Reno-protective Effects Of Neutral CB1 Receptor Antagonists AM6545 And AM4113 On Metabolic Syndrome

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PART Ⅱ: Interference with TGFβ1-Mediated Inflammation and Fibrosis Underlies Reno-Protective Effects of the CB1 Receptor Neutral Antagonists AM6545 and AM4113 in a Rat Model of Metabolic Syndrome

Basma G.Eid， Thikryat Neamatallah， Abeer Hanafy， Hany M.El-Bassossy & et al.

Abstract

The role of cannabinoid receptors in nephropathy is gaining much attention. This study investigated the effects of two neutral CB1 receptor antagonists, AM6545 and AM4113, on nephropathy associated with metabolic syndrome (MetS). MetS(Metabolic syndrome) were induced in rats by high-fructose high-salt feeding for 12 weeks. AM6545, the peripheral silent antagonist, and AM4113, the central neutral antagonist were administered in the last 4 weeks. At the end of the study, blood and urine samples were collected for biochemical analyses while the kidneys were excised for histopathological investigation and transforming growth factor-beta 1(TGFβ1) measurement. MetS(Metabolic syndrome) were associated with deteriorated kidney function as indicated by the elevated proteinuria and albumin excretion rate. Both compounds equally inhibited the elevated proteinuria and albumin excretion rate while having no effect on creatinine clearance and blood pressure. In addition, AM6545 and AM4113 alleviated the observed swelling and inflammatory cells infiltration in different kidney structures. Moreover, AM6545 and AM4113 alleviated the observed histopathological alterations in the kidney structure of Met rats. MetS(Metabolic syndrome) were associated with a ten-fold increase in urine uric acid while both compounds blocked this increase. Furthermore, AM6545 and AM4113 completely prevented the collagen deposition and the elevated expression of the TGF1 seen in MetS(Metabolic syndrome) animals. In conclusion, AM6545 and AM4113, possess reno-protective effects by interfering with TGFβ1-mediated renal inflammation and fibrosis, via peripheral action.

Keywords: metabolic syndrome; cannabinoids; AM6545; AM4113; kidney; rats

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3. Discussion

The development of nephropathy is a serious complication of MetS(Metabolic syndrome) that is currently increasing worldwide. AM6545 and AM4113 are both neutral antagonists for the CB1 receptor, which has been shown to be hyper-activated in MetS(Metabolic syndrome) [27]. In this study, we have examined the effect of these drugs on MetS(Metabolic syndrome)-induced nephropathy. The increased risk of developing kidney disease in MetS(Metabolic syndrome) has been well documented [6-8]. We have recently demonstrated that eight weeks of high-fructose and high-salt loading in Wistar rats was sufficient to induce a state of MetS(Metabolic syndrome) manifested by obesity, hyperinsulinemia, hyperuricemia and dyslipidemia [28]. Furthermore, we have demonstrated that pre-treatment of these animals with either AM6545 or AM4113 caused an alleviation of the developed metabolic syndrome. This was demonstrated by inhibition of insulin resistance, decreased bodyweight, anti-dyslipidemic, anti-hyperuricemic as well as anti-inflammatory actions. We have demonstrated that the area under the curve of the oral glucose tolerance test following an oral challenge of glucose was higher in the metabolic syndrome group relative to the controls. Effects on blood glucose levels were ameliorated after either AM6545 or AM4113 administration [28]. Moreover, studies by members of our group have shown that both AM6545 and AM4113 reduce food intake in rodents and thus can be useful in the treatment of obesity [22,29].

The high-fructose high-salt model of MetS(Metabolic syndrome) was shown to induce morphological changes in the kidneys and marked elevation in renal injury markers [10]. In the current study, we have found that MetS(Metabolic syndrome) rats had an increased endocannabinoid tone as shown by the elevated anandamide and 2-AG amounts in the kidney. Therefore, we hypothesized that using the CB1 antagonists AM6545 and AM4113 could relieve the enhanced tone and ameliorate kidney functional parameters. Although metabolic syndrome rats had significantly higher blood pressures compared with control rats, the administration of neither AM6545 nor AM4113 had no further effect on blood pressure in the present model. This is consistent with a recently published study by members of our group, which reported that AM6545 did not cause any change in blood pressure when administered to streptozotocin-induced diabetic mice [30]. Some studies have reported that blocking CB1 receptors systemically improved blood pressure in hypertensive, insulin resistant and obese rats [31,32]. However, the effects of AM6545 and AM4113 on blood pressure and the cardiovascular system are an area requiring further investigation. The findings of our study and of Barutta et al. (2018)suggest that AM6545 and AM4113 may be producing their effects on the kidney independently of blood pressure [18].

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In the present study, assessment of kidney morphology revealed that untreated MetS(Metabolic syndrome) rats displayed glomerular, tubular, and stromal injuries in the renal cortex. In accordance, Alderson et al. (2004)attributed the enlarged glomeruli which were found in our study to structural and functional adaptations. This eventually leads to hyperfiltration in the nephrons and is commonly reported in focal segmental glomerulosclerosis [33]. Additionally, there were several glomeruli that displayed atrophy and enlarged capsular space. This is in accordance with other studies, which have classified this glomerulus as shrunken and sclerotic [34].

This study also looked at the effect on the PCT, particularly the tubular cells. This is where the fructose transporters (GLUT-2 and GLUT-5) are found as well as the location in which the enzyme keto kinase is expressed. Ketokinase has been reported as a major enzyme in fructose metabolism [35]. The dilation of the tubules and cytoplasmic vacuolation were consistent with previous findings, which found that chronic tubulointerstitial nephropathy leads to the formation of cysts and dilatation of the cortex tubules [34]. Perhaps the infiltration of cells reported in this study represents a mechanism by which the kidney defends itself and rapidly removes necrotic tissues. In addition, there is a halt in the circulation due to congestion, which ultimately increases the blood capillaries' permeability causing red blood cell extravasation. Furthermore, fructose is known to cause inflammation in the rat renal tissues as well as to stimulate inflammation in the endothelium[36].

In the current study, there was an excess presence of collagen fibers in the MetS(Metabolic syndrome) rats. This is in agreement with other findings which suggest that collagen fiber accumulation is present in patients having protein urea. They also reported that this is classically seen in patients with glomerulosclerosis and could lead to glomerular degeneration. Furthermore, it was reported that very few glomerular capillaries were found in sclerotic lesions, with collagen deposition and accumulation of mesangial matrix [37]. CB1 receptor activation was previously shown to affect renal fibrogenesis [38]. Consistent with this notion, MetS(Metabolic syndrome) rats in the current study exhibited tissue fibrosis in the renal stroma relative to control rats (Figure 4). This was further explained by the significant elevation in TGFβ1 levels(Figure 5). TGFβ1 is an inflammatory and profibrogenic cytokine, which is involved in the pathological process of kidney fibrosis [39,40]. Importantly, treatment of MetS(Metabolic syndrome) rats with AM6545 and AM4113 significantly reduced renal TGFβ1 levels, indicating that CB1 could be a possible downstream mediator of TGFβ1 signaling. Collectively, the reduction in TGFβ1 is tied with reduced inflammatory responses reversing the fibrotic responses and morphological renal changes discussed above, A correlation was found between AER and levels of TGFβ1 in the kidney in untreated animals and in animals treated with AM6545 but not AM4113. This suggests that AM4113 could be acting slightly differently than AM6545.

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Another significant finding is that treatment of MetS(Metabolic syndrome) rats with AM6545 and AM4113 significantly improved renal function via the reduction in renal fibrosis. This included a significant reduction in urinary uric acid excretion, albumin excretion rate and urinary protein excretion. These findings are in accordance with previous studies, indicating that CB1 blockade with rimonabant had significantly improved renal functional parameters in obese fa/fa Zucker rats [14]. Briefly, Janiak et al. (2007) showed that long-term rimonabant administration significantly improves creatinine clearance, slows proteinuria development and lowers glomerular and tubular lesions severity. In the present study, we report that AM6545 and AM4113 inhibited the elevated proteinuria and albumin excretion rate while having no effect on creatinine clearance.

Alternatively, the therapeutic effects of AM6545 and AM4113 antagonists in renal function might also result from the correction of the underlined MetS(Metabolic syndrome) disorder. Several reports have demonstrated that dyslipidemia, including high triglycerides and high serum cholesterol levels, may contribute to kidney disease [41,42]. In our previous study, CB1 blockade with both antagonists produced a marked decrease in serum cholesterol and triglycerides, serum insulin, body weight, serum uric acid and liver TNFα together with increased adiponectin levels in the same MetS(Metabolic syndrome) rat model [28]. Therefore, these beneficial effects could indirectly be preserving renal function. In the future, it would be interesting to examine whether these CB1 antagonists could be therapeutically useful in kidney disease unrelated to MetS(Metabolic syndrome).

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4. Conclusions

In summary, the CB1 receptor neutral antagonists, AM6545 and AM4113, inhibited the raised proteinuria and albumin excretion rate without affecting creatinine clearance or blood pressure. We have shown that blockade of the CB1 receptor with both drugs reversed the histopathological changes induced by high-fructose high-salt feeding in renal tissue. Furthermore, both drugs produced a significant decrease in the renal pro-inflammatory cytokine TGFβ1. Collectively, these results indicate that AM6545 and AM4113 have renal protective effects in this MetS(Metabolic syndrome) animal model, raising hope for their future potential therapeutic intervention in a variety of kidney diseases.

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