In the present study, we showed that açai extract (EA), rich in polyphenols, reduced cell proliferation, NO levels and ROS production in MiMCs treated with high glucose. In contrast, EA in these cells was able to increase the expression of proteins from the antioxidant defense system and reduce inflammatory factors. To our knowledge, this is the first study to use açai as a nonpharmacological way to control the deleterious effects of high glucose in mesangial cells.
Açai has been studied due to its high content of anthocyanins and flavonoids; some studies have shown beneficial effects of açai in some experimental models of diseases. In vitro and in vivo studies showed that açai has antioxidant, anti-inflammatory, antiproliferative and antitumorigenic properties, as well as pro-apoptotic and cholesterol lowering action [24, 39, 40]. The benefits of açai on health are already partially proven, and this antioxidant effect was also found in other fruits and vegetables ; Pacheco-Palencia et al.  showed that polyphenolic extracts of various fruits and vegetables have an inhibitory effect on cell proliferation.
Açai, in addition to being known for its antioxidant potential, has approximately 2.4% protein and 5.9% lipids; monounsaturated oleic acid is the primary fat (56.2%), palmitic acid (24.1%) and linoleic acid (12.5%) [20, 43]. Anthocyanins are generally considered the main contributors to the antioxidant activity of the pulp; the pulp has 48.6 µmol Trolox equivalents/ml of antioxidant capacity. In addition, other phenolic compounds are present in the fruit, such as 3-glycosides of anthocyanin, ferulic acid, epicatechin, p-hydroxybenzoic acid, gallic acid, protocatechuic acid, catechin, ellagic acid, vanillic acid, and p-coumaric acid. and gallotannins , which confers its high polyphenolic content.
Similar to other fruits, such as cranberries, blueberries, and strawberries, açai has a high antioxidant capacity. A study evaluated the total polyphenol content (w/w% GAE) and antioxidant activity (DPPH, IC50; g/mL) of crude berry fruit extracts rich in polyphenols and anthocyanins, such as blackberry (6.8% /1968.6), black raspberry (4.1% /2865.9), blueberry (9.8% /381.1), cranberry (7.7% /392.6), red raspberry (6.4%/ 268.5), and strawberry with 3.8% of total polyphenols .
Another study with carcinogenic cells treated with açai hydroalcoholic extract in three doses (10, 20, or 40 µg/mL) for 24, 48 or 72 h did not show cytotoxicity in 24 or 48 h , corroborating our data, once we did not observe cytotoxicity after treatment with EA, in MiMC at these periods of time.
High glucose induces cell proliferation and collagen synthesis through mechanisms involving the increase in ROS and transforming growth factor beta 1 (TGF-β1) in mesangial cells . Liu et al  found increased proliferation of rat mesangial cells in HG medium compared to NG. In our study, exposure to high glucose also promoted cell proliferation, independent of osmolarity (as shown in the mannitol group), and increased ROS generation; these effects were suppressed by EA, suggesting that ROS are important mediators of high glucose-induced mesangial cell activation.
Song et al  treated mesangial cells with high glucose medium and delphinidin (an anthocyanin) and obtained similar results, in which anthocyanin reduced cell proliferation, ROS generation, suppressed NOX-1 mRNA, and mitochondrial superoxide. Hogan et al  also showed an inhibitory effect of açai extract (50, 100 or 200 µg/mL) on the proliferation of C6 rat brain glioma cells, showing that the higher the dose, the greater the reduction of cell proliferation. The açai extract was recently reported as a dose-dependent inhibitor of proliferation in HT-29 colon carcinoma cells. The polyphenol fractions of açai pulp have shown a reduction in leukemic HL-60 cell proliferation by activating caspase-3, inducing cell apoptosis .
Our results demonstrated that high glucose generated oxidative stress through increased ROS production and extracellular and intracellular measured NO overproduction in MiMC, probably via NF-κB and iNOS stimuli. El Remessy et al  suggested that NO generation can be stimulated by an increase in glucose levels. Hyperglycemia, via glucose oxidation, can stimulate the production of ROS, including the superoxide anion, which reacts with NO to form peroxynitrite, a highly cytotoxic nitrogen reactive species. By increasing intracellular oxidative stress, AGEs activate the transcription factor NF-κB, promoting the upregulation of various target genes controlled by this factor and increasing NO production, which is believed to be a mediator of β-cell damage . Other researchers showed that exposure of mouse mesangial cells to HG (25 mM) stimulated iNOS gene and protein expression and enhanced NO synthesis ; similar data were shown in our study, demonstrating in turn that EA reduced the inflammatory stimuli in 72 h.
The activation of the NF-κB pathway may trigger pro- or anti-apoptotic cascades, predominantly pro-apoptotic cascades . Inhibition of this pathway protects pancreatic β-cells from cytokine-induced apoptosis in vitro and in vivo and can be a potential strategy for protecting these cells . High glucose generates ROS in mesangial cells and upregulates NF-κB, as shown by the elevation of the p-NF-κB/NF-κB ratio. NF-κB-dependent pathways play an important role in the infiltration of macrophages and kidney damage . In addition, a study showed that NF-κB is also regulated by angiotensin II; this regulates cell proliferation, apoptosis, fibrosis and the inflammatory response through NF-κB-dependent pathways . Furthermore, açai pulp extracts rich in anthocyanins showed a reduction in oxidative stress and inflammation via inhibiting iNOS, cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinase (p38-MAPK), TNFα and NF-κB, attenuating the release of extracellular NO .
TNF-α, an inflammatory cytokine, activates the proinflammatory signaling pathway of NF-κB . There are two pathways for NF-κB activation: the classic pathway (regulated by activation IkB kinase β - IKKβ), which is triggered by toll-like receptors (TLRs) and proinflammatory cytokines such as TNF-α and interleukin-1, leading to activation of RelA , and the alternative pathway that is activated by lymphotoxin β, resulting in activation of the RelB/p52 complex but not TNF-α. Another study showed that supplementation with anthocyanins inhibited NF-κB activation induced by lipopolysaccharides (LPS) in monocyte culture and further reduced TNF-α in healthy volunteers . Our results showed a significant increase in the expression of NO, iNOS, TNF-α and the p-NF-κB/NF-κB ratio in the HG group, characterizing the activation of inflammatory mediators; however, all these markers were reduced after treatment with EA, possibly via NF-κB inactivation.
In our study, EA increased the expression of catalase, both cytosolic Cu-Zn-SOD (SOD-1) and mitochondrial Mn-SOD (SOD-2), which are regulated by Nrf2 activation. Under normal conditions, Nrf2 is linked with Keap1 (the protein in the cytoplasm), but under oxidative and inflammatory conditions, Nrf2 decouples from Keap1 and translocates to the nucleus, where it activates the expression of several cytoprotective proteins and enzyme genes, increasing cellular survival. Therefore, Keap1-Nrf2-ARE signaling plays an important role in maintaining the cellular redox balance .
Açai modulated ROS production by neutrophils in the liver of rats with diabetes and increased the antioxidant defense system . In another study, açai reduced the levels of malondialdehyde and carbonyl protein in hypertensive rats, recovered the levels of SOD, CAT and GPx and the expression of SOD-1 and SOD-2, preventing vascular remodeling; these findings suggest that açai produced an antihypertensive effect and prevented endothelial dysfunction and vascular structural alterations . Additionally, an anthocyanin extract study showed an increase in antioxidant defense system enzymes (SOD, CAT and GPx) providing protection against H2O2 and glucose toxicity in β-pancreatic cells , corroborating our study.
Cyanidin-3-glucoside, a component present in açai, reduced light-induced retinal oxidative stress by activating the Nrf2 antioxidant pathway and attenuated the expression of inflammation-related genes by suppressing NF-κB activation in an animal experimental model . In another study, rats fed a açai-enriched diet presented Nrf2 modulation and ubiquitin-proteasomal pathway regulation in the hippocampus and frontal cortex, resulting in benefits to cognitive function during brain aging . Our data agree with these studies, since EA treatment promoted an upregulation of Nrf2, resulting in a significant increase in all antioxidants, suggesting that açai was able to modulate the Nrf2 antioxidant response in conditions of increased oxidative stress and inflammation via NF-κB activation.
Consistent with previous reports [21, 65], we found that oxidative stress was elevated in DN. In the present study, the oxidative damage assessed by TBARS was markedly increased in the DM group, and NO and ROS production was elevated in the HG group. EA significantly reduced these oxidative stress markers, indicating an important antioxidant effect that could contribute to renal protection.
Due to hyperglycemia, the reabsorption of excess filtrate leads to renal overload, triggering podocyte detachment, death of epithelial and mesangial cells and intense production of the ECM, which can cause glomerular sclerosis and contribute to the progression of DN . In our study, alterations in parameters of renal function, such as elevated plasma creatinine and urea, in association with proteinuria characterized DN; however, the decline in renal function was prevented by EA, which was observed through the reduction of histological markings in the DMEA group.
Açai extract protected the kidneys of spontaneously hypertensive rats with STZ-induced diabetes, reducing albuminuria excretion, serum levels of urea and creatinine and renal fibrosis, evidenced by decreased TGF-β and collagen IV . In another study, açai preserved kidney morphology and function in hypertensive rats with chronic ischemic renal injury (2K1C), preventing albuminuria and increasing serum urea and creatinine levels, contributing to the reduction of glomerular damage ; these findings agree with our research, since we observed a significant improvement in renal function in diabetic animals that consumed EA. As a limitation of this study, we think that the creatinine and urea levels in urine would need to be determined in these animals.
Our findings allow us to conclude that EA was beneficial to delay diabetic kidney disease, as observed by the preservation of both renal function and morphology, suggesting that the mechanisms underlying these effects of EA may involve the reduction of cell proliferation, oxidative stress, ROS, NO, lipid peroxidation and inflammatory mediators via NF-κB inactivation and upregulation of the Nrf2 antioxidant response pathway.