor-a (TNF-a), lipopolysaccharide (LPS), andendoplasmic reticulum (ER) stress (71).ASK1 activates downstream terminalMAPK kinases p38 and c-Jun N-terminalkinase (JNK), which promote insulinresistance, cell death, proinflammatorycytokine/chemokine production, and fi-brogenesis (71) (Fig. 3).

Data have implicated ASK1 activationin oxidative stress–induced inflamma-tion and fibrogenesis in NASH and CKD,and pharmacological ASK1 inhibitionprevented diet-induced NASH andhalted progression of diabetic and non-diabetic experimental CKD (72,73). Onthis basis, the highly selective oralASK1 inhibitor GS-4997 is being evalu-ated in patients with NASH with moder-ate advanced fibrosis (clinical trial reg. no.NCT02466516, www.clinicaltrials.gov)and in patients with diabetes with stage3/4 nephropathy (clinical trial reg. no.NCT02177786, www.clinicaltrials.gov)(Table 2). Several issues need to be clari-fied, however. ASK1 inhibition did not im-prove podocyte loss and albuminuria inexperimental diabetic CKD, suggestingthat this kinase is not central for glomer-ular injury (73). Furthermore, the impactof ASK1 inhibitors in nondiabetic CKD isunknown.

Nrf2 is a transcription factor expressedubiquitously in human tissues and mostabundantly in the liver (74), where itregulates the expression of several anti-oxidant and detoxifying enzymes and hasdirect metabolic, anti-inflammatory, andproautophagic actions (74) (Fig. 3).

Figure 2—Continued.

care.diabetesjournals.org Musso and Associates 1835

Under basal conditions, Nrf2 is kept tran-scriptionally inactive through binding toits inhibitor, Kelch-like ECH-associatedprotein 1 (KEAP1) (74). ROS and reactivenitrogen species interact with KEAP1 andcause the loosening of Nrf-2, whichtranslocates to the nucleus and modu-lates transcription of its target genes(Fig. 3). The relevance of Nrf2 in thepathogenesis of liver and kidney injuryhas emerged in diverse diet-inducedmodels of NASH and CKD, where diseaseprogression was accelerated by Nrf2 de-letion and prevented by Nrf2 activators

(75,76). On this basis, several natural andsynthetic small-molecule Nrf2 activatorsare currently being evaluated in patientswith NASH and CKD (Table 2). Followingearly encouraging data, clinical develop-ment of bardoxolone methyl, a syntheticNrf2 activator, was interrupted for safetyconcerns related to heart failure (77). Areanalysis of the potential risk/benefit ra-tio of the drug for a Japanese populationand the observation that most of the se-vere adverse effects occurred in the firstmonthof therapyprompted initiationof adose-escalating RCT in Japan (clinical trial

reg. no. NCT02316821, www.clinicaltrials.gov). Another potent synthetic Nrf2 acti-vator, oltipraz, is being evaluated in pa-tients with NAFLD (clinical trial reg. no.NCT01373554, www.clinicaltrials.gov).

TARGETING MOLECULAREFFECTORS OF INFLAMMATIONAND FIBROSIS

Chemokines are small proteins that reg-ulate leukocyte migration into tissuesand consequent inflammation, tissueremodeling, and fibrosis (78). Amongthe.40 chemokine ligands and 20 che-mokine receptors currently identified,chemokine (C-C motif) ligand 2 (CCL2or MCP-1) and its receptor CCR2 havebeen implicated in the pathogenesis ofNASH and CKD. In NAFLD, hepatic cellsand adipocytes secrete CCL2, which at-tracts proinflammatory cells to the liverto promote NASH development (79,80),whereas genetic or pharmacological in-hibition of the CCL2/CCR2 axis reversessteatohepatitis and advanced hepaticfibrosis (80). In the kidney, tubule cellsand podocytes secrete chemokinesCCL2 and CCL5 in response to diverseproinflammatory stimuli to promotetubulointerstitial inflammation andfibrosis, which are reversed by chemokineantagonists (81). On this basis, chemokineantagonists are advancing to the clini-cal stage of development: The small-molecule CCR2 antagonist CCX140-Bhas reduced albuminuria and slowedeGFR decline in diabetic nephropathy(82), whereas the dual chemokine recep-tor CCR2/CCR5 antagonists BMS-813160,PF-04634817, and cenicriviroc are be-ing evaluated in diabetic nephropathy(clinical trial reg. nos. NCT01752985and NCT01712061, www.clinicaltrials.gov) and in NASH (clinical trial reg. no.NCT02217475, www.clinicaltrials.gov)(Table 2).

Galectin-3 is a lectin broadly expressedby immune and epithelial cells where itlocalizes mainly in the cytoplasm as wellas in the nucleus, cell surface, and extra-cellular space (83). Galectin-3 regulatescell proliferation, apoptosis, cell adhe-sion, and affinity for advanced glycationend products (AGEs), exerting multipleand sometimes contrasting effects, de-pending on its cellular location, cell type,and mechanisms of injury (83) (Fig. 3).Galectin-3 is upregulated in the liver andkidney of patients with NASH and CKDand correlates with the severity of liver

Figure 3—Role of effectors of redox regulation ASK1 and Nrf2 and role of galectin-3 in the path-ogenesis of NAFLD and CKD. ASK1, Nrf2, and galectin-3 are reported in the text box at the center ofthe scheme and aremarkedwith a superscript number. The variousmolecular pathways affected bythese three molecules are shown in the boxes surrounding the central box, with the superscriptnumber referring to the corresponding factor affecting the pathway. COX, cyclooxygenase; eNOS,endothelial nitric oxide synthase; ERK, extracellular signal–related kinase; G6PD, glucose-6-phosphatedehydrogenase; Glt-Px, glutathione peroxidase; Glt-R, glutathione reductase; GST, glutathioneS-transferase; HPC, hepatic progenitor cells; iNOS, inducible nitric oxide synthase; IRS, insulin re-ceptor substrate; NKT, natural killer T; NOS, nitric oxide synthase; PAI-1, plasminogen activatorinhibitor 1; TXN-R, thioredoxin reductase.

1836 NAFLD and Chronic Kidney Disease Diabetes Care Volume 39, October 2016

and renal disease (26,84). Furthermore,circulating galectin-3 levels predict renalfunction decline and cardiovascular andall-cause mortality in patients with CKD(85). Consistent with epidemiologicaldata, functional galectin-3 manipulationhas shown important proinflammatoryand profibrotic effects of this lectin(26,83,84). On this basis, pharmacologicalgalectin-3 inhibition with small-moleculecompetitive inhibitors, including GR-MD-02(galactoarabino-rhamnogalaturonan),GM-CT-01 (galactomannan), and N-acetyllactosamine, prevents hyperten-sive nephropathy (86) and reversesdiet- induced NASH and cirrhosis (87).GR-MD-02 was well tolerated and im-proved markers of hepatic fibrosis in aphase I RCT enrolling patients with NASHand advanced fibrosis (clinical trial reg. no.NCT01899859, www.clinicaltrials.gov),whereas a phase IIa RCT is exploring theeffect of the galectin-3 antagonist GCS-100 on eGFR in CKD (clinical trial reg. no.NCT01843790, www.clinicaltrials.gov)(Table 2).Data on galectin-3 inhibition are not

univocal, however, and galectin-3 de-letion exacerbates systemic inflam-mation, hyperglycemia, and liver andkidney injury in diet-induced obeserodents (88). It has been suggested thatinhibition of AGE uptake by the liver,which clears.90% of these end productsfrom the circulation, promotes the sys-temic accumulation of AGEs and receptorfor AGE (RAGE)–mediated uptake by othertissues, thereby aggravating extrahepatictoxicity of these molecules. Becauseboth NASH and CKD are characterized byAGE accumulation, a better understandingof the impact of galectin-3 inhibitorson AGE-mediated tissue injury in vivo iswarranted.

THE GUT CONNECTION:TARGETING INCRETINS AND GUTMICROBIOTA FOR THETREATMENT OF NAFLD AND CKD

Incretin-based therapies, including GLP-1mimetics and dipeptidyl peptidase 4(DPP-4) inhibitors, increase insulin re-lease from the pancreas, reduce gluca-gon production, and possess numerousextrapancreatic metabolic benefits,which prompted the evaluation for thetreatment of NAFLD and CKD (89,90)(Table 3). Preliminary human data sug-gested that incretin mimetics improveNAFLD. A meta-analysis of six RCTs

from the Liraglutide Effect and Actionin Diabetes (LEAD) program found a sig-nificant improvement in biochemicaland radiological features of steatosis(91). Furthermore, in the Liraglutide Ef-ficacy and Action in NASH (LEAN) trial,liraglutide 1.8 mg/day for 48 weeks in-duced NASH resolution and improvedmarkers of lipotoxicity, inflammation,and metabolic dysfunction comparedwith placebo (92) (Table 2).

Incretin-based therapies also havethe potential for nephroprotection in-dependent of improved glycemic controlthrough several mechanisms (Table 3).GLP-1 administration induced natriure-sis through inhibition of proximal tu-bular Na-H exchanger 3 (NHE3) andreduced activation of the angiotensinII (AngII) axis (93). These actions coun-teract the increase in proximal tubularsodium reabsorption, which has beenhypothesized to trigger glomerular hyper-filtration, the functional defect believedto be central to obesity-associated anddiabetic CKD (93). Furthermore, GLP-1 mi-metics have demonstrated direct renalanti-inflammatory, antifibrotic, and anti-oxidative actions (27,94). Collectively,these properties may explain the attenu-ated progression of overt diabetic ne-phropathy in two small preliminary RCTsof treatment with liraglutide (27,94). Inaddition to inactivating GLP-1, DPP-4cleavesmultiple other peptides, includingbrain-derived natriuretic peptide, neuro-peptide Y, peptide YY, and stromal-derived factor 1a (Table 3). Thus, the effectof DPP-4 inhibition depends on the actionsof the various substrates inactivated ineach tissue and organ. This may theoreti-cally explain the slightly lower antihyper-tensive effect observed with DPP-4inhibitors compared with GLP-1 mimetics(28). Although the impact of DPP-4 inhib-itors on NAFLD must be assessed, resultsfrom the Saxagliptin Assessment of Vascu-lar Outcomes Recorded in Patients withDiabetes Mellitus-Thrombolysis in Myo-cardial Infarction (SAVOR-TIMI) 53 trial(29) and from four other RCTs (95) havesuggested that saxagliptin and linagliptinreduce the development and progres-sion of albuminuria. Renal effects oflinagliptin are currently being investi-gated in the MARLINA-T2D (Efficacy,Safety, & Modification of Albuminuriain Type 2 Diabetes Subjects With RenalDiseaseWith Linagliptin; clinical trial reg.no. NCT01792518, www.clinicaltrials

.gov) and CARMELINA (Cardiovascularand Renal Microvascular Outcome StudyWith Linagliptin in Patients With Type 2Diabetes Mellitus; clinical trial reg. no.NCT01897532, www.clinicaltrials.gov)trials. In conclusion, incretin-based ther-apies address several pathophysiologicalmechanisms common to experimentalNAFLD and CKD, but their impact on re-nal disease in nondiabetic CKD is un-known. Furthermore, incretin-basedtherapies did not affect the risk of CVD,a major cause of death in NAFLD andCKD (96).

The capacity for gut microbiota to in-teract with host metabolic and immuneresponses and to contribute to the de-velopment of obesity-associated disor-ders has been increasingly recognized(97). PatientswithNAFLD andCKD exhibitan altered gut microbiota composition,with a relative decrease in healthy Bac-teroidetes, Lactobacillaceae, and Prevo-tellaceae families and disruption of thenormal gastrointestinal barrier (98,99).The resulting accumulation of gut-derived toxins induces inflammation(100), insulin resistance, and ectopic fatdeposition in liver and muscle throughseveral mechanisms (Table 3). Some ofthese molecules, including endotoxin,indoxylsulfate, p-cresyl sulfate, and trime-thylamine-N-oxide (TMAO), have docu-mented clinical relevance for thedevelopment and progression of CKD(101–104).

CKD may also aggravate gut dysbiosisand systemic inflammation through ac-cumulation of uremic toxic metabolites(URMs) normally eliminated by the kid-neys, including urea and p-cresyl sulfate.The accumulation of urea may lead toinflux into the gastrointestinal lumenwhere it is hydrolyzed to ammonia bymicrobial urease and then convertedto ammonium hydroxide. Ammonia andammonium hydroxide promote thegrowth of urea-metabolizing bacteria atthe expense of carbohydrate-fermentingstrains and disrupt intestinal epithelialtight junctions, enhancing passage ofLPS and other toxic luminal compoundsinto the circulation (105). Further high-lighting the relevance of this mechanismto systemic inflammation, administra-tion of oral activated charcoal absorbentAST-120 improves intestinal barrierfunction and reduces systemic oxidativestress, inflammation, and endotoxemiain rodent models of CKD (105). Gut

care.diabetesjournals.org Musso and Associates 1837

Table 3—Role of incretin-based therapies and gut microbiota in the pathogenesis of NAFLD and CKD

Biological effect

Incretin analogs: GLP-1 receptor agonistsCellular mechanismHypothalamus ↓ Calorie intake

↓ AppetiteSkeletal myocyte ↑ Insulin sensitivity

↑ Glucose uptake↑ Glycogen synthesis

Hepatocyte ↑ Insulin sensitivity↑ Glycogen synthesis and ↓ gluconeogenesis↑ Autophagy ↓ Hepatic steatosis↑ cAMP→↑AMPK and SIRT-1 activity↓ FGF21 secretion

Adipocyte ↑ Insulin sensitivity↑ Lipolysis↑ Glucose uptake ↓ Adipose tissue↑ Lipogenesis (↑ FFA synthesis, uptake, and reesterification)↑ LPL activity Dysfunction↑ Adiponectin secretion

Proximal tubule cell ↑ Na delivery to distal tubule→↓ NHE3 activity→↑ Na uresis→↓ Na hyperreabsorption ↓ Tubuloglomerular feedback→↑ PPAR-a→↑ FFA oxidation ↓ Glomerular hyperfiltration

Glomerular endothelial cell, mesangial cell, tubule cell ↓ Blood pressure↓ Apoptosis ↓ Fat infiltration↓ AGE/RAGE axis activation ↓ Glomerular sclerosis↓ IL-1/MCP-1/TGF-b secretion→↓ monocyte recruitment and fibrogenesis ↓ Inflammation and fibrosis↑ NOS activity ↓ Endothelial dysfunction↑ cAMP→ NOX downregulation→↓ ROS generation ↑ Insulin sensitivity↓ AngII activity→↓ IRS-1 phosphorylation→↑ IRS-1 signaling

Incretin analogs: DPP-4 inhibitorsLiver, kidney↑ GLP-1 activity

Kidney Same effects of GLP-1R agonists↑ BNP→↑ natriuresis and vasodilation, ↓ RAAS and sympathetic activity↑ NPY and PYY→↑ AngII-mediated vasoconstriction↑ SDF-1a→↑ mesenchymal stem cells recruitment Ultimate effect depends on local tissue activity of

various substrates inactivated by DPP-4

Altered gut microbiota composition and intestinal barrier disruptionCellular mechanismHepatocyte, macrophage, HSC, renal vascular endothelial cell, podocytes

↑ LPS-TLR-4 axis activation→ ↑ proinflammatory cytokines/TGF-b Hepatic and renal inflammation, hepatic and renalfibrogenesis

↑ LPS-TLR-4 axis activation→ ↑ NOX type II activity→ ROS production Endothelial dysfunctionEnterocyte Gut barrier disruption

↓ SCFA production→↑ epithelial injury and ↓ GLP-1 secretionHepatocyte

↑ Conversion of intestinal TMA to TMAO by flavin-containing monooxygenases Hepatic steatosisHepatic necroinflammation

Tubule cells Atherosclerosis↑ TMAO-induced TGF-b/SMAD3 axis activation Tubule-interstitial fibrosis

Skeletal myocyte Insulin resistance↑ URM-induced phosphorylation of IRS-1→↓ IRS-1 signaling

Hepatocyte Insulin resistance↑ URM-induced de novo lipogenesis Hepatic steatosis↓ URM-induced VLDL secretion

Adipocyte↑ URM-induced leptin secretion Insulin resistance↓ URM-induced de novo lipogenesis Adipose tissue dysfunction↑ URM-induced zinc-a2-glycoprotein and ↓ perilipin expression→↑ lipolysis

Macrophage Adipose tissue and systemic inflammation↑ LPS-induced NF-kB activation

BNP, brain-derived natriuretic peptide; HSC, hepatic stellate cell; IL, interleukin; IRS-1, insulin receptor substrate 1; LPL, lipoprotein lipase; NOS,nitric oxide synthase; NOX, NADPH oxidase; NPY, neuropeptide Y; PYY, peptide YY; SCFA, short-chain fatty acid; SDF-1a, stromal-derived factor 1a;SIRT, sirtuin; TLR, Toll-like receptor; TMA, trimethylamine.

1838 NAFLD and Chronic Kidney Disease Diabetes Care Volume 39, October 2016

microbiota manipulation with probioticsor prebiotics improved surrogate mark-ers of NAFLD in small RCTs of short du-ration (106) and reduced URM levels inpatients with CKD (107). The impactof synbiotic administration on renalfunction in CKD is being investigatedin the Synbiotics Easing Renal Failureby Improving Gut Microbiology (SYN-ERGY) trial (clinical trial reg. no.ACTRN12613000493741, www.anzctr.org.au).

NAFLD AS A DETERMINANT OFCKD: TARGETING THE LIVER TOIMPROVE CKD

In NAFLD, liver disease per se contrib-utes to kidney injury through severalmechanisms. The liver contains up to80% of all macrophages of the body,and the steatotic liver may represent amore relevant source of proinflamma-tory cytokines than adipose tissue (30).Furthermore, the liver is a central regu-lator of lipoprotein metabolism and se-cretes hepatokines like fibroblastgrowth factor 21 (FGF21), which canmodulate whole-body metabolism andinflammation.

Hepatic Secretion of VLDL,Cholesteryl Ester Transfer Protein,and Syndecan-1 in the Pathogenesisof Atherogenic Dyslipidemia andKidney InjuryAtherogenic dyslipidemia is the mostcommon lipid abnormality in CKD andan independent predictor of the inci-dence and progression of CKD and ofCVD in patients with CKD (108,109). Ath-erogenic dyslipidemia promotes CKDthrough receptor-mediated uptake ofqualitatively abnormal lipoproteins byglomerular and tubulointerstitial cells(110). NAFLD may promote atherogenicdyslipidemia through several mecha-nisms, which represent potential thera-peutic targets. Liver fat accumulationper se proportionally increases the he-patic secretion rate of large VLDL1,which exchanges triglycerides with cho-lesterol contained in circulating LDL andHDL particles, resulting in small LDL andHDL3 formation (55). Furthermore, re-cent studies demonstrated that circulat-ing cholesteryl ester transfer protein(CETP) derives largely from hepaticKupffer cells, and its levels parallel theseverity of histological necroinflammationsin NASH (111). Of note, CETP inhibitors

alleviate high-fat diet–induced steatohepa-titis and fibrosis, possibly by reducingoxidized LDL uptake by hepatic Kupfferand stellate cells (112), and represent apotential therapeutic target for thetreatment of both NAFLD and CKD(Table 2).

Syndecan-1 is another key mediatorof hepatic clearance of triglyceride-richlipoproteins (TRLPs) (113). It is a trans-membrane heparan sulfate proteogly-can constitutively bound to hepatocytemembrane, where it binds lipoproteinlipase and apolipoprotein (apo) E throughits heparan sulfate chains and internalizesapoE-containing lipoproteins (31). Sulfa-tion by hepatic sulfotransferases andbinding to hepatocyte membrane are re-quired for syndecan-1 biological activity.NAFLD is characterized by an increasedshedding of syndecan-1 (114), as a resultof increased hepatic metalloproteinaseactivity, and by a defective syndecan-1sulfation, as a result of defective hepaticsulfotransferase activity (115,116). Thesechanges in hepatic syndecan-1 metabo-lism impair TRLP clearance and, accord-ingly, predicted atherogenic dyslipidemiain CKD (117). Beside mediating hepaticTRLP clearance, syndecan-1 is a key con-stituent of the endothelial glycocalyxlayer, and its shedding has been associ-ated with loss of endothelial barrier integ-rity and endothelial dysfunction acrossprogressive CKD stages (118). Inhibitorsof syndecan-1 shedding, including thephospholipid sphingosine-1-phosphate,restored endothelial integrity experi-mentally (119) and may represent a po-tential therapeutic tool for the treatmentof CKD.

FGF21FGFs are signaling proteins that regulateembryonic development, tissue regen-eration, and diverse metabolic functionsby binding extracellularly to four cellsurface tyrosine kinase FGF receptors(FGFRs 1–4) (120). FGF21 is mainly se-creted by the liver and exerts its multi-ple beneficial metabolic effects bybinding to FGFRs in the presence ofcoreceptor b-Klotho (120). FGF21 ad-ministration ameliorates adipose andhepatic insulin sensitivity, suppresseshepatic gluconeogenesis and lipogenesis,and enhances free fatty acid (FFA) oxida-tion and mitochondrial function, at leastin part by activating the AMPK-SIRT1-PGC-1a pathway (32,120). Furthermore,

FGF21 has recently been demonstratedto direct anti-inflammatory and antifi-brogenic activity by inhibiting the keynuclear factor kB (NF-kB) and transform-ing growth factor-b (TGF-b)/mothersagainst decapentaplegic homolog (SMAD)2/3 signaling pathways (121). By virtue ofthese properties, FGF21 administrationhas improved experimental NASH andCKD (32,120,121). The clinical develop-ment of FGF21, however, is hamperedby its short half-life (0.5–5h) and by tissueFGF21 resistance, which is subtended byFGFR1 and b-Klotho downregulation(122) and is overcome by pharmacolog-ical doses of FGF21. To this aim, engi-neering of the native molecule yieldedFGF21 analogs with improved biophys-ical properties, and one of these FGF21analogs, LY2405319, ameliorated ath-erogenic dyslipidemia, insulin resistance,and adiponectin in obese patients withdiabetes (123).

TARGETING THE RENAL TUBULETO IMPROVE CKD AND NAFLD

Sodium-glucose cotransporter-2 (SGLT2)inhibitors block the activity of the SGLT2protein, which is expressed in the S1segment of the renal proximal tubule,leading to substantial glucosuria and a re-duction in plasma glucose levels. Experi-mental evidence has demonstrated thatSGLT2 inhibitors confer nephroprotectionindependently of their glucose-loweringor blood pressure–lowering properties.SGLT2 inhibitors attenuate glomerular hy-perfiltration, which is believed to be theinitial pathogenic alteration in diabeticand obesity-related CKD. In diabetes, hy-perglycemia induces an increase inSGLT2-mediated proximal tubule NaClreabsorption. The consequent reductioninNaCl distal delivery to themacula densadecreases tubuloglomerular feedback–mediated afferent arteriolar vasocon-striction, thereby increasing glomerularafferent-to-efferent arteriolar tone, in-traglomerular ultrafiltration pressure,and glomerular filtration rate (124).Proximal tubule SGLT2 upregulation andincreased NaCl reabsorption also havebeen documented in obesity-relatedCKD as a result of enhanced sympatheticactivity (33,125) and TGF-b1/SMAD3axis activation (126). By virtue of theseactions, SGLT2 inhibitors synergize withrenin-angiotensin-aldosterone system(RAAS) inhibitors, and their combinationmay confer incremental renal benefits

care.diabetesjournals.org Musso and Associates 1839

(127). Simultaneous SGLT2-RAAS block-ade induces afferent arteriole constric-tion (SGLT2 inhibition) and efferentarteriole vasodilatation (RAAS blockade),thereby more thoroughly counteractingearly intrarenal hemodynamic abnormal-ities underlying glomerular hyperfiltrationin CKD. Additionally, SGLT2 inhibitors havedecreased inflammatory and fibrogenicresponses, oxidative stress, and cell apo-ptosis in diverse experimental models ofCKD (128).Available data on the impact of SGLT2

inhibitors on CKD have derived from ananalysis of RCTs conducted with efficacy

and safety end points in a diabetic popu-lationwhere SGLT2 inhibitors slowed renalfunction decline and reduced albuminuriaindependently of glycemic control (129),and dedicated nephroprotection trialsare under way (Evaluation of the Effectsof Canagliflozinon Renal and Cardiovascu-lar Outcomes in ParticipantsWith DiabeticNephropathy [CREDENCE]; clinical trial reg.no. NCT02065791, www.clinicaltrials.gov).Besides nephroprotection, SGLT2 inhibi-tors have the potential for cardiovascularprotection. In the EMPA-REG OUTCOME[BI 10773 (Empagliflozin) CardiovascularOutcome Event Trial in Type 2 Diabetes

Mellitus Patients] trial, empagliflozinadded to standard care was associatedwith a lower rate of the primary compos-ite outcome of cardiovascular and all-cause mortality (130).

SGLT2 inhibitors may also ameliorateNAFLD. In a post hoc analysis of an RCTand a small RCT, remogliflozin etabonateand luseogliflozin improved liver fat accu-mulation and liver enzymes in patientswith diabetes (131,132). The conclusionsof these RCTs are supported by preclinicaldata, whereby SGLT2 inhibitors pre-vented diet-induced hepatic steatosis, in-flammation, and fibrosis independently

Figure 4—Molecular pathwaysmediating the interplay among the liver, kidney, gut, andadipose tissue in thepathogenesis ofNAFLDandCKDand theeffect oftheirmodulation.A: InNAFLD-associatedCKD, the gut promotes liver andkidney injury by reduced incretinGLP-1 andbyenhancedmicrobial productionof LPSandURMs, the excretion of which is reduced in CKD, further increasing their systemic levels. Further injuriousmechanisms contributing to NASH and CKD at asystemic level include inappropriate activation of SREBP-2, which promotes intracellular cholesterol accumulation; cellular sensors HIF-1 and mTORC1; andoxidative stress–activated ASK1. Collectively, these factors induce insulin resistance, ectopic lipid accumulation that triggers lipoperoxidative stress andenhances secretion of proinflammatory cytokines, chemokines CCL2 andCCL5, andprofibrogenic factors such asmiRNA21and galectin-3. The liver promotesCKD through enhanced secretion of uric acid and proatherogenic factors, including VLDL1, CETP, and syndecan-1, which promote atherogenic dyslipidemia,endothelial dysfunction, and renal vascular disease. Additionally, in NAFLD, there is an impaired action of hepatokine FGF21, the systemic levels of which areelevated because of tissue FGF21 resistance and impaired renal FGF21 excretion in CKD. B: Besides directly antagonizing the action of noxious factors describedin A, NAFLD and CKD can be ameliorated at intestinal levels by incretin mimetics and modulation of gut microbiota composition with prebiotics, probiotics, orsynbiotics and at systemic levels by activating cellular energy sensor AMPK and several nuclear transcription factors, including PPAR-a, PPAR-d, PPAR-g, FXR,and Nrf2. Tables 1–3 and the text provide a detailed description of molecular mechanisms underlying the gut-liver-kidney connection. Chol, cholesterol; IR,insulin resistance.

1840 NAFLD and Chronic Kidney Disease Diabetes Care Volume 39, October 2016

of antihyperglycemic action (34,35). Po-tential mechanisms underlying liver-related benefits of these drugs includeinsulin sensitizing (131) and body fatloss–inducing properties mediated byenhanced lipolysis and fatty acid oxida-tion (34,35), attenuation of adipose tis-sue dysfunction and inflammation (133),increased ACE2 activation (134), andintrinsic drug-specific antioxidant andanti-RAGE axis activation properties(36). Furthermore, SGLT2 mRNA ex-pression has been documented in theliver, where its biological and clinicalsignificance remain unknown (135).Several issues with SGLT2 inhibitorswarrant assessment, including the re-noprotective effects in patients withCKD but without diabetes, the impacton liver histology, long-term safety inpatients with various degrees of renaland hepatic impairment, and risk of ke-toacidosis, which led the U.S. Food andDrug Administration to issue a safetywarning in 2015 (136).

CONCLUSIONS AND FUTUREPERSPECTIVES

Despite progress made in the past de-cade, CKD still remains a major healthproblem for two reasons. CKD oftengoes unrecognized, and the currenttherapeutic armamentarium has limitedeffectiveness in retarding disease pro-gression. NAFLD is the most commonchronic liver disease and, given thelack of an effective treatment, is becom-ing the leading indication for liver trans-plantation in theWestern world (5). Theshared unmet needs of NAFLD and CKD,therefore, are boosting research onnovel therapeutic targets in these twoconditions.

Epidemiological data suggest a tightrelationship between the presence andseverity of NAFLD and the presence andstage of CKD and place NAFLD as an im-portant contributor to the developmentand progression of CKD independentlyof traditional risk factors. When analyz-ing the pathophysiological basis for this

association, striking analogies can befound between fatty liver and CKD.Like NAFLD, CKD is characterized by aderanged cellular substrate metabo-lism; ectopic fat deposition, which trig-gers oxidative stress; and inflammatoryand profibrotic responses to drive theprogression of both disease processes.This review shows a wealth of cellularpathways and mechanisms that repre-sent key contributors to liver and kidneyinjury and potential therapeutic targets(Fig. 4). Most of these targets currentlyare being evaluated in phase II RCTs, andsome of them, such as PPAR-a/d ago-nists, FXR agonists, and incretin analogs,have promising results (137), althoughfew of them have advanced to the samedevelopmental stage in both NAFLD andCKD, reflecting a continued low aware-ness of the similarities in the pathogenicmechanisms underlying these two condi-tions. Besides shared pathogenic mecha-nisms that promote both liver and kidneyinjury, the fatty liver per se may promote

Figure 4—Continued.

care.diabetesjournals.org Musso and Associates 1841

kidney injury and vice versa, with poten-tially relevant therapeutic implications.For example, SGLT2 inhibitors targetthe renal tubule but may improve bothCKD and NAFLD. Whether the relativemerits of the various therapeutic ap-proaches will translate into a clinicalbenefit needs assessment in adequatelypowered, larger RCTs of longer durationwith clinical end points. A key challengeto therapeutic success of these variousapproaches will be the selection of theoptimal therapeutic strategy for eachpatient. NAFLD and CKD progression islikely a multifactorial process, involvingvaried molecular pathways that may op-erate in different patient subsets and atdifferent stages of disease. Within thiscontext, recent developments in meta-bolic phenotyping with metabolomicsand systems biology technologies willhopefully enable individualized treat-ment tailored to individual profiles.Given the increasing prevalence of CKDand NAFLD and their direct effects onand acceleration of CVD, strategies to re-duce the incidence, progression, andcomplications of these twin conditionsare an important priority in health care.

Duality of Interest. No potential conflicts ofinterest relevant to this article were reported.Author Contributions. G.M. contributed tothe study conception and design, literaturesearch, data acquisition, critical analysis of theresults, and drafting and final approval of themanuscript.M.C., S.C., F.D.M., S.P., F.S., andR.G.contributed to the literature search, data acqui-sition, critical analysis of the results, anddraftingand final approval of themanuscript. G.M. is theguarantor of this work and, as such, had fullaccess to all the data in the study and takesresponsibility for the integrity of the data andthe accuracy of the data analysis.

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