In-vivo Testing of a Bio-Artificial KidneyStolyar R.; Rojas Pena A., MD; Cook K., PhD; Humes H. D., MD

AbstractTitle: Bioartificial Renal Cell System: Developing an alternative method of renal replacement therapy in a large animal model.

Over 200,000 Americans suffer from end stage renal disease (ESRD) and this number increases ~9 percent/year. Current therapies for ESRD include renal transplantation and dialysis (hemodialysis –HD, or peritoneal dialy-sis –PD). Organ transplantation is limited due to organ scarcity, dialysis is efficient for filtration, but lacks other renal functions (immune modulation, endocrine, and metabolic), and they are also associated with complica-tions such as bleeding, patient compliance, high cost, and infection. A Bioartificial Renal Epithelial Cell System (BREC-d) is being developed as a potential alternative. BREC-d provides secondary renal function that dialysis can not offer. To test the BREC-d before clinical trials, we have developed a novel uremic sheep model, supplemented by PD, with the objective to asses BREC-d suitability during PD, and to identify the effects of BREC-d compared to standard PD techniques.

Methods: 45-60kg sheep sustained bilateral nephrectomies under anesthesia in two surgeries. Surgery one, left nephrectomy and placement of PD catheters. Second surgery takes place after 2 week recovery period, the neck lines for hemodynamic control (carotid artery) and i.v. medication administration (external jugular vein) a placed, followed by right nephrectomy. Three groups were studied, control (5 animals with acellular BREC-d units); study group L (6 animals with lamb cellular BREC-d units); and study group H (5 animals with human cellular BREC-d units). Pain medications are administered immediately after surgery. In all animals, PD with BREC-d therapy is begun within 24 hours of surgery 2 dialysis exchanges of 3-5L are performed once daily. Data Collection: Hemodynamics, arterial blood gases, PD fluid flow rates, and blood samples for chemistry. Animals from both study groups L and H (n=11) were lumped together, and evaluated against the control group (n=5).

Results:On average, both groups are able to maintain heart rates within normal range of 60-120 beats/min. Mean arterial pressure was maintained within the acceptable range of 80-130 mmHg by the lumped study group, however control group experienced hypotension within 6 days. No difference in central venous pressure was observed initially, the control group spiked towards end of study due to external attempts to improve hypo-tension. Respiration rate for both groups was elevated after surgery 2 due to pain and discomfort caused by large quantity of dialysis fluid in the peritoneum. 6 days of exchanges were required before K+ values could be reduced to non life-threatening levels. PD flow for control group was, on average higher than study group, due to a change in protocol at advanced stages of study. Post surgery 2 average values of blood urea nitrogen and creatine was 58 mg/dL and 5.8 mg/dL indication of uremic status of all the animals before treatment. Recovered BREC-d cells consume, on average 39.58 nMoles of oxygen/min in-vivo, compared to 19.24 nMoles in-vitro, indication of system viability throughout study.

Conclusions:Cellular BREC-d units are able to maintain adequate mean arterial pressures. PD is suitable in this large animal model if no problems such as occlusions or leaks occur with the catheters within the first 48 hours. BREC-d environment is suitable as demonstrated by recovered BREC-d cells oxygen consumption. Continuous PD isn’t as efficient on this model demonstrated by high values of BUN, Creatine and K+ and the need to perform 2-3 dialysate exchanges through the day. Future work will be done via extending study periods (>2 weeks), increasing the PD exchanges (>1/daily), analyzing sheep vs. human BREC-d performance.

BackgroundEnd Stage Renal Disease (ESRD) is a serious medical condition and it’s incidence and prevalence is increasing. It’s estimated that the number of patients with ESRD will in-crease to more than 770,000 by the end of 2020 in the US. (1)

ESRD is treated with kidney transplantation, or renal replacement therapies such as peritoneal dialysis (PD) and hemodialysis (HD). Renal transplantation is limited due to organ shortage, and dialysis therapies are effective at replacing primary renal functions such as filtration, fluid balance, metabolite and electrolyte regulation. However, they are not capable of replacing the secondary immunological, endocrine, and metabolic func-tions of the kidney.

The Bioartificial Renal Epithelial Cell System (BREC-d) developed by Innovative Bio-therapies, Inc (Ann Arbor, MI) may be able to partially replace these secondary functions when used in conjunction with PD or HD. The BREC-d contains live renal cells obtained from either donated human kidneys unfit for transplantation, or lamb kidneys.

The main goal of this study is to assess the feasibility and biocompatibility of the BREC-d unit in the setting of peritoneal dialysis in a large uremic animal model. Simulta-neously, this study will test effectiveness of the BREC-d unit to reduce inflammation as-sociated with ESRD.

Reference

1. U.S. Renal Data System, USRDS 2009 Annual Data Report: Atlas of Chronic Kidney Dis-ease and End-Stage Renal Disease in the United States, National Institutes of Health, Na-tional Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2009; (2): 231-241

Table 1

Chart 5

Results

Viability of Renal Cell System Oxygen Consumption in vivo

Average oxygen consumption in vitro is 19.24 mmoles of Oxygen/ min

This data illustrates that renal cells can survive in BREC-d environment.

Animal Oxygen Consumption of Recovered Renal Cells

Sheep 16 49.76 +/- 3.25 mmoles of Oxygen/ minSheep 17 35.88+/- 7.86 mmoles of Oxygen/ minSheep 18 33.08 +/- 4.18 mmoles of Oxygen/ min

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Experimental Time (Hrs)

Average Sheep Central Venous Pressure per Treatment Group

Error bars = SEMAcellular-BREC (n=5) Cellular- BREC (n=11)

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Pre Brecs Day 0

Brecs Day 1

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Sheep 18 Metabolic Waste

BUN mg/dL (Reference Range 5.0-20.0) Creatine mg/dL (Reference Range 0.6-1.5)

mg/

dl

Results and DiscussionUremic status of animals is confirmed by Day 1 potassium concentrations of 7.4mEq/L (Reference Range 3.5-4.5mEq/L) at the beginning of the study (Chart 1), as well as by metabolite (BUN and Creatinine) concentrations up to four times higher than the normal upper ranges (20.0 and 1.5 mg/dL respec-tively) (Chart 2)

Sheep heart rate (HR)(Reference Range 60-120 bpm) is not significantly differ-ent between groups until day 6. Instability in the acellular group at later stages may be due to ventricular tachycardia induced by uremia, and fluid im-balance caused by poor PD management.

Chart 3 represents mean arterial pressures (MAP). MAP in the cellular group had acceptable values (Reference Range, 73-120 mmHg) throughout the course of the study. The acellular group became hypotensive (MAP< 73mmHg) in later stages of the experiment.

Central venous pressure (CVP) is represented in Chart 4. After 6 days, a differ-ence in CVP is observed between the acellular and cellular group. This may be due to poor PD management.

Table 1, summarizes activity of renal cells in the BREC-d units during the length of the study. Recovered renal epithelial cells have post experiment oxygen consumption levels of ~39.6 mmoles/min, more than twice as high as in-vitro cells (19.24 mmoles/min), demonstrating viability of PD as a source of oxygen and nutrients.

Respiratory rate (RR)(Reference Range 10-20bpm), is faster than the normal upper range for both groups, but stabilizes as experiment progresses. Sheep use their abdominal muscles to breathe, and the amount of PD dialysate re-quired for this study is large quantity (3-5 liters), and the animals become tachypneic.

The average life span of an animal from the cellular group is 7.9 days with a SEM of 1.075 days, and the average life span on an animal from the acellular group is 6.9 days with a SEM of 1.45 days.

MethodsAll animals were cared for by the standards of the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.

Uremic Animal Model: Sixteen (~50kg) female sheep were anesthetized to surgically place two PD catheters in the abdominal cavity, followed by left nephrectomy in all ani-mals. After two weeks of recovery time, the right kidney was removed, and neck lines to monitor hemodynamics (mean arterial pressure, heart rate, central venous pressure) were placed in the carotid artery and the external jugular vein.

Study Groups: Two study groups were investigated, a cellular BREC-d group (n=11) and an acellular study group (n=5)

Data Collection: Electrolytes (K) and metabolite (BUN and Creatininie) concentration in the blood, hemodynamics (heart rate, mean arterial pressure, central venous pressure), respiration rate, and PD circuit main flow rate was collected every hour for up to ten days.

Data Analysis: Cellular BREC-d study groups were evaluated as one against the control group A. Excel 2007 was used to analyze numerical data collected. Averages, standard deviations, and standard error of the means (SEM) were performed on all variables. Av-erages and SEM were graphed.

Chart 1 Chart 2

Chart 3

ConclusionsRenal epithelial cells are viable for the entire course of study in this setting of PD. Cells consume oxygen at higher rates than cells from in-vitro studies.

The cellular BREC-d units appear to maintain hemodynamics within normal values as compared to the acellular units, possibly due to a cardioprotection effect by the renal cells and better regulation of the inflammatory cascade.

Unfortunately complications such as catheter occlusions and infections lead to the early terminations of certain animal studies. Poor PD management led to problems with dangerously high K+ values.

Future research will analyze the effects of the BREC-d for longer periods of time (>two weeks), increase the frequency of dialysis exchanges, and analyze the differences in performance between human and lamb renal cells.

Explanation of PD CurcuitFigure 1: 3-5 liters of PD fluid (dialysate) was infused into the abdominal cavity. PD fluid was pumped continuously out of the animal at ~100mL/min, while another pump is re-moving 120mL/min of dialysate (waste), and third pump is adding new dialysate at 140mL/min in order to compensate fluid absorption by the animals abdominal surface. After, the main circuit pump dialysate passes through an F-80 (filter) which filters out toxins that can damage the live renal cells in the BREC-d. Then a roller pump, pumps a constant 50mL/min of fluid through the BREC unit, while the rest of the dialysate by-passes the BREC-d unit, to avoid higher resistance in the F-80 filter. Then the dialysate is filtered by the F-40, and pumped back into the sheep.

Reference Range 3.5-4.5mEq/L

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Study DayPD Fluid Exchange Rate: 1/Day

Avg Acellular K+ Values (n=3)

Avg K+ Before PD Exchange Avg K+ After PD Exchange

Normal Range for Sheep (80-130 mmHg)

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Experimental Time (Hrs)

Average Mean Arterial Blood Pressure: Acellular vs CellularError bars = SEM

Acellular-BREC (n=5) Cellular-BREC (n=11)

Chart 4

Figure 1

Acknowledgments: Rojas Pena A., MD

To request a copy of this poster, contact rodionst@umich.edu