Name: a rat orthotopic renal transplantation model for renal allograft rejection
Uploaded: 2022-02-02T15:00:00
Description: Watch this Scientific Journal Video about A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection at JoVE.com

This work was supported by the National Natural Science Foundation of China (81870304) to Jun Li and by the Else Kr&#246;ner-Fresenius-Stiftung (Nr. 2017_A28) to Marcus Groettrup.

Inbred 8-10 weeks old male F344 and Lewis rats (200 g to 250 g) were commercially obtained. Allogeneic left kidney transplantation was performed between male F344 and Lewis rats. F344 rats were used as donors and syngeneic recipients, and Lewis rats served as allogeneic recipients. All animal handling procedures were conducted in compliance with guidelines for the Care and Use of Laboratory Animals published by NIH, and all animal experimental protocols were approved by the Animal Care and Use Committee of Chongqing Univ...

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Renal transplantation in the rat is a challenging work requiring a high level of micro-surgery techniques and the operation techniques have been optimized several times. From the beginning, Gonzalez et al. implanted the donor kidney into the neck of the recipient and anastomosed the donor ureter to the skin9. However, because of the high incidence of urinary infection and stenosis of the donor ureter, the operation was abandoned in a short time. Subsequently, the implant operation of the donor&#39...

Renal transplantation has become the most effective therapeutic approach for patients with end-stage renal function failure. However, T cell-mediated acute rejection and alloantibody-mediated humoral immune rejection result in the pathologic injury of renal allografts and limit the short-term and long-term survival of patients after kidney transplantation1,2,3. Unfortunately, the effective pharmaceuticals that prevent the rejection of renal allografts are still lacking, because the exact mechanisms of immune and pathologic rejection of renal allografts are not clear. Consequently, the preclinical studies that elucidate the mechanisms of immune and pathologic rejection of renal allografts contribute to finding novel targets and developing relevant effective pharmaceuticals to prevent the rejection of renal allografts and eventually prolong the survival of patients.
Many potential immunological and pathophysiological mechanisms of renal allograft rejection have been proposed recently in rat model studies of orthotopic renal transplantation4,5,6,7,8. These findings propose several novel targets and relevant interfering approaches as promising therapeutics to suppress renal allograft rejection, such as complement regulatory factors and anti-CD59 antibodies6, immunoproteasome, and epoxyketone inhibitors7,8. Thus, rat orthotopic renal transplantation is an ideal preclinical model to investigate the mechanisms of immune rejection and pathologic injury of renal allografts after kidney transplantation.
Rat kidney transplantation has gradually shifted from heterotopic implantation of donor&#39;s kidneys9 to orthotopic renal implantation using end-to-side anastomosis of vessels or using end-to-end anastomosis of the ureter using a cuff method10,11,12. The present study describes detailed protocols of the orthotopic renal transplantation between rats using end-to-end anastomosis to recipients&#39; renal artery and renal vein, and an end-to-side &#34;tunnel&#34; method of ureter-bladder anastomosis, which avoids the ischemia of the lower body and the thrombosis of inferior vena cava and reduces postoperative urine leakage and the twist of the ureter.

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			<td>&#160;10-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
			<td>G0090781</td>
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			<td>&#160;4-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
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			<td>&#160;8-0 Polyamide Monofilament suture</td>
			<td>B.Braun Medical Inc.</td>
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			<td>Buprenorphine</td>
			<td>US Biological life Sciences</td>
			<td>352004</td>
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			<td>Electrocoagulator</td>
			<td>Electrocoagulator</td>
			<td>ZJ1099</td>
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			<td>F344 and Lewis rats</td>
			<td>Center of Experimental Animals (Tongji Medical College, Huazhong University of Science and Technology, China)</td>
			<td>NA</td>
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			<td>Gauze</td>
			<td>Henan piaoan group Co., LTD</td>
			<td>10210402</td>
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			<td>Heating pad</td>
			<td>Guangzhou Dewei Biological Technology Co., LTD</td>
			<td>DK0032</td>
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			<td>Heparin</td>
			<td>North China Pharmaceutical Co., LTD</td>
			<td>2101131-2</td>
			<td></td>
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			<td>Injection syringe (1 ml and 10 ml)</td>
			<td>Shandong weigao group medical polymer Co., LTD</td>
			<td>20211001</td>
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			<td>Isoflurane</td>
			<td>RWD Life Science Co., LTD</td>
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			<td>Penicillin G Sodium</td>
			<td>Wuhan HongDe Yuexin pharmatech co.,Ltd</td>
			<td>69-57-8</td>
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			<td>RWD Life Science</td>
			<td>R500</td>
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			<td>Small Animal Surgery Kit</td>
			<td>Beyotime</td>
			<td>FS500</td>
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			<td>Sodium chloride injection</td>
			<td>Southwest pharmaceutical Co., LTD</td>
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			<td>Surgical operation microscope</td>
			<td>Tiannuoxiang Scientific Instrument Co. , Ltd, Beijing, China</td>
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a rat orthotopic renal transplantation model for renal allograft rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

In this rat orthotopic renal transplantation model, the recipient rats move normally after operation. To observe the chronic rejection of renal allograft, recipient rats are raised for 10 weeks after transplantation, and the total survival rate of recipient rats at this time point is approximately 90%. The major causes of death are bleeding and leakage of urine post-operation. The other major complications include bleeding during the operation, thrombosis in renal vessels, and hydronephrosis, whose respective incidence i...

Watch this Scientific Journal Video about A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection at JoVE.com

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...

Rat orthotopic renal transplantation is an important model to investigate the mechanism of renal allograft rejection in preclinical studies and could aid in developing novel approach to improving the long-term survival of renal allografts. End-to-end anastomosis of renal vessels in this surgery avoids ischemia of the lower body and the thrombosis of inferior vena cava, while anastomosis of ureter in an end-to-side tunnel method reduces postoperative urine leakage and the twist of the ureter. This method provides insight into investigating the mechanisms of immune rejection and injury of renal allografts after kidney transplantation.Begin by incising the abdomen using a surgical scissor along the midline from the symphysis pubis to the subxiphoid. Then pull open the incision using a retractor. Take out the intestines from the right side of the incision and wrap them with moistened gauze to prevent them from drying out.Then expose the left kidney. Dissociate the left renal artery and vein using micro-forceps and cotton swabs under 20 times magnification. Ligate and transect the adrenal vein and genital vein if necessary.Dissociate the ureter with the help of micro-forceps, preserving the peripheral connective tissues to ensure blood supply to the ureter. Dissociate and clip or ligate the aorta approximately five millimeters above the left renal artery with a vascular clamp or 4-0 polyamide monofilament suture. Then transect the left renal vein distal to the conjunction of the left genital vein and adrenal vein.Using a 24 gauge scalpel needle, flush the kidney from the aorta below the left renal artery with ice cold UW solution supplemented with 100 units per milliliter Heparin until blood fades in color. Transect the left renal artery next to the aorta, then transect the ureter next to the bladder and dissociate the kidney with the help of micro-forceps and scissors. Preserve the donor left kidney in ice cold UW solution.Under 20 times magnification, dissociate the left renal artery and renal vein of the recipient rat using cotton swabs and micro-forceps. Ligate the adrenal vein and genital vein if necessary. Clip the left renal artery and renal vein at the root using microvascular clamps, then transect the renal artery and renal vein three millimeters away from the microvascular clamp.Dissociate and ligate the left ureter approximately two to three centimeters below the kidney with an 8-0 polyamide monofilament suture and transect it at the ligation. Resect the recipients native left kidney by dissociating the kidney with the help of micro-forceps and scissors. Implant the donor kidney into the left renal fossa of the recipient rat and put ice around the implanted donor kidney.Switch to 45 times magnification, then anastomose the donors renal artery and renal vein to the recipients renal artery and renal vein in an end-to-end pattern using 10-0 polyamide monofilament sutures. To anastomose the renal artery with interrupted sutures, place the stay sutures at 12 and six o'clock positions of anastomosis. Equidistantly suture one side of the anastomosis between the two stay sutures with two to three stitches.Turn over the stay sutures and similarly suture the other side of the anastomosis between the two stay sutures with two to three stitches. To anastomose the renal vein with continuous sutures, place the stay sutures at the six and 12 o'clock positions of the anastomosis. Suture one side of the anastomosis from the 12 o'clock position with four to five stitches using the running sutures.Turn over the stay sutures and similarly, suture the other side of the anastomosis from the six o'clock position. Reperfuse the donor kidney by releasing the non-invasive microvascular clamp of the renal vein, identify the bleeding sites and make additional stitches if necessary. Next, release the non-invasive microvascular clamp of the renal artery, identify the bleeding sites and make additional stitches if necessary.Under 20 times magnification, ligate or stitch the end of the donor ureter with a 4-0 polyamide monofilament suture as a tow to drag the end through the tunnel of the recipients bladder. Fix the donor ureter with the recipients bladder by stitching the adventitia of the donor ureter with the muscle layer of the recipients bladder outside at four equidistant positions using an 8-0 polyamide monofilament suture. Afterward, transect the end of the donor ureter with the suture outside the recipients bladder and allow the donor ureter to shrink back into the recipients bladder.Place the intestines back into the abdominal cavity and close the abdominal incision with continuous sutures in the muscle layer and then the skin layer with a 4-0 polyamide monofilament suture. At ten weeks after transplantation, hematoxylin and eosin staining and periodic acid shift staining revealed glomerular sclerosis, interstitial fibrosis, tubular atrophy and interstitial arterial sclerosis in the renal allograft in contrast to the isograft kidney. Silver staining showed the thickening of the glomerular basement membrane in the allograft kidney when compared with the isograft kidney, indicating the success of the rat orthotopic renal transplantation model.Because the vascular hole of the renal is very thin, to avoid the wrong suturing, remember to perform the anastomosis on the high magnification field.

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