Scientific Poster presented at UROP symposium at the University of Michigan-Ann Arbor

1. In-vivo Testing of a Bio-Artificial Kidney
Stolyar R.; Rojas Pena A., MD; Cook K., PhD; Humes H. D., MD
Abstract Figure 1 Results and Discussion
Title: 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- Uremic status of animals is confirmed by Day 1 potassium concentrations of
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.
7.4mEq/L (Reference Range 3.5-4.5mEq/L) at the beginning of the study
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
(Chart 1), as well as by metabolite (BUN and Creatinine) concentrations up to
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
four times higher than the normal upper ranges (20.0 and 1.5 mg/dL respec-
from both study groups L and H (n=11) were lumped together, and evaluated against the control group (n=5).
Results:
tively) (Chart 2)
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
Sheep heart rate (HR)(Reference Range 60-120 bpm) is not significantly differ-
in-vitro, indication of system viability throughout study.
Conclusions:
ent between groups until day 6. Instability in the acellular group at later
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.
stages may be due to ventricular tachycardia induced by uremia, and fluid im-
balance caused by poor PD management.
Background Explanation of PD Curcuit
Chart 3 represents mean arterial pressures (MAP). MAP in the cellular group
Figure 1: 3-5 liters of PD fluid (dialysate) was infused into the abdominal cavity. PD fluid had acceptable values (Reference Range, 73-120 mmHg) throughout the
End Stage Renal Disease (ESRD) is a serious medical condition and it’s incidence and was pumped continuously out of the animal at ~100mL/min, while another pump is re-
prevalence is increasing. It’s estimated that the number of patients with ESRD will in- course of the study. The acellular group became hypotensive (MAP<
moving 120mL/min of dialysate (waste), and third pump is adding new dialysate at
crease to more than 770,000 by the end of 2020 in the US. (1) 140mL/min in order to compensate fluid absorption by the animals abdominal surface.
73mmHg) in later stages of the experiment.
After, the main circuit pump dialysate passes through an F-80 (filter) which filters out
ESRD is treated with kidney transplantation, or renal replacement therapies such as toxins that can damage the live renal cells in the BREC-d. Then a roller pump, pumps a Central venous pressure (CVP) is represented in Chart 4. After 6 days, a differ-
peritoneal dialysis (PD) and hemodialysis (HD). Renal transplantation is limited due to constant 50mL/min of fluid through the BREC unit, while the rest of the dialysate by- ence in CVP is observed between the acellular and cellular group. This may be
organ shortage, and dialysis therapies are effective at replacing primary renal functions passes the BREC-d unit, to avoid higher resistance in the F-80 filter. Then the dialysate is due to poor PD management.
such as filtration, fluid balance, metabolite and electrolyte regulation. However, they are filtered by the F-40, and pumped back into the sheep.
not capable of replacing the secondary immunological, endocrine, and metabolic func-
Table 1, summarizes activity of renal cells in the BREC-d units during the
tions of the kidney.
length of the study. Recovered renal epithelial cells have post experiment
The Bioartificial Renal Epithelial Cell System (BREC-d) developed by Innovative Bio- Results oxygen consumption levels of ~39.6 mmoles/min, more than twice as high as
therapies, Inc (Ann Arbor, MI) may be able to partially replace these secondary functions Chart 1 Chart 2 in-vitro cells (19.24 mmoles/min), demonstrating viability of PD as a source of
when used in conjunction with PD or HD. The BREC-d contains live renal cells obtained oxygen and nutrients.
from either donated human kidneys unfit for transplantation, or lamb kidneys. Reference Range 3.5-4.5mEq/L Sheep 18 Metabolic Waste
90
Avg Acellular K+ Values (n=3)
Avg K+ Before PD Exchange Avg K+ After PD Exchange
80 Respiratory rate (RR)(Reference Range 10-20bpm), is faster than the normal
The main goal of this study is to assess the feasibility and biocompatibility of the 8 70
upper range for both groups, but stabilizes as experiment progresses. Sheep
60
BREC-d unit in the setting of peritoneal dialysis in a large uremic animal model. Simulta- 7
6
50
use their abdominal muscles to breathe, and the amount of PD dialysate re-
mg/dl
neously, this study will test effectiveness of the BREC-d unit to reduce inflammation as- 40
sociated with ESRD.
5
30 quired for this study is large quantity (3-5 liters), and the animals become
mEq/L
4
20
3 10
tachypneic.
2 0
Reference 1 Pre Pre Brecs Brecs Day Brecs Day Brecs Day Final
0 Chart 5 Surgery 1 Day 0 1 2 3
The average life span of an animal from the cellular group is 7.9 days with a
0 1 2 3 4 5 6 7 8
1. U.S. Renal Data System, USRDS 2009 Annual Data Report: Atlas of Chronic Kidney Dis- Study Day
PD Fluid Exchange Rate: 1/Day
BUN mg/dL (Reference Range 5.0-20.0) Creatine mg/dL (Reference Range 0.6-1.5)
SEM of 1.075 days, and the average life span on an animal from the acellular
ease and End-Stage Renal Disease in the United States, National Institutes of Health, Na- group is 6.9 days with a SEM of 1.45 days.
tional Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2009; (2): Chart 3 Chart 4
231-241 Average Sheep Central Venous Pressure per Treatment
Normal Range for Sheep (80-130 mmHg) Group
Conclusions
Error bars = SEM
Methods
Acellular-BREC (n=5) Cellular- BREC (n=11)
Average Mean Arterial Blood Pressure: Acellular vs Cellular
Error bars = SEM 35
Acellular-BREC (n=5) Cellular-BREC (n=11) 30
140
25
All animals were cared for by the standards of the University Committee on Use and
120
20
Renal epithelial cells are viable for the entire course of study in this setting of
mmHg
100
Care of Animals (UCUCA) at the University of Michigan. 15 PD. Cells consume oxygen at higher rates than cells from in-vitro studies.
mmHg
80
10
60
5
40
Uremic Animal Model: Sixteen (~50kg) female sheep were anesthetized to surgically 0
20
place two PD catheters in the abdominal cavity, followed by left nephrectomy in all ani- 0
0 24 48 72 96 120 144 168 192 216 240
The cellular BREC-d units appear to maintain hemodynamics within normal
Experimental Time (Hrs)
mals. After two weeks of recovery time, the right kidney was removed, and neck lines to 0 24 48 72 96 120 144 168 192 216 240
values as compared to the acellular units, possibly due to a cardioprotection
Experimental Time (Hrs)
monitor hemodynamics (mean arterial pressure, heart rate, central venous pressure) effect by the renal cells and better regulation of the inflammatory cascade.
were placed in the carotid artery and the external jugular vein. Table 1
Study Groups: Two study groups were investigated, a cellular BREC-d group (n=11) and Unfortunately complications such as catheter occlusions and infections lead
an acellular study group (n=5) Viability of Renal Cell System to the early terminations of certain animal studies. Poor PD management led
 Oxygen Consumption in vivo
to problems with dangerously high K+ values.
Data Collection: Electrolytes (K) and metabolite (BUN and Creatininie) concentration in
Animal Oxygen Consumption of
the blood, hemodynamics (heart rate, mean arterial pressure, central venous pressure), Recovered Renal Cells Future research will analyze the effects of the BREC-d for longer periods of
respiration rate, and PD circuit main flow rate was collected every hour for up to ten Sheep 16 49.76 +/- 3.25 mmoles of Oxygen/ min
time (>two weeks), increase the frequency of dialysis exchanges, and analyze
Sheep 17 35.88+/- 7.86 mmoles of Oxygen/ min
days. Sheep 18 33.08 +/- 4.18 mmoles of Oxygen/ min the differences in performance between human and lamb renal cells.
 Average oxygen consumption in vitro is 19.24 mmoles of
Data Analysis: Cellular BREC-d study groups were evaluated as one against the control Oxygen/ min
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-  This data illustrates that renal cells can survive in
BREC-d environment.
erages and SEM were graphed. Acknowledgments: Rojas Pena A., MD
To request a copy of this poster, contact rodionst@umich.edu