Obesity is perceived as both an initiator and accelerator of CKD (Othman, Kawar, & El Nahas, 2009). Interventions to prevent the development and/or progression of CKD to end‐stage renal disease have great potential to save lives as well as decrease health care‐associated costs, specifically with the increased prevalence of obesity and its co‐morbidities (Zandi‐Nejad & Brenner, 2005). Nonetheless, existing data suggest that the incidence of CKD differs significantly across different global regions, which have different prevalence of obesity, indicating that obesity is an important risk factor for CKD (Chalmers, Kaskel, & Bamgbola, 2006; Hallan et al., 2006). Consequently, the search for novel therapeutic targets/agents against obesity‐induced CKD has a crucial significance.

Obesity may cause deleterious effects on the kidney via inducing two main molecular signalling pathways. The first modulator, which was identified by us as a crucial player in renal lipotoxicity, is the CB₁ receptor (Drori et al., 2019; Udi et al., 2017). Its overactivation in the kidney during obesity may lead to chronic inflammatory responses and alter the normal functioning parenchyma to exhibit more fibrogenic characteristics. These alternations impair RPTC function and, consequently, may lead to kidney dysfunction. Interestingly, this cascade of events is attributed to a halted beta‐oxidation signalling pathway governed by the LKB1/AMPK/ACC signalling pathway (Udi et al., 2017). Moreover, activating CB₁ receptors in RPTCs results in a parallel signalling pathway that involves recruiting the canonical fission protein, dynamin‐related protein 1, to the mitochondria to induce mitochondrial fragmentation, reduced oxygen consumption and ATP production, elevated ROS and cellular lactate levels, and a decline in mitochondrial biogenesis (Drori et al., 2019), contributing to renal tubular injury associated with lipotoxicity.

The second obesity‐related regulator that may affect the kidney is the iNOS signalling pathway, which is well known to promote renal dysfunction and fibrosis (Correia‐Costa et al., 2016) via producing the free radical NO by iNOS (Ruggiero, Ehrenshaft, Cleland, & Stadler, 2011). In fact, obesity provokes elevated oxidative stress by up‐regulating beta‐oxidation (Marseglia et al., 2014) and decreasing mitochondrial content (Jeon et al., 2012). Moreover, this oxidative stress is mainly derived from the production of NO by iNOS, which makes this enzyme to be considered as a fundamental perpetrator of autotoxicity (Bogdan, 1998).

An up‐regulation in the expression of both regulators in the kidney under diabetic conditions was reported previously (Barutta et al., 2010; Barutta et al., 2014; Chang et al., 2004; Cosenzi et al., 2002; Jenkin, McAinch, Zhang, Kelly, & Hryciw, 2015; Jourdan et al., 2014; Lim et al., 2011; Lim & Park, 2012; Nam et al., 2012; Slyvka et al., 2016; Veelken et al., 2000), and our current findings support these observations. However, we report here, for the first time, a strong up‐regulation of both CB₁ receptors and iNOS in kidney samples from patients with FSGS and ORG, suggesting their pathogenic role in these CKDs. These findings in ORG patients are supported by our recent study demonstrating increased eCB “tone” in DIO mice (Udi et al., 2017) and by the study by Kang and colleagues (Kang et al., 2015) indicating that expression of two genes involved in anandamide degradation (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1400 and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1402) are significantly reduced in renal samples collected from CKD patients, whereas https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=3084, a key enzyme implicated in the biosynthesis of anandamide (Liu et al., 2006), is increased. Taken together, these changes could result in a marked CKD‐associated increase in eCB “tone” in humans as well.

Orally available peripherally restricted CB₁ receptor antagonists , which are potentially able to ameliorate the deleterious effects of obesity, via the first signalling mechanism previously mentioned, have been preclinically tested (Tam et al., 2010; Tam et al., 2012) and have been further developed into the clinic (Chorvat, 2013). However, most iNOS inhibitors, targeting the second signalling mechanism, lack oral bioavailability. Therefore, obesity‐induced kidney fibrosis, oxidative stress, and dysfunction currently have no effective antifibrotic therapy approved for clinical use. By using a novel, orally bioavailable, dual‐targeted drug, MRI‐1867, which can simultaneously inhibit CB₁ receptors and iNOS in peripheral organs (Cinar et al., 2016), we demonstrated here, for the first time, its beneficial effect in mitigating obesity‐induced CKD. This molecule does not penetrate the brain and shows negligible occupancy of CB₁ receptors in the CNS, as shown by PET analysis, leading to no central CB₁ receptor‐associated side effects (Cinar et al., 2016). In fact, this novel molecule holds promise in blocking both CB₁ receptor and iNOS signalling in the kidney. For instance, with regard to CB₁ receptors, its peripherally restricted inhibition creates a “positive” metabolic shift in DIO mice by decreasing body mass and food intake, as well as enhancing total energy expenditure and fat oxidation. These general metabolic effects are in accordance with a previous work published by Tam et al. (2012), which investigated the efficacy of the well‐characterized, peripherally restricted CB₁ receptor inverse agonist, JD5037, whose structure is quite similar to MRI‐1867, and which was also generated, based on the parent compound SLV‐319 (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9234; Cinar et al., 2016). The positive effect of MRI‐1867 was further demonstrated here in restoring renal morphological and functional parameters in DIO mice. Whereas the morphological improvements in glomerulus size, Bowman's space area, and mesangial expansion, as well as the reversal of the kidney‐to‐body weight ratio and BUN, are indistinguishable from the positive effect observed in DIO mice lacking CB₁ receptors in the RPTCs (Udi et al., 2017) or in obese animals treated chronically with JD5037 tested here, the ability of MRI‐1867 to restore kidney function, assessed by measuring the urinary levels of albumin, creatinine, KIM‐1, and TIMP‐1, is most likely mediated via its dual action to block both CB₁ receptors and iNOS. The renal effects of MRI‐1867 were not replicated by pair feeding, which rules out the role of a transient reduction in food intake by MRI‐1867 as their cause. This, of course, further suggests that renal CB₁ receptors and iNOS are key modulators of nephropathy during obesity.

Notably, these improvements in DIO mice were associated with decreased inflammation and fibrosis in renal parenchyma, characterized by reduced levels of fibrogenic and proinflammatory markers such as collagen‐1, CCL2, and TIMP‐1. These modalities of MRI‐1867 are in accordance with previous studies done by Cinar and colleagues underscoring the anti‐fibrogenic role of MRI‐1867 in mitigating both liver (Cinar et al., 2016) and lung (Cinar et al., 2017) fibrosis. As MRI‐1867 induces weight loss and improves metabolic features, we could not exclude the possibility that the reversal of obesity‐induced CKD by this compound may be related to an extrinsic effect that modulates renal function indirectly and not via CB₁ receptors and/or iNOS. Nevertheless, the additive effect of reversing obesity‐induced renal oxidative stress in DIO mice and more specifically in RPTCs can be attributed to the iNOS inhibitory modality of MRI‐1867, as its chronic administration was able to completely reduce the levels of the oxidative stress marker 4‐HNE. In addition, the up‐regulation of iNOS protein expression in the kidney of DIO mice and its reversal by MRI‐1867 strongly suggest that one should consider its involvement in obesity‐induced nephropathy. In fact, its ability to normalize the fatty acid flux‐induced up‐regulation in 4‐HNE, ROS production, and PDGFa and FGF2 expression emphasizes the distinctive role of MRI‐1867 in iNOS inhibition and resolution of renal oxidative stress, independently of CB₁ receptor inhibition. Nevertheless, we could not completely rule out the obligatory role of CB₁ receptors in modulating renal oxidative stress as well, because knocking it out in RPTCs protected the cells from O:P‐induced lipid oxidation and oxidative stress. These results support our previous findings in mice lacking CB₁ receptors specifically in RPTCs, which are completely protected from the deleterious effects of HFD feeding and fatty acid flux‐induced renal dysfunction, inflammation, mitochondrial damage, oxidative stress, and fibrosis (Drori et al., 2019; Udi et al., 2017).

Previous studies have shown that adiponectin has renoprotective effects in rodent experiments and that knocking it out in mice results in microalbuminuria and elevated oxidative stress (Ohashi et al., 2007). This led us to test the involvement of adiponectin signalling in the reversal of obesity‐induced CKD by MRI‐1867. Interestingly, activation of either CB₁ receptors or iNOS, independently of each another, decreases adiponectin expression (Bensaid et al., 2003; Dallaire et al., 2008; Jeon et al., 2012) and mitigates adiponectin signalling via its receptors, Adipo1 and Adipo2 (Kabir et al., 2015; Tam et al., 2014). As reported previously (Perri et al., 2013), we were also able to identify adiponectin and its receptors within the kidney and more specifically in RPTCs. Interestingly, the dual blockade of CB₁ receptors and iNOS, achieved by MRI‐1867, ameliorated obesity‐induced down‐regulation in renal adiponectin signalling both in vivo and in vitro. Others (Rutkowski et al., 2013; Sharma et al., 2008) have reported the causative relationship of adiponectin with albuminuria and oxidative stress in the kidney, most likely to be mediated via activating the energy sensor AMPK. Our previous findings, that CB₁ receptors modulate AMPK activity to induce lipotoxicity in RPTCs and renal dysfunction (Udi et al., 2017), may link our two findings, suggesting that simultaneous targeting of CB₁ receptor and iNOS signalling by MRI‐1867 could contribute to kidney protection during obesity via the induced activity of AMPK by increasing adiponectin signalling in RPTCs (Figure 8i).

In conclusion, this study presents the beneficial effects of a novel, orally bioavailable dual‐targeted molecule, MRI‐1867, which possesses the ability to inhibit both the CB₁ receptor and iNOS signalling pathways, and it pharmacologically ameliorates obesity‐induced CKD. After the Food and Drug Administration recently approved testing clinically novel molecules that block CB₁ receptors in peripheral organs for the treatment of obesity and its co‐morbidities, one should consider further emphasizing the therapeutic benefit obtained by simultaneously acting on more than one target.