| Background and Objectives: In recent years, with the high speed of the development of the society, chronic kidney disease(CKD) has become the main disease that threats people's health all over the world. In China, CKD is a high prevalence in morbidity and mortality. Renal tubular interstitial fibrosis(TIF) caused by chronic kidney disease is a major pathological factor to cause end stage renal failure. The main pathological feature of TIF is the deposition of extracellular matrix and accumulation of large numbers of myofibroblasts. The normal structure of renal tubules and renal interstitium was destroyed or disappeared when people suffered TIF. For patients who suffered end stage renal failure(ESRD) caused by TIF, they have to choose dialysis treatment or renal transplantation. The rate of illness was not just harming the quality of life for those afflicted, but it was having a debilitating economic and social impact. However, up to now, the mechanism of TIF was unclear and whether TIF is a reversible process is still controversial. How to delay or even reverse the progression of TIF has become a global urgent problem. So it is urgent to provide an ideal animal model to study the mechanism of TIF and probe novel targets for intervention, which will bring us great extensive economic and social benefits. As we known, the injured kidney cells possess the ability to repair and regenerate. However, whether TIF is reversible, it is controversial. Until now, rodent unilateral ureteral ubstrution(UUO) model is well-established and has been extensively applied to study the pathogenesis of renal fibrosis. Furthermore, it has been reported that kidney has the propensity to regenerate and restore renal parenchyma following the relief of unilateral ureteral obstruction(R-UUO). While much work has focused on the UUO model, few have observed the alteration induced by relief of UUO in rodents. Although previous studies examining TIF have been carried out in rodents, their kidney size and physiological character differ with humans, and the difference among diverse individuals before and after damage was obvious. Thus an experimental animal model to simulate human kidney disease was urged to be established. In order to increase the applicability of the model and eliminate individual difference, the pig R-UUO model was constructed. The renal pathological changes were observed in the same pig and the same kidney through renal biopsies before and after renal injuries at different time points in order to clarify whether TIF is reversible, and the exact time points that the kidney has the capacity to be repaired.Materials and methods:First, the pig R-UUO model was successfully constructed through repeated demonstration testing. Thirty-two three-month-old female Guangxi BA-MA mini pigs weighing 8–10 kg were purchased from training base of Jiangsu province in China. Preoperative preparation was proceeded and then pigs were treated with a combined general anesthesia. Surgical process followed the principal of aseptic technique. When make modeling, a 2-cm polyethylene slice was encompassed around the ureter. Two ligations were made around the polyethylene slice. When the obstruction was due to be relieved, the same procedure was followed as above, and the polyethylene slice, along with the ligature, was identified and removed. After unilateral ureteral obstruction surgery, pig kidneys displayed obvious macroscopic changes. Compared to controls, kidneys undergoing UUO were larger in size and had rough surfaces. Kidneys also displayed uronephrosis and hydronephrectasia as well as decreased blood supply. Additionally, UUO-treated animals displayed circuitous and thickened ureters. Following R-UUO, the ureters became straightened and thinner. The models showed excellent structural reversal at the gross anatomical level following R-UUO, which showed successful modeling.Secondly, in order to explore the time points of renal repair following R-UUO, a series of preliminary experiments were carried out. Pigs undergoing 2 or 3 weeks of UUO demonstrated serious tubular injury in the renal interstitium and large areas of irreversible renal interstitial fibrosis. We therefore chose the time points of UUO at 5, 7, and 10 days in order to observe reversible renal repair. When UUO was carried out for 10 days, the time for lesions to form was increased to over 7 days and therefore the kidney needed a longer time to recover. Subsequently, R-UUO was adaptively prolonged to 56 days.Then in order to clarify whether TIF is reversible, and the exact time points that the kidney has the capacity to be repaired, we further divided the pigs into 3 groups:Group 1: UUO 5 day group(animals are performed R-UUO after 5 days of UUO i.e., UUO 5 days, R-UUO 7 days, R-UUO 14 days, and R-UUO 21 days); Group 2: UUO 7-day group(animals are performed R-UUO after 7 days of UUO i.e., UUO 7 days, R-UUO 7 days, R-UUO 14 days, and R-UUO 21 days); Group 3: UUO 10-day group(animals are performed R-UUO after 10 days of UUO i.e., UUO 10 days, R-UUO 14 days, R-UUO 28 days, and R-UUO 56 days). Each group has five time points and UUO 0 day was regarded as a control. Renal function, morphology, and immuno-biomarkers were used to evaluate the levels of kidney damage and repair. Blood samples were collected from the precaval vein and bilateral renal veins, and serum creatinines were measured. Renal tissues were collected through repeated open renal biopsies and a semi-quantitative pathological scoring method was used to evaluate renal damage and repair. The expressions of biomarkers of renal fibrosis-a-smooth muscle actin(?-SMA), vimentin and epithelial cells-E-cadherin were determined.Results: The results showed that after 5, 7, or 10 days of UUO, compared to control, serum creatinine levels were significantly decreased and the kidneys showed lesions to a diverse extent. The expressions of ?-SMA and vimentin were increased and E-cadherin expression was decreased(p<0.05). Following R-UUO after 5 and 7 days of UUO, compared to UUO, serum creatinine levels were significantly decreased. Renal pathological tissue damage was repaired. The expressions of a-SMA and vimentin were decreased and E-cadherin expression was increased(p<0.05). However, during R-UUO 14, 28, and 56 days after 10 days of UUO, serum creatinine was not decreased significantly. The expressions of ?-SMA and vimentin consistently remained at high levels. Renal damage was unable to be restored and resulted in chronic lesions. Kidney damage induced by UUO can be reversed after 5 or 7 days of UUO in the early stages. However, longer time such as 10 days of UUO with significant levels of TIF showed limited reversibility but changed to chronic lesions.Besides, according to the expression of biomarkers of epithelial cells- E-cadherin and mesenchymal cells-?-SMA or vimentin, it can reasoned that pig R-UUO correlates with EMT and its reversal. Indeed, reversal of EMT may participate in the repair of TIF.Conclusions: we constructed the pig R-UUO model successfully and clarified the injury degree of TIF at different time points of UUO and R-UUO in the same kidney and the same pig. We found the time points when the kidney could be repaired after injury in this animal model. TIF could be repaired or even reversed in the early stage of injury. However, long terms of injury showed limited reversibility. We preliminarily discussed the mechanism of renal injury and repair and suggested that reversal of EMT may participate in the repair of renal injury and TIF which will provide the theoretical basis for the study of the mechanism of TIF. The pig R-UUO model provides an ideal tool for determining the time course of treatment and evaluating the effect of intervention. This may, subsequently, allow for clinicians to provide novel treatment regimes to prevent renal fibrosis. |
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