From MC1R to MC5R, a new horizon for the podoprotective effect of melanocortin

Abstract

The successful use of ACTH in treating proteinuric glomerulopathies attached new importance to the melanocortin receptors (MCRs). While MC1R was originally identified as the receptor mediating melanocortins’ podoprotection, Chen et al.’s work in this issue highlights the role of MC5R in maintaining podocyte integrity in the face of stressors, by modulating GSK3β signaling. These findings fill in a gap about melanocortin signaling in podocytes, supporting the therapeutic potential of MC5R agonism, while prompting questions for discussion.

Melanocortin receptor family members (MC1–5Rs) are G protein-coupled receptors that can be activated by melanocortin peptide hormones including Adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormones. Depending on the expression patterns of MCRs, they either mediate skin pigmentation, CNS-related functions (food intake and sexual function), adrenal steroidogenesis in response to stress, or a variety of other functions in peripheral tissues. Therefore, with the emerging synthetic analogues of the melanocortins, MCR agonism has attracted interest as potential therapy for diverse medical conditions like obesity, and sexual and dermatological disorders.

Since 1952, an FDA-approved melanocortin peptide, ACTH, has proved effective in treating many refractory, proteinuric glomerulopathies (including steroid resistant minimal change disease, focal segmental glomerulosclerosis [FSGS], membranous nephropathy [MN], and immunoglobin A nephritis) with fewer side effects compared to steroids. This success inspired a wide interest in investigating the MCRs in mediating the steroidogenic-independent, renoprotective effects of melanocortin in kidney homeostasis. To date, two MCRs, MC1R and MC5R have been reported to play such roles in protecting against podocyte injuries. In 2010, Lindskog et al. first identified MC1R as the most abundantly expressed MCR in podocytes,¹ with upregulated expression in human podocytopathies.² In rodent models of podocytopathy (for example, Heymann nephritis of experimental MN^1,3 and puromycin aminonucleoside nephropathy [PAN]²), selective MC1R agonism was able to protect podocytes in some of these models, suggesting a role for MC1R in mediating the renoprotective effect of melanocortin. The downstream protective mechanisms described include stabilization of the actin cytoskeleton by antagonism of EGFR signaling, activation of ERK1/2- and Rho small GTPase-mediated stress fiber formation, and reductions of oxidative stress and apoptosis in podocytes. However, it was reported that ADR-induced podocytopathy could not be attenuated by MC1R agonists.³

Subsequently, the singular role of MC1R in renoprotection was challenged by the work of Gong’s group, who found that podoprotection by pan-MCR agonists in mouse models of ADR or lipopolysaccharide-elicited nephropathy was not blunted in MC1R-null mice,^4,5 indicating a “non-MC1R-mediated melanocortinergic signaling” in protecting against podocyte injury. The idea of an MC1R-independent mechanism in mediating the podoprotective effect of melanocortin was further supported by clinical studies of patients with MN or FSGS who happen to carry dominant-negative MC1R mutations; nonetheless, these patients still responded well to ACTH monotherapy.⁵ The existence of redundancy to MC1R-mediated renoprotection prompted the group to test other MCRs in podocyte homeostasis, and they found that an MC5R agonist could ameliorate proteinuria and podocyte loss in a rodent model of PAN, increasing the interest in the role of MCR5.⁶

To clarify the distinct role of MC5R in mediating melanocortin’ s effects on podocytes, here Chen et al. tested the hypothesis that MC5R by itself plays a key role in maintaining podocyte integrity and survival, specifically under conditions of immune-inflammatory (nephrotoxic serum nephrosis model, NTS) or apoptotic (Adriamycin induced nephrosis model, ADR) insults in transgenic mice with selective MC5R knockout. Furthermore, they tested the anti-proteinuric and podoprotective effects of MC5R agonism in these nephrosis models.

First, the authors demonstrated a podocyte-selective expression of MC5R and revealed a prominent reduction of MC5R in podocytes of wildtype mice subjected to ADR or NTS-induced nephrosis. The knockout of MC5R in mice caused no spontaneous podocyte defect (at least in mice as old as reported in the study) but exacerbated the ADR or NTS-induced podocyte injury, suggesting that even if MC5R is not required for podocyte survival/health, it is essential for podocytes to fight against stresses induced by ADR (ROS-induced apoptotic stresses) or NTS (immunoinflammatory stress). The proposed role of MC5R in protecting podocytes from post-stress injury is supported by similar findings in hematopoietic stem cells and skin cells, where MC5R was also found dispensable for steady-state hematopoiesis in MC5R null mice, but rather promoted cell regeneration in response to stresses by ionizing radiation.⁷ Chen et al. subsequently reported that, although diminished, the remaining MC5R in ADR or NTS-stressed podocytes was able to mediate the melanocortinergic effect of an MC5R agonist, rescuing podocyte morphology and function, and ameliorating albuminuria, in an MC5R-dependent manner.

In delineating how MC5R mediated the melanocortinergic podoprotection against ADR- and NTS-induced nephrosis, the authors found that MC5R is essential in mitigating multiple Glycogen synthase kinase-3 β (GSK3β)-dependent signal cascades underlying podocyte pathophysiology, including NFκB-driven inflammation reflected by increased MCP-1, B7–1 and Cathepsin L expression, and paxicillin-related actin remodeling and apoptosis. MC5R signaling mitigates GSK3β-driven podocyte injury by negatively regulating GSK3B activity, partially through PI3K/Akt signaling (an upstream kinase that suppresses GSK3β).

In their study, Chen et al also showed that ADR- or NTS-induced podocyte injury in MC5R null mice could be alleviated by the transient reconstitution of MC5R by hydrodynamic gene delivery to the podocytes, supporting direct podoprotection by MC5R. However, work in MC1R null mice had demonstrated that melanocortinergic podoprotection through MC1R is primarily through systemic immunomodulation via MC1R-dependent hematopoietic cell signaling that acts on immune regulatory cells. MC5R is abundantly expressed in circulating immune cells.

Accordingly, to clarify whether podoprotection by MC5R is through an immunomodulation on immune cells, Chen et al. subsequently transplanted bone-marrow-derived immune cells from wildtype mice to MD5R null mice subjected to ADR-induced nephrosis. Unlike the efficacy of hematopoietic MC1R in restoring the renoprotection of melanocortin in MC1R null mice,⁸ transfer of hematopoietic cells expressing MC5R was unable to rescue ADR nephrosis in MC5R null mice. The mechanistic distinction between MC5R and MC1R outlined in Figure 1 is further supported by additional experiments with ADR, which is a direct podocyte toxin. Expression of MC5R in podocytes exerts a renoprotective effect in ADR-induced nephrosis, while analogous MC1R expression offers no protection against ADR nephrosis.

Figure 1.

Schematic summary of MC1R and MC5R in mediating renoprotection by melanocortin in mouse models of nephrosis. MC5R and MC1R are two major melanocortin receptors expressed by both podocytes (in situ podocyte MCRs) and circulating immune cells (hematopoietic MCRs). MC5R primarily mediates the direct podocyte-protective effect of the melanocortin, because MC5R agonism or podocyte-specific genomic reconstitution of MC5R rescues nephrosis in both immune- (nephrotoxic serum) and non-immune-(Adriamycin) mediated nephrosis. MC5R agonism suppresses the activation of GSK3β that is induced after injury and the downstream signal cascades for inflammation (via NFκB), apoptosis and hypermobility of podocytes. Hematopoietic expression of MC1R primarily mediates steroidogenic-independent renoprotection through systemic immune modulation, as evidenced by the observation that an MC1R agonist was only protective in immune-mediated nephrosis models and not in models of nephropathy induced by direct podocyte toxins.

Although direct podoprotection mediated by the in situ podocyte MC5R may play a key role against nephrosis, one can put forth the argument that the dose of the cell therapy with MC5R⁺ hematopoietic cells used in this study was too low to make a difference, especially in a non-immune mediated nephrosis model like induction by ADR, instead of the NTS-induced immune-mediated nephrosis model tested in MC1R null mice. Therefore, the outcome of this work still cannot exclude a renoprotective role of MC5R through modulation of immune cells, in addition to the in-situ protection of podocytes. An optimized dosing regimen of the cell therapy in more typical immune-mediated nephrosis models will better address this question.

The study by Chen et al sparks several other interesting questions to be further investigated and discussed:

First, although this work showed a nice pattern of podocyte-specific expression of MC5R, another work showed quite a different expression pattern: only weak MC5R around the interstitial cells.⁹ MC5R was found diminished in the nephrosis models, presumably due to loss/apoptosis of podocytes; while MC1R was reported to be increased in proteinuric glomerulopathies, suggesting a function different from MC5R.¹ Therefore, interest remains in the expression of these two MCRs, especially in normal human glomeruli as well as in kidney biopsy specimens of patients with proteinuric glomerulopathy, to provide pivotal information to distinguish their functions in maintaining kidney homeostasis. Moreover, although mutations in MCRs have not been reported to directly cause kidney disease, dominant-negative mutations in MC1R were reported in rare cases of patients with both skin/hair phenotypes and glomerulopathy (MN and FSGS). Therefore, further exploration of the correlation between the MC1/5R genotype and kidney disease phenotypes may provide insights in identifying candidate patients for melanocortin treatment.

It is not unusual for either MC1R or MC5R null mice of the C57BL/6J background to show no podocytopathy phenotype, because this strain of mice is known for its resistance to developing glomerular diseases. Could these MCR null mice develop a spontaneous podocyte defect at an advanced age or could MCR1/5R null phenotypes be uncovered in another strain of mice? Alternatively, is the lack of a podocytopathy phenotype because the intact (or even upregulated) MC1R expression functionally complements the MC5R null background to maintain kidney health? Also, there has not been a study in parallel to compare the protective effect of a pan-MCR agonist on nephrosis induced in MC5R null mice, similar to what was done in the MC1R null mice. Such an experiment could clarify the degree to which MC5R alone can mediate melanocortinergic podoprotection. If such an experiment provided evidence of MC5R-independent podoprotection in this context, further studies would be warranted to clarify what unique roles these two differently expressed MCRs, MCR5 and MCR1, play and how they work reciprocally to regulate podocyte health in response to melanocortin.

Finally, despite the non-selectivity of ACTH, it is the only FDA-approved MCR-agonist for therapy of kidney disease, with efficacy and side effect profiles superior to the mainstay of steroid treatment. Although MC5RA showed promising post-stress podoprotection in preclinical research for counteracting inflammation and apoptosis-related kidney injury, the impact and safety profiles of this MC5R agonist on the global health of healthy, wildtype animals or humans have not been studied, challenging its potential for therapeutics. On the other hand, the coexpression of MC5R in immune cells and adipose tissue may expand the MC5R-based therapeutic spectrum to nephrosis secondary to autoimmune-inflammation etiologies (lupus) and even metabolic disorders like diabetes, complementing its benefit in direct podoprotection.