Nutrition profile and potency of RGD motif in protein hydrolysate of green peas as an antifibrosis in chronic kidney disease

Document Type : Original Article

Authors

1 Faculty of Medicine, Universitas Kristen Maranatha, Jalan Prof. Drg. Suria Sumantri 65 Bandung 40163, Indonesia

2 Research Center for Molecular Biotechnology and Bioinformatics, Universitas Padjadjaran, Jalan Singaperbangsa No. 2, Bandung 40133, Indonesia

3 Faculty of Medicine, Universitas Jenderal Achmad Yani, Jalan Terusan Jenderal Sudirman, Cibeber, Kec. Cimahi Selatan, Cimahi 40531, Indonesia

Abstract

Objective(s): Fibrosis is the major cause of chronic kidney injury and the primary etiology in diabetic glomerulosclerosis. The initial study of protein hydrolysate of green peas hydrolyzed by bromelain (PHGPB) considered it to improve kidney function parameters and showed no fibrosis in histopathology features in gentamicin-induced nephrotoxicity rats. In the current study, we aimed to assess the nutrition profile and potency of RGD in PHGPB as antifibrosis in chronic kidney disease (CKD).
Materials and Methods: Green peas (Pisum sativum) were hydrolyzed by bromelain from pineapple juice to obtain PHGPB. The amino acid content of PHGPB was measured using the UPLC method, while the primary structure used LC-MS/MS. Bioinformatic analysis was conducted using the Protease Specificity Predictive Server (PROSPER). The potency of RGD in PHGPB was characterized by determining the levels of Fibronectin (FN) and TGF-β1 in mesangial SV40 MES 13 cell lines of diabetic glomerulosclerosis.
Results: The level of lysine was 364.85 mg/l. The LC-MS/MS data showed two proteins with 4–15 kDa molecular weight originated from convicilin (P13915 and P13919) which were predicted by PROSPER proteolytic cleavage, resulted in RGD in the LERGDT sequence peptide. PHGPB increased SV40 MES 13 mesangial cell proliferation that died from high-glucose levels (diabetic glomerulosclerosis model). PHGPB and RGD reduced the levels of FN and TGF-β1 in mesangial cell lines of diabetic glomerulosclerosis.
Conclusion: The nutrition profile and RGD motif in PHGPB show great potential as antifibrosis in CKD.

Keywords


1. Pozzi A, Zent R. Integrin in kidney disease. J Am Soc Nephrol 2013; 24:1034–1039.
2. Coresh J, Selvin E, Stevens LA. Prevalence of chronic kidney disease in the United States. JAMA 2017; 298: 2038-2047.
3. Mundel P. Cell biology and pathology of podocytes. Ann Rev Phys 2012; 74:299-323.
4. Reiser J, Sever S. Podocyte biology and pathogenesis kidney disease.  Ann Rev Med 2013; 64:357-366.
5. Ma LJ, Yang H, Gaspert A, Carlesso G, Barty MB, Davidson JM, et al. Transforming growth factor-beta-dependent and -independent pathways of induction of tubulointerstitial fibrosis in beta6 (−/−) mice. Am J Pathol 2003; 163:1261-1273.
6. Jin DK, Fish AJ, Wayner EA, Mauer M. Distribution of integrin subunits in human diabetic kidneys. J Am Soc Nephrol 1996; 7:2636-2645.
7. Nishimura SL. Integrin-mediated transforming growth factor-beta activation, a potential therapeutic target in fibrogenic disorders. Am J Pathol 2009; 175:1362–1370.
8. Sureshbabu A, Muhsin SA, Choi ME. TGF-beta signaling in the kidney: pro-fibrotic and protective effects. AJP-Ren Physiol 2016; 310:596–606.
9. Henderson NC, Arnold TD, Katamura Y, Giacomini MM, Rodriguez JD, McCarty JH, et al. Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nature Med 2013; 19:1617-1624.
10. Guyton AC, Hall JE. The body fluids and kidneys. Guyton and Hall Textbook of Medical Physiology 2020. Elsevier, Philadelphia P.A. 19103-2899.
11. Pradnja D. Normal Physiology of Kidneys 2021. Dissertation of Post-Doc. Aarhus University.
12. Anthea M, Hopkins J, McLaughlin CW, Johnson S, Warner MQ; LaHart D, et al. 1993. Human Biology and Health. Englewood Cliffs, New Jersey, USA: Prentice Hall.
13. Sherwood, L. Human Physiology: From cells to systems. Human Physiology, Yolanda Cossio 2010. Cencage Learning, Boston MA 02210, USA.
14. Prairie NP. The cardio-renal effect of pea protein hydrolysate in a chronic kidney disease rat model. 2011. Thesis. Department of Human Nutritional Sciences. The University of Manitoba, Winnipeg.
15. Krefting J. The appeal of pea protein. J Ren Nutr 2017; 27:31-33.
16. Chauveau P, Koppe L, Combe C, Lasseur C, Trolonge S, Aparicio M. Vegetarian diets and chronic kidney disease. Nephrol Dial Trans 2019; 34:199–207.
17. Stewart D, Rose SD, Strombom AJ. A plant-based diet prevents and treats chronic kidney disease. JOJ Uro Nephron 2019; 6:49-64.
18. Joshi S, Shah S, Kalantar-Zadeh K. Adequacy of plant-based proteins in chronic kidney disease. J Ren Nutr 2018; 29:112-117.
19. Hidayat M, Prahastuti S, Riyani DU, Suliska N, Garmana A, Soemardji AA, et al. Therapeutic potential of peptides derived from the bromelain hydrolysis of green peas protein. Iran J Basic Med Sci 2019; 22:1016-1025.
20. Hidayat M, Prahastuti S, Wahyudianingsih R, Wargasetia TL, Ferdinand V, Soemardji AA, et al. Role of pea protein hydrolysates as anti-nephrotoxicity. J Pharm Res Sci 2019; 8:55-60.
21. Merrifield R. Solid phase peptide synthesis: the Synthesis of tetrapeptide. J Am Chem Soc 1963; 85:2149-2154.
22. Maharani R, Hidayat AT, Sabana IR, Firdausi A, Aqmarina A, Kurnia DY, et al. Synthesis, anti-oxidant activity, and structure–activity relationship of SCAP1 analogues. Int J Pep Res Ther 2020; 27:17-23.
23. Sabana IR, Naufal M, Wiani I, Zainuddin A, Hidayat AT, Harneti D, et al. Synthesis of anti-oxidant peptide SCAP1 (Leu-Ala-Asn-Ala-Lys). Egypt J Chem 2020; 63:921–926.
24. Harlow E, Lane D. Bradford Assay. Cold Spring Harbor Protocols, 1999. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
25. Hale LP, Greer PK, Trinh CT, James C. Proteinase activity and stability of natural bromelain preparations. Int Imm Pharmacol 2005; 5:783–793.
26. Kunitz M. Crystalline desoxyribonuclease: isolation and general properties spectrophotometric method for the measurement of desoxyribonuclease activity. J Gen Physiol 1950; 33:349-363.
27. Hidayat M,  Universitas Kristen Maranatha. Copyright pembuatan dan pengujian Protein Hidrolisat Kacang Polong Hijau untuk Terapi Perbaikan Fungsi Ginjal. Preparation and Measurement of Green Peas Hydrolysate Protein for Therapy to Improve Kidney Function. Directorate General of Intellectual Property, Republic of Indonesia 2018.
28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.
29. Yesmine BH, Antoine B, da Silva Ortência Leocádia NG. Identification of ACE inhibitory cryptides in Tilapia protein hydrolysate by UPLC–MS/MS coupled to database analysis. J Chrom B 2017; 1052:43-50.
30. Rohman A, Gandjar IG. Chapter III in Metode Kromatografi untuk Analisis Makanan. 2007. Pustaka Pelajar Publisher. Yogyakarta, Indonesia.
31. Schaafsma G. The protein digestibility-corrected amino acid score. J Nutr 2000; 130:1-3.
32. Song J, Tan H, Perry AJ, Akutsu T, Webb GI, Whisstock JC, et al. Prosper: an integrated feature-based tool for predicting protease substrate cleavage sites. PLos One 2012; 7:1-23.
33. Widowati W, Widyanto RM, Laksmitawati DR, Erawijantari PP, Wijaya L, Ferry Sandra F. Phytochemical, free radical scavenging and cytotoxic assay of Cucumis melo L extract and β-carotene. J Adv Agric Tech 2015; 2:114–119.
34. Widowati W, Wijaya, L, Murti H, Widyastuti H, Agustina D,  Laksmitawati DR,  et al. Conditioned medium from normoxia (WJMSCs-norCM) and hypoxia treated WJMSCs (WJMSCs-hypoCM) in inhibiting cancer cell proliferation. Biomark Gen Med 2015; 7:8–17.
35. Prahastuti S, Hidayat M, Hasiana ST, Widowati W, Amalia A, Qodariah RL, et al. Ethanol extract of jati belanda (Guazuma ulmifolia L.) as therapy for chronic kidney disease in in vitro model. J Rep Pharm Sci 2019; 8:229–235.
36. Walther C, Döring K, Schmidtke M. Comparative in vitro analysis of inhibition of rhinovirus and influenza virus replication by mucoactive secretolytic agents and plant extracts. BMC Compl Med Ther 2020; 20:380-392.
37. Pankov R, Yamada KM. Non-radioactive quantification of fibronectin matrix assembly. Current protocols in cell biology. Chapter 10, Unit 10.13. 2004. John Wiley & Sons: Hoboken, NJ, USA.
38. Liu Y, Lu S, Zhang Y, Wang XD, Kong F, Liu Y, et al. Role of caveolae in high glucose and TGF-β1 induced fibronectin production in rat mesangial cells. Int J Clin Exp Pathol 2014; 7:8381-8390.
39. Universitas Kristen Maranatha, Meilinah Hidayat. Patent P00201907647. Preparation and Assesment of Green Peas protein hydrolyzate (Pisum sativum) as Antifibrosis for the Therapy of Chronic Kidney Disease. Directorate General of Intellectual Property, Ministry of Law and Human Rights, Republic of Indonesia.
40. Huisman J, Heinz Th, Van Der Poel AB, Van Leeuwen, Souffrant WB, Verstegen MWA. True protein digestibility and amounts of endogenous protein measured with the 15N dilution technique in piglets fed on peas (pisum sativum) and common beans (phaseolus vulgaris). Br J Nutr 1992; 68:101-110.
41. Chewcharat A, Takkavatakarn K, Wongrattanagorn S, Kittikulsnam P, Eiam-Ong S, Susantitaphong P, et al. The effects of restricted protein diet supplemented with ketoanalogue on renal function, blood pressure, nutritional status, and chronic kidney disease-mineral and bone disorder in chronic kidney disease patients: a systematic review and meta-analysis. J Ren Nutr 2019; 30:30291-30292.
42. Ko GJ, Obi Y, Tortoricci AR, Kalantar-Zadeh K. Dietary protein intake and chronic kidney disease. Curr Op Clin Nutr Met Care 2017; 20:77–85.
43. Kabi F. Clinical Trials.gov. ClinicalTrials.gov Identifier: NCT03077048. Short-term Metabolic Effects of Ketosteril® Supplemented Low Protein Diet in Pre-dialysis Chronic Kidney Disease (CKD) Patients. 2020. U.S. National Library of Medicine.
44.  Tzekov VD, Tilkian EE, Pandeva SM, Nikolov DG, Kumchev EP, Manev EI, et al. Low protein diet and ketosteril in predialysis patients with renal failure. Folia Med 2000; 42:34-37.
45.  Milovanova SY, Milovanov YS, Taranova M, Dobrosmyslov IA. Effects of keto/amino acids and a low-protein diet on the nutritional status of patients with Stages 3B-4 chronic kidney disease. Terapevticheskiĭ arkhiv 2017; 89:30-33.
46. Kalantar-Zadeh K, Kramer H.M, Fouque D. High-protein diet is bad for kidney health: unleashing the taboo. Nephrol Dial Trans 2019; 35:1–4.
47. Merck Sharp and Dohme Corp Patents.: Compositions Methods for Treating Chronic Kidney Disease. Patent US20120003201A1 2016.
48.  Miller LM. The development of small molecule inhibitors for fibrosis drug discovery. Awarding body: University of Strathclyde. Thesis (PhD) 2016. British Library.
49. Goligorsky MS, Dibona GF. Pathogenetic role of Arg-Gly-Asp recognizing integrins in acute renal failure. Proc Natl Acad Sci USA 1993; 90:5700-5704.
50. Noiri E, Gailit J, Sheth D, Magazine H, Gurrath M, Mulle G, et al. Cyclic RGD peptide ameliorates Ischemic Acute Renal Failure in Rats. Kidney Intl 1994; 46:1050-1058.
51. Goligorsky MS, Noiri E, Kessler H, Romanov V. Therapeutic potential of RGD peptides in acute renal injury. Kidney Intl 1997; 51:1487-1492.
52. García AJ, Schwarzbauer JE, Boettiger D. Distinct activation states of α5β1 integrin show differential binding to RGD and synergy domains of fibronectin. Biochemistry 2002; 41:9063-9069.
53. Okuda S, Languino LR, Ruoslahti E, Border WA. Elevated expression of transforming growth factor-beta and proteoglycan production in experimental glomerulonephritis possible role in expansion of the mesangial extracellular matrix. J Clin Invest 1990; 86:453-462.
54  Wang Q, Wang Y, Minto A.W, Wang J, Shi Q, Li X, et al. MicroRNA-377 is up regulated and can lead to increased fibronectin production in diabetic nephropathy. FASEB J 2008; 22:4126-4135.
55. Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-β1: the master regulator of fibrosis. Nat Rev Nephrol 2016; 12:325–338.
56.  Shi ML, Zhu J, Wang R, Chen X, Mi L, Walz T, et al. Latent TGF-β structure and activation. Nature 2011; 474:343–349.
57. Hidayat M, Witjaksono AO, Natalia J. Green peas protein hydrolysate as functional food for kidney disease, antifibrotic effects and safety aspects. Lambert Academic Publishing: Mauritius 2020.