In their manuscript entitled, “Cannabinoid receptor 1 inhibition in chronic kidney disease: a new therapeutic toolbox?” the authors provide a comprehensive review of the involvement of the endocannabinoid system in metabolic and non-metabolic chronic kidney disease (CKD), focusing mainly on CB1R and the therapeutic potential of its inhibition.

Dao and Francois provide a wealth of data supporting the involvement of the endocannabinoid system (ECS) in metabolic and non-metabolic CKD. They expended their knowledge on the regulatory role of the ECS in non-metabolic CKD and specifically its main characteristics in hypertension, blood pressure, and fibrosis. Additionally, in a slightly moderate proportion they emphasize the involvement of the ECS in metabolic CKD, although most of the clinical development of CB1R antagonists/blockers is mainly focused on treating metabolic disorders.

The perspective on non-metabolic CKD reveals the contradictions and complexity of the ECS in regulating blood pressure in the kidney, which may very well have implications in the development and clinical testing of peripheral CB1R antagonists.

However, evaluating metabolic CKD through the fibrogenesis point of view is misleading. Fibrotic renal tissue usually represents advanced feature of CKD and in most cases is irreversible. The main goal in treating metabolic CKD is to reverse the metabolic aberrations (glucotoxicity, lipotoxicity and hypertension) that affect many cellular regions in the kidney and are reversible by nature. Therefore, the focus in metabolic CKD should be the ability of CB1R antagonists to attenuate metabolic abnormalities rather than fibrosis.

In addition, the topic of this manuscript emphasizes the general lack of knowledge regarding the role of CB1R in the normal kidney physiology. The extremely low expression levels of CB1R in the kidney make it hard to investigate its role using reverse transcription, rtPCR and antibodies (which their integrity is a major problem by itself). The best methodological approach would be to use genetic manipulations in which specific deletion of CB1R in a diverse cell types within the kidney is mandated, as the authors suggested by themselves.

I recommend acceptance of this article after addressing the few minor comments listed below: 1. Since the current evidence supports the potential of targeting the CB1R for the treatment of CKD, removing the question mark at the end of the title would make it a statement and not a question. 2. The sentence that is written in the abstract and section 2.1.1 saying that “in the normal kidney, CB1R is mainly expressed in the vasculature” is not fully correct. CB1R is present in many cell type within the kidney also under normal conditions. 3. The study by Udi et al.1 should be cited in Table 1 (reference 168) 4. In the introduction - SGLT2 inhibitors are authorized for CKD treatment at the US. 5. With the current view of using GLP-1 agonist for metabolic abnormalities, you may mention their beneficial effects in attenuating diabetic kidney disease2,3
6. The figures were mixed up as they appear at the end of the manuscript [Figure1 in the manuscript is Figure 3, Figure 2 in the manuscript is Figure 1 and Figure 3 in the manuscript is Figure 2] Whereas Figures 1 and 3 are excellent and highly informative, Figure 2 is a bit confusing, since the increased Na+/K+ ATPase activation in RPTCs by WIN and HP inhibiting effect are not mentioned. 7. In section 2.3 Sampaio et al.4 should be cited. More recent results in renal ischemia and reperfusion (IR) in rats or ATP-depletion in LLC-PK1 cells (IR model in cells), which implies that CB1R and CB2R activation by WIN can restores ECS reduced levels and Na+/K+ ATPase down regulation in IR model. Although this is not a CKD model, it connects the ECS to Na+ reabsorption in the proximal tubules. 8. In section 2.4 Rozenfeld et al.5 should also be cited. They suggested that CB1R has the ability to form a heterodimer with type 1 angiotensin II receptor (AT1R) and amplify its activity, which connects it to Jourdan et al.6. The latter showed that administering Losartan, an AT1R antagonist, was found to attenuate diabetic kidney disease by downregulating the expression of CB1R in podocytes. 9. Add CB2R to the title of Section 3, as half of it discusses the role of CB2R on renal fibrosis. 10. In section 3.2, is there any mechanistic insight how the elevated levels of CB1R in chronic kidney allograft dysfunction promotes collagen production? 11. Hirsch et al.7 Should be cited in section 4.2 as they also summarize the peripheral CB1R antagonists that are currently under preclinical development. 12. In section 4.2, MRI-1967 should be termed MRI-1867. 13. MRI-1867, described in Cinar et al.,8 is absent from Table 2

1 Udi, S. et al. Dual inhibition of cannabinoid CB1 receptor and inducible NOS attenuates obesity-induced chronic kidney disease. British journal of pharmacology 177, 110-127, doi:10.1111/bph.14849 (2020). 2 Greco, E. V. et al. GLP-1 Receptor Agonists and Kidney Protection. Medicina 55, doi:10.3390/medicina55060233 (2019). 3 Kristensen, S. L. et al. Cardiovascular, mortality, and kidney outcomes with GLP-1 receptor agonists in patients with type 2 diabetes: a systematic review and meta-analysis of cardiovascular outcome trials. The lancet. Diabetes & endocrinology 7, 776-785, doi:10.1016/S2213-8587(19)30249-9 (2019). 4 Sampaio, L. S. et al. Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na(+)/K(+) ATPase expression and activity in kidney proximal tubule cells. Biochemical pharmacology 154, 482-491, doi:10.1016/j.bcp.2018.06.005 (2018). 5 Rozenfeld, R. et al. AT1R-CB(1)R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II. EMBO J 30, 2350-2363, doi:10.1038/emboj.2011.139 (2011). 6 Jourdan, T. et al. Overactive cannabinoid 1 receptor in podocytes drives type 2 diabetic nephropathy. Proc Natl Acad Sci U S A 111, E5420-5428, doi:10.1073/pnas.1419901111 (2014). 7 Hirsch, S. & Tam, J. Cannabis: From a Plant That Modulates Feeding Behaviors toward Developing Selective Inhibitors of the Peripheral Endocannabinoid System for the Treatment of Obesity and Metabolic Syndrome. Toxins 11, doi:10.3390/toxins11050275 (2019). 8 Cinar, R. et al. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis. JCI insight 1, doi:10.1172/jci.insight.87336 (2016).

I have no conflicts of interest