In this paper we describe a murine model of acute kidney injury induced by ischemia reperfusion injury, and we'll highlight how this can be a very useful model of both innate injury to the kidney, but also Regeneration of the kidney. This schematic cartoon shows the great And renal blood vessels together with kidneys and ureters. After a midline laparotomy, the right ureter is ligated and the right nephrectomy performed.
The left renal artery and vein are clamped by an a traumatic clamp, and following a period the clamp is removed and kidney perfusion is restored. Here is a representation of a typical experiment. The right nephrectomy tissue serves as a useful controlled tissue.
My are typically sacrificed at the 24 hour time point to study acute kidney injury. Kidney function can be assessed using plasma creatinine or blood urea nitrogen. The tissue can be assessed for both injury as in acute tubular necrosis, but also regeneration, which is more prominent at later time points.
A very important feature of this model is that it can be used in mice at different age, different strains, and Also in both male and female mice. Here we see a male eight week Old B mouse that has been anesthetized with an intraperitoneal injection of ketamine, hydrochloride, and meine. The surgical area has been shaved and then sanitized using a dilute chlorhexidine solution.
Following this, the mouse has been placed on a sterile draped heated surgical pad in a supine position with the upper and lower extremities fixed to the pad using low adhesive tape. At this point, eye ointment is applied to prevent the corneas from drying and analgesics are administered via a subcutaneous injection of buprenorphine hydrochloride. The procedures performed using sterile autoclave surgical instruments position within easy reach.
In a sterile surgical field, the operator will wear a sterile gown, mask and gloves, and if a series of surgeries are being performed, then each procedure should be performed with Sterile autoclave instruments using tissue separating scissors. A midline laparotomy is made. The skin is bluntly separated from the peritoneum to allow ease of skin and peritoneal closure.
Later following this, an incision is made along the linear alba using a scissors or scalpel. A Collibra retractor is inserted into the incision and the mouse is covered in sterile drapes. The intestines have been displaced to the right and covered with moist and gores to expose the right kidney and ureter.
As shown here, the first stage of the right nephrectomy is the isolation and division of the right ureter. The right ureter is isolated using angled forceps and ligated twice using six oh silk braided suture before division. As shown in this clip, The connective tissue along The dorsal side of the right kidney are bluntly dissected up to the renal artery and vein.
Once the renal artery and vein is accessible, forceps are carefully slid underneath to create a channel that will enable a hemostatic clip to be applied. Here you can see the hemostatic clip being applied and this will prevent any bleeding from the cut renal vessels. Following the nephrectomy procedure, the occluded renal artery and vein is then divided close to the kidney, which is then removed.
Minor loss of blood derived from the kidney is evident and this is removed using sterilized cotton wool Buds. The Connected tissues anterior and posterior to the left renal artery and vein are dissected in a careful fashion. A channel underneath the left renal vessels is created in a similar manner to that described for the right nephrectomy.
Renal ischemia is then induced by the application of a micro cellophane clip to occlude both the renal artery and vein. The successful ischemia can be visually confirmed by the gradual uniform darkening of the kidney. Following the induction of ischemia, the retractor is removed and the peritoneum closed with a single suture.
The mouse is then immediately placed on a homeo themic blanket with a rectal TER attached to a control unit to ensure that the body temperature is maintained at 37 degrees for the required duration of ischemia. After the period of ischemia, the micro clip is exposed and removed immediately following clip removal, the kidney will rapidly transition from a dark purple to a healthy pink color indicating successful reperfusion after carefully replacing the intestines. The peritoneum is sutured using six oh braided silk suture.
The skin is then closed using metallic clips and iodine applied to the surgical area to minimize the risk of postoperative infection. The anesthesia is partially reversed by the subcutaneous administration of aamaz. All hydrochloride and fluids were administered by a subcutaneous injection of one mil of warm sterile normal saline.
Mice are then carefully monitored until they have recovered consciousness appear alert and able to write themselves moist and food can be given to encourage fluid intake. The mice are allowed to recover in a heated box kept at 29 degrees centigrade located in a quiet environment for 24 hours for long-term experiments. Ongoing analgesia is administered and the skin clips are removed seven days following surgery presented.
Next is data that indicates the functional and structural injury occurring in biopsy. Mice sacrificed 24 hours following renal ischemia. Renal function is typically assessed by measurement of plasma or serum creatinine or blood urea nitrogen.
The graphs indicate increasing renal failure with increasing duration of the length of ischemia. Renal structure is examined by histopathology on palin embedded tissue sections stained with either H and E or PAS. A simple binary system was adopted to score acute tubular necrosis or a TN in the outer stripe of the outer medulla of the kidney On the basis of the cell.
Morphology and cell integrity tubals are designated as either viable with a healthy cell, monolayer or necrotic if the cells are disrupted or lost. The number of necrotic tubals is then expressed as a percentage of the total number of tubals in the field. As the graphical data illustrates renal ischemia induce significant acute tubal necrosis in ischemic kidneys compared to the non-injured controls.
The following data illustrates the regeneration that occurs over seven days following renal ischemia in eight to 10 week old male FVB mice. Renal function gradually recovers over the course of seven days following ischemia, both plasma creatinine and blood urea Nitrogen steadily decline with plasma creatinine returning to basal levels, illustrating the improved renal function to assess structural tissue regeneration following ischemic injury. Another scoring system is adopted in brief on the basis of cell integrity and cell number tubals are designated as either healthy, recovering, injured or necrotic.
These are then expressed as a percentage of the total tubal number. The scoring system is outlined in detail in the accompanying protocol. As this data illustrates.
Tissue injury begins to show signs of recovery seven days following renal ischemia with an increased number of tubals designated as healthy tubular cell proliferation is an important indicator of regeneration. Mice can be injected with BRDU before sacrifice and BRDU incorporation and expression by dividing cells quantified and assessed by immunohistochemistry on patin embedded kidney sections. The images and graphical data show dramatic tubular cell proliferation and accompanying BRDU incorporation at day four following ischemia which lessens Markedly by day seven.
The mouse model of renal Ischemia fusion injury presented here uses a midline laparotomy approach enabling both the right nephrectomy and clamping of the left renal pedicle via one incision. As a model of acute kidney injury, a high level of control over the severity of injury is afforded by titrating the length of ischemia. It is a highly versatile model and applicable to a broad range of study of the molecular and cellular mediators of acute kidney injury and subsequent tissue regeneration.
Furthermore, our lab has successfully utilized this model across a wide range of mouse strains of both genders and a variety of ages.
