Construction And Application Of Parabiosis Animal Models To Study The Environmental Impact On Kidney Aging And Aged Kidney Injury

Background:Aging in most species associates with impaired adaptive and homeostatic mechanisms that leave an individual susceptible to environmental or internal stress followed by increasing rates of disease and death. The aging population has become a global public health problem. With the increasing number of elderly people, the number of elderly patients with kidney disease also increases. Kidney is a typical target organ of age-associated tissue damage, and the increased incidence of chronic kidney disease (CKD) and acute kidney injury (AKI) in the elderly is a health problem worldwide. However, there is little or no information on the mechanisms underlying age-associated kidney damage. It is very important to explore the key factors in the occurrence and the pathogenesis of aging and elderly kidney disease. It may provide useful clues for clinical treatment. Our previous study found that the internal environment has an important impact on the kidney aging process. Changes of the state of the environment can accelerate or reverse the kidney aging process.Objective:Based on previous studies of our laboratory, we present original scientific hypothesis:there may be some humoral factors that can promote or inhibit aging, through the regulation of autophagy, apoptosis and inflammatory cytokines genes expression, to promote organ senescence or make aging getting younger. This study mainly used parabiosis animal models and renal ischemia/reperfusion injury (IRI) model to explore the internal environment on kidney aging and elderly kidney ischemia/reperfusion injury in mice.Methods:3-month-old and20-month-old male C57BL/6mice were used. First, we established parabiosis mouse model and made sure sharing the blood circulation between two mice after parabiosis lweek and2week through small animal in vivo imaging. Groups in the research of environment on kidney aging:(1) young single con6mice,(2) young-young parabiosis7pairs,(4) young-old parabiosis3pairs,(5) old-old parabiosis4pairs,(6) old single con6mice; feeding and observation for five weeks after parabiosis. Finally, all mice were sacrificed. Mice were anesthetized via intraperitoneal injection of sodium pentobarbital (40mg/kg). Blood samples were collected from angular vein of each mouse for the measurement of biochemical efficacy markers. The renal tissues from each group of mice were removed and perfused with ice-cold, isotonic phosphate-buffered saline (PBS; pH7.4) to remove any remaining blood. A portion was immersed into OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA, USA) for SA-(3-gal staining. The remaining tissue was immediately frozen in liquid nitrogen and stored at-80C until further processing. Aging index p16, apoptotic index cleaved caspase-3, autophagy indicators LC3, p62, polyubiquitin were measured by western blot. Groups in the research of environment on elderly acute kidney injury were:(1) young mice sham operated (Y:SO),(2) young mice with bilateral renal ischemia-reperfusion injury model (Y:IRI),(3) recipient young mice with bilateral renal ischemia-reperfusion injury model of young-young parabiosis (Y-Y:IRI),(4) recipient old mice with bilateral renal ischemia-reperfusion injury model of young-old parabiosis (Y-O:IRI),(5) recipient old mice with bilateral renal ischemia-reperfusion injury model of old-old parabiosis (Y-O:IRI),(2) old mice with bilateral renal ischemia-reperfusion injury model (O: IRI),(7) old mice sham operated (O:SO); IRI was conjoined after parabiosis3weeks. Blood samples and renal tissues were collected after ischemia30min and reperfusion24h. Blood of each mouse was for the measurement of serum creatinine and urea nitrogen. The renal tissues were PAS and TUNEL staining. Two investigators who were masked to sample identity performed the image analysis. Quantitative analysis of histopathology score and TUNEL-positive cell counts was done with Image-Pro software from20random fields per mice at a total magnification of X200or X400. Microarray analysis was used for filter out cytokines that had a significant difference among all groups. Cluster analysis of cytokines was to identify key factors that may affect renal aging and elderly acute kidney injury.Results:(1) Successfully constructed parabiosis animal model; and applied anatomy observation, in vivo small animal imaging scans, and green fluorescent protein transgenic mice to verify sharing the blood circulation between the pairs. This part layed the foundation for the next studies.(2) The survival rate of mice in heterochronic parabiosis (HP) was significantly lower than their controls. There was no significant change in the old mice of HP on kidney morphology and function, and the HP-old mice showed similar as the isochronic parabiosis (IP)-old mice. Aging-related indicators p16and SA-β-gal in the HP-old mice had no a significant improvement, but in an improvement trend. Apoptosis marker cleaved caspase-3decreased in the HP-old mice, but not statistically different. Autophagic activity increased in the HP-old mice, but there was no significantly difference. We have found6kinds of cytokines that may be closely associated with aging using high-throughput screening methods in the blood circulatory system; namely IGFBP-2, Eotaxin-2, Fractalkine, CD27, LIX and SCF. When aged, these six cytokines increased, while they decreased in the HP-old mice.(3) Established kidney IRI model on the basis of parabiosis animal model, and constructed sham controls. We first found that IRI24h serum creatinine and serum urea levels of HP-old mice were significantly lower than that of IP-old mice and old single mice. The degree of renal tissue injury was less than that of IP-old mice and old single mice. The TUNEL positive tubular cells were less than that of IP-old mice and old single mice. Our results suggested that the youth environment can significantly reduce the aged mice with acute ischemia/reperfusion renal injury. Moreover, we also used a high-throughput screening method to screen120kinds of cytokines of blood. We found7kinds of cytokines that may be closely associated with aged kidney injury; namely IGFBP-2, Fractalkine, EGF, SDF-lalpha, CXCL16, TNF RII and ACE. When AKI occurred, these7kinds of cytokines changed in HP-old mice basicly same with the young mice.Conclusions:(1) Successfully constructed parabiosis animal model; and applied anatomy observation, in vivo small animal imaging scans, and green fluorescent protein transgenic mice to verify sharing the blood circulation between the pairs.(2) Completed the5weeks’observation on the young-young, young-old and old-old parabiosis models. Established kidney IRI model on the basis of parabiosis animal model.(3) Found that there were improvement trends in aging-related indicators, apoptosis and autophagy activity.(4) We found that the degree of renal tissue injury and the TUNEL positive tubular cells were both less than that of IP-old mice and old single mice, suggesting that the youth environment can significantly reduce the aged mice with acute ischemia/reperfusion renal injury.(5) Using high-throughput screening methods in the blood circulatory system, we found that there were6kinds of cytokines that may be closely associated with aging and7kinds of cytokines that may be closely associated with aged kidney injury.