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Brazilian Journal of Nephrology

Print version ISSN 0101-2800On-line version ISSN 2175-8239

J. Bras. Nefrol. vol.40 no.4 São Paulo Out./Dec. 2018  Epub Aug 16, 2018 

Original Articles

Inflammation and kidney injury attenuated by prior intake of Brazil nuts in the process of ischemia and reperfusion

Maria Fernanda Ribeiro Cury1 

Estéfany Queiroz Olivares1 

Renata Correia Garcias1 

Giovana Queda Toledo1 

Natassia Alberici Anselmo1 

Leticia Colombo Paskakulis1 

Fernanda Fortuci Resende Botelho1 

Natiele Zanardo Carvalho1 

Analice Andreoli da Silva1 

Camila Agren1 

Carla Patrícia Carlos1

1Laboratório de Pesquisa Experimental, FACERES Faculdade de Medicina, São José do Rio Preto, SP, Brasil.



Ischemia and reperfusion (IR) is a process inherent to the procedures involved in the transplantation of organs that causes inflammation, cell death and cell injury, and may lead to rejection of the graft. It is possible that the anti-inflammatory properties of the Brazil nuts (BN) can mitigate the renal injury caused by IR.


To investigate whether the previous intake of BN reduces the expression of markers of inflammation, injury, and cell death after renal IR.


Male Wistar rats were distributed into six groups (N = 6/group): SHAM (control), SHAM treated with 75 or 150 mg of BN, IR, and IR treated with 75 or 150 mg of BN. The IR procedure consisted of right nephrectomy and occlusion of the left renal artery with a non-traumatic vascular clamp for 30 min. BN was given daily from day 1 to 7 before surgery (SHAM or IR), and maintained until sacrifice (48 h after surgery). Inflammation was evaluated by renal expression of COX-2 and TGF-β, injury by the expression of vimentin, and cell death by apoptosis through caspase-3 expression (immunohistochemistry).


Pretreatment with 75 mg of BN reduced renal expression of the COX-2, TGF-β, vimentin, and caspase-3. The dose of 150 mg caused increased expression of COX-2.


In experimental IR, the damage can be minimized with a prior low-dose intake of BN, improving inflammation, injury, and cell death.

Keywords: Ischemia; Reperfusion; Acute Kidney Injury; Inflammation; Bertholletia; Rats


Ischemia and reperfusion (IR) is a major cause of acute renal failure and graft rejection. This condition occurs during the procedures involved in the transplantation of organs, and the damages in kidney are associated with reactive oxygen species produced after blood reperfusion. A cascade of cellular responses leads to inflammation, cell death and organ failure.1-4 Therefore, the understanding of the mechanisms involved in this injury is essential to minimize the consequences of the procedures involved in renal transplantation. Thus, our study proposed an investigation of the protective action of Brazil nut (BN), Bertholettia excelsa, in inflammation and cell death caused by acute renal injury during experimental IR in rats.

It is known that the regular consumption of BN improves the lipid profile and microvascular function, and reduces oxidative stress in obese teens,5 an effect also observed in subjects with metabolic syndrome,6 reducing the atherogenic risk in obese women, with increased activity of glutathione-peroxidase.7 Healthy volunteers taking BN present reduced inflammation markers, such as IL-1 (interleukin 1), IL-6, TNF-α (tumor necrosis factor alpha), and IFN-γ (gamma interferon),8 and improved lipid profile for a period exceeding 30 days.9 Thus, it is clear that BN has a protective effect in diseases related to oxidative stress and inflammation. Also, this benefit may be related to its bioactive compounds selenium, tocopherol, phenolic compounds, folate, magnesium, and mono/polyunsaturated fatty acids.10-11

Given the increased risk of mortality due to acute renal failure and the fact that the anti-inflammatory activity of BN have been poorly explored in IR-induced kidney injury, the aim of the present study was to investigate whether the previous intake of this nut reduces the expression of the markers of inflammation, injury, and cell death after renal IR.



All procedures performed in this study were in accordance with the ethical standards approved by the Committee for Animal Experiments and the Ethics Committee of FACERES School of Medicine (approval number 001/2015).


Male Wistar rats (200-220 g) were randomly distributed into six groups (N = 6/group): SHAM (control), SHAM treated with 75 or 150 mg of BN (SHAM+BN), IR, and IR treated with 75 or 150 mg of BN (IR+BN). The animals were housed under a 12:12 h light-dark cycle and allowed access to food and water ad libitum.

The IR procedure consisted of right nephrectomy and occlusion of the left renal artery with a non-traumatic vascular clamp for 30 min under anesthesia (xylazine 10 mg/Kg + ketamine 85 mg/Kg).12-14

BN (75 or 150 mg/animal, Belém do Pará, Brazil) were given daily and individually from day 1 to 7 before surgery (SHAM or IR) and maintained until animal sacrifice (48 h after surgery). The doses were selected according to previous studies of humans who ingested nuts in doses without nephrotoxic and hepatotoxic effects.5-9 The amount of BN was adjusted daily according to the weight of the animal. Each rat was allocated to a single cage, and BN was offered separate from the feed. As it is very palatable, the nut had no rejection and was consumed almost immediately by the animals, even in the postoperative period.

Animals from the SHAM groups were submitted to the same anesthesia and surgical procedures described above but without the renal artery clamped. The animals were euthanized 48 h after reperfusion under overdose of anesthetic.

At the end of the surgery, all the animals were given tramadol 2 mg/Kg by gavage for postoperative pain control, kept in individual cages, and received food and water ad libitum for 48 hours. Nut intake was kept, according to the group, until the moment of sacrifice.


After 2 days of IR or SHAM procedure, the rats were euthanized by an overdose of the anesthetic (thiopental 100 mg/kg) and renal tissue samples were collected, fixed in a 4% paraformaldehyde in PBS 0.1 M (pH 7.4) for 24 h at 4°C, and embedded in paraffin. The study was performed by immunohistochemistry method as described before15-16 with immunoperoxidase reaction. The tissue fragments were incubated overnight at 4°C with primary antibodies. The inflammation was studied using anti-COX-2 (1:500, ab62331, ABCAM, Cambridge, UK) and anti-TGF-β (1:80, SC-7892 policlonal, Santa Cruz, CA, USA) markers. The markers for apoptosis and injury were anti-caspase-3 (1:1000, 9662, Cell Signaling Technology, Danvers, MA, USA) and anti-vimentin (1:500, M0725, Dako, Denmark). Twenty-five fields of the renal juxtamedullary region were evaluated in one slide from each animal to obtain an average of the scores performed as previously described.14-16


The data were previously subjected to descriptive analysis and determination of normality using the Kolmogorov-Smirnov test. We applied analysis of variance (ANOVA), followed by the Tukey's post-hoc test for multiple comparisons of samples with a normal distribution. The Kruskal-Wallis test followed by Dunn's test was used for samples with a non-normal distribution. A p value ≤ 0.05 was considered significant.



The animals of the IR group exhibited increased expression of caspase-3 (Figure 1 D/H) compared with IR+BN75 (Fig.1 E/H). A similar result was observed for the expression of vimentin (Fig. 1 L/O), compared to IR groups treated with 75 (Fig. 1 M/O) or 150 mg of BN (Fig. 1 N/O).

Figure 1 Injury and cell death in renal tissue after ischemia and reperfusion (IR) and pretreatment with Brazil nuts (BN): expression of caspase-3 (A-H) and vimentin (I-O). IR group shows increased expression of caspase-3 (black arrows, D/H) and vimentin (L/O) in the tubules and interstitium. The pretreatment with 75 mg (M/O) and 150 mg (N/O) reduced the cellular injury. [L] Detail of vimentin expression in the IR group. Red arrows indicate vimentin expression normally displayed in the blood vessels and glomerulus. Cell death was also mitigated by the BN dose of 75 mg (E/H), but not with the 150 mg dose (F/H). [G] Control reaction (CR). [H/O] Average score. Counterstain: Hematoxylin. Bars: 100 µm. *p<0.05, IR vs. IR+BN75; +p<0.05 IR vs. IR+BN150 (ANOVA + Tukey's post-test). Data presented as medians, quartiles 25-75%, minimum and maximum values. 


The IR groups presented elevation in COX-2 (Figure 2 D/H)) compared with SHAM group (Fig. 2 A/H); this marker was higher in the IR group treated with 150 mg of BN (Fig. 2 F/H) compared with all groups. The expression of TGF-β was higher in the IR group (Fig. 2 L/O) compared to IR groups treated with 75 (Fig. 2 M/O) or 150 mg of BN (Fig. 2 N/O).

Figure 2 Inflammation in the renal tissue after ischemia and reperfusion (IR) and pretreatment with Brazil nuts (BN): expression of COX-2 (A-H) and TGF-β (I-O). Treatment with 150 mg of BN (F/H) 7 days before IR surgery increased the expression of cyclooxygenase-2 (COX-2) compared to other groups. The IR group had increased expression of COX-2 (D/H) and TGF-β (L/O), but treatment with 75 mg (E/H and M/O) mitigated this effect. [G] Control reaction (CR). [H/O] Average score. Counterstain: Hematoxylin. Bars: 100 µm. #p<0.05, IR vs. SHAM, and *p<0.05, IR+BN150 vs. all groups (Kruskal-Wallis + Dunn's post-test); +p<0.05 IR vs. IR+BN75 and IR+BN150 (ANOVA + Tukey's post-test). Data presented as medians, quartiles 25-75%, minimum and maximum values. 


IR-induced renal injury causes the release of reactive oxygen species (ROS), proinflammatory mediators, and adhesion molecules, and leukocyte recruitment; together, these processes induce kidney dysfunction and mortality.1,4,17-18 In a previous study, we verified that treatment with 75 mg of BN seven days before the IR process attenuated the deleterious effects of IR on renal function, such as reduction of plasmatic urea and proteinuria, and increased creatinine clearance and urine volume. These improvements are related to the inhibition of macrophage infiltration and oxidative stress. However, kidney injury was not different among the IR groups treated or not with 75 mg and 150 mg of BN.14 Thus, in the present study, we tested whether the intake of this nut reduces the expression of the markers of kidney inflammation, injury, and cell death after renal IR.

We studied the expression of vimentin in juxtamedullary region of IR kidney rats. Under physiologic conditions, renal vimentin is found in the arterial smooth muscle cells and in the glomerulus, but not in the tubular cells.15-16,19 Kidney injury induces changes in the vimentin expression. Renal epithelial tubular cells may modify their phenotype, adopting characteristics of mesenquimal cells such as fibroblasts, which are involved in extracellular matrix deposition and fibrosis development. This process is called epithelial-mesenchymal transition, or EMT,20-21 and is mediated by TGF-β1.20-23 EMT has been described in IR, and may contribute to the genesis of late fibrosis observed in this condition.24-27 Vimentin is useful as marker of injury and EMT development, and change in its expression pattern indicates proximal tubular injury.15-16,19,28 In our study, consistent with previously published data,24-27,29-30 IR-induced enhancement of vimentin expression in the renal tubule and interstitium in the juxtamedullary region was observed, suggesting proximal tubule cell damage. Both treatments, 75 and 150 mg of BN seven days before the IR procedure, reduced the tubular injury with lower expression of vimentin. The changes attenuated by BN corroborate the protective role of the nut observed previously.14

As reported before, IR condition causes elevation of kidney macrophage influx.14 Here, we verified that IR caused elevation in COX-2 expression, confirming kidney inflammation. One of the cytokines released by the macrophages is the TGF-β,31 and its release may be induced by the IR process,24,26,30 as was confirmed in this study. TGF-β is largely involved in the development of interstitial fibrosis and progression of chronic kidney disease.24,26,31-32 As reported, it is related to the EMT process.20-23 Kidney macrophage influx14 and TGF-β expression after IR procedure are reduced by previous treatment with both BN doses of 75 and 150 mg. These results are corroborated by the anti-inflammatory action demonstrated by studies of BN consumption by healthy humans8 and by renal disease patients33-35, and indicate that low intake of BN is a promising alternative during IR procedure. It would be interesting to verify its effect on the progression of chronic kidney disease. However, the dosage of 150 mg of BN elevated the kidney COX-2 expression after IR, which confirms the findings reported earlier that the highest dose of BN is harmful to the IR procedure.14

In addition to the inflammatory mediators, we studied the kidney cell death by apoptosis after IR and BN treatment by checking the expression of caspase-3. The kidneys of the IR group presented elevation of apoptosis in accordance with other authors.4,36-37 The IR process induces nitric oxide expression in tubule cells generating ROS14,36, which cause renal tubule cell injury by oxidation of proteins, peroxidation of lipids, damage to DNA, and induction of apoptosis.1,4,36 As proposed by Devarajan,36 there is evidence that apoptosis is the main mechanism of early tubule cell death in contemporary clinical acute renal failure, and the inhibition of apoptosis and inflammation at this stage may represent a powerful therapeutic approach. In fact, our study demonstrates that the previous treatment with 75 mg of BN reduces the caspase-3 expression in kidneys after IR, confirming its protective effect against cell death.

Thus, our study shows that BN exerts a beneficial effect on renal injury and inflammation and improves the function harmed by the IR as demonstrated previously.14

However, unlike treatment with 75 mg of BN, the kidney COX-2 expression was higher in the BN150 group. Corroborating these data, the kidney function of IR animals got worse with the intake of 150 mg of BN. The plasmatic creatinine, urea, and phosphorus were elevated compared to the other groups.14 As explained, this effect may be related to elevated phosphorus and amino acids content of the BN.11 Electrolytes disorders are commonly encountered in kidney diseases, and nutritional support is frequently needed38 such as the low intake of protein.39-40 Therefore, a higher consumption of nuts could harm this nutritional management. In fact, the ingestion of only one Brazil nut per day is recommended for hemodialysis patients to get the anti-inflammatory and antioxidant protective effect.33 Our study reinforces this daily maximum recommendation of nuts for patients with kidney diseases. Moreover, it indicates a possible pro-inflammatory effect of 150 mg of BN. Thus, the mechanism involved in this pro-inflammatory effect needs to be better investigated. In addition, it would be interesting to verify the effect of BN in chronic kidney disease to address the protective effect on renal injury caused by ischemia and reperfusion.


In summary, the results point to a beneficial effect of a prior intake of a low dose of BN on kidney damage caused by the IR procedure, improving inflammation, injury, and cell death.


This project was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, 2015/25616-0 and 2017/07138-9).


1 Kinsey GR, Li L, Okusa MD. Inflammation in acute kidney injury. Nephron Exp Nephrol 2008;109:e102-7. [ Links ]

2 Rubinstein I, Abassi Z, Milman F, Ovcharenko E, Coleman R, Winaver J, et al. Hyperbaric oxygen treatment improves GFR in rats with ischaemia/reperfusion renal injury: a possible role for the antioxidant/oxidant balance in the ischaemic kidney. Nephrol Dial Transplant 2009;24:428-36. [ Links ]

3 Kleikers PW, Wingler K, Hermans JJ, Diebold I, Altenhöfer S, Radermacher KA, et al. NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury. J Mol Med (Berl) 2012;90:1391-1406. [ Links ]

4 Malek M, Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J Renal Inj Prev 2015;4:20-7. [ Links ]

5 Maranhão PA, Kraemer-Aguiar LG, de Oliveira CL, Kuschnir MC, Vieira YR, Souza MG, et al. Brazil nuts intake improves lipid profile, oxidative stress and microvascular function in obese adolescents: a randomized controlled trial. Nutr Metab (Lond) 2011;8:32. [ Links ]

6 López-Uriarte P, Nogués R, Saez G, Bulló M, Romeu M, Masana L, et al. Effect of nut consumption on oxidative stress and the endothelial function in metabolic syndrome. Clin Nutr 2010;29:373-80. [ Links ]

7 Cominetti C, de Bortoli MC, Garrido AB Jr, Cozzolino SM. Brazilian nut consumption improves selenium status and glutathione peroxidase activity and reduces atherogenic risk in obese women. Nutr Res 2012;32:403-7. [ Links ]

8 Colpo E, Dalton D A Vilanova C, Reetz LG, Duarte MM, Farias IL, Meinerz DF, et al. Brazilian nut consumption by healthy volunteers improves inflammatory parameters. Nutrition 2014;30:459-65. [ Links ]

9 Colpo E, Vilanova CD, Brenner Reetz LG, Medeiros Frescura Duarte MM, Farias IL, Irineu Muller E, et al. A single consumption of high amounts of the Brazil nuts improves lipid profile of healthy volunteers. J Nutr Metab 2013;2013:653185. DOI: ]

10 Ros E. Nuts and novel biomarkers of cardiovascular disease. Am J Clin Nutr 2009;89:1649S-56S. [ Links ]

11 Tabela Brasileira de Composição de Alimentos - TACO. 4ª ed. Campinas: NEPA/Universidade Estadual de Campinas (UNICAMP); 2011 [cited 2018 Jun 20]. Available from: ]

12 Pereira BJ, Castro I, Burdmann EA, Malheiros DM, Yu L. Effects of sirolimus alone or in combination with cyclosporine A on renal ischemia/reperfusion injury. Braz J Med Biol Res 2010;43:737-44. [ Links ]

13 Facio FN Jr, Sena AA, Araújo LP, Mendes GE, Castro I, Luz MA, et al. Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats. J Mol Med (Berl) 2011;89:51-63. [ Links ]

14 Anselmo NA, Paskakulis LC, Garcias RC, Botelho FFR, Toledo GQ, Cury MFR, et al. Prior intake of Brazil nut attenuates renal injury induced by ischemia and reperfusion. J Bras Nefrol 2018;40(1):10-17 [ Links ]

15 Coimbra TM, Janssen U, Gröne HJ, Ostendorf T, Kunter U, Schmidt H, et al. Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 2000;57:167-82. [ Links ]

16 Carlos CP, Mendes GE, Miquelin AR, Luz MA, da Silva CG, van Rooijen N, et al. Macrophage depletion attenuates chronic cyclosporine A nephrotoxicity. Transplantation 2010;89:1362-70. [ Links ]

17 Versteilen AM, Blaauw N, Di Maggio F, Groeneveld AB, Sipkema P, Musters RJ, et al. ρ-Kinase inhibition reduces early microvascular leukocyte accumulation in the rat kidney following ischemia-reperfusion injury: roles of nitric oxide and blood flow. Nephron Exp Nephrol 2011;118:e79-86. [ Links ]

18 Wan X, Huang WJ, Chen W, Xie HG, Wei P, Chen X, et al. IL-10 deficiency increases renal ischemia-reperfusion injury. Nephron Exp Nephrol 2014;128:37-45. [ Links ]

19 Gröne HJ, Weber K, Gröne E, Helmchen U, Osborn M. Coexpression of keratin and vimentin in damaged and regenerating tubular epithelia of the kidney. Am J Pathol 1987;129:1-8. [ Links ]

20 Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest 2003;112:1776-84. [ Links ]

21 Liu Y. Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 2004;15:1-12. [ Links ]

22 Slattery C, Campbell E, McMorrow T, Ryan MP. Cyclosporine A-induced renal fibrosis: a role for epithelial-mesenchymal transition. Am J Pathol 2005;167:395-407. [ Links ]

23 McMorrow T, Gaffney MM, Slattery C, Campbell E, Ryan MP. Cyclosporine A induced epithelial-mesenchymal transition in human renal proximal tubular epithelial cells. Nephrol Dial Transplant 2005;20:2215-25. [ Links ]

24 Favreau F, Thuillier R, Cau J, Milin S, Manguy E, Mauco G. Anti-thrombin therapy during warm ischemia and cold preservation prevents chronic kidney graft fibrosis in a DCD model. Am J Transplant 2010;10:30-9. [ Links ]

25 Lan R, Geng H, Polichnowski AJ, Singha PK, Saikumar P, McEwen DG, et al. PTEN loss defines a TGF-β-induced tubule phenotype of failed differentiation and JNK signaling during renal fibrosis. Am J Physiol Renal Physiol 2012;302:F1210-23. [ Links ]

26 Wang M, Weng X, Guo J, Chen Z, Jiang G, Liu X. Metformin alleviated EMT and fibrosis after renal ischemia-reperfusion injury in rats. Ren Fail 2016;38:614-21. [ Links ]

27 Zeng F, Miyazawa T, Kloepfer LA, Harris RC. ErbB4 deletion accelerates renal fibrosis following renal injury. Am J Physiol Renal Physiol 2018;314:F773-87. [ Links ]

28 Floege J, Alpers CE, Sage EH, Pritzl P, Gordon K, Johnson RJ, et al. Markers of complement-dependent and complement-independent glomerular visceral epithelial cell injury in vivo. Expression of antiadhesive proteins and cytoskeletal changes. Lab Invest 1992;67:486-97. [ Links ]

29 Zhang Y, Li Q, Liu D, Huang Q, Cai G, Cui S, et al. GDF11 improves tubular regeneration after acute kidney injury in elderly mice. Sci Rep 2016;6:34624. [ Links ]

30 Abassi Z, Hamoud S, Hassan A, Khamaysi I, Nativ O, Heyman SN, et al. Involvement of heparanase in the pathogenesis of acute kidney injury: nephroprotective effect of PG545. Oncotarget 2017;8:34191-204. [ Links ]

31 Burdmann EA, Bennett WM. Nephrotoxicity of calcineurin and mTOR inhibitors. In: De Broe MF, Porter GA, eds. Clinical Nephrotoxins - Renal Injury from Drugs and Chemicals. New York: Springer US; 2008. p. 617-82. [ Links ]

32 López-Hernández FJ, López-Novoa JM. Role of TGF-β in chronic kidney disease: an integration of tubular, glomerular and vascular effects. Cell Tissue Res 2012;347:141-54. [ Links ]

33 Stockler-Pinto MB, Mafra D, Moraes C, Lobo J, Boaventura GT, Farage NE, et al. Brazil nut (Bertholletia excelsa, H.B.K.) improves oxidative stress and inflammation biomarkers in hemodialysis patients. Biol Trace Elem Res 2014;158:105-12. [ Links ]

34 Stockler-Pinto MB, Malm O, Moraes C, Farage NE, Silva WS, Cozzolino SM, et al. A follow-up study of the chronic kidney disease patients treated with Brazil nut: focus on inflammation and oxidative stress. Biol Trace Elem Res 2015;163:67-72. [ Links ]

35 Cardozo LF, Stockler-Pinto MB, Mafra D. Brazil nut consumption modulates Nrf2 expression in hemodialysis patients: A pilot study. Mol Nutr Food Res 2016;60:1719-24. [ Links ]

36 Devarajan P. Update on mechanisms of ischemic acute kidney injury. Clin J Am Soc Nephrol 2006;17:1503-20. [ Links ]

37 Liu F, Ni W, Zhang J, Wang G, Li F, Ren W. Administration of Curcumin Protects Kidney Tubules Against Renal Ischemia-Reperfusion Injury (RIRI) by Modulating Nitric Oxide (NO) Signaling Pathway. Cell Physiol Biochem 2017;44:401-11. [ Links ]

38 Langston C. Managing Fluid and Electrolyte Disorders in Kidney Disease. Vet Clin North Am Small Anim Pract 2017;47:471-90. [ Links ]

39 Robertson LT, Treviño-Villarreal JH, Mejia P, Grondin Y, Harputlugil E, Hine C, et al. Protein and Calorie Restriction Contribute Additively to Protection from Renal Ischemia Reperfusion Injury Partly via Leptin Reduction in Male Mice. J Nutr 2015;145:1717-27. [ Links ]

40 Shah SR, Tunio SA, Arshad MH, Moazzam Z, Noorani K, Feroze AM, et al. Acute Kidney Injury Recognition and Management: A Review of the Literature and Current Evidence. Glob J Health Sci 2015;8:120-4. [ Links ]

Received: January 07, 2018; Accepted: June 11, 2018

Correspondence to: Carla Patrícia Carlos. E-mail:

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