The Study Of Nephrotoxicity And Primary Mechanism Induced By The Gardenia And Its Main Component Geniposide

Gardenia(Zhi Zi),the fruit of Gardenia jasminoides Ellis, was widely used in traditional Chinese Medicine as the hepatoprotective cholagogue drug. Gardenia is of cold-natured and tasted bitter, and has the functions of purging fire, heat-clearing and diuretic, blood-cooling and detoxifying.Geniposide as the major active constituent of Gardenia, was decided as the quality standard of Gardenia in the 2015 edition of Chinese Pharmacopoeia. The functions of the Geniposide are anti-inflammatory, antipyretic choleretic and laxative.It was reported that high-dosage of Gardenia and Geniposide induced hepatotoxicity nephrotoxicity in the animal experiment. However, meanwhile there is only limited research conducted on its nephrotoxicity, and the mechanism of Geniposide is unclear.ObjectiveThis research focus on the nephrotoxicity mediated via both short term and long term administration of Geniposide, the potential toxic dosage and toxic machanism, and offering secure information for clinical medicine.MethodsThere are two parts of the nephrotoxicity test: one was conducted for three days as short-term experiment and another one was 90 days as long term experiment.1.Short-term experiment: Male Sprague-Dawley(SD) rats were randomly divided into four groups: three groups were intragastrically administrated with Geniposide of 50, 100, 300 mg·kg-1(Geniposide-50, Geniposide-100,Geniposide-300), and one group were administrated with water. Geniposide of 50,100 and 300 mg·kg-1 are equally to 5, 10 and 30 times of clinical dose respectively.Each group contains 8 rats. The rats were administered for three days consecutively.2.Long-term experiment: SD rats were randomly divided into 6 groups: three groups were intragastrically administrated with Geniposide of 25, 50 and100mg·kg-1(Geniposide-25, Geniposide-50, Geniposide-100), two group were intragastrically administrated with Gardenia extract, and Gardeniae and Sojae Praeparati Decoction(both of their the content of geniposide equal to 50mg·kg-1 of geniposide group) and one group were administrated with water. Geniposide of 25, 50 and 100mg·kg-1 are equally to 2.5, 5 and 10 times of clinical dose. Gardenia extract,and, Gardeniae and Sojae Praeparati Decoction are equally to 50mg·kg-1 of geniposide. Each group contains 5 female and male rats. The rats were administered for 90 days consecutively.Serum samples were collected to analyze the biochemical alterations including BUN and CRE. Urine samples were collected to detect content of Kim-1 and Ngal by ELISA. Kidney tissues were used to pathological examination and, RNA and protein anlysis. The mRNA expression levels of Oat1, Oat3, Ngal, Timp-1 and Clusterin were analyzed by qRT-PCR. The protein expression levels of Oat1 and Oat3 were analyzed by estern-blot.Results1.The short-term experiment.Geniposide of 300mg·kg-1 induced renal injury after 3 days of Geniposide treatment. Compared with control rats, Geniposide-300 rats showed that renal tubules slightly swell and glomerular mesangial hyperplasia by pathological examination,increased expression of Kim-1 and NGAL in urine through ELISA analysis. While serum BUN and CRE did not change in geniposide-300 rats compared with control rats. However, Geniposide of 50,100 mg·kg-1 have no side-effect to rats kidney.2.The long-term experiment.The kidney tissues of Geniposide-100 rats turned to dark green, showed renal tubular cell cytoplasm with drug pigment deposition and slightly swell through pathological analysis. In addition, the expression of Ngal in urine increased in female Geniposide-100 rats. Therefore these results showed that Geniposide of 100 mg·kg-1caused minious nephrotoxicity on rats. However, Geniposide of 25 and 50mg·kg-1had no effect on rats. Gardenia extract and, Gardeniae and Sojae Praeparati Decoction caused slightly renal tubules swell and drug pigment deposition on kidney.3.Effect of renal transporters and other related molecules:a.The short-term experiment The expression of Oat1 and Oat3 decreased in Geniposide-50, Geniposide-100 and Geniposide-300 groups of rats both in mRNA and protein level. The mRNA expression levels of Ngal, Timp-1, clusterin increased in Geniposide-50,Geniposide-100 and Geniposide-300 groups of rats.b.The long-term experiment The protein expression levels of Oat1 and Oat3 decreased in Geniposide-50,Geniposide-100, Gardenia extract and, Gardeniae and Sojae Praeparati Decoction groups of rats.Conclusion1. Geniposide induces renal injury in the dose 300 mg·kg-1 but not in the dose of mg·kg-1 traeted for 3 days. Geniposide of 300 mg·kg-1 increases the expression ofKim-1 and Ngal, but does not changes the traditional markers of renal function including BUN and CRE. Therefore, it is highly recommended that urine Kim-1 and Ngal might be the new renal injury biomarkers.2.Geniposide of 100 mg·kg-1 induces slightly tubular cell lesion in rats after 90 days of administration. However, Geniposide of 25 mg·kg-1 and 50 mg·kg-1 does not induce renal injury in rats after 90 days of administration.3.There is a certain relationship between Geniposide mediated renal iunjury and the inhibition of renal tubular cell transporters. Geniposide of 300 mg/kg/3d decreases the expression of Oat1 and Oat3, affecting the excretion of toxic substances in the kidneys. Geniposide of 300 mg/kg/3d increases the expression of matrix metalloproteinase inhibitor Timp-1, affecting the synthesis and degradation of extracellular matrix.4.Gardenia extract and, Gardeniae and Sojae Praeparati Decoction do not induce renal injury in rats after 90 days of administration.