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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CaNANObinoids (From Peripheralized to Cell- and Organelle-Targeted Medicine: The 3rd Generation of Cannabinoid-1 Receptor Antagonists for the Treatment of Chronic Kidney Disease)

Teaser

Summary

More than 347 million people worldwide live with diabetes, with about 60 million cases in Europe. In 2012, an estimated 1.5 million people died from diabetic complications. Unless a cure is found, death due to diabetes is predicated to reach double this number between 2000 and 2030. Chronic kidney disease (CKD), a distinct manifestation of diabetic renal disease that is seen in patients with both type 1 and type 2 diabetes, is also a significant epidemiologic problem and is thought to affect around 8% of the European population. A decade ago, most attention was focused on glomerular changes related to CKD. Since then, there is growing evidence that proximal tubular and mitochondrial injuries are key features in the pathogenesis of this disease. This shift in paradigms will lead to new approaches for a cure.
This ERC-funded study introduces the novel hypothesis that activation of the cannabinoid-1 receptor (CB1R)/endocannabinoid (eCB) system explicitly in renal proximal tubule cells (RPTCs) and their mitochondria may lead to kidney dysfunction and to the development of CKD. Therefore, specific blockade of CB1Rs on these cells or organelles could serve as novel therapeutic strategies to treat CKD and its comorbidities. This study tackles a complex biological question by means of three independent but complementary objectives, each bearing its own importance and interest. The merging of all three aspects will provide a complete picture of the involvement of the renal CB1R/eCB system in the pathogenesis of CKD â€“ one that cannot be attained by the pixelated view of any single piece of data.
The specific objectives are:
1. Characterize the contribution of RPTC CB1R to the pathogenesis of CKD
2. Elucidate the mechanism by which CB1R modulates mitochondrial function in RPTCs
3. Develop efficient and selective CB1R antagonists that target RPTCs and their mitochondria

Specifically, when this work is completed, we will have obtained answers to previously unresolved questions, such as: To what extent the improved metabolic effects by CB1R blockers are mediated globally, peripherally or via a specific cell type within the kidney? To what extent the observed improvements in renal damage by CB1R antagonists are mediated by a specific organelle within the renal cells? Would it be possible to use novel methodologies to directly affect cell- and organelle-specific CB1Rs? By providing answers to these questions, we stand to enrich our knowledge of central aspects of the etiology of CKD.
This research project is of great relevance for the society specifically at this time as both academic institutions and pharmaceutical companies worldwide are channelling enormous efforts to basic and clinical research to develop new strategies to combat diabetes and its comorbidities. The current research project is uniquely poised to address the molecular mechanisms underlying the role of the tubular eCB system in the development of CKD. Hence, this project will further support the rationale for the development and clinical testing of novel methodologies to block CB1Rs for the treatment of diabetes and its sequelae.

Work performed

To prove the hypothesis that proximal tubular CB1Rs are critically involved in the pathogenesis of obesity- or diabetes-induced CKD, we have utilized our novel mouse strain that lacks CB1R in the RPTCs (RPTC-CB1R-/-), and subjected the KO mice and their littermate controls to either obesity or diabetes (by maintaining the mice on a high-fat diet or injecting them with streptozotocin, respectively). In both cases, the null mice develop the same degree of obesity or diabetes as their littermate controls, BUT their kidney was completely protected from the development of dysfunction, inflammation and fibrosis.
Next, we elucidated the downstream cellular pathways involved in CB1R-induced CKD during obesity or diabetes. Our findings show that during obese conditions CB1R governs intracellular lipid accumulation in the RPTCs by modulating the LKB1/AMPK/ACC signaling pathway. On the other hand, during diabetes conditions we found that CB1R regulates Ca2+-dependent PKC-Î²1 activation, which, in turn, modulates the expression and translocation of the facilitative glucose transporter 2 (GLUT2) in RPTCs. This effect results in changes in glucose reabsorption in the kidney, which hampers kidney function.
In addition, we have found that stimulating CB1R results in mitochondrial fragmentation in RPTCs both in vivo and in vitro, and this effect is associated with the progression of obesity-induced CKD, and most likely related to the increased phosphorylation of dynamin-related protein 1 (DRP-1) that regulates mitochondrial fission. More specifically, we found that CB1R regulates the phosphorylation of DRP-1 on both S637 and S616 residues. CB1R-induced mitochondrial fission was found to be associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we also documented that exposure of RPTCs to a fatty acid flux induced CB1R-depended mitochondrial fission, lipotoxicity, and cellular dysfunction.
Taken together, we have established the first complete view of the involvement of proximal tubular CB1R in the development of CKD, and delineated the cascade of events underlying the activation of CB1R in RPTCs that lead to diabetic renal dysfunction. In addition, we have uncovered a new role for CB1R as a direct modulator of mitochondrial function in RPTCs by regulating mitochondrial shape, biogenesis integrity and membrane physiology.

Final results

Our achievements so far have paved the way for us to try to use novel methodologies to identify new molecules and/or drug delivery systems which would allow us to target the CB1R for the treatment of CKD. Therefore, we are currently trying to design novel, efficient and selective CB1R antagonists that specifically target the diseases organ or cell by utilizing two methodologies: (i) Nanotechnology (e.g. polymeric nanoparticles, liposomes, micelles), as nanoscaled drug delivery systems (DDSs). These have the ability to deliver insoluble lipophilic drugs (such as CB1R blockers), increase drug stability, and provide sustained drug release directly to the organ/cell, and (ii) Virtual Screening, the need for methods enabling faster discovery of effective drug candidates is obvious. In the current era of drug discovery process, computational methods play a vital role in the identification of novel hits, which could be optimized further into clinically useful therapeutic agents. Towards that goal, we use a computerized platform for screening and finding novel and diverse bioactive molecules that interact with the CB1R in peripheral organs. The work described above is currently undergoing and once finalizing the preparation, characterization and validation of each of the nanoscaled DDSs, and in parallel identifying novel drug candidates using the virtual screening methodology, we will test their pharmacological, behavioral and metabolic efficacy against the metabolic syndrome and its comorbidities in vitro and in vivo in our well-established cutting-edge methodologies and models.