leveraging melanocortin pathways to treat glomerular diseases

Leveraging Melanocortin Pathways to TreatGlomerular DiseasesRujun Gong

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cine, R

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134

Themelanocortin system is a neuroimmunoendocrine hormone system that constitutes the fulcrum in the homeostatic control

of a diverse array of physiological functions, including melanogenesis, inflammation, immunomodulation, adrenocortical ste-

roidogenesis, hemodynamics, natriuresis, energyhomeostasis, sexual function, and exocrine secretion. The kidney is a quintes-

sential effector organ of the melanocortin hormone system with melanocortin receptors abundantly expressed by multiple

kidney parenchymal cells, including podocytes, mesangial cells, glomerular endothelial cells, and renal tubular cells. Converg-

ing evidence unequivocally demonstrates that the melanocortin-based therapy using themelanocortin peptide adrenocortico-

tropic hormone (ACTH) is prominently effective in inducing remission of steroid-resistant nephrotic syndrome caused by

various glomerular diseases, including membranous nephropathy, minimal change disease and focal segmental glomerulo-

sclerosis, suggesting a steroidogenic-independent mechanism. Mechanistically, ACTH and other synthetic melanocortin ana-

logues possess potent proteinuria-reducing and renoprotective activities that could be attributable to direct protection of

glomerular cells and systemic immunomodulation. Thus, leveraging melanocortin signaling pathways using ACTH or novel

synthetic melanocortin analogues represents a promising and pragmatic therapeutic strategy for glomerular diseases. This re-

view article introduces the biophysiology of the melanocortin hormone systemwith an emphasis on the kidney as a target or-

gan, discusses the existing data on melanocortin therapy for glomerular diseases, and elucidates the potential mechanisms of

action.

Q 2014 by the National Kidney Foundation, Inc. All rights reserved.Key Words: Glomerulopathy, Nephrotic syndrome, Melanocortin, Adrenocorticotropic hormone, Podocyte

Glomerular disease is the third leading cause of end-stage kidney failure in the United States.1 Treatment

of glomerular disease depends on its cause and type, butit is currently limited largely to the use of blockades of therenin-angiotensin-aldosterone system and immunosup-pressants, including glucocorticoids, alkylating agents,calcineurin inhibitors, antimetabolites, and more.2-4

However, these treatments are of limited utility withunsatisfying therapeutic efficacy. Indeed, although 95%of children5 and 50% to 75% of adults6 with minimalchange disease (MCD) achieve complete remission ofproteinuria after an 8-week course of prednisone therapy,more than 70% of all patients who are initially prednisoneresponsive go on to experience relapses of the nephroticsyndrome, and almost half of them show frequent re-lapses or steroid dependence and require further immu-nosuppression.5,6 For other glomerular diseases such asidiopathic membranous nephropathy (iMN)7,8 and focalsegmental glomerulosclerosis (FSGS),9 initial immuno-suppressive treatments usually have much lowerresponse rates than for MCD, and additional immuno-supressants are essential to induce remission. In actuality,

m Division of Kidney Disease and Hypertension, Department of Medi-

hode Island Hospital, Brown University School of Medicine, Providence,

ancial Disclosure: R.G. received research grants and speaker honorariauestcor and served on scientific advisory boards for Questcor.

dress correspondence to RujunGong,MD, PhD,Division of KidneyDis-

d Hypertension, Department ofMedicine, Rhode IslandHospital, Brown

sity School of Medicine, 593 Eddy Street, Providence, RI 02903. E-mail:[email protected]

014 by the National Kidney Foundation, Inc. All rights reserved.

8-5595/$36.00p://dx.doi.org/10.1053/j.ackd.2013.09.004

Advances in Chronic Kidney Disease, Vol

neither the target cells nor the mechanism of action ofthese immunosuppressants for glomerular disease hasbeen clearly understood.4 Most of these therapeutic strat-egies were borrowed from transplant immunosuppres-sive regimens and thus are believed to be effective inglomerular diseases also via immunosuppression; how-ever, some such as cyclosporine A10 are found to function,at least in part, through nonimmune mechanisms10

whereas some others, such as levamisole,11 might be ef-fective via immune stimulatory mechanisms. Further-more, a considerable number of patients suffer from thecomplications of overimmunosuppression, including op-portunistic infection, neoplasia formation, and growth re-tardation.2,3 Therefore, it is imperative to develop noveland more effective therapeutic modalities with minorside effects to satisfactorily ameliorate glomerular injuryand induce remission of proteinuria in patients withrefractory glomerular diseases. Recently, a plethora ofevidence suggests that melanocortins possess potentantiproteinuric and renoprotective activities and mightserve in this role.12-17

Melanocortin System: A MultitaskingNeuroimmunnoendocrine Hormone System

Themelanocortin system is a set of hormonal, neuropepti-dergic, and immune signaling pathways that play an inte-gral role in the homeostatic control of a diverse array ofphysiological functions, includingmelanogenesis, inflam-mation, immunomodulation, adrenocortical steroidogen-esis, hemodynamics, natriuresis, energy homeostasis,sexual function, and exocrine secretion.17 The melanocor-tin hormone system comprises multiple components, in-cluding the 5 guanine protein-coupled melanocortin

21, No 2 (March), 2014: pp 134-151

Delta:1_given name

mailto:[email protected]

http://dx.doi.org/10.1053/j.ackd.2013.09.004

Melanocortin Therapy for Glomerular Diseases 135

receptors (MCRs); peptide ligands derived from theproopiomelanocortin preprohormone precursor; and en-dogenous antagonists, agouti signaling protein, andagouti-related protein (Table 1).18,19

The endogenous agonist ligands of the melanocortinhormone system, also knownasmelanocortins, are a groupof hormonal neuropeptides that include adrenocorticotro-pic hormone (ACTH) and the different forms ofmelanocyte-stimulating hormone (MSH). Melanocortinpeptides are produced by corticotrophs in the anteriorlobe of pituitary gland, constituting 15% to 20% of the cellsin the anterior lobe of the pituitary gland.20 Melanocortinsare synthesized from the precursor peptide pre-proopiomelanocortin, which is encoded by a single-copygene on chromosome 2p23.3 that is over 8 kb in length.20

The removal of the signal peptide during translationproduces the 267-amino-acid polypeptide proopiomelano-cortin (POMC), which undergoes a series of post-

CLINICAL SUMMARY

� The melanocortin system is a set of hormonal,

neuropeptidergic, and immune signaling pathways that

play an integral role in the homeostatic control of

a diverse array of physiological functions, including

inflammation, immunomodulation, and adrenocortical

steroidogenesis.

� The kidney is a quintessential effector organ of the

melanocortin hormone system with melanocortin receptors

abundantly expressed by multiple kidney parenchymal

cells, including podocytes, mesangial cells, glomerular

endothelial cells, and renal tubular epithelial cells.

� Converging clinical and experimental evidence suggests

that the melanocortin-based therapy using ACTH and

other melanocortin analogues confers potent proteinuria-

reducing and kidney protective effects in various

glomerular diseases possibly through both direct protec-

tion of glomerular cells and systemic immunomodulation.

translational modificationssuch as phosphorylation andglycosylation before it is pro-teolytically cleaved by pro-hormone convertase (PC)enzymes PC1 and PC2 toyield a chemically and bioge-netically related family ofpolypeptides with varyingphysiological activities, in-cluding endorphin, lipotro-pins, and melanocortins20

(ACTH and a-, b-, andg-MSH) (Fig 1A).

Plasma melanocortinshave a diurnal variation innormal subjects21,22 and canbe induced by physicalor psychological stress viahypophysiotropic hormonesincluding corticotropin-
releasing hormone and arginine vasopressin secreted bythe hypothalamus. Conversely, melanocortin synthesisand release are negatively controlled by slow/intermedi-ate or fast feedbacks by many substances secreted withinthe hypothalamic-pituitary-adrenal axis. Glucocorticoids(cortisol in human) secreted from the adrenal cortex in re-sponse to ACTH stimulation generate a negative feed-back.21 Thus, patients treated with a high dose ofsynthetic glucocorticoids for a long period are likely tohave a very low plasma level of melanocortins and de-velop a clinical constellation of symptoms that highlymimic the phenotypes of POMC deficiency syndrome,a rare genetic disease, including hyperphagia, central obe-sity, pale skin, and adrenal insufficiency.23
The melanocortins exert their biological functions bybinding to and activating the cognate MCRs with differ-ent affinity.24 So far, 5 MCRs have been cloned and char-

acterized. All of the 5 MCRs are highly conservativeacross different species and share many homologs.19,25

The MCRs are all members of the rhodopsin family(class A) of 7-transmembrane guanine protein-coupledreceptors, which intracellularly mediate their effectsmainly by activating adenylate cyclase, leading to stimu-lation of the cyclic AMP (cAMP)-dependent cell signalingpathways.24 The 5MCRs have distinct tissue distribution,convey signaling of different melanocortins, and mediatevarying biological effects.24

MC1R exhibits high affinity forACTHandmostMSH. Itis highly expressed inmelanocytes and is theprincipalMCRin the skin,where itmediates pigmentation as 1 of themajorbiological functions of most melanocortin peptides.19,25

MC1 R is also widely expressed in other organ systems,including adrenals, lung, lymph nodes, ovary, testis, brain,placenta, spleen, and uterus.19,25 It is also present invascular endothelial cells and immune-competent cells in-

cluding leukocytes,dendriticcells, andmacrophages, sug-gesting a role ofMC1R in theregulation of inflammatoryreaction and immune re-sponse.19,25 Indeed, a-MSH26 or ACTH27 treatmenthas been shown to preventacute and chronic inflamma-tion in animal models ofmultiple diseases, includingacute kidney inflammation27

and injury.26,28 Direct ev-idence of the important roleof MC1 R in inflammationand immunomodulationwas recently shown in micewith a nonfunctionalMC1 R.29 These mice dem-onstrated a dramatic ex-acerbation of experimental

inflammation,29 confirming a general anti-inflammatoryeffect of the MC1 R signaling pathway.

MC2 R is the primary and exclusive receptor for ACTHthat is expressedmainly in the adrenal gland and binds toACTH with strong affinity but does not bind to the MSHpeptides.19,25 Activation of MC2 R initiates a cascade ofevents affecting multiple steps in steroidogenesis andgrowth of adrenal cortex. Of note, although MC2 R ispredominantly expressed in adrenal cortex, recentstudies indicate that it is also present in some othertissues including adipocytes, where MC2 R mediatesstress-induced lipolysis via central ACTH release.19,25

MC3 R and MC4 R regulate energy expenditure.19,25

MC3 R is expressed in the brain, predominantly in thearcuate nucleus in the hypothalamus and limbic systemand in a few regions of the brain stem, as well as inthe periphery, where it has been found in the placenta

Table 1. Components of the Melanocortin Hormone System

The Melanocortin System

Ligands ACTH, a-MSH, b-MSH, g-MSH

Receptors MCRs 1-5 (Class A G-protein-coupled receptors)

Antagonists ASIP

AgRP

Abbreviations: ACTH, adrenocorticotropic hormone; AgRP, agouti-related protein; ASIP, agouti signaling protein; MCR, melanocortinreceptor; MSH, melanocyte stimulating hormone.

Gong136

and gut tissues; it is also abundantly expressed in theheart and in renal distal tubules and thus has beenpostulated to regulate hemodynamics, natriuresis, andcardiovascular functions. Indeed, g-MSH promotesurinary salt excretion via MC3 R in the kidneys.30 Miceeither with MC3 R knockout or lacking g-MSH becauseof a defect in PC2, when fed a high-salt diet, consistentlydeveloped severe hypertension.30 MC4 R is predomi-nately found in the central nervous system, where it isrepresented in almost every brain region, including thecortex, thalamus, hypothalamus, brain stem, and spinalcord.19,25 MC3 R and MC4 R can both bind to ACTH ora-MSH with high affinity, but evidence suggests thatthey are not redundant in regulating food intakebehavior and energy homeostasis, such as hunger and

A

B

Figure 1. Biosynthesis and molecular structures of melanocortianocortins are synthesized in the anterior pituitary gland from thpost-translational modifications such as phosphorylation and gPC1 and PC2 to yield a chemically and biogenetically relateda-, b-, and g-MSH. (B) Molecular sequences of natural humanACTH is a linear nonatriacontapeptide straight-chain polypeptidoccupying positions 25 to 33 may vary among species. Howeverand actually identical in all species. The only different amino acid31 is serine for human and lysine for porcine). All melanocortinwhich is theminimal sequence required for selectivity and stimuthe address sequence that permits MC2 R recognition and is unimone; CLIP, corticotropin-like intermediate peptide; HFRW, Hismelanocortin receptor; MSH, melanocyte-stimulating hormonenocortin.

satiety signaling, because the MC4 R receptor knockoutmice manifest hyperphagia, obesity, insulin resistance,and increased linear growth whereas the MC3 Rreceptor knockout animals are not hyperphagic but arestill obese.19,25 In addition, MC3 R has been foundto have thermoregulatory and immunomodulatoryeffects, whereas MC4 R could additionally governmood effects, control erectile function, and mediateaphrodisiac actions.19,25

MC5 R was the last of the MCR gene family to bediscovered by homology screening from genomic DNAin the early 1990s.19,25 High expression levels of MC5 Rcan be found in exocrine glands in addition to variousother organs, including adrenal glands, fat cells, liver,lung, lymph nodes, bone marrow, thymus, mammaryglands, testis, ovary, pituitary testis, uterus, esophagus,stomach, duodenum, skin, lung, skeletal muscle,exocrine glands, and kidney.19,25 MC5-R-mediated physi-ological functions are not well understood. Targeted dis-ruption of the MC5 R gene in mice resulted in a severedefect in water repulsion and thermoregulation becauseof decreased production of sebaceous lipids.19,25 MC5 Rhas similar affinity to ACTH and a-MSH and is believedto be responsible for the biological functions such assebaceous gland secretion, thermoregulation, and im-

n peptides. (A) Pituitary biosynthesis of melanocortins. Mel-e precursor peptide pre-POMC, which undergoes a series oflycosylation before it is proteolytically cleaved by enzymesfamily of melanocortin polypeptides, including ACTH andmelanocortin peptides, porcine ACTH, and synthetic ACTH.e containing 39 amino acids. The sequence of amino acids, the N-terminal 24-amino-acid segment is highly conservedbetween porcine and human ACTH is underscored (residualpeptides share the same core tetrapeptide sequence HFRW,lation of the cognateMCRs (exceptMC2R). The KKRRmotif isque to ACTH. Abbreviations: ACTH, adrenocorticotropic hor--Phe-Arg-Trp; KKRR, Lys-Lys-Arg-Arg; LPH, lipotropin; MCR,; PC, prohormone convertase; pre-POMC, pre-proopiomela-


munomodulation.19,25 It has been suggested that MC5 Ragonists may perhaps alleviate conditions such as dryeyes and mouth, and MC5 R antagonists might be usefulin the treatment of acne. In addition, MC5 R is widelyexpressed on B and T lymphocytes and might mediateimmunomodulatory effects of melanocortin peptidessuch as a-MSH and ACTH.19,25

Receptor binding studies revealed that ACTH is able totarget all of the 5 types of MCRs, implying a close associa-tion among multiple melanocortin-directed physiologicalprocesses, including stress response, regulation of food in-take, control of energy balance, immunoregulation, andexocrine gland functions.19,25 The other 3 MSH peptidesare pan agonists of the MCRs with different potency,except MC2 R. The molecular basis for varying affinitiesof melanocortins to different MCRs lies in the molecularstructures of the melanocortin peptides.19,25 ACTH andall of the 3 MSHs share the same core tetrapeptidesequenceHis-Phe-Arg-Trp, which is theminimal sequencerequired for selectivity and stimulation of the cognateMCRs (except MC2 R).19,25 The Lys-Lys-Arg-Arg motif isthe address sequence that permits MC2 R recognition andis unique to ACTH. This explains why ACTH is the pri-mary melanocortin that binds to MC2 R (Fig 1B).19,25

Recent data consistently proved the presence of variousMCRs in kidney parenchymal cells, signifying the kidneyas a prototypical target organ of the melanocortin system.

Kidney Is an Important Effector Organ of theMelanocortin Hormone System

As small peptides with very lowmolecular weights (�2.2-4.5 kDa), melanocortins could circulate throughout thewhole body and exert biological activities on most organsystems expressing the cognate MCRs. It has been knownfor a long time thatmelanocortin peptides possess a signif-icant effect in the kidney.30 As a matter of fact, ACTH hasbeen used since a half century ago for the treatment of ne-phrotic glomerular disease.31 In addition, a general protec-tive effect conferred bymelanocortins, in particular a-MSHandACTH, has been reproducibly demonstrated in animalmodels of multiple kidney diseases, including membra-nous nephropathy (MN),32 FSGS,33 acute kidney injury(AKI) due to ischemia or sepsis,26,28 kidney toxicity,34 andobstructive nephropathy.27,35 Furthermore, g-MSH hasbeen found to have a prominent natriuretic and diureticeffect on the kidney.30 These extensive kidney effects sug-gest that the kidney serves as an important effector organof the melanocortin hormone system. However, althoughMCRs have been extensively investigated in many organsystems inanimals andhumans, their expression in thekid-ney has been less studied and what kind of MCRs the kid-ney parenchymal cells actually express remainscontroversial (Table 2). For instance, although Ni andcolleagues36 found thatMC3 R, MC4 R, andMC5 R are ex-pressed in the cortex and medulla of murine kidneys; Lee

and colleagues37 only demonstratedMC1R andMC3R ex-pression in rat kidney. Our group recently found thatMC1 R is expressed abundantly and predominantly by re-nal tubules andweakly by glomeruli,whereasMC5R is ex-pressedweakly and sporadically by kidney interstitial cellsbut strongly by podocytes in rodents in vivo andin vitro.17,28 Moreover, strong expression of MC5 R andweak expression of MC2 R have been demonstratedin human kidney by polymerase chain reactionamplification of human-kidney-specific cDNA.38 To a cer-tain extent, this is consistent with the observation in seabass in which MC5 R was abundantly expressed in theanterior andposteriorkidneys.39 Incontrast, ina latelypub-lished study,32 only MC1 R was detected by reverse-transcriptase polymerase chain reaction as the majorMCR in human kidney and in cultured human podocytes,glomerular endothelial cells, mesangial cells, and tubularepithelial cells. However, in depth studies indicate thatbasal expression of MC1 R is low in murine podocytes,and upon stimulation by selective MC1 R agonists thecAMP responsewas not significantly induced.40 These dis-crepancies couldreflect speciesdifference inkidneyexpres-sionofMCR,but aremore likelydue to thepotential pitfallsin the nature of the detection technologies. Future studiesshould adopt more conclusive assays such as proteomicidentification, and results should be cross-validated bymultiple traditional detection technologies. Nevertheless,despite the existing controversy on the exact subtypes ofMCRs expressed in the kidney, a growing body of evidencehas clearly and consistently proved that the kidney is in-deed an important target organ of the melanocortin hor-mone system (Table 2). In line with this view, a deficiencyofmelanocortins such as ACTHhas been found to be asso-ciated with nephrotic syndrome caused by FSGS,41 in-ferring that the melanocortin hormone system might beessential for kidney homeostasis and melanocortindeficiency might predispose to kidney disease.41

Collectively, kidney parenchymal tissues and cells, in-cluding podocytes, mesangium, glomerular endothelia,tubular epithelia, and tubulointerstitium, are potentialtargets of melanocortin-mediated actions. Accordingly,melanocortin-based therapeutic regimens might have po-tential effects on diseases or injuries of these target kidneytissues or cells, including podocytopathies such as MCDand FSGS, mesangial diseases such as immunoglobulin Anephropathy (IgAN) andmesangial proliferative glomeru-lonephritis (MsPGN), glomerular endotheliosis caused bytransplant glomerulopathy, preeclampsia, or thromboticmicroangiopathy due to hemolytic-uremic syndrome orthrombotic thrombocytopenic purpura, and possibly tubu-lar injuries such as acute tubular necrosis.

Currently Available MCR Agonists

A couple of natural and synthetic melanocortin peptidesare already available for clinical use. Some novel

Table 2. The Kidney Is an Important Effector Organ of the Melanocortin Hormone System With Evident Expression of Various MCRs in Parenchymal Kidney Cells

MCRs

MC1 R MC2 R MC3 R MC4 R MC5 R

Agonist preference and

affinity

ACTH ¼ a-MSH . b -MSH .g-MSH

ACTH only ACTH ¼ a-MSH ¼ b-MSH ¼g-MSH

ACTH ¼ a-MSH . b-MSH .g-MSH

ACTH ¼ a-MSH . b-MSH .g-MSH

Signaling pathways � cAMP pathway

� MAPK/ERK pathway

� cAMP pathway � cAMP pathway

� IP3-Ca21 pathway

� cAMP pathway � cAMP pathway

� IP3-Ca21 pathway

� MAPK/ERK pathway

Biological functions � Melanogenesis

� Antipyresis

� Anti-inflammation/

immunomodulation

� Antiapoptosis/prosurvival

effects

� Steroidogenesis � Energy homeostasis

� Hemodynamic regulation

� Energy homeostasis

� Appetite regulation

� Aphrodisia


immunomodulation

� Regulation of exocrine

functions


effects

Expression in the kidney Human kidney

Mouse kidney

Rat kidney

Human kidney

(weak)

Human kidney

Mouse kidney

Rat kidney

Rat kidney Human kidney

Mouse kidney

Rat kidney

Distribution in the kidney Glomeruli

Tubular epithelia

Unknown Inner medullary collecting

duct

Renal cortex and medulla Glomeruli

Interstitium

Expression in kidney cells Podocyte

Mesangial cell

Glomerular endothelial cell

Tubular epithelial cell

Unknown Inner medullary

collecting duct cell

Glomerular

endothelial cell

Tubular epithelial cell

Inner medullary

collecting duct cell

Podocyte

Inner medullary collecting

duct cell

Kidney effects � Protection of podocytes

and other glomerular

cells from injury

� Protection of tubular cells

from injury

� Anti-inflammation

Unknown Enhanced sodium excretion

in collecting ducts

Unknown � Podocyte protection

� Anti-inflammation

Clinical effects on kidney

disease

� Proteinuria-reducing

effects

� Renoprotection in AKI

Unknown � Natriuresis/diuresis Unknown � Proteinuria-reducing

effects

Abbreviations: ACTH, adrenocorticotropic hormone; AKI, acute kidney injury; cAMP, cyclic AMP; IP3, inositol 1,4,5-triphosphate; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase; MCR, melanocortin receptor; MSH, melanocyte-stimulating hormone.

Gong

138


synthetic melanocortin analogues have been success-fully developed and are under clinical or preclinical tri-als for the purposes of sunless tanning, potentialantiobesity drugs, aphrodisiacs, improvement of erectiledysfunction, and possibly treatment of kidney diseases(Table 3).

New Approaches with Available Drugs

ACTH is a linear straight-chain nonatriacontapeptidecontaining 39 amino acids.42 The sequence of amino acidsoccupying positions 25 to 33 may vary among species.42

However, the N-terminal 24-amino-acid segment ishighly conserved and actually identical in all species. Inaddition, this segment is required and sufficient for theadrenocorticotropic activity. As a key component of thehypothalamic-pituitary-adrenal axis, ACTH is the onlymelanocortin peptide that is capable of activatingMC2 R and executing the steroidogenic action. Thus,ACTH possesses dual physiological functions, namelyMC2-R-mediated steroidogenesis in the adrenal glandsand a potent melanocortin effect in extra-adrenal organsystems (Fig 2). There are currently 2 forms of ACTHthat are commercially available: natural and syntheticforms. The natural ACTH, Acthar gel, is a proprietarymixture isolated from porcine pituitary extracts that isformulated with 16% gelatin gel. The major componentof ACTH gel is porcine ACTH1-39, which is only differ-ent from human ACTH in 1 amino acid residual (residual31 is serine for human and lysine for porcine) (Fig 1B).The ACTH gel has been approved by the U.S. Food andDrug Administration (FDA) since 1952 to induce diuresisor remission of proteinuria in the nephrotic syndromewithout uremia of the idiopathic type or that due to lupuserythematosus.43 An active synthetic analogue ofACTH(1-24) that is not naturally occurring consists ofthe first 24 amino acids of the native ACTH and is avail-able in the form of tetracosactide, including Cosyntropinas a short-acting formulation and Synacthen (aqueoussuspension of tetracosactide with zinc hydroxide) asa long-acting formulation.43 So far, Cosyntropin hasbeen used solely to assess adrenal gland functionwhereas repository ACTH, either ACTH gel or Syn-acthen, has been commonly applied to treat infantilespasm, multiple sclerosis, gouty arthritis, autoimmunediseases, and nephrotic glomerular diseases.42,43 It isgenerally believed that short-acting ACTH has strongand acute steroidogenic activities whereas slow-release,long-acting ACTH, either ACTH gel or Synacthen, dis-rupts the diurnal rhythm in adrenal steroidogenesisand thus has minor steroidogenic effects44 but potentiallygreater melanocortin effects in extra-adrenal organ sys-tems, including the kidney.

Because of the high cost and potential concerns ofanaphylactic reactions, natural ACTH as comparedwith synthetic ACTH has been less frequently chosen

for various clinical or experimental purposes. However,although it is claimed that synthetic ACTH(1-24) pos-sesses the same efficacy as natural ACTH with regardsto all of its biological activities, a growing body ofevidence suggests that they might be distinguishablydifferent in terms of pharmacokinetics and pharmaco-dynamics. Of note, the biological function of the 15amino acids (25-39) in the C-terminus of ACTH is un-certain and has been largely ignored for a long time.Recent data suggest that amino acids 25 to 39 primarilyconfer stability of the circulating ACTH, thus determin-ing the half-life and efficacy of ACTH.42 In support ofthis, site-specific amino acid substitution of phenylala-nine (F) 39 of ACTH by cysteine (C), which has a freesulfhydryl group that can react specifically with iodoa-cetamide derivatives of lipophilic groups, significantlyenhanced the biological activities of lipophilizedACTH(F39 C) as compared with native ACTH.45

Lipophilized ACTH(F39 C) bound more tightly to se-rum albumin and cell membranes in vitro and had lon-ger serum half-lives in vivo than native ACTH.45 Thedocumented plasma elimination half-life times of differ-ent types of ACTH have been shown to vary remarkablybetween 4.9 and 28.6 minutes. A study in healthy hu-mans indicated that the half-life of native ACTH is ap-proximately 15 minutes.46 By contrast, the half-life fortetracosactide (the active ingredient of Synacthen) is sig-nificantly shortened to 7 minutes. Consistently, in an-other study, the half-life of tetracosactide in thecirculation of fetal sheep was only 0.27 minutes,47 sug-gesting that the elimination rate of Synacthen in vivo issubstantially accelerated as comparedwith that of naturalACTH. Therefore, it is speculated that natural ACTHmight have more potent extra-adrenal actions than syn-thetic ACTH because of a longer duration of action.In addition, post-translational modification is an impor-tant mechanism that regulates the biological activity ofendocrine hormones, including ACTH. Such modifica-tions as glycosylation, phosphorylation, C-terminal ami-dation, and N-terminal acetylation have been found tobe present in natural ACTH48 but be totally absentfrom the synthetic ones. These distinct molecular com-positions and peptide structures might incur differentbiological activities and efficacies.48 Moreover, ACTHis known to stimulate lipostasis49 and insulin secre-tion.50,51 The b-cell tropic and insulin-releasing activityhas been confirmed to reside in the C-terminal part ofACTH.51 Therefore, it is conceivable that syntheticACTH in which the C-terminal amino acids 25 to 39have been truncated would substantially lack theb-cell tropic and insulin-releasing activities. Despitethese differences in pharmacokinetics and pharmacody-namics, ACTH gel and Synacthen have recently beenused, respectively, in the United States and Europe forlong-term treatment of steroid-resistant nephrotic glo-merulopathies.

Table 3. Currently Available Melanocortin Receptor Agonists, Their Biological Functions, and Potential Benefits for Kidney Diseases

MCR Agonists Classification MCR Affinity Biological Functions Benefits for Kidney Diseases Proprietor

Acthar gel Long-acting natural ACTH

gel; FDA approved

Pan agonist � Anti-inflammation/

immunomodulation


effects

� Steroidogenesis

� Antipyresis

� Melanogenesis

� Induce remission of

steroid-resistant nephrotic

syndrome

� Antiproteinuric effects in

iMN, MCD, FSGS, IgAN,

LN, DN, TG


experimental postadap-

tive FSGS

� Kidney protection in ex-

perimental septic AKI and

obstructive nephropathy

Questcor Pharmaceuticals

Synacthen Long-acting synthetic ACTH;

approved outside of the

United States

Pan agonist � Anti-inflammation/

immunomodulation

� Steroidogenesis

� Antipyresis

� Melanogenesis

� Induce remission of

steroid-resistant nephrotic

syndrome

� Antiproteinuric effect in

iMN, MCD, FSGS, MsPGN,

MPGN, LN, DN


experimental MN

Questcor Pharmaceuticals

a-MSH Synthetic a-MSH peptide Pan agonist

(no MC2 R)


immunomodulation


effects

� Antipyresis

� Melanogenesis


perimental AKI caused by

sepsis, ischemia, nephro-

toxins, and ureteral

obstruction


experimental MN

Generic API

Afamelanotide

(melanotan I)

Synthetic a-MSH analogue Pan agonist

(no MC2 R)

� Melanogenesis


immunomodulation

Unknown Clinuvel Pharmaceuticals

Melanotan II Synthetic a-MSH analogue Pan agonist

(no MC2 R)

� Melanogenesis

� Aphrodisia

Unknown mondoBiotech Laboratories

AG

Bremelanotide Synthetic a-MSH analogue MC1 R

MC4 R

� Aphrodisia

� Melanogenesis


immunomodulation

Unknown Palatin Technologies

AP214 Synthetic a-MSH analogue Pan agonist

(no MC2 R)


immunomodulation


effects


perimental AKI caused by

sepsis, ischemia, nephro-

toxins;

Abbott Pharmaceuticals

RM-493 Synthetic peptide agonist MC4 R � Regulation of appetite and

satiety

� Antiobesity

Unknown Rhythm Pharmaceuticals

MS05 Synthetic a-MSH analogue MC1 R � Anti-inflammation/

immunomodulation


experimental MN

Action Pharma A/S

Abbreviations: ACTH, adrenocorticotropic hormone; AKI, acute kidney injury; API, active pharmaceutical ingredients; DN, diabetic nephropathy; FDA, U.S. Food and Drug Admin-istration; FSGS, focal segmental glomerulosclerosis; IgAN, immunoglobulin A nephropathy; iMN, idiopathic membranous nephropathy; LN, lupus nephritis; MCD, minimal changedisease;MCR,melanocortin receptors;MN,membranous nephropathy;MPGN,membranoproliferative glomerulonephritis; MSH, a-melanocyte stimulating hormone;MsPGN,me-sangial proliferative glomerulonephritis; TG, transplant glomerulopathy.

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Figure 2. Melanocortin-based therapy is effective in glomerular diseases via direct protection of glomerular cells and sys-temic immunomodulation. Melanocortins and their synthetic analogues induce remission of glomerular diseases mainlyby targeting the glomerular cells (podocytes, mesangial cells, and glomerular endothelial cells) and immune-competencecells via the expressed melanocortin receptors (pathways in blue), thereby exerting a direct cellular protective effect on glo-merular cells and a systemic immunomodulatory and anti-inflammatory effect. ACTH, the onlymelanocortin peptidewith ste-roidogenic activities, exerts an additional cortisol-dependent effect that might also contribute to the remission of glomerulardiseases (pathways in yellow). Abbreviations: adrenocorticotropic hormone; APC, antigen-presenting cells; BC, Bowman’scapsule; C, glomerular capillary lumen; E, glomerular endothelial cells; M, mesangium; MCR, melanocortin receptor; MSH,melanocyte-stimulating hormone; P, podocytes. Republishedwith permission of Company of Biologists Ltd., fromSchematicof a normal glomerulus is reproduced with permission from Quaggin SE and Kreidberg JA. Development. 2008;135(4):609-620; permission conveyed through Copyright Clearance Center, Inc.


Novel MCR Agonists

a-MSH

a-MSH is a naturally occurring melanocortin peptidewith a tridecapeptide structure.52 It is the most importantMSH involved in stimulating melanogenesis, a processthat is responsible for hair and skin pigmentation inmammals. It also plays a key role in the control of feedingbehavior, energy homeostasis, and sexual activities.52

a-MSH is a nonselective pan agonist of MC1 R, MC3 R,MC4 R, and MC5 R but not MC2 R, which is exclusivefor ACTH. Activation of the MC1 R is responsible forits effect on pigmentation, whereas its regulation of ap-petite, metabolism, and sexual behavior is mediatedthroughMC3 R andMC4 R. a-MSH is generated as a pro-

teolytic cleavage product from ACTH (1-13). Unfortu-nately, natural a-MSH has too short of a half-life in thebody to be practical as a therapeutic drug.53 Moreover,the potential impurity of the product of natural a-MSHdue to contamination by other dominant melanocortinsor neuropeptides is another concern. So far, synthetica-MSH (acetate salt) has been mainly used in preclinicalstudies and demonstrated remarkable beneficial effectsthat ameliorate acute, chronic, or systemic inflammationand injuries in various organ systems, including skin,lung, liver, and kidney.26,54 Since 1999, the FDA Officeof Orphan Products Development has been sponsoringa Phase I clinical trial (ClinicalTrials.gov identifiernumber NCT00004496) to assess the maximal tolerateddose and effects of a-MSH on acute kidney failure andto determine the safety and pharmacokinetics of a-MSH

http://ClinicalTrials.gov

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in patients with established ischemic acute kidney failureor at high risk of acute kidney failure after kidneytransplantation.

Synthetic a-MSH Analogues

Several synthetic analogues of a-MSH have been devel-oped and investigated as medicinal drugs. These includeafamelanotide55 (previously known as melanotan I),melanotan II,56 bremelanotide,57 AP214,26,58 RM-493,59

MS05,32 and more. Most of these chemical compoundsare derived from the chemical modification of the molec-ular structure of a-MSH, and most are also pan agonistsof the MCRs (no MC2 R), except RM-493 and MS05,which respectively target MC4 R and MC1 R with highspecificity. All of these a-MSH mimetics have sig-nificantly greater potencies than a-MSH, along withimproved pharmacokinetics and distinctive MCR selec-tivity profiles. Because of the difference in their molecularstructures, these analogues possess different agonizingactivities for different MCRs and thus display distinct bi-ological functions and clinical effects.

Afamelanotide or Melanotan I is a potent and longer-lasting synthetic analogue of naturally occurring a-MSHwith the molecular structure modified ([Nle4-D-Phe7]-a-MSH) to increase binding affinity to its main receptorMC1 R.55 Afamelanotide is approximately 1000 timesmore potent than natural a-MSH. Afamelanotide is cur-rently in Phase II clinical trials in Europe and in PhaseIII in the United States for skin diseases including vitiligo,erythropoietic protoporphyria, and polymorphic lighteruption and prevention of actinic keratoses in organtransplant recipients.55

Melanotan II is a cyclic lactam analog of a-MSH. It hasweaker melanogenic activities as compared with melano-tan I but exhibits much greater aphrodisiac effects.56 Mela-notan II was found to enhance libido and erections inmostmale test subjects and sexual arousal with correspondinggenital involvement in most female test subjects.56

Bremelanotide, a metabolite of melanotan II that lacksthe C-terminal amide functional group, is under drug de-velopment as a treatment for female sexual dysfunction,hemorrhagic shock, and ischemia/reperfusion injury.60 Itfunctions by activating MC1 R and MC4 R to modulateinflammation and limit ischemia-associated injury. Itwas originally tested for intranasal administration intreating female sexual dysfunction, but this applicationwas temporarily discontinued because of concerns ofside effects of elevated blood pressure.60

AP214, another synthetic analogue of a-MSH anda panMCR agonist (noMC2 R), was developed by ActionPharma and is now owned by Abbott Pharmaceuticals.AP214 has been shown by numerous preclinical stud-ies26,58 to have potent anti-inflammatory activities in ex-perimental sepsis and arthritis. A Phase II clinical trialhas been completed using AP214 to prevent AKI in pa-tients undergoing cardiac surgery.

In summary,more andmore synthetica-MSHmimeticshave been developed for the purpose of melanocortin-based therapies for various human diseases. Because thekidney expresses all MCRs targeted by these synthetic ag-onists, the melanocortin-based treatments using thesenovel drugsmay also bepromising therapeuticmodalitiesfor glomerular diseases.

Melanocortin-Based Therapy for GlomerularDiseases: Clinical and Experimental Evidence

A plethora of experimental evidence lately supports thatmelanocortins or their synthetic analogues have a greatbeneficial effect in animal models of kidney diseases, in-cluding MN, FSGS, and AKI.17,61 However, the clinicalexperience of melanocortin-based therapy for kidney dis-eases is largely limited to the use of ACTH because so farall of the MSH and MSH analogues have not been ap-proved for clinical use, and ACTH is the only FDA-approved drug with melanocortin activities. ACTH hasbeen used since a half century ago for the treatment of ne-phrotic glomerular disease, and it exhibited great clinicalbenefits in inducing remission of proteinuria and ne-phrotic syndrome.17,61 Because ACTH has bothsteroidogenic effects in the adrenal gland andmelanocortin activities in extra-adrenal organs, the clini-cal effects of ACTH could be easily confounded andmasked by the glucocorticoid-mediated effects. Indeed,in early times, it was believed that ACTH exerts theseproteinuria-reducing effects indirectly through the adre-nal steroidogenic action because corticosteroids some-times also induce similar response.17 Some clinicalstudies recently applied ACTH therapy in patients withglucocorticoid-resistant glomerular diseases and shedlight on the mechanism of action of the antiproteinuric ef-fect of ACTH. It seems that ACTHmight have a protectiveeffect on the kidney that is independent of its steroido-genic activity. This notion is based on the following clin-ical observations. First, the plasma levels of cortisolsdetected in ACTH-treated patients13,14,44 were muchlower than circulating glucocorticoid levels in patientsundergoing standard glucocorticoid therapy.62 However,the ACTH therapy still resulted in a comparable or evenbetter outcome than steroid treatments in terms of remis-sion of proteinuria. Second, for some glomerular diseasessuch as iMN, corticosteroids as the sole treatment, evenwhen administered at high doses, are unable to modifythe natural disease course and have unproven benefit,as demonstrated by multiple randomized controlled clin-ical trials7,8; in contrast, ACTH monotherapydemonstrated striking beneficial effects and inducedremission of proteinuria and nephrotic syndrome,16 argu-ing that the effects of ACTH are not mediated via steroidmechanisms. Third, in some nephrotic patients that hadbeen resistant to previous glucocorticoid therapy,ACTH treatment could still produce satisfactory


improvement in proteinuria, again suggesting a steroid-independent mechanism of action. Collectively, aburgeoning body of evidence supports that the renopro-tective effect of ACTH cannot be fully explained by itssteroidogenic action. The extra-adrenal melanocortin sig-naling mechanism might be, at least in part, responsiblefor the antiproteinuric effect of ACTH in nephrotic glo-merular diseases.

Idiopathic Membranous Nephropathy

After decades of neglect, the therapeutic effect of ACTHon nephrotic syndrome was first reexamined by Berg andcolleagues.13 In their first study to optimize the dose ofACTH for adults with nephrotic syndrome, 14 patientswith biopsy-proven iMN received intramuscular injec-tion of synthetic ACTH (1-24) at increasing dose during8 weeks. The optimal dose was estimated to be 1 mgtwice per week, taking the therapeutic effects and themodest side effects of ACTH into consideration. ACTHtherapy attained a concomitant improvement of dyslipi-demia, proteinuria, and kidney dysfunction. Among the14 patients, 5 who had severe steroid-resistant nephroticsyndrome responded remarkably well to ACTH therapy,implying that the therapeutic effect of ACTH might beascribed not to steroidogenesis but to its melanocortinactivities. It is important to note that a quick relapsewas observed in all cases when ACTH was discontinuedafter a short treatment duration of 8 weeks. By contrast,patients who received ACTH therapy for 1 year were stillin remission even 18 months after cessation of treatment,suggesting that an extended term of ACTH treatment isrequired to achieve a sustained remission. In support ofthis, Hofstra and colleagues63 recently reported a high in-cidence of relapse of nephrotic syndrome after ACTHtreatment for 9 months. In their nonrandomized study re-ported as a conference abstract, 75% of the iMN patients,who achieved a remission after twice-weekly syntheticACTH therapy for 9 months, experienced a relapse dur-ing 3 months of follow-up after discontinuation of thetherapy. To assess the effectiveness and safety of ACTHtherapy for nephrotic syndrome, Ponticelli and col-leagues16 conducted an open-label, prospective, random-ized controlled trial to compare the 12-month course ofsynthetic ACTH monotherapy (1 mg, twice weekly intra-muscular injection) with the 6-month course of Ponticelliimmunosuppressive regimen (methylprednisolone plusa cytotoxic agent [either chlorambucil or cyclophospha-mide]) in 32 nephrotic patients with biopsy-proveniMN. ACTH therapy resulted in a similar remissionrate (83%) as compared with the Ponticelli immunosup-pressive regimen (75%).16 Furthermore, there were nosignificant differences in the relapse rate between the 2therapeutic regimens after a median follow-up of24 months.16 This study at least demonstrated that thetherapeutic efficacy of ACTHmonotherapy is not inferior

to that of the standard Ponticelli immunosuppressiveregimen for iMN. Consistently, Picardi and colleagues64

reported a sustained remission of nephrotic syndromein 5 iMN patients in a follow-up period up to 26 monthsafter receiving a 12-month course of synthetic ACTH(1 mg twice weekly) monotherapy.

Similar antiproteinuric and renoprotective effects werealso observed lately with natural ACTH in patients withrefractory iMN. In a retrospective case series study, Bom-back and colleagues15 recruited 21 patients with refrac-tory nephrotic syndrome due to various glomerulardiseases. Among the 21 patients, 11 were diagnosed ashaving iMN and most of them were previously treatedand failed to respond to a mean of 2 immunosuppressiveregimens. After ACTH gel treatment at a most commonlyused dose of 80 IU twice a week for 5 to 12 months, 9 ofthe 11 patients (82%) with iMN achieved partial (55%) orcomplete (27%) remission of proteinuria.15

To understand the mechanism of action underlyingthe proteinuria-reducing effect of ACTH in MN, Lind-skog and colleagues32 tested the effect of syntheticACTH in a rat model of passive Heymann’s nephritis.Synthetic ACTH (10 mg/day) therapy for 4 weeks signif-icantly reduced proteinuria (�50% reduction in urine al-bumin to creatinine ratio). This antiproteinuric effect wasmimicked with an equal efficacy by the nonsteroidogenicmelanocortin peptide a-MSH or its analogue MS05,a highly selective MC1 R agonist, accompanied withimproved glomerular morphology and podocyte ultra-structure as well as diminished urine excretion ofthiobarbituric acid-reactive substances, a marker of oxi-dative stress,32 suggesting that the proteinuria-reducingand kidney protective effects of ACTH are mediatedmainly by MC1 R signaling and are less likely due tosteroid effects. It is surprising to note that glomerular de-position of the complement C5b-9 membrane attack com-plex remained unchanged as estimated by fluorescentimmunohistochemistry, inferring that the proteinuria-reducing effect was achieved via direct podocyte protec-tion.32Thishypothesiswas further supportedby successfuldetection ofMC1R inpodocytes in human and rat glomer-uli.32 Thus, melanocortins including ACTH and a-MSHas well as synthetic melanocortin analogues could poten-tially protect the podocytes from injury possibly by acti-vating the MC1 R signaling in podocytes.

In addition to a direct podocyte-protective effect,ACTH and other melanocortins might induce improve-ment of proteinuria in iMN via systemic immuno-modulatory mechanisms. This has been supported bymultiple clinical studies. In a prospective study by Bom-back and colleagues,14 5 patients with resistant iMN, whopreviously failed 2 to 4 immunosuppressive regimens,were recruited as 1 of the studied groups and receivedACTH gel therapy (80 IU twice a week) for 6 months.By the end of the treatment, 2 of 5 patients (40%) attainedpartial remission of proteinuria. Mechanistically, by

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4 months of ACTH gel therapy, 3 of 5 patients (60%) dem-onstrated the disappearance in the serum of the anti-phospholipase A2 receptor (PLA2 R) autoantibody,14 anetiological factor recently characterized for the pathogen-esis of iMN, thus denoting an immunologic remission.Consistently, ACTH-induced remission of proteinuria iniMN is also observed to correlate with a reduction inanti-PLA2 R levels by another 2 collaborative studies re-ported as conference abstracts.65,66 One study examinedserum samples from iMN patients treated withsynthetic ACTH by Berg and colleagues. 65 Of the 9 pa-tients who were positive for anti-PLA2 R at baseline, allhad either absent or decreased anti-PLA2 R in theirpost-treatment serum samples, with a mean decrease of86% (range 19-100%) from the initial anti-PLA2 R levels.In 7 patients, a disappearance or a substantial decreasein anti-PLA2 R correlated, respectively, with complete(4 patients) or partial (3 patients) remission of protein-uria.65 In the other study,66 12 patients that were positivefor anti-PLA2 R at baseline experienced a reduction inanti-PLA2 R autoantibody (17-100%) after 6 months ofACTH gel treatment with a complete disappearance ofanti-PLA2 R in 5 cases. Another pilot study67 lately re-ported as an abstract was designed to determine thedose and effectiveness of ACTH gel in nephrotic patientswith iMN and revealed a similar effect of ACTH on thedepletion of anti-PLA2 R autoantibody: ACTH gel treat-ment induced a significant remission of proteinuria alongwith improvement in serum albumin and total choles-terol levels67; anti-PLA2 R was positive at baseline in 15of the total 20 patients, and the levels were found tochange prior to and in correlation with the proteinuria re-sponse to ACTH gel therapy.67 Taken together, it seemsthat ACTH therapy may exert the therapeutic effects ad-ditionally by suppressing autoantibody production.

In agreement with the above clinical evidence, immu-nological research has evidently indicated that B lympho-cytes express abundant MCRs, including MC1 R andMC3 R. Melanocortins such as a-MSH possess a potentinhibitory effect on human immune responses to exoge-nous antigens likely via activating these MCRs on B lym-phocytes.68 Moreover, these melanocortin-inducedimmunomodulatory effects on B lymphocytes seem dis-tinct from the immunosuppressive effect conferred byglucocorticoids because most of the patients with refrac-tory iMN recruited in the aforementioned studies hadbeen previously treated with other immunosuppressiveregimens, including glucocorticoids, and failed toachieve clinical or immunologic remissions. Thus, theautoantibody-suppressing activity of ACTH is appar-ently not steroidogenic dependent but possibly mediatedby a unique MCR-directed mechanism.

Clinical and experimental evidence collectively provesthat melanocortin therapy is able to induce remissionof proteinuria in patients with iMN through a directpodocyte-protective mechanism and a steroidogenic-

independent systemic autoantibody-suppressing mech-anism that might be ascribed to the MCR-mediatedimmunomodulatory effects on B lymphocytes (Fig 2).

MCD and FSGS

As 1 of the 4 native melanocortin peptides, ACTH hasbeen widely used since the early 1950s for the treatmentof nephrotic syndrome possibly caused by lipoid nephro-sis, an older term for nil lesion or MCD.31,69,70 ACTHtherapy was particularly provided for childhoodnephrotic syndrome in lieu of steroids with the hope ofless growth retardation because it does not suppress theadrenal glands.31 ACTH therapy was quite effective forpediatric and adult patients with nephrotic syndromein the 1950s and 1960s, but since then it has been largelyabandoned and replaced by synthetic glucocorticoidtherapy because (1) the mechanism of action of ACTHwas believed at that time to be mediated solely via ste-roidogenesis; and (2) ACTH (naturally or syntheticallyderived) is a peptide andmust be administered by subcu-taneous or intramuscular injection, which has been con-sidered to be less convenient as compared with oralsynthetic glucocorticoid therapy. Recent clinical studiesusing ACTH in patients with steroid-resistant MCDand FSGS revealed the extra benefit of ACTH and shedlight on melanocortin mechanisms. In an observationalstudy by Berg and colleagues,12 2 patients with MCDand 1 patient with FSGS had nephrotic-range proteinuriaand demonstrated no response to previous steroid andcytotoxic treatments. After the patients were convertedto synthetic ACTH monotherapy for 2 to 7 months, the2 MCD patients achieved complete remission and theFSGS patient attained partial remission. The patients allremained in remission after they were followed for 4 to28 months after cessation of ACTH therapy.12 In anotherretrospective case series abstract report,71 12 patientswith primary FSGS and nephrotic syndrome resistant toprior immunosuppressive therapies (median 3 therapies)were administered ACTH gel (median dose 80 IU subcu-taneous injection twice weekly) for a median of 26 (range12-56) weeks. Five of 12 patients (42%) experienced par-tial remission at last follow-up (median follow-up time58 weeks after stopping ACTH). The median time to re-mission was 6 (range 6-24) weeks. This ACTH-inducedremission rate in patients with refractory FSGS is consis-tent with the finding by another prospective trial14 inwhich 5 patients with MCD or FSGS failed 2 to 4 previousimmunosuppressive treatments and were converted toACTH gel monotherapy. After 6 months of treatment, 2of 5 patients (40%), 1 MCD and 1 FSGS, achieved partialremission. Because the development and progression ofFSGS might involve multiple pathogenic mechanisms,one would assume that the combination of ACTH anda second immunosuppressive agent that simultaneouslytargets different pathogenic pathways might confer


better benefit for refractory FSGS. Indeed, in a recentstudy, Berg and colleagues72 recruited 10 patients withsevere FSGS that failed to respond to prior treatmentswith prednisolone in combination with other immuno-suppressants, including cyclosporine, tacrolimus, myco-phenolate mofetil, and/or cyclophosphamide. Afterprednisolone was replaced with synthetic ACTH fora median 18 months while continuing therapy with a sec-ond immunosuppressive agent, 8 of 10 patients reachedcomplete (3 patients) or partial remission (5 patients),again suggesting a steroidogenic-independent mecha-nism mediating such a robust therapeutic efficacy.

As more and more physician scientists and kidney pa-thologists agree, regardless of the original etiology, thepathological basis of MCD and FSGS is essentially a dis-ease of podocytes or podocytopathy characterized bymassive podocyte foot process effacement, microvilloustransformation, and podocytopenia as revealed by elec-tron microscopy of the diseased glomeruli.73 In an effortto understand if the antiproteinuric activity of ACTH ob-served in steroid-resistant MCD and FSGS is attributableto a possible melanocortin-mediated podocyte protec-tion, ACTH gel therapy was performed in rats with sub-total nephrectomy,33 a standard model of postadaptiveFSGS (classic variant) and progressive CKD.74 After sub-total kidney ablation, rats received ACTH gel treatmentevery other day for 5 weeks. ACTHmarkedly diminishedproteinuria (.50% reduction in 24-hour urine protein ex-cretion) and significantly preserved kidney function asmeasured by increased kidney plasma flow, improved in-ulin clearance rate, and lowered serum creatinine levels.Histologically, glomerulosclerosis and tubulointerstitialfibrosis, kidney inflammation, tubular atrophy, and tubu-lar epithelial to mesenchymal transdifferentiation wereall reduced after ACTH therapy. Of note, ACTH had nosignificant effects on mean arterial pressure or kidney-to-body weight ratio, suggesting that the effect ofACTH is less likely due to hemodynamic regulatoryactivities or antihypertrophic mechanisms. Because glu-cocorticoid therapy exacerbates proteinuria and glomer-ulosclerosis in this model,75 steroidogenesis is alsounlikely to contribute to the protective effect of ACTH.Instead, evidence for direct podocyte protection was dis-covered in remnant glomeruli. This included reduction infoot process effacement, diminished podocytic apoptosis,and lesser reductions in glomerular expression of podo-cyte markers including vimentin, nephrin, podocin, andWT-1 in ACTH-treated rats. These data suggest thatACTH reduces proteinuria and injury in progressive glo-merulosclerosis via direct podocyte protection. BecauseMC1 R and MC5 R have been evidently identified in po-docytes in vivo and in vitro, ACTH and a-MSH as well assynthetic a-MSH analogues that possess agonizing activ-ities on these MCRs might be useful for the treatment ofpodocytopathic glomerular diseases such as MCD andFSGS (Fig 2).

Other Glomerular Diseases

Experimental evidence proves that human glomerularcapillary endothelial cells and mesangial cells also ex-press abundant MCR in vivo and in vitro,32 suggestingthat glomerular endothelium and mesangium may serveas targets of the melanocortin system, and melanocortintherapy might have effects on mesangial diseases, glo-merular endothelial injuries, and mesangiocapillary dis-eases. Thus far, preclinical data are very scarce on theeffects of melanocortins in these glomerular diseases.However, clinical experience from ACTH therapy insteroid-resistant glomerular diseases, including MsPGN,IgAN, mesangiocapillary (also known as membranopro-liferative) glomerulonephritis, and glomerular endothe-liosis suggests that melanocortin-based therapy isindeed beneficial for these glomerular diseases.

Berg and colleagues12 and Bomback and colleagues14,15

reported in their case series studies that patients withsteroid-resistantMsPGN, IgAN, ormesangiocapillary glo-merulonephritis responded very well to ACTH therapy interms of remission of proteinuria, inferring steroidogenic-independentmelanocortinmechanismsmediating a reno-protective benefit. In a prospective study by Bomback andcolleagues,14 5 patients with IgAN, 3 of who had previ-ously failed other immunosuppressive regimens includ-ing prednisone, were recruited as 1 of the studied groupsand receivedACTHgel therapy (80 IU twice aweek).After6 months of treatment, 2 of 5 patients, 1 previously un-treated and 1 resistant to steroids, achieved remarkableproteinuria remission. The experience of melanocortin-based therapy in glomerular endotheliosis is even scarcerand only limited to a couple of case reports as conferenceabstracts. Patel and colleagues76 tried ACTH gel therapyin a patient with biopsy-proven transplant glomerulop-athy, a prototypical glomerular endotheliosis possiblycaused by antibody-mediated rejection, hepatitis C infec-tion, or calcineurin inhibitor toxicity. After 4 months ofACTH treatment, the patient demonstrated 50% reductionin proteinuria, associated with improvement in serum al-bumin levels and kidney function. Noteworthily, as earlyas the 1950s, ACTHwas proposed as a promising therapyfor thrombotic microangiopathy.77 However, more experi-mental and clinical studies are required to validate ifmelanocortin therapeutics are really beneficial for diseasesof the glomerular endothelium, including thromboticmicroangiopathy, transplant glomerulopathy, and pre-eclampsia.

In addition to direct melanocortin effects on kidney pa-renchymal cells, melanocortins including ACTH are alsoable to target leukocytes and generate potent immuno-modulatory effects. Therefore, in addition to direct protec-tion of podocytes, mesangial cells, and glomerularendothelial cells in primary glomerular diseases,melanocortin-based therapy might also have a beneficialeffect in glomerular diseases that are secondary to

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systemic autoimmune disorders, such as lupus nephritis.In fact, 1 of the FDA-approved indications for ACTH ther-apy is to induce remission of nephrotic syndrome causedby lupus erythematosus. In a recent study by Berg and col-leagues78 that was presented as a conference abstract, 5 pa-tients with lupus nephropathy received synthetic ACTHtreatment for an average of 6 months. Urinary albumin ex-cretion decreased from an average of 4278 mg/mL to285 mg/mL, associated with significant improvement inhypoalbuminemia and stabilization of kidney function.Three of the 5 patients previously failedmultiple immuno-suppressive regimens including glucocorticoids, againsuggesting a steroidogenic-independent melanocortinmechanism mediating the renoprotective effect in lupusnephritis. To further verify the efficacy of melanocortin-based therapy in systemic autoimmune diseases, morerandomized controlled studies are merited to evaluatethe improvements in not only kidney parameters butalso the activity of systemic autoimmunity.

Molecular Mechanisms of Melanocortin-BasedTherapy for Glomerular Diseases

TheMCRs are prototypical G-protein-coupled receptors.18

Upon activationbymelanocortins,MCRswill activate the 2principal signal transduction pathways involving MCRs:the cAMP signal pathway and the phosphatidylinositolsignal pathway.79 These 2 signal pathways will ignitemany other downstream signaling cascades that are re-sponsible for various cellular effects, ultimately leading torenoprotection and remission of proteinuria.

Antiapoptosis and Prosurvival Activities

Evidence suggests that the cAMP signaling pathway trig-gered by the MCR, in particular MC1 R and MC5R, in-teracts and cross-talks with the mitogen-activatedprotein kinase/extracellular signal-regulated kinasepathway,80-83 which is a key signaling cascade thatrelays prosurvival/antiapoptotic signals.84 Podocyteshave been proven to express MCRs. In cultured murinepodocytes expressing MC1 R, a selective agonist ofMC1 R strongly triggered the cAMP signaling pathway.40

Thus, MCR agonists such as ACTH and a-MSH mighthave a direct antiapoptosis/prosurvival effect on podo-cytes and protect podocytes from death through thecrosstalk between the MCR/cAMP signaling pathwayand the mitogen-activated protein kinase/extracellularsignal-regulated kinase pathway (Fig 3). This mechanismpresumably explains why ACTH treatment had a directpodocyte protective effect and prevented podocytopeniain animal models of postadaptive FSGS.33

Anti-Inflammation and Immunomodulation

As neuroimmunopeptides, melanocortins possess a po-tent immunomodulatory activity in circulating leuko-

cytes, including monocytes, T and B lymphocytes,natural killer cells, and antigen-presenting cells.85-87 Inaddition, in parenchymal cells of most organ systems,including the kidney, melanocortins have a prominentanti-inflammatory effect.88 This anti-inflammatory/immunomodulatory effect could be mediated by multi-ple MCRs, including MC1 R, MC3 R, and MC5 R, andachieved through suppression of the transactivationactivities of nuclear factor kB (NFkB),54,88 a pivotaltranscription factor that governs the expression ofnumerous proinflammatory mediators implicated in theimmune response and inflammatory reaction, such aschemokines, lymphokines, adhesion molecules, andmore.89 The exact mechanisms for NFkB inhibition in-duced by MCR signaling pathways remain unclear, butevidence suggests that the MCR-activated cAMP signal-ing pathway is involved.88 Protein kinase A (PKA) is sub-stantially activated after the MCR-triggered induction ofintracellular cAMP levels. As an important substrate ofPKA, glycogen synthase kinase 3b (GSK3b) will be phos-phorylated and its activity suppressed. GSK3b has beenshown to be an important signaling transducer that con-trols the phosphorylation specificity of NFkB RelA/p65and directs the selective transcription of NFkB-depen-dent proinflammatory molecules.90 Thus, inhibition ofGSK3b activity by the MCR-cAMP-PKA signaling path-way will selectively obliterate the expression of proin-flammatory cytokines responsible for inflammatoryreactions and T helper (Th)1-mediated immune response,including tumor necrosis factor-a, interleukin (IL)-1b,and IL-12 (Fig 3).87 In addition, activation of PKA and in-hibition of GSK3b could increase the activity of cAMP re-sponse element binding protein (CREB), a transcriptionfactor that controls the expression of anti-inflammatorycytokines such as IL-10 that are essential for immunotol-erance.91 Besides, CREB also accounts for differential reg-ulation of Th1, Th2, and Th17 responses.86,87,91 CREBactivation directs regulatory T-cell lineage commitmentand thus favors immune tolerance91 (Fig 3). Indeed, treat-ment of primed T cells with a-MSH could induce toler-ance/anergy in primed CD41 Th cells and mediateinduction of CD251CD41 regulatory T cells through anMC5-R-dependent mechanism, thus resulting in toler-ance to experimental autoimmune injuries.92 Further-more, a-MSH has also been demonstrated to haveimmunosuppressive activities in humans, likely actingin part via MC1 R on monocytes and MC1,3R on B lym-phocytes.68 It is tempting to speculate that the therapeuticeffects of melanocortin treatments in immune-mediatedglomerular diseases such as iMN and podocytopathiessuch as MCD and FSGS might be attributable, at leastin part, to systemic immunomodulation, which could(1) reinstate the immune balance between the subsets ofTh lymphocytes, (2) reduce the production of autoanti-body, and (3) diminish the release of pathogenic lympho-kines that possess glomerular permeability activities.93,94

Figure 3. Molecularmechanisms responsible for the effect ofmelanocortin-based therapy in glomerular diseases.Whenmel-anocortin peptide ligands bind to MCRs, which are prototypical G-protein-coupled receptors, they cause a conformationalchange in the MCR that allows it to act as a guanine nucleotide exchange factor. The MCR can then activate an associatedG-protein by exchanging its boundGDP for aGTP. TheG-protein’s a subunit, togetherwith theboundGTP, can thendissociatefromthebandg subunits to further affect intracellular signalingproteins, includingadenylyl cyclaseandphospholipaseC, thustriggering the 2 principal signal transduction pathways involvingMCRs: the cAMP signal pathway and the phosphatidylinosi-tol signal pathway. The cAMP-PKApathway regulates thedownstreamsignalingcascades, including theMAPK/ERK-mediatedprosurvival/antiapoptosis pathway that might protect the glomerular cells, such as podocytes and glomerular endothelialcells, from injury and death; the CREB/NFkB pathway that balances the pro- and anti-inflammatory reaction as well as the im-mune tolerance and autoimmune response; and the cytoskeleton regulatory signaling pathway that might be involved in cy-toskeleton remodeling in glomerular cells such as podocytes. Abbreviations: cAMP, cyclic AMP; CREB, cAMP responseelement binding protein; GSK3b, glycogen synthase kinase 3b; MAPK/ERK, mitogen-activated protein kinase/extracellularsignal-regulated kinase; MEK, MAPK kinase; MCR, melanocortin receptor; NFkB, nuclear factor kB; PKA, protein kinase A.


Regulation of Cytoskeletons

G-protein signaling ignited by MCRs, which are proto-typical G-protein-coupled receptors, is closely involvedin fast-acting cellular responses, including cytoskeletonrearrangement and cell shape changes. Activation ofMC1 R has been shown to regulate the activities of mul-tiple cytoskeleton regulatory signaling transducers, in-cluding Rac, Cdc42, and Rho through the cAMP-PKAsignaling pathway.95,96 MC1 R-cAMP-PKA signalingenhances the activity of the GTP-binding protein Rac,quickly resulting in altered lamellipodia and filopodiaformation and increased cellular arborization and den-dritogenesis95 (Fig 3). This effect might protect the podo-cytes from the cytoskeleton dysorganization induced byvarious immune- or nonimmune-mediated injuries andmight account for the improvement in the podocytefoot process effacement and podocyte shape changes ob-

served after ACTH treatment in animal models of posta-daptive FSGS.33

Side Effects of Melanocortin-Based Therapy

According to the existing experience of clinical use ofACTH as well as ongoing clinical trials of other melano-cortin analogues, the side effects of melanocortin-basedtherapy seem mild, tolerable, and reversible.

Allergy and Anaphylaxis

All of the melanocortins that are currently available formedicinal use are peptides and require injection asa way of administration. Among these drugs, naturalACTH is prepared from porcine pituitary glands whereassynthetic ACTH and other synthetic melanocortin ana-logues are oligopeptides structurally derived from natural

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ACTH or a-MSH. As foreign proteins or synthetic oligo-peptides, these macromolecular drugs might potentiallyhave antigenic/immunogenic activity. Thus, allergy andanaphylaxis are reasonable concerns for melanocortin-based therapy. However, in the past 60 years there wereonly several case reports onmild allergic/anaphylactic re-actions to clinical use of natural or synthetic ACTH andmost were limited to pediatric patients,97,98 suggestingthat the antigenicity and immunogenicity of themelanocortin peptides might be minor.

Cushingoid Symptoms

The steroidogenic melanocortin peptide ACTH has beenlabeled to potentially generate steroid-like side effectsand incur Cushingoid symptoms, including visceralobesity, hyperglycemia, osteoporosis, avascular osteonec-rosis, and more. However, multifaceted evidence indi-cates that ACTH as compared with glucocorticoidsmight play a distinct or even opposing role in the devel-opment of some of these side effects. It is known that glu-cocorticoid treatment or overproduction of endogenouscortisol could reduce bone mineral density and result inosteoporosis.99 However, patients with adrenal Cush-ing’s syndrome, in which ACTH levels are suppressed,experience greater bone loss than those with pituitaryCushing’s with high serum ACTH levels.100 Likewise,patients with familial glucocorticoid deficiency withelevated ACTH levels, due to hypothalamic feedback,have a higher bone mass than age-matched controls.101

Taken together, these findings are consistent with ananabolic effect of ACTH, which seemingly counteractsthe bone loss due to cortisol. Lately, through an animalmodel of glucocorticoid-induced osteonecrosis, it is con-firmed that ACTH treatment actually protected frommethylprednisolone-induced avascular osteonecrosis ofthe femoral head,102 and this effect was attributed tothe ACTH-promoted maintenance and regeneration offine vascular networks surrounding the highly remodel-ing bone.102

In addition, hyperglycemia is another common side ef-fect of glucocorticoid therapy because of impaired insulinproduction103 or insulin resistance.104 By contrast, ACTHhas been demonstrated to have opposing effects andprominently induce b-cell production of insulin.105-107

In actuality, natural ACTH possesses a potent b-celltropic and insulinogenic activity, which has beenproven to reside in the C-terminal part ACTH(22-39) ofACTH.51 Consistently, in a recent prospective random-ized trial, 18 patients with advanced diabetic nephropa-thy were treated with low doses of ACTH gel (16 or 32IU daily, subcutaneous injection) for 6 months. The aver-age 24-hour urine protein excretion was significantly re-duced from 7.8 to 2.3 g, associated with stabilizedkidney function and no deterioration of diabetes. Only2 of 18 patients (11%) required reduction in ACTH dose

secondary to hyperglycemia,108 suggesting that ACTHtherapy, totally different from glucocorticoid therapy, issafe for patients with diabetes, although large-scale trialswith long-term follow-up are required to validate thisfinding. Taken together, the steroid-like side effects orCushingoid symptoms seem to be mild at the clinicaldoses of ACTH. It is also conceivable that clinical use ofnonsteroidogenic melanocortins such as a-MSH or itssynthetic analogues should be able to avoid thesesteroid-like side effects.

Hypertension

Hypertension is another occasional side effect observedin melanocortin-based therapy. For nonsteroidogenicmelanocortins or analogues, mildly elevated blood pres-sure has been reported during Phase I or II clinical trials,possibly because of the hemodynamic actions subse-quent to MC3 R activation in the cardiovascular system30

or because of the central nervous actions.109 In addition,ACTH therapy through the cortisol effect might also in-duce mild hypertension. As a type of natural glucocorti-coid, cortisol may directly elevate blood pressure byenhancing vascular tone.110 On the other hand, cortisolhas a fair amount of activity on the minerocorticoid re-ceptor111 and may cause sodium and water retention, po-tassium wasting, and expansion of effective circulatingvolume, thus resulting in mild hypertension or even con-gestive heart failure in some high-risk patients.111 Uncon-trollable malignant hypertension after ACTH therapy isvery rare, but it could be seen in patients with glucocor-ticoid remediable aldosteronism (GRA).112 GRA is a rare(prevalence: 0.66%) autosomal-dominant disorder113 inwhich the promoter region of the 11-hydroxylase geneis fused to the coding region of aldosterone synthasebecause of an unequal gene crossing-over so that adre-nal production of aldosterone is not only regulated bythe renin-angiotensin-aldosterone system, but is largelyunder the control of ACTH.112 After ACTH treatment,GRA patients may rapidly manifest with fatigue, a dras-tic increase in blood pressure, and severe headache.112

The most serious side effect of ACTH therapy in GRApatients is very severe hypertension resulting in hem-orrhage stroke.112 Therefore, for patients with uncon-trollable hypertension after ACTH treatment, GRAshould be suspected and ACTH therapy should be dis-continued.

Other Side Effects

Other possible side effects of melanocortins include skinpigmentation and increased risk of melanoma because ofMC1 R activation.114–116 Therefore, melanocortin-basedtherapy should be avoided in patients with current oreven antecedent malignant melanoma. Additional sideeffects may include anorexia, mood changes, sleep disor-ders, and behavioral disturbances due to activation of


MC3 R andMC4 R in the central nervous system.18 Sebor-rheic dermatitis and acne vulgaris are also possible con-cerns because of overfunction of sebaceous glands afteractivation of MC5 R on sebocytes.117 Pan MCR agonistsmay potentially increase libido and activate the yawning,stretching, and erection reflexes because of activation ofMC4 R in the central nervous system.118

Conclusions

In the past several years, a growing body of clinical andexperimental evidence consistently indicates that melano-cortin treatments have prominent antiproteinuric andkidney-protective effects in various glomerular diseases.Although, the existing clinical findings are encouraging,most were made in single-center observational studieswith small sample sizes, poor controls, and short-term fol-low-up. Thus, additional large-scale multicenter random-ized controlled trials are warranted to validate the safetyand efficacy of the melanocortin-based therapy for glo-merular diseases. Moreover, an extended follow-up dura-tion is merited to assess whether the melanocortin-basedtherapy can actually retard the progression of CKD andstabilize kidney function in addition to its proteinuria-reducing effect. In summary, the melanocortin-based ther-apy might represent a novel, promising, and pragmatictherapeutic strategy for glomerular diseases.

Acknowledgments

This work was made possible, in part, by support from theFoundation for Health, the Rhode Island Foundation, and theU.S. National Institutes of Health grant R01 DK092485.

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