Studies On Pathogenesis Of Kidney And Testis Injury Caused By Hepatitis E Virus Infection

In this research, two mature animal models Mongolian gerbils and rabbits were used to study the extrahepatic lesions caused by Hepatitis E virus (HEV) infection. Swine HEV is intraperitoneally inoculated to Mongolian gerbil and rabbit HEV to the rabbit. RT-PCR, Immunohistochemistry (IHC) staining and Western blotting (WB) techniques were adopted to characterize HEV quantification, localization and dynamic changes in the kidney and testis of gerbils. Histopathological changes in kidney and testis of gerbil and rabbit were determined via microscopic and ultramicroscopic analysis. Cytochemistry and IHC assays were conducted to detect the protein expression associated with endoplasm reticulum stress in kidney and mitochondrion apoptosis in both kidney and testis in order to preliminarily elucidate the mechanism of kidney and testis lesions caused by HEV infection. The results are as follows.1. Observation results for gerbil kidneyReal-time PCR result revealed that HEV RNA was detectable at 7 dpi in the kidney. The viral load peaked at 14 dpi (7.18 logs g-1), which is supported by the IHC staining and WB analysis targeting on HEV ORF2 and ORF3 antigen respectively. The histopathological changes of the kidney were characterized as glomerular atrophy, degeneration, edema, and renal tubular epithelial cell necrosis under microscopic observation. Mallory and Sirius red staining indicated collagen fiber proliferation. Ultramicroscopic studies of kidney demonstrated rough basal membrane of tubular, vacuolated and swelling mitochondrion in the epithelial cells. TUNEL staining revealed significantly increased amount of positive cells in gerbil kidney from experimental group compared with the control group (P<0.01).With IHC staining analysis, the parameters associated with cell apoptosis including Bax, ratio of Bax io Bcl-2, Fas and Caspase-3 in kidney from HEV infected gerbils were detected with significantly higher value than the control group at each time point. Meanwhile, the level of HSP70, GRP78, CHOP and Caspase-12 related to the endoplasmic reticulum stress in HEV positive kidney were obviously higher than the control group. Real-time PCR detection for the mRNA levels of parameter in accordance with the IHC assays showed same trend, along with the JNK, Caspase-3 (P<0.05 or P<0.01), which is coincide with the WB results. The findings above indicated that the mitochondrion apoptosis and endoplasmic reticulum stress pathways were both activated after HEV infection, and this might be the trigger mechanism of kidney damage.2. Observation results for gerbil testisHEV RNA was detected in gerbil testis at 14 dpi and the viral load peaked during 28-42 dpi (3.12 logs g-1-6.23 logs g-1)-HEV ORF2 protein was tested positively by IHC staining and WB assays. Microscopic and ultramicroscopic analysis revealed varying degrees of pathological changes in gerbil testis from experimental group. Clinical blood biochemical indexes detection indicated that the testosterone of the HEV inoculated gerbils decreased while the BUN increased, and the TUNEL positive cell count was significantly higher in comparison with control group (P<0.01). IHC staining results showed that there is a significant increase in Fas-L, Fas, Caspase-3, Bcl-2 and Bax expression in experimental group testis, and this is supported by WB and real-time PCR assays (P<0.05 or P<0.01). These results identified that HEV RNA, ORF2 and ORF3 proteins was detectable in experimentally infected gerbil testis, and the replication of HEV in the testis cells was able to cause pathological lesions. HEV infection is capable of activating the mitochondrion apoptosis pathway and promotes the process of cell apoptosis in testis.3. Observation results for rabbit kidneyHEV RNA and ORF2 protein were detected in rabbit kidney via PCR, IHC, and WB methods at 14 days post inoculation with rabbit HEV strains. Apparent histopathological changes were observed including glomerular atrophy, glomerulus capsule enlargement, massive tubular epithelial cells degeneration and necrosis, protein cast formation, interstitial edema and hemorrhage, lymphocyte infiltration and focal fibroplasias. The AST, ALT and TBIL levels of inoculated rabbits were significantly higher than the control group (P<0.05, P<0.01). TUNEL staining results showed that positive cell count in inoculated rabbit kidney was larger than control group between 7 dpi to 42 dpi (P<0.05, P<0.01). The observations confirmed that HEV is able to replicate in the rabbit kidney and produce pathological changes, and promote cell apoptosis.In conclusion, swine HEV RNA, ORF2 and ORF3 antigens was identified and to be detectable in extrahepatic organs including kidney and testicle in experimentally infected gerbils, and HEV is able to replicate in the kidney and testis tissue cells and cause pathological changes. This is also proved in the rabbit model infected with rabbit HEV strain. HEV infection can induce obvious damages to mitochondrion structure in renal tubular epithelial cells and testicular spermatogenic epithelial cells, activate the mitochondrion apoptosis pathway, and promote renal and testicular tissue cell apoptosis.