Studies on Molecular and Pathophysiological Mechanism of Phytate-induced Chronic Kidney Disease

김옥희 인천대학교 2014 국내박사

Phytate (myo-inositol hexakisphosphate, IP6) represents up to 80% of phosphorus in oilseed meals, nuts, legumes, and staple foods, such as cereal grains. Phytate binds essential minerals, including calcium, magnesium, and iron, with high affinity, producing indigestible mineral-phytate salts. To further understand the chemistry of phytate binding with biologically important cationic minerals, thermodynamic parameters such as the stoichiometry, affinity, and thermodynamics of these interactions were determined by isothermal titration calorimetry. The results suggest that phytate forms stable complexes with several trivalent and divalent cations such as Fe3+, Ca2+, Mg2+, and Zn2+ because of the strong negative charges on its six phosphate groups, especially showing that phytate has a strong binding affinity for multiple Ca2+-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). However, systematic studies into the effects of dietary phytate on calcium malabsorption and renal dysfunction are far from being completely understood. Thus, the long-term effects of dietary phytate on calcium homeostasis were investigated. The results show that phytate, a major but indigestible form of dietary phosphorus, caused rats to excrete excessive calcium into feces, indicating malabsorption of intestinal calcium. Calcium malabsorption triggered a dramatic increase in intact parathyroid hormone (PTH) in a time- and dose-dependent manner. High PTH levels predisposed rats to develop intratubular nephrocalcinosis, glomerular and tubulointerstitial nephritis, and acquired renal cysts. These defects accelerated renal calcium leak and serum phosphate retention, leading to secondary hyperparathyroidism in association with hypocalcemia, hyperphosphatemia, severe bone loss, and peritrabecular osteitis. Importantly, these pathological features were ameliorated by phytate-hydrolyzing enzyme, phytase, suggesting that dysregulation of calcium homeostasis is one of the major causes rather than a consequence of CKD. Thus, restriction of dietary phytate is a potential therapeutic option for controlling calcium-phosphate homeostasis in CKD. Finally, proteome and immunohistochemical analyses of kidney tissues showed that multi-phosphorylated osteopontin (OPN) was significantly increased in the kidney of phytate-fed rats. Multi-phosphorylated OPN deposits in the kidney were further activated downstream NF-B pathway, which may cause glomerular and interstitial nephritis in the kidney of phytate-fed rats. These results suggest that circulating multi-phosphorylated OPN derived from excessive bone resorption is one of the main mediators of phytate-induced nephrocalcinosis and CKD. Thus, circulating multi-phosphorylated OPN may be a unique pathological biomarker for early diagnosis of CKD, and a novel and promising therapeutic target for the patients with CKD.

The Mechanism of High Phytate-Diet on Hypertension : High Phytate – Diet의 고혈압에 대한 영향 기전

정윤신 가천의과학대학교 대학원 2011 국내석사

Phytate is known as the principal storage form of phosphorus in plant seeds and strongly binds divalent cations such as Ca2+, Fe2+, Mg2+, and Zn2+. Phytate is not hydrolyzed in monogastric animals because they lack the hydrolyzing enzyme phytase and therefore acts as an anti-nutritional factor by chelating with important divalent cations. High phytate diet in monogastric animals decreases mineral bioavailability and may cause chronic mineral deficiency. In high phytate diet animal model, calcium deficiency was observed and resulted in low level of vitamin D status followed by hypertension. Therefore this study was aimed to investigate the mechanism of how high phytate diet induces hypertension through Ca2+ deficiency. After high phytate diet for 12 weeks on Sprague Dawley rat, the analysis of plasma calcium and parathyroid hormone level clearly indicated that dietary phytate causes calcium deficiency with hyperparathyroidism on the animal model. To determine whether the phytate is involved in hypertension on animal models, this study has investigated to the level of mRNA and protein of the key enzymes in renin-angiotensin systems in normal rat and rat with dietary phytate. The results indicated that the phytate diets strongly increase the expression level of angiotensin converting enzyme (ACE and ACE2) in the rat fed with high phytate. For mechanism study, in vitro Renin and ACE promoter assay were performed with intracellular calcium enhancing drug and PTH in 293T. Upon intracellular calcium enhancing drug treatment, both Renin and ACE promoter activity were increased while vitamin D treatment decreased the promoter activity of Renin and ACE. These results strongly suggested that calcium deficiency may be one of the causes for hypertension. While further investigation is required if calcium dependent transcriptional factor regulates Renin and ACE transcriptional activity, this study provides a novel insight into link Ca2+ deficiency to hypertension. Therefore, it can be concluded that high phytate diet may induce hypertension through Ca2+ deficiency. Phytate는 곡물의 인의 주요 저장원으로 칼슘, 철, 마그네슘, 아연과 같은 2가 양이온들과 강하게 결합한다. 단위동물은 phytate 가수분해효소가 존재하지 않아 phytate 과량 섭취는 미네랄 이온의 이용성을 감소시켜 미네랄 이온 결핍을 일으킬 수 있다. Phytate 섭취 동물 모델을 통하여 혈중 칼슘결핍현상을 관찰하였고, 비타민 D 결핍과 고혈압이 나타났다. 따라서 이 연구는 phytate의 섭취가 어떻게 고혈압을 일으키는 지에 대하여 알아보는데 목적을 두었다. 12주 동안 랫드에 phytate 섭취 후, 혈액분석을 통하여 phytate 섭취 동물에서 칼슘 결핍과 함께 부갑상선항진증을 유발하는 것을 확인하였다. Phytate와 고혈압 발병과의 상관관계를 밝히기 위하여 phytate를 섭취한 랫드와 일반사료를 섭취한 랫드에서 Renin- Angiotensin 시스템에 중요한 효소의 유전자 및 단백질 발현을 조사하였다. Phytate를 섭취한 동물모델에서 고혈압 발병에 중요한 Angiotensin Converting Enzyme (ACE와 ACE2)의 유전자 및 단백질 발현을 증가시켰다. In vitro 전사활성분석을 통하여, 세포내 칼슘 결핍 약물 및 부갑상선 호르몬이 Renin과 ACE의 promoter 활성을 증가시켰으며, 비타민 D 처리에 의해서는 Renin과 ACE promoter 활성도가 감소하였다. 이 결과를 통하여 칼슘결핍이 Renin 및 ACE의 발현율을 증가시켜, 혈중 Angiotensinogen의 함량을 증가시킴으로 고혈압을 유발하는 요인으로 작용함을 암시하였다. 향후 어떠한 전사인자가 세포내 칼슘변화에 따라 Renin과 ACE의 발현조절에 대한 연구가 필요할 것으로 사료된다. 이 연구는 칼슘 결핍과 고혈압과의 상관관계를 밝히는 새로운 장을 열었다는데 의미가 있으며, 따라서 높은 함량의 phytate 섭취는 칼슘 결핍을 유발하며, 고혈압의 원인인자로 작용할 수 있음을 암시한다.

The Mechanism studies of High Phytate Diet on hypouricemia : High phytate diet 에 의해 유발되는 저요산혈증에 대한 영향 기전 연구

고선주 가천의과학대학교 일반대학원 2012 국내석사

Phytate는 칼슘, 마그네슘, 철, 그리고 아연 과 같은 2가 무기질 이온을 강하게 결합하여 복합체를 형성하는 것으로 알려져 있다. 특히 사람, 돼지, 닭, 쥐 와 같은 단위동물은 phytate 를 분해하는 효소가 존재하지 않아 phytate -무기이온 복합체를 소화 흡수 하지 못하고 분변으로 배출한다. 본 연구에서는 phytate를 함유한 사료를 섭취한 동물 모델에서의 무기이온의 흡수와 억제가 동물모델의 혈중 요산 농도에 미치는 영향에 관한 연구를 수행하였다. 동물의 혈청 생화학 분석을 통하여 무기이온 함유량을 분석한 결과, phytate 함유 사료를 섭취한 동물 모델의 혈청에서 여러 mineral중에서도 칼슘이 결핍된 것을 확인하였다. 또한 phytate 식이사료는 혈중요산이 감소한 결과를 확인하고, 혈중요산 대사에 주요한 효소인, ADA, PNP, AMPD, XDH의 유전자와 단백질 발현을 동물모델의 여러 장기를 이용하여 분석하였다. 그 결과, phytate 함유 사료를 섭취한 Rat 모델의 소장에서 Adenosine deaminase(ADA)와 Purine nucleoside phosphorylase(PNP)의 발현이 증가하고 혈중요산의 생성에 주요한 XDH의 발현이 감소한 것을 확인하였다. 또한 Immunohistochemistry 기법을 이용하여, Xanthine dehydrogenase(XDH)의 발현이 감소한 것을 확인하였다. 혈중 요산 대사에 중요한 ADA, PNP 그리고 XDH가 유전자발현을 조절하는 인자를 규명하기 위하여 이들 유전자의 promoter luciferase assay를 이용하여 연구를 수행한 결과 칼슘 결핍이 ADA와 PNP의 유전자 발현은 증가시키는 반면, XDH 유전자 발현을 감소시킴을 확인하였다. 따라서 Uric acid pathway의 최종산물인 uric acid의 생성이 억제됨을 확인하였다. 결과를 종합하면 phytate 식이 사료를 섭취한 동물모델의 혈중 칼슘결핍은 동물모델의 혈중요산의 농도를 저하시켜 동물모델의 자가면역이 활성화되어 Hypouricemia가 유발되었음을 확인하였고 kidney의 병변이 악화 되는 것을 확인하였다. Phytate is an anti-nutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. The insolubility of these phytate salts is a major cause of reduced bioavailability of minerals in monogastric animals, such as humans, pigs, poultry, and fish. Monogastric animals secrete insufficient quantities of enzymes into their digestive tracts to fully hydrolyze phytate salts and instead eliminate the mineral phytate compounds in their feces. The loss of phytate salts contributes to the anti-nutritional impact of phytate and may result in mineral deficiencies in monogastric animals whose staple diet includes foods with high phytate content. In high-phytate diet animal model, calcium and iron deficiency were observed and resulted in low level of vitamin D status as well as iron deficiency anemia followed by hypouricemia (low level of blood uric acid). Therefore, this study was aimed to investigate the mechanism of high phytate diet-induced hypouricemia through mineral deficiency. To determine whether the phytate causes hypouricemia on the animal model, this study has performed the analysis of mRNA and protein expression of the enzymes that are involved in uric acid metabolism at the animal tissues of liver, kidney, and small intestine by qRT-PCR and Western blot/Immunohistochemistry. The results indicated that high-phytate diets strongly increase the expression level of adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP) but dramatically decrease the expression level of Xanthine oxidase (XDH). For mechanisms study, in vitro ADA, PNP, and XDH promoter assay were performed with intracellular calcium enhancing drug. Upon intracellular calcium enhancing drug treatment, ADA and PNP promoter activity were increase while XDH promoter activity was dramatically decreased. The results strongly suggested that high-phytate diet induced mineral deficiency may one of the causes for blood hypouricemia. While further investigation is required what kinds of transcriptional factor regulates ADA, PNP and XDH gene expressions, this study provides a novel insight into the connection between mineral delicacy and blood hypouricemia. In conclusion, high-phytate diets may cause blood hypouricemia through mineral deficiency.

Studies on Metabolic Characterization of β propeller Phytase Knock in Mice Model : β propeller Phytase Knock in Mice Model 의 대사적 특징에 관한 연구

Kim Jun Sik 가천대학교 일반대학원 2014 국내석사

Phytate (myo-inositol 1,2,3,4,5,6-hexakiphosphate, IP6) is the principal storage form of phosphorus in plant seeds and forms strong metal-phytate complexes with di- or trivalent metal ions such as Ca2+, Mg2+, Zn2+, Mn2+ and Fe3+. Monogastric animals, such as human, swine, chickens and rodents, lack the abilities to hydrolyze these metal-phytate complexes, which may cause mineral deficiency in monogastric animals. To address the effects of β-propeller phytase on phytate-fed animal models, a mouse model was developed a knock-in gene targeting strategy, which introduced a gene of β-propeller phytase (BPP) from Bacillus amyloliquifacience DS11 by Cre-loxP system. The BPP knock-in mice model was generated by β-actin Cre mice, which was expressed BPP in the whole body of the mice. After the expression levels of BPP by immunoblot in the gastrointestinal systems such as salivary gland, intestine, and pancreas confirmed, BPP knock-in mice were fed on control diet (control) and diets containing 3% sodium phytate (3 % phytate) for 12 weeks. The BPP knock-in mice showed higher bone mineral density (BMD) than that of the wild type mice in each feeding group. Especially,, BMD of BPP knock-in mice was increased about 35 % compared with the wild-type mice in control diet groups. Furthermore, BPP knock-in mice were more higher trabecular and cortical bone density than those of wild type mice. These results suggested that BPP knock-in mice were efficiently hydrolyzed the metal-phytate complexes and increased calcium uptake from the gastrointestinal systems through the increased bioavailability of calcium ion. Moreover, the analysis of renal histopathology showed that the kidneys of BPP knock-in mice fed high phytate decreased the number of renal cyst formation and glomerulonephritis compared to wild type mice. Therefore, these results strongly suggest that increased bioavailability of dietary calcium by phytate hydrolysis of BPP is a potential therapeutic options for controlling body calcium homeostasis.

쌀 품종의 phytate 함량 분석 및 phytate 저감화 연구

백지은 계명대학교 대학원 2012 국내석사

국내에서 육종된 9가지 품종의 쌀에 대한 phytate 함량을 분석하였고, 발아 및 발효를 통해 쌀의 phytate 저감화 효과를 조사하였으며, 가공 과정의 저감화 효과를 높이기 위해 발효 첨가물에 대해 연구하였다. 도정하지 않은 볍씨는 Goamibyeo, Milyang260, Chenmaai의 phytate 함량이 가장 높았고, Hanareumbyeo가 가장 낮았다. 도정된 쌀은 Suweon517이 가장 높았으며 Goamibyeo, Milyang261 순으로 많이 함유되어 있었고, Seolgaengbyeo와 Manmibyeo의 phytate 함량이 가장 낮았다. Goamibyeo는 볍씨와 도정한 쌀 모두에서 phytate 함량이 높았다. Phytate의 함량이 도정 여부에 관계없이 높은 Goamibyeo를 이용하여 phytate의 함량을 저하시키기 위한 가공 방법을 연구한 결과, 볍씨를 발아 시키면 phytate 함량이 유의적으로 감소되었으며, 발아된 볍씨를 24시간 발효하였을 때 phytate의 함량이 가장 많이 감소되었다. 발효 온도는 30℃보다 50℃에서 효과가 높았고, 발효 첨가물은 ammonium sulfate가 가장 감소 효과가 높았다. 이상의 결과, 발아한 볍씨에 ammonium sulfate를 넣고 50℃에서 24시간 발효하면 대부분의 phytate가 감소됨을 확인할 수 있었다. This study analyzed phytate content in 9 rice cultivars that were bred in Korea, and investigated the effects of germination, fermentation, and fermentation additives on reducing the phytate content during processing. In the rice seeds that were not milled, the phytate content was highest in Goamibyeo, Milyang260 and Chenmaai and lowest in Hanareumbyeo. In the polished rices, Suweon517 was the highest, then Goamibyeo and then Milyang261 in order, and Seolgaengbyeo and Manmibyeo were the lowest. Goamibyeo was high in phytate content both in rice seed and polished rice. Using Goamibyeo that had high phytate content both before and after milling, there was a meaningful reduction in phytate content when the rice seed was germinated. There was the biggest phytate content reduction when the germinated rice seed was fermented at 50°C for 24 hours. Ammonium sulfate was most effective as a fermentation additive to reduce phytate content of fermented rice flour. In conclusion, most of the phytate can be reduced when ammonium sulfate is added to germinated rice seeds and when they are fermented at 50°C for 24 hours.

참깨종실 단백질로부터 Phytate 제거에 관한 연구

이현주 漢陽大學校 大學院 1986 국내석사

대두 식품의 Phytate 함량 및 단백질 소화율에 관한 연구

김희승 이화여자대학교 대학원 1994 국내석사

들깨 종실 단백질중의 Phytate 제거에 관한 연구

박진희 漢陽大學校 1989 국내석사

韓國産 平地種實 蛋白質의 Phytate 除去에 관한 硏究

허채옥 漢陽大學校 大學院 1985 국내박사

Mechanism Studies of Phytate-Diet on Vitamin D pathway : Phytate 식이사료가 Vitamin D 경로에 미치는 기전 연구

정우진 가천의과학대학교 대학원 2011 국내석사

파이테이트는 영양학적으로 중요한 2가의 양이온인 칼슘, 마그네슘, 아연, 망간, 철과 같은 무기이온과 강하게 결합하여 무기이온-파이테이트 복합체를 형성한다. 돼지, 닭, 사람과 같은 단위동물은 파이테이트를 분해하는 효소의 결핍으로 무기이온-파이테이트 복합체를 소화 흡수하지 못하고 분변으로 배출한다. 본 연구에서는 파이테이트 식이사료가 동물모델에서 미네랄이온의 흡수와 억제에 관한 역할을 연구하였다. 동물의 배설물과 혈청 생화학 분석을 통하여 미네랄이온 함유량을 분석한 결과, 파이테이트 함유사료는 칼슘, 마그네슘과 철 같은 2가의 양이온의 흡수를 억제하여 렛드 동물모델에서 미네랄 결핍을 유발하였다. 게다가 파이테이트 식이사료는 부갑상선 기능항진 유발 및 혈중 비타민D의 함량이 감소하는 원인으로 작용하였다. 본 연구는 정상 렛드와 파이테이트 식이사료를 섭취한 렛드에서 비타민D 경로에 중요한 효소의 mRNA와 단백질 발현에 대한 영향을 조사하였다. 그 결과, 파이테이트 사료는 신장조직에서 1-hydroxylase (CYP27B1)와 24-hydroxylase (CYP24A1)의 발현을 강하게 증가시킴을 확인하였다. 동물모델의 혈중 칼슘결핍은 신장에서 비타민D 합성에 중요한 1-hydroxylase (CYP27B1)와 24-hydroxylase (CYP24A1)의 발현을 증가시켜 비타민D의 이화작용이 증가하여 비타민D 결핍을 유발시키는 것으로 사료된다. 또한 본 연구에서 칼슘결핍 및 부갑상선 호르몬이 비타민D 경로에서 중요한 효소인 CYP27B1과 CYP24A1의 promoter 활성을 증가시키는 것을 확인하였다. 특히 고함량의 파이테이트 식이사료는 동물모델의 혈중칼슘 결핍을 유발하며, 이로 인하여 신장의 1-hydroxylas와 24-hydroxylase의 효소활성을 증가시켜, 비타민D 이화작용이 증가하여 비타민D 결핍을 유발함을 증명하였다. Phytate is a strong chelator for nutritionally important divalent cations such as Ca2+, Mg2+, Zn2+, Mn2+ and Fe2+ into a stable metal-phytate complex. Monogastric animals such as pig, chicken and human have a limited ability to hydrolyze phytate, and they excrete out most of it as metal-phytate complex. To address the roles of phytate in reducing mineral bioavailability on animal model, the animals were fed the dietary phytate for 12 weeks. The analysis of mineral contents in animal feces and serum biochemistry indicated that dietary phytate strongly decrease the uptake of divalent cations such as Ca2+, Mg2+, and Fe2+ and eventually causes mineral deficiency of the animals. Furthermore, the dietary phytate causes the animal with low Vitamin D status with hyperparathyroidism. To determine whether the phytate is involved in vitamin D deficiency on animal models, this study has investigated to the level of mRNA and protein of the key enzymes in vitamin D pathway in normal rat and rat with dietary phytate. The results suggested that phytate diets strongly increased the expression level of 1-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1) in the kidney tissue of rat with phytate diets. Elevated CYP27B1 and CYP24A1 expression levels in phytate diets in combination with low level calcium and vitamin D status may reflect accelerated catabolism of vitamin D3 by CYP27B1 and CYP24A1. The promoter analysis of CYP27B1 and CYP24A1 further confirmed that calcium deficiency and parathyroid hormone increases the expression level of the key enzymes of vitamin D pathway. These findings, in particular, the strong expression of CYP27B1 and CYP24A1 in kidney tissues from phytate diet animals, suggest that high amounts of dietary phytate causes calcium deficiency and low level of vitamin D status.