Browsing by Author "Wade, Michael"
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Item Colloid-Enhanced Flush Limits Initial Edema but Exacerbates Subsequent Edema During Hypothermic Machine Perfusion of Porcine Kidneys(2023) Tamayo, Jesus; Mohammad, Moath; Wade, Michael; Tran, Amanda; O'Hara, Collin; Yurvati, Albert; Mallet, Robert T.Purpose: The epidemic of end-stage renal disease (ESRD) has steadily increased demand for transplantable kidneys, and the widening disparity between organ supply and demand is a major public health concern. Hypothermic machine perfusion (HMP) is widely used to preserve deceased donor kidneys for transplantation. Kidneys are harvested and flushed with crystalloid solution before HMP. This study tested the hypothesis that flushing kidneys with solution containing a colloid, hydroxyethyl starch (HES), minimizes edema and improves organ perfusion during subsequent HMP. Methods: Kidneys harvested from anesthetized Yorkshire swine were flushed for 15 min with ice-cold Ringer’s solution ± 50 g/l HES, and then either biopsied or installed in a LifePort organ preservation system for 72 h hypothermic (2-4°C) machine perfusion (HMP) before biopsy. ATP contents in renal cortical biopsies were analyzed by UV-Vis spectrophotometry. Results: Kidneys gained 49 ± 5% of initial mass during flush with control solution, but only another 3% to 52 ± 5% of initial mass over 72 h HMP. Kidneys flushed with HES-enriched solution gained only 18 ± 3% of initial mass (P<0.001 vs control) during flush, but over 72 h HMP gained another 72% to 90 ± 7% above initial mass (P<0.001 vs. control). Tissue water contents paralleled the respective weight gains (Figure). The HES-flushed kidneys experienced steeper declines in perfusion during HMP than the controls. Cortical ATP content (mmol/g dry mass) fell over 72 h HMP from 2.36 ± 1.40 to 0.44 ± 0.44 (mean ± SD) in control kidneys, and from 1.36 ± 1.31 to 0.38 ± 0.16 in HES-flushed kidneys (both groups: P<0.05, pre- vs. post-HMP). Conclusion: Flushing kidneys with HES-enriched solution minimized edema before HMP, but exacerbated edema during subsequent machine perfusion, failed to preserve ATP, and was associated with a steeper decline in organ perfusion during HMP. The mechanisms responsible for edema exacerbation by HES-enhanced flush are under investigation.Item Hydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys(2022) Wade, Michael; Ramos, Katherine; Reyad, Ashraf; Williams, Arthur; Yurvati, Albert; Mallet, Robert T.; Horani, CaseyHydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys. Michael Wade, Katherine Ramos, Casey Horani, Ashraf Reyad, Arthur G. Williams Jr., Albert H. O-Yurvati, Robert T. Mallet. PURPOSE: Improved preservation of explanted kidneys is essential to narrow the supply vs. demand disparity for transplantable human kidneys. Edematous expansion of the explanted organ may make explanted kidneys unsuitable for transplant. This study evaluated the impact of initial flushing of porcine kidneys with a solution containing hydroxyethyl starch (HES), an osmolyte commonly used in preservation solutions for kidney transplantation, on edema during subsequent hypothermic machine perfusion METHODS: This study utilized kidneys from Yorkshire swine due to the anatomical and functional similarities of porcine and human kidneys. Left kidneys were harvested from isoflurane-anesthetized pigs via laparotomy. The renal artery was cannulated and the organ was flushed for 10 min with 400 mL of control Ringer's solution (group A) or with Ringer's solution containing 50 g/L hydroxyethyl starch (group B). Kidneys then underwent hypothermic machine perfusion (2-4°C) for 21-72 hr in a LifePort organ preservation system, with flow rates and resistance recorded throughout perfusion. The kidneys were weighed before and after flush of the organ, weighed again after machine perfusion, and then biopsied for histological analysis of renal cortex and medulla. RESULTS: Group A kidney mass increased by 49 ± 13% (mean ± standard deviation) during initial flush (n =7) and by 52 ± 14% after flush and 72 h machine perfusion (n=8); thus, 95% of the organ expansion occurred during the initial flush. In contrast, Group B kidney mass increased by 19 ± 9% during initial organ flush (n=6), a 60% reduction vs. Group A (P < 0.001), but total weight gain was 83 ± 21% after machine perfusion (n=3). Machine perfusion of 2 of the 3 Group B kidneys failed at 21 hr and 23 hr perfusion, and only 1 was perfused for the entire 72 hours. In both groups, histology revealed preserved tubular and glomerular architecture, but appreciable cellular edema following machine perfusion. Accumulation of debris in the tubular lumina and vacuolization of tubular epithelial cells was evident in Group B, but not Group A. CONCLUSION: In explanted kidneys flushed with colloid-free Ringer's and machine-perfused for 72 h, almost the entire increase in mass occurred during the initial organ flush. As hypothesized, the addition of HES colloid sharply lowered the initial organ expansion, but unexpectedly exacerbated organ expansion during subsequent hypothermic machine perfusion, such that the HES-flushed kidneys gained more mass than the controls flushed without HES. Thus, the potential benefit of including HES in the initial flush solution was lost during machine perfusion. Arguably, the excessive expansion may have contributed to the impaired machine perfusion of these kidneys. Animal Use Protocol: IACUC-2020-0011Item Iatrogenic Ureter Injury and Repair: Comparing Suture Versus Suture and Glue in Ureter End-to-End Anastomosis(2023) Iloani, Nwamaka Amy; Wade, Michael; Joseph, Matthew; Martinez, Maria; Ntekim, Nedeke; Mallet, Robert T.; Yurvati, AlbertFunding: Dallas Southwest Osteopathic Physicians The ureter is a delicate structure due to its size and anatomical location. It is found deep within the retroperitoneal space and the lower third of the ureter lies adjacent to numerous pelvic structures such as the uterine artery, cervix, vagina, colon, and iliac vessels, and due to its proximity, the ureter is subject to unintentional accidental injury during diagnostic or medical procedures, termed iatrogenic. This paper and associated aim to explore the odds of iatrogenic ureteral injuries (IUI) when comparing different surgical methods, discuss the best preventative options and review the current repair measures, as well as any associated complications. The aim of this study focused on ureter transection that require surgical repair using a ureterouretal anastomosis, a procedure that ligates the free ends of the transected ureter together. Ureters were obtained from anesthetized Yorkshire pigs via standard laparotomy [protocol: IACUC-20-0011]. Ureters were transected laterally midway along the length of the harvested ureter. In Group A ureters, 2 standard stay sutures were placed 180˚ degrees opposite one another to anchor the ureter and ligated using a simple continuous running suture technique. In Group B, ureters were anchored in a similar fashion with stay sutures and ligated using glue to perform the anastomosis. 500 mL saline was then flushed through the ureter to assess the structural integrity of the ureterouretal anastomosis. Group A ureters showed less stricture formation and fluid leakage at the site of anastomoses, indicating that the standard suture technique used in ureter repair is a safe and reasonable gold standard. In contrast, Group B ureters showed stricture formation and some fluid leakage at suture/glue line, indicating the method of ureter anastomoses was inferior. When IV saline was pushed in Group B ureters, the fluid often exited the ureter at both the suture site and the end portion of the ureter, more so than the Group A ureters. Future study needs to be conducted in suture/glue method as it is promising. However, it is not at the level where it can replace current ureter repair end to end anastomoses gold standard. *These authors are solely listed in alphabetical order Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R25HL125447. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Item A Modified Kidney Perfusion System for Improved Data Acquisition and Assessment of Renal Function and Metabolism(2023) Wade, Michael; Farmer, George; Yurvati, Albert; Mallet, Robert T.PURPOSE In the United States, there are over 88,000 candidates on the waitlist for kidney donation as of December 2022 with less than 42,0000 transplant procedures being recorded in the same year. Coinciding with this continued disparity has been a growing interest in the technology to preserve and improve the function of kidneys for transplantation, most notably machine perfusion (MP). Studies have shown that MP is superior to static cold storage, the most common method of kidney preservation used globally, which has allowed transplant programs to expand the current donor pool by use of marginal donor kidneys. Despite these advancements, machine perfusion technology used clinically is limited in its use for obtaining critical information regarding the real-time assessment of renal function, fluid dynamics, and metabolism. Here we discuss the development of a modified kidney perfusion system that allows for enhanced data acquisition of perfusion parameters, renal arterial and venous sampling, and organ monitoring within a controlled environment. METHODS The modified kidney perfusion system was assembled to house and monitor a singular organ. The organ is placed in a chamber that allows for maximal cooling to 2˚C - 6˚C or temperature regulation up to room temperature. Renal artery and vein cannulation allow for accurate perfusate sampling in different parts of the system without disruption of organ perfusion. Continuous monitoring of pressure, flow, and resistance is performed using pressure and flow transducers in line with the polyethylene tubing. In line with the system includes an air trap to prevent gas emboli, modifiable filter, and screen for large particulate matter. A specialized rotor allows for pulsatile perfusion into the kidney while a servomotor is used to set a desired pressure or flow into the system. Values for flow, pressure, resistance are recorded in real time using Spike2 software and the corresponding waveforms displayed in the program. RESULTS & CONCLUSION The limitations of machine perfusion technology currently used clinically hinder the ability to obtain data for interpretation of pre-transplant renal function. This modified kidney perfusion system improves upon many of these limitations and provides entirely new methods to gain insight on kidney function. Assessment of renal metabolite production and injury markers can be obtained from multiple sites throughout the perfusion circuit and instantaneous recording of both pressure and flow waveforms throughout the duration of machine perfusion.Item Optimization of machine perfusion to preserve structure and function of porcine kidney(2021) Wade, Michael; Mallet, Robert T.; Yurvati, Albert; Williams, Arthur; Hare, Richard; Rayad, Ashraf; Chaudhari, SarikaPurpose: Improved preservation of explanted kidneys is essential to narrow the supply vs. demand disparity for transplantable human kidneys. This study evaluated whether the use of buffer containing mannitol, a non-toxic osmolyte that minimizes cellular edema, improves kidney preservation during prolonged hypothermic perfusion. Methods: Left kidneys were harvested from anesthetized Yorkshire pigs via laparotomy, followed by cold (2-4°C) machine perfusion for 72 h. Flow rate and resistance were recorded throughout perfusion to assess renal vascular function, then biopsy for histological analysis of hematoxylin and eosin-stained renal cortex and medulla. Results: Kidneys with comparable initial flows and resistances received standard (A) or mannitol-enriched (B) perfusion buffer. Flows (ml/min)/resistances (mm Hg/ml/min) were 65/0.40 (A) vs. 65/0.38 (B) at 5 h, 27/1.03 (A) vs. 54/0.46 (B) at 24 h, and 13/1.99 (A) vs. 49/0.50 (B) at 48 h perfusion. The mass of Kidney A increased by 47%, and that of kidney B by 39%, over 72 h. Histology revealed improved preservation of tubular and glomerular architecture in B vs. A. Conclusion: The addition of mannitol afforded appreciable improvement in renal vascular function during machine perfusion and reduced tubular necrosis when compared to mannitol-free perfusion. Preservation of renal structure using mannitol-enhanced perfusion buffer was superior to the current standard of cold machine perfusion buffer for preserving human kidneys for transplant. By reducing cellular damage and maintaining renal perfusion, the optimization of cold machine perfusion with mannitol holds the potential to increase the availability of kidneys suitable for transplantation.Item Optimizing Preservation Solutions for Hypothermic Machine-Perfused Porcine Kidneys(2022) Ramos, Katherine; Wade, Michael; Williams, Arthur; Mallet, Robert T.; Yurvati, AlbertBackground: With the mounting incidence of hypertension, type 2 diabetes and other risk factors, end-stage renal disease (ESRD) is increasingly prevalent in the U.S. and worldwide, especially among racial minorities including Hispanics and African Americans. Kidney transplant is the most effective treatment for ESRD, but the supply of transplantable kidneys is inadequate to meet the increasing demand. Before transplantation, kidneys from deceased donors are maintained in a device (LifePort) that pumps ice-cold preservation solution through the organ. Although this hypothermic machine perfusion (HMP) is the most effective kidney preservation method available, kidneys from deceased donors often fail to resume function after transplant, especially when HMP is prolonged. Consequently, any kidneys not transplanted within 30 hours of harvest are discarded. We propose that optimizing the composition of the preservation solution may prevent post-transplant kidney failure and thereby improve kidney transplant outcomes, and potentially extend the useful lifespan of explanted kidneys beyond 30 hours, thereby increasing the pool of available kidneys for transplant. Hypothesis: Hypothermic perfusion of kidneys with solutions containing pyruvate, an energy substrate, anti-oxidant and anti-inflammatory compound, will better maintain perfusion flow and lower vascular resistance, indicating improved stability of the explanted organs. Methods: Kidneys obtained from thirteen anesthetized female Yorkshire pigs were fluid-flushed and then perfused for 72 h with modified, cold (2-4?C) Ringer's solution containing 20 mM glucose, 30 mM mannitol, 50 g/l hydroxyethyl starch ? 20 mM pyruvate in a LifePort organ preservation device while flow and renal vascular resistance were monitored and recorded. Results: Kidneys maintained adequate perfusion for 72 hours despite a 50 ? 4% (mean ? SEM) increase in kidney mass indicating edema. Flows plateaued within the first 6 hours of perfusion and then declined slowly with prolonged perfusion. At 6 h perfusion, flows (ml/min*g) were 0.54 ? 0.06 in kidneys receiving control solution, and 0.61 ? 0.06 in kidneys perfused with pyruvate-enriched solution. At 24 and 72 h, flows were 0.49 ? 0.06 and 0.38 ? 0.04 in the control kidneys, and 0.49 ? 0.03 and 0.37 ? 0.02 in the kidneys perfused with pyruvate enriched solution, respectively. The increases in mass of the control (52?4%) and pyruvate (46?6%) over 72 h perfusion did not differ. Conclusions: Pyruvate augmentation of preservation solution did not affect hypothermic kidney perfusion. The possibilities that pyruvate may affect the kidney's energy and/or antioxidant metabolism without altering organ perfusion or edema, and that measures limiting edema could improve kidney perfusion during HMP, remain to be tested.Item Pyruvate-enriched solution limits lactate and creatine accumulation in hypothermic machine-perfused kidney(2023) Mohammad, Moath; Tamayo, Jesus; Wade, Michael; Tran, Amanda; O'Hara, Collin; O-Yurvati, Albert; Mallet, Robert T.With a 5% annual increase in incidence, end-stage renal disease (ESRD) is a mounting epidemic. Kidney transplantation is the only definitive treatment for ESRD, but the growing demand for transplantable kidneys greatly exceeds the supply, thus improved methods of organ preservation are urgently needed. Recently, renewed interest in hypothermic machine perfusion (HMP) has prompted refinements of perfusion solutions to improve graft performance and transplant outcomes. In previous studies, solutions containing the intermediary metabolite pyruvate prevented ATP depletion, detoxified reactive oxygen species, suppressed inflammation and optimized protection of ischemic myocardium and brain. This study aimed to examine the hypothesis that pyruvate-enriched preservation fluids provide robust preservation of histological structure and energy metabolism in porcine kidneys during 72 hours HMP. The study used 7 Yorkshire swine kidneys, which were harvested and immediately flushed with ice-cold Ringer's solution and then perfused with either control or 20 mM pyruvate-enriched Ringer's solution for 72 hours in a LifePort organ preservation system. Values for renal artery flow and resistance were recorded over time. After 72 hours HMP, the renal cortex was biopsied and analyzed for metabolite content via spectrophotometry, while the cortex and medulla were biopsied for histological evaluation. The results showed that pyruvate-enriched preservation fluids lowered creatine content by 89% (P < 0.05), and surprisingly lowered lactate content while increasing glucose-6-phosphate content, a source for maintaining antioxidant reducing power. Flow and resistance were comparable between the two groups, and histological analysis revealed interstitial and intracellular edema, varying stages of acute tubular necrosis and variable loss of overall glomerular architecture in both groups. In conclusion, pyruvate-enriched preservation fluids stabilized the cellular energy state, supplied glucose-6-phosphate for sustaining antioxidants, and shunted metabolites from the glycolytic pathway leading to lower lactate accumulation in the renal cortex. Further research is warranted to understand pyruvate's impact on oxidative stress and inflammation during HMP.