Reconstruction of the urinary tract at the appropriate time reduces fibrosis of the metanephros in rats as judged by imaging

Chronic kidney disease leads to high morbidity rates among humans. It is a serious disease that requires curative treatments other than kidney transplantation. Recently, we successfully established the iPS-derived generated kidney, which might produce urine. The urine can be directed to the native bladder with a stepwise peristaltic ureter system, followed by anastomosis with the recipient ureter for reconstruction of the urinary tract. However, the growth of the regenerated kidney varies significantly, whereas the time window of the anastomosis is quite narrow. Therefore, this study was conducted to evaluate the growth of transplanted metanephros with bladder periodically and noninvasively using computed tomography and ultrasonography. Ultrasonographic findings showed high correlations with computed tomographic findings and clearly evaluated metanephros with bladder. We found that the degree of growth of the metanephros with bladder after the transplantation differed in each individual. However, most of them reached the appropriate period for urinary tract reconstruction within 3 weeks after transplantation. Optimizing the stepwise peristaltic ureter system anastomosis by ultrasonography reduced long-term tubular dilation of the metanephros, thereby decreasing fibrosis caused by transforming growth factor-β. This may be significantly related to long-term maturation of fetal grafts. These results provide new insights into transplanting regenerated kidneys in higher animals. We are one step closer to the first human trial of kidney generation.


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The morbidity rate of end-stage renal disease (ESRD) remains high. Although  There is a need treatment for alternatives to kidney transplantation and dialysis, 51 which must be a fundamental treatment. There has been an attempt to make a kidney 52 from pluripotent stem cells de novo. Takasako et al. [3,4] examined a method for 53 producing renal organoids in vitro by aggregating nephron progenitor cells and ureteric 54 bud. Organoids include differentiating nephrons, interstitial, and vasculature, which 55 have matured in a culture. In addition, the use of human stem cells has made it possible 5 56 to produce organoids that resemble human fetal kidneys. However, the size of organoids 57 was smaller than 2 mm, and could not gain enough ability for the production of urine, 58 such as functional maturation of tubules, glomerular neovascularization, and urinary 59 excretion pathway. 60 To overcome these issues, we made an attempt to generate a kidney from induced 61 pluripotent stem cells (iPS cells) using nephrogenic niche of xeno-animal as the scaffold 62 to generate the kidney [5,6]. In this system, iPS cell-derived nephron progenitor was 63 injected into the nephrogenic zone of xeno-embryo and cultured in the nephrogenic 64 environment. We confirmed that injected cells continued to develop further to form a 65 nephron, and it started producing urine following transplantation in vivo [7]. By 66 eliminating the native nephron progenitor cells (NPCs) in the nephrogenic zone during 67 development using genetic manipulation, pure nephron from external NPCs can be 68 successfully generated [8]. We also confirmed that this system can generate interspecies 69 chimeric nephron between rats and mice [9], and also iPS cells from hemodialysis 70 patients can be used without deterioration compared with those from healthy controls 71 (Tajiri Sci Rep). Based on this success, we are currently conducting the scale up 72 experiment using bigger animals to proof the efficacy and safety for human clinical use. 73 However, one big hurdle remains for the next stage. Transplantation alone does not 6 74 provide a route for excretion of the produced urine. Thus, metanephros can cause 75 hydronephrosis and renal insufficiency [11,12]. This may be solved using Stepwise 76 Peristaltic Ureter (SWPU), which (S1 Fig.) comprises anastomosis of the ureters of the 77 recipient rats to the bladder using a developed metanephros with bladder (MNB) [11]. 78 This new method made it possible to continuously excrete urine produced from the 79 MNB to the recipient bladder via the recipient ureter [11]. However, the timing of 80 anastomosis with the ureter of the recipient after transplantation is crucial and owing to 81 individual differences in the growth of MNB, hydronephrosis may occur at ambiguous 82 anastomosis times [11,13]. Postrenal nephropathy due to hydronephrosis imposes a 83 heavy burden on the kidneys, and the delayed release of obstruction has substantial 84 effects on the kidneys [14][15][16]. Ureteral primordia obstruction during the fetal stage has 85 been shown to cause dysplastic metanephros [17]. Therefore, we believe that early 86 released obstruction is significantly involved in subsequent renal functions, even with 87 fetal-derived grafts. In the case of xenotransplantation and MNB transplantation in large 88 experimental animals, the effects of individual differences are considered to be greater.  19-085). The rats were housed in cages under temperature and light-controlled 115 conditions in a 12-hour cycle and were provided with fresh food and water ad libitum. 116 In Experiment 1, we used three pregnant female Lewis rats on gestation day 15 117 (E15) (Japan Charles River Laboratories, Kanagawa, Japan) to obtain fetal MNB. As 118 recipient rats (organ recipient animals), we used 12 male Lewis rats (Japan Charles 119 River Laboratories) aged 11 weeks, with a body weight of 309.0 ± 11.4 g.

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In Experiment 2, we used three pregnant female Lewis rats on E15 (Japan Charles 121 River Laboratories), and as recipient rats, 18 male Lewis rats (Japan Charles River 122 Laboratories) aged 9 weeks, with a body weight of 243.0 ± 7.5 g.

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In Experiment 3, we used three pregnant female Lewis rats on E15 (Japan Charles 124 River Laboratories), and as recipient rats, 9 male Lewis rats (Japan Charles River 125 Laboratories), aged 10 weeks, with a body weight of 292.8 ± 7.6 g.   The wound was closed using conventional methods. The animals were divided into two 146 groups. The first group comprised randomly selected rats that had the left recipient 147 kidney removed 4 weeks after MNB1 transplantation and underwent urinary tract 148 reconstruction by anastomosing the recipient ureter to the MNB (n = 5: anastomosis 149 group). The second group consisted of randomly selected rats that did not undergo    performed the examination to randomly selected rats. To carry out these tests, the rats 202 were anesthetized and maintained until the end of the procedure using 2.5% isoflurane.

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Their abdomens were shaved, and the animals were kept in the supine position. The 204 ultrasound device LOGIQ S8 (GE Healthcare Japan K.K., Tokyo, Japan) was used.

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After anesthetizing the rats with 2.5% isoflurane, we made a midline abdominal 219 incision and removed the MNB, which was used for histopathological examinations. were determined by measuring healthy adult rats (Table S). vessels around the MNB by the color Doppler method (Fig. 1 D). Additionally, it was 272 possible to confirm the metanephros (Figs 1B and C).   (Fig. 3). Furthermore, the amount of urine collected from the MNB1 showed a strong The MNBs removed 21 or more days after transplantation had significantly 342 milder tubular dilation than those removed less than 21 days after transplantation (p 343 <0.01) (Fig. 5 A). There was no difference in fibrosis in MNB removed 21 days prior 344 and 21 days after transplantation. (Fig. 5 B).   An image of the extracted MNB is shown as an example (Fig. 6 A). In the SP 364 group, the color of the surface of the metanephros could be visually confirmed to have 365 blood flow, and the SP group grew without hydronephrosis. As shown in Fig. 6 B, in 366 the 28UR group, the observed shape of metanephros was irregular. In some cases, the 367 metanephros was hydronephrotic without liquid storage in the MNB bladder. Fig. 7 368 shows a micrograph of HE staining for the evaluation of tubular dilatation. The 28UR 369 group tended to expand compared to the SP group, but no significant difference was 370 observed between the two groups ( Fig. 7 C).  The measurements of fibrosis marker are shown in Fig. 9. TGF-β1 was strongly 396 expressed in the 28UR group; mainly in the tubular cells, interstitial, and glomeruli, and interstitial. The SP group was significantly milder in all evaluations than the 28UR 399 group (p <0.01) (Fig. 9 G).   is thought that a certain amount of tissue was present. Urinary tract reconstruction for 542 these metanephros was performed mainly at a time determined by naked eye and may 543 have been exposed to long-term obstruction. Prolonged obstruction results in the fact that GFR could be measured in tissues, approximately, 60 days after transplantation 559 would be a great knowledge.

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There are some limitations to this study. First, we only performed diagnostic 561 imaging-based assessments and did not assess aspects such as renin and erythropoietin 562 activities. Second, because we assessed the MNB only for a short period (60 days after 563 transplantation), we did not perform long-term assessment of function and morphology 564 of the transplants. These issues need to be elucidated further in future studies.

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In conclusion, this is the first study to successfully observe the time course of