Leveraging Melanocortin Pathways to Treat Glomerular Diseases

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 melanogenesis, inflammation, immunomodulation, adrenocortical steroidogenesis, hemodynamics, natriuresis, energy homeostasis, sexual function and exocrine secretion 17 . The melanocortin hormone system is comprised of multiple components, including the five guanine protein-coupled melanocortin receptors; peptide ligands derived from the proopiomelanocortin preprohormone precursor; and endogenous antagonists, agouti signaling protein and agouti-related protein ( Table 1 ) 18 , 19 . The endogenous agonist ligands of melanocortin hormone system, also known as melanocortins, are a group of hormonal neuropeptide that include adrenocorticotropic hormone (ACTH) and the different forms of melanocyte-stimulating hormone (MSH). Melanocortin peptides are produced by corticotrophs in the anterior lobe of pituitary gland, constituting 15–20% of the cells in the anterior lobe of the pituitary gland 20 . Melanocortins are synthesized from the precursor peptide pre-pro-opiomelanocortin (pre-POMC), which is encoded by a single-copy gene on chromosome 2p23.3 that is over 8 kb in length 20 . The removal of the signal peptide during translation produces the 267 amino acid polypeptide POMC, which undergoes a series of post-translational modifications such as phosphorylation and glycosylation before it is proteolytically cleaved by prohormone convertase (PC) enzymes PC1 and PC2 to yield a chemically and biogenetically related family of polypeptides with varying physiological activity, including endorphin, lipotropins and melanocortins 20 (ACTH, α, β, γ-MSH) ( Figure 1a ). Plasma melanocortins have a diurnal variation in normal subjects 21 , 22 and can be induced by either physical or psychological stress, via hypophysiotropic hormones including corticotropin-releasing hormone and arginine vasopressin secreted by hypothalamus. Conversely, melanocortin synthesis and release are negatively controlled by slow/intermediate or fast feedbacks by many substances secreted within the hypothalamic-pituitary-adrenal (HPA) axis. Glucocorticoids (cortisol in human) secreted from the adrenal cortex in response to ACTH stimulation generate a negative feedback 21 . Thus patients treated with a high dose of synthetic glucocorticoids for a long period are likely to have a very low plasma level of melanocortins and develop a clinical constellation of symptoms that highly mimic the phenotypes of POMC deficiency syndrome, a rare genetic disease, including hyperphagia, central obesity, pale skin and adrenal insufficiency 23 . The melanocortins exert their biological functions by binding to and activating the cognate melanocortin receptors (MCRs), with different affinity 24 . So far five MCRs have been cloned and characterized. All of the five MCRs are highly conservative across different species and share many homologs. 19 , 25 The MCRs are all members of the rhodopsin family (class A) of seven-transmembrane guanine protein-coupled receptors, which intracellularly mediate their effects mainly by activating adenylate cyclase leading to stimulation of the cAMP-dependent cell signaling pathways 24 . The five MCRs have distinct tissue distribution, convey signaling of different melanocortins and exert varying biological activities 24 . MC1R exhibits high affinity for ACTH and most MSH. It is highly expressed in melanocytes and is the principal melanocortin receptor in the skin where it mediates pigmentation as one of the major biological functions of most melanocortin peptides 19 , 25 . MC1R is also widely expressed in other organ systems, including adrenals, lung, lymph node, ovary, testis, brain, placenta, spleen and uterus 19 , 25 . It is also present in vascular endothelial cells and immune competent cells including leukocytes, dendritic cells and macrophages, suggesting a role of MC1R in the regulation of inflammatory reaction and immune response 19 , 25 . Indeed, α-MSH 26 or ACTH 27 treatment has been shown to prevent acute and chronic inflammation in animal models of multiple diseases, including acute kidney inflammation 27 and injury 26 , 28 . Direct evidence of the important role of MC1R in inflammation and immunomodulation was recently shown in mice with a nonfunctional MC1R 29 . These mice demonstrated a dramatic exacerbation of experimental inflammation 29 , confirming a general anti-inflammatory effect of the MC1R signaling pathway. The MC2R is the primary and exclusive receptor for ACTH that is expressed mainly in the adrenal gland and binds to ACTH with strong affinity but does not bind to the MSH peptides 19 , 25 . Activation of the MC2R initiates a cascade of events affecting multiple steps in steroidogenesis and growth of adrenal cortex. Of note, even though MC2R is predominantly expressed in adrenal cortex, recent studies indicate that

A couple of natural and synthetic melanocortin peptides are already available for clinical use. Some novel synthetic melanocortin analogues have been successfully developed and are under clinical or pre-clinical trials for the purposes of sunless tanning, potential anti-obesity, aphrodisiac, improvement of erectile dysfunction and possibly treatment of kidney diseases ( Table 3 ). New approaches with available drugs ACTH is a linear straight-chain nonatriacontapeptide containing 39 amino acids 42 . The sequence of amino acids occupying positions 25 to 33 may vary among species 42 . However, the N-terminal 24-amino acid segment is highly conserved and actually identical in all species. In addition, this segment is required and sufficient for the adrenocorticotropic activity. As a key component of the HPA axis, ACTH is the only melanocortin peptide that is capable of activating MC2R and executing the steroidogenic action. Thus, ACTH possesses dual physiological functions, namely MC2R mediated steroidogenesis in the adrenal glands and a potent melanocortin effect in extra-adrenal organ systems ( Figure 2 ). Currently there are two forms of ACTH that are commercially available, natural and synthetic ones. The natural ACTH, Acthar gel, is a proprietary mixture isolated from porcine pituitary extracts that is formulated with 16% gelatin gel. The major component of ACTH gel is porcine ACTH1-39, which is only different from human ACTH in one amino acid residual (residual 31 is serine for human and lysine for porcine) ( Figure 1b ). The ACTH gel has been approved by the FDA since 1952 to induce diuresis or remission of proteinuria in the nephrotic syndrome without uremia of the idiopathic type or that due to lupus erythematosus 43 . An active synthetic analogue of ACTH(1–24) that is not naturally occurring consists of the first 24 amino acids of the native ACTH and is available in the form of tetracosactide, including Cosyntropin as short acting formulation and Synacthen (aqueous suspension of tetracosactide with zinc hydroxide) as long acting formulation 43 . So far Cosyntropin has been used solely to assess adrenal gland function, whereas repository ACTH, either ACTH gel or Synacthen has been commonly applied to treat infantile spasm, multiple sclerosis, gouty arthritis, autoimmune diseases and nephrotic glomerular diseases 42 , 43 . It is generally believed that short acting ACTH has strong and acute steroidogenic activities while slow release long acting ACTH, either ACTH gel or Synacthen, disrupts the diurnal rhythm in adrenal steroidogenesis and thus has minor steroidogenic effects 44 but potentially greater melanocortin effects in extra-adrenal organ systems, including the kidney. Due to the high cost as well as potential concerns of anaphylactic reactions, natural ACTH as compared with synthetic ACTH has been less frequently chosen for various clinical or experimental purposes. However, even though it is claimed that synthetic ACTH (1–24) possesses the same efficacy as natural ACTH with regards to all its biological activities, a growing body of evidence suggests that they might be distinguishably different in terms of pharmacokinetics and pharmacodynamics. Of note, the biological function of the 15 amino acids (25–39) in the C-terminus of ACTH is uncertain and has been largely ignored for a long time. Recent data suggests that amino acids 25–39 primarily confer stability of the circulating ACTH, thus determining the half-life and efficacy of ACTH 42 . In support of this, site-specific amino acid substitution of phenylalanine (F) 39 of ACTH by cysteine (C), which has a free sulfhydryl group that can react specifically with iodoacetamide derivatives of lipophilic groups, significantly enhanced the biological activities of lipophilized ACTH(F39C) as compared with native ACTH 45 . Lipophilized ACTH(F39C) bound more tightly to serum albumin and cell membranes in vitro and had longer serum half-lives in vivo than native ACTH 45 . The documented plasma elimination half-life times of different types of ACTH has been shown to vary remarkably between 4.9 and 28.6 minutes. A study in healthy man indicated that the half-life of native ACTH is about 15 min 46 . By contrast, the half-life for tetracosactide (the active ingredient of Synacthen) is significantly shortened to 7 min. Consistently, in another study, the half-life of tetracosactide in the circulation of foetal sheep was only 0.27 min 47 , suggesting that the elimination rate of Synacthen in vivo is substantially accelerated as compared to that of natural ACTH. Therefore, it is speculated that natural ACTH might have more potent extra-adrenal actions than synthetic ACTH due to longer duration of action. In addition, post-translational modification is an important mechanism that regulates the biological activity of endocrine hormones, including ACTH. Such modifications as glycosylation, phosphorylation, C-terminal amidation and N-terminal acetylation, have been

Several synthetic analogues of α-MSH have been developed and investigated as medicinal drugs. These include afamelanotide 55 (previously known as melanotan I), melanotan II 56 , bremelanotide 57 , AP214 26 , 58 , RM-493 59 , MS05 32 and more. Most of these chemical compounds are derived from the chemical modification of the molecular structure of α-MSH and most are also pan agonists of the MCRs (no MC2R) except RM-493 and MS05, which respectively target MC4R and MC1R with high specificity. All of these α-MSH mimetics have significantly greater potencies than α-MSH, along with improved pharmacokinetics and distinctive MCR selectivity profiles. Because of the difference in their molecular structures, these analogues possess different agonizing activities for different MCRs and thus display distinct biological functions and clinical effects. Afamelanotide or Melanotan I is a potent and longer-lasting synthetic analogue of naturally occurring α-MSH with the molecular structure modified ([Nle4-D-Phe7]-α-MSH) to increase binding affinity to its main receptor MC1R 55 . Afamelanotide is approximately 1,000 times more potent than natural α-MSH. Currently, Afamelanotide is in phase II clinical trials in Europe and in phase III in the US for skin diseases including vitiligo, erythropoietic protoporphyria, polymorphic light eruption and prevention of actinic keratoses in organ transplant recipients 55 . Melanotan II is a cyclic lactam analog of α-MSH. It has weaker melanogenic activities as compared to Melanotan I but exhibits much greater aphrodisiac effects 56 . Melanotan II was found to enhance libido and erections in most male test subjects and sexual arousal with corresponding genital involvement in most female test subjects 56 . Bremelanotide, a metabolite of melanotan II that lacks the C-terminal amide functional group, is under drug development as a treatment for female sexual dysfunction, hemorrhagic shock and ischemia/reperfusion injury 60 . It functions by activating MC1R and MC4R to modulate inflammation and limit ischemia associated injury. It was originally tested for intranasal administration in treating female sexual dysfunction but this application was temporarily discontinued due to concerns of side effects of elevated blood pressure 60 . AP214, another synthetic analogue of α-MSH and a pan MCR agonist (no MC2R), was developed by Action Pharma and is now owned by Abbott Pharmaceuticals. AP214 has been shown by numerous preclinical studies 26 , 58 to have potent anti-inflammatory activities in experimental sepsis and arthritis. A phase-II clinical trial has been completed using AP214 to prevent acute kidney injury in patients undergoing cardiac surgery. In summary, more and more synthetic α-MSH mimetics have been developed for the purpose of melanocortin-based therapies for a variety of human diseases. As the kidney expresses all MCRs targeted by these synthetic agonists, the melanocortin-based treatments by using these novel drugs may also be a promising therapeutic modality for glomerular diseases.

As one of the 4 native melanocortin peptides, ACTH has been widely used since early 1950s for the treatment of nephrotic syndrome possibly caused by lipoid nephrosis, an older term for nil lesion or MCD 31 , 69 , 70 . ACTH therapy was particularly provided for childhood nephrotic syndrome in lieu of steroids with the hope of less growth retardation since it does not suppress the adrenal glands 31 . ACTH therapy was quite effective for both pediatric and adult patients with nephrotic syndrome in 1950s and 1960s, but later has been largely abandoned and replaced by the synthetic glucocorticoid therapy, due to the following reasons: 1) the mechanism of action of ACTH was believed at that time to be mediated solely via steroidogenesis; and 2) ACTH, either naturally or synthetically derived, is a peptide and has to be administered by subcutaneous or intramuscular injection, which has been considered less convenient as compared with the oral synthetic glucocorticoid therapy. Recent clinical studies using ACTH in patients with steroid resistant MCD and FSGS revealed extra benefit of ACTH and shed light on the melanocortin mechanisms. In an observational study by Berg et al 12 , 2 patients with MCD and 1 patient with FSGS had nephrotic range proteinuria and demonstrated no response to previous steroid and cytotoxic treatments. After the patients were converted to synthetic ACTH monotherapy for 2 to 7 months, the 2 MCD patients achieved complete remission and the FSGS patient attained partial remission. The patients all remained in remission after they were followed up for 4 to 28 months following cessation of ACTH therapy 12 . In another retrospective case series abstract report 71 , 12 patients with primary FSGS and nephrotic syndrome resistant to prior immunosuppressive therapies (median 3 therapies) were administered ACTH gel (median dose 80 IU subcutaneous injection twice weekly) for a median of 26 (range 12–56) weeks. Five of 12 patients (42%) experienced partial remission at last follow-up (median follow-up time 58 weeks after stopping ACTH). The median time-to-remission was 6 (range 6–24) weeks. This ACTH induced remission rate in patients with refractory FSGS is consistent with the finding by another prospective trial 14 , in which 5 patients with either MCD or FSGS failed 2 to 4 previous immunosuppressive treatments and were converted to ACTH gel monotherapy. After 6-month treatment, 2 of 5 patients (40%), 1 MCD and 1 FSGS, achieved partial remission. Because the development of FSGS might involve multiple pathogenic mechanisms, one would assume that combination of ACTH and a second immunosuppressive agent that simultaneously targets different pathogenic pathways might confer better benefit for refractory FSGS. Indeed, in a recent study reported as a conference abstract, Berg et al 72 recruited 10 patients with severe FSGS that failed to respond to prior treatments with prednisolone in combination with other immunosuppressants, including cyclosporine, tacrolimus, mycophenolate mofetil and/or cyclophosphamide. After prednisolone was replaced with synthetic ACTH for a median 18 months while continuing therapy with a second immunosuppressive agent, 8 of 10 patients reached either complete (3 patients) or partial remission (5 patients), again suggesting a steroidogenic independent mechanism mediating such a robust therapeutic efficacy. As more and more physician scientists and renal pathologists agree, regardless of the original etiology, the pathological basis of MCD and FSGS is essentially a disease of podocytes or podocytopathy, characterized by massive podocyte foot process effacement, microvillous transformation and podocytopenia as revealed by electron microscopy of the diseased glomeruli 73 . In an effort to understand if the anti-proteinuric activity of ACTH observed in steroid resistant MCD and FSGS is attributable to a possible melanocortin mediated podocyte protection, ACTH gel therapy was performed in rats subtotal nephorectomy 33 , a standard model of postadaptive FSGS (classic variant) and progressive chronic kidney disease 74 . After subtotal renal ablation, rats received ACTH gel treatment every other day for 5 weeks. ACTH markedly diminished proteinuria (over 50% reduction in 24 hour urine protein excretion) and significantly preserved kidney function as measured by increased renal plasma flow, improved inulin clearance rate and lowered serum creatinine levels. Histologically, glomerulosclerosis and tubulointerstitial fibrosis, renal inflammation, tubular atrophy, and tubular epithelial to mesenchymal transdifferentiation were all reduced after ACTH therapy. Of note, ACTH had no significant effects on mean arterial pressure or kidney-to-body weight ratio, suggesting that the effect of ACTH is less likely due to hemodynamic regulatory activities or anti-hypertrophic mechanisms. Because glucocorticoid therapy exacerbates proteinuria and glomerulosclerosis in this model 75 , steroidogenesis is als

The MCRs are prototypical G protein coupled receptors 18 . Upon activation by melanocortins, MCRs will activate the two principal signal transduction pathways involving MCRs: cAMP signal pathway and phosphatidylinositol signal pathway 79 . These two signal pathways will ignite many other downstream signaling cascades that are responsible for various cellular effects, ultimately leading to renoprotection and remission of proteinuria. 1. Anti-apoptosis and pro-survival activities Evidence suggests that the cAMP signaling pathway triggered by the MCR, in particular MC1R, interacts and cross-talks with the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway 80 – 83 , which is a key signaling cascade that relays pro-survival/anti-apoptotic signals 84 . Podocytes have been proved to express MCRs. In cultured murine podocytes expressing MC1R, selective agonist of MC1R strongly triggered cAMP signaling pathway 40 . Thus MCR agonists like ACTH and α-MSH might have a direct anti-apoptosis/pro-survival effect on podocytes and protect podocytes from death through the crosstalk between the MCR/cAMP signaling pathway and the MAPK/ERK pathway ( Figure 3 ). This mechanism presumably explains why ACTH treatment had a direct podocyte protective effect and prevented podocytopenia in animal models of postadaptive FSGS 33 .

G protein signaling ignited by MCRs, prototypical GPCR, is closely involved in fast acting cellular responses, including cytoskeleton rearrangement and cell shape changes. Activation of MC1R has been shown to regulate the activities of multiple cytoskeleton regulatory signaling transducers, including Rac, Cdc42 and Rho through the cAMP-PKA signaling pathway 95 , 96 . MC1R-cAMP-PKA signaling enhances the activity of GTP-binding protein Rac, quickly resulting in altered lamellipodia and filopodia formation and increased cellular arborization and dendritogenesis 95 ( Figure 3 ). This effect might protect the podocytes from cytoskeleton dysorganization induced by various immune or non-immune mediated injuries, and might also account for the improvement in podocyte foot process effacement and podocyte shape changes observed after ACTH treatment in animal models of postadaptive FSGS 33 .

The steroidogenic melanocortin peptide ACTH has been labeled to potentially generate steroid like side effects and incur cushingoid symptoms, including visceral obesity, hyperglycemia, osteoporosis, avascular osteonecrosis and more. However, multifaceted evidence indicates that ACTH as compared with glucocorticoids might play a distinct or even opposing role in the development of some of these side effects. It is known that glucocorticoid treatment or overproduction of endogenous cortisol could reduce bone mineral density and result in osteoporosis 99 . However, patients with adrenal Cushing’s syndrome, where ACTH levels are suppressed, experience greater bone loss than those with pituitary Cushing’s with high serum ACTH levels 100 . Likewise, patients with familial glucocorticoid deficiency with elevated ACTH levels, due to hypothalamic feedback, have a higher bone mass than age-matched controls 101 . Taken together, these findings are consistent with an anabolic effect of ACTH, which seemingly counteracts the bone loss due to cortisol. Lately, through an animal model of glucocorticoid-induced osteonecrosis, it is confirmed that ACTH treatment actually protected from methylprednisolone induced avascular osteonecrosis of femoral head 102 . And this effect was attributed to the ACTH promoted maintenance and regeneration of fine vascular networks surrounding the highly remodeling bone 102 . In addition, hyperglycemia is another common side effect of glucocorticoid therapy due to impaired insulin production 103 or insulin resistance 104 . By contrast, ACTH has been demonstrated to have opposing effects and prominently induce β cell production of insulin 105 – 107 . In actuality, natural ACTH possess a potent β-cell tropic and insulinogenic activity, which has been proved to reside in the C-terminal part ACTH(22–39) of ACTH 51 . Consistently, in a recent prospective randomized trial presented as a conference abstract, 18 patients with advanced diabetic nephropathy were treated with low doses of ACTH gel (16 or 32 IU daily subcutaneous injection) for 6 months. The average 24 hour urine protein excretion was significantly reduced from 7.8 to 2.3 gram, associated with stabilized kidney function and no deterioration of diabetes. Only 2 of 18 patients (11%) required reduction in ACTH dose secondary to hyperglycemia 108 , suggesting that ACTH therapy, totally different from glucocorticoid therapy, is safe for patients with diabetes, although large-scale trials with long-term follow-up are required to validate this finding. Collectively, the steroid like side effects or Cushingoid symptoms seem to be mild at the clinical doses of ACTH. It is also conceivable that clinical use of non-steroidogenic melanocortins such as α-MSH or its synthetic analogues should be able to avoid these steroid like side effects.

Other possible side effects of melanocortins include skin pigmentation and increased risk of melanoma due to MC1R activation 114 – 116 . Therefore, melanocortin based therapy should be avoided in patients with current or even antecedent malignant melanoma. Additional side effects may include anorexia, mood changes, sleep disorders and behavioral disturbances due to activation of MC3R and MC4R in the central nervous system 18 . Seborrheic dermatitis and acne vulgaris are also possible concerns due to over-function of sebaceous glands following activation of MC5R on sebocytes. 117 Pan MCR agonists may potentially increase libido and activate the yawning, stretching and erection reflexes due to activation of MC4R in central nervous system 118 .