Bioartificial Kidney Ameliorates Gram-Negative Bacteria-Induced Septic Shock in Uremic Animals
William H. Fissell,
Liandi Lou,
Simin Abrishami,
Deborah A. Buffington and
H. David Humes
Departments of Medicine, Veterans Administration Medical Center and The University of Michigan, Ann Arbor, Michigan.
Correspondence to Dr. H. David Humes, Department of Internal Medicine, University of Michigan Medical School, 7220 MSRB III, Box 0644, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0726. Phone: 734-647-8018; Fax: 734-763-4851;
ABSTRACT. The bioartificial kidney (BAK) consists of a conventionalhemofiltration cartridge in series with a renal tubule assistdevice (RAD) containing 109 porcine renal proximal tubule cells.BAK replaces filtration, transport, and metabolic and endocrinologicactivities of a kidney. Previous work in an acutely uremic dogmodel demonstrated that BAK ameliorated endotoxin (lipopolysaccharide[LPS])-induced hypotension and altered plasma cytokine levels.To further assess the role of BAK in sepsis in acute renal failure,dogs were nephrectomized and 48 h later administered intraperitoneallywith 30 x 1010 bacteria/kg of E. coli. One hour after bacterialadministration, animals were placed in a continuous venovenoushemofiltration circuit with either a sham RAD without cells(n = 6) or a RAD with cells (n = 6). BP, cardiac output, heartrate, pulmonary capillary wedge pressure, and systemic vascularresistance were measured throughout the study. All animals testedwere in renal failure, with blood urea nitrogen and serum creatinineconcentrations greater than 60 and 6 mg/dl, respectively. RADtreatment maintained significantly better cardiovascular performance,as determined by arterial BP (P < 0.05) and cardiac output(P < 0.02), for longer periods than sham RAD therapy. Consistently,all sham RAD-treated animals, except one, expired within 2 to9 h after bacterial administration, whereas all RAD-treatedanimals survived more than 10 h. Plasma levels of TNF-, IL-10,and C-reactive protein (CRP) were measured during cell RAD andsham RAD treatment. IL-10 levels were significantly higher (P< 0.01) during the entire treatment interval in the RAD animalscompared with sham controls. These data demonstrated in a pilotlarge animal experiment that the BAK with RAD altered plasmacytokine levels in acutely uremic animals with septic shock.This change was associated with improved cardiovascular performanceand increased survival time. These results demonstrate thatthe addition of cell therapy to hemofiltration in an acutelyuremic animal model with septic shock ameliorates cardiovasculardysfunction, alters systemic cytokine balance, and improvessurvival time. E-mail: dhumes@umich.edu
The development of acute renal failure (ARF) in a hospitalizedpatient results in a 5- to 8-fold higher risk of death (1,2).Although hemodialysis or hemofiltration treatment with its smallsolute and fluid clearance function has prevented death fromhyperkalemia, volume overload, and uremic complications, suchas pericarditis, patients with ARF still have mortality ratesexceeding 50%.
Acute renal failure secondary to ischemic and/or nephrotoxiccauses arises from acute tubular necrosis (ATN), predominantlyto renal proximal tubule cells. Replacement of the functionsof these cells during the episode of acute tubular necrosiswould provide almost full renal replacement therapy in conjunctionwith hemofiltration. The addition of metabolic activity, suchas ammoniagenesis and glutathione reclamation; endocrine activity,such as vitamin D3 activation; and cytokine homeostasis mayprovide additional physiologic replacement activities to changethe current natural history of this disease process (3).
Our laboratory has developed an extracorporeal device usinga standard hemofiltration cartridge containing approximately109 renal tubule cells grown as confluent monolayers along theinner surface of the fibers (4–8). The nonbiodegradabilityand the pore size of the hollow fibers allow the membranes toact as scaffolds for the cells and as an immunoprotective barrier.In vitro studies of this renal tubule assist device (RAD) haveshown that the cells retain differentiated active transportproperties, differentiated metabolic activities, and importantendocrine processes (6). Further studies have shown that theRAD, when incorporated in series with a hemofiltration cartridgein an extracorporeal blood perfusion circuit to formulate abioartificial kidney (BAK), replaces filtration, transport,metabolic, and endocrine functions of the kidney in acutelyuremic dogs (7). Studies from our laboratory have also shownthat the RAD ameliorates endotoxin shock secondary to intravenousinfusion of lipopolysaccharide (LPS) in acutely uremic animals(9).
To further assess the role of the RAD and the BAK in sepsisin acute renal failure, nephrectomized dogs were challengedwith intraperitoneal administration of 30 x 1010/kg body weightof E. coli bacteria followed by treatment with a cell containingRAD or sham RAD cartridge. This report summarizes an initialpilot animal study to assess the influence of the RAD on variouscardiovascular and biochemical parameters in this model andtests the hypothesis that renal cell therapy in a BAK wouldadd metabolic renal function, be associated with changes insystemic cytokine balance, and provide a survival advantagein a uremic animal model of septic shock.
Mongrel dogs weighing approximately 20 to 25 kg were fed a low-proteindiet for seven days before being used for this experimentalprotocol, as previously reported (7–9). Animals underwentbilateral nephrectomy to produce an acute uremic state. Aftersurgery, they were permitted to eat and drink ad libitum for48 h. The water and salt ingestion during this recovery periodwas no different between the two groups, because the fillingpressures to both the left and right ventricles were similarin both groups at baseline at the beginning of the experimentalprotocol. Blood samples for electrolytes and complete bloodcounts were obtained before the low-protein diet, before surgery,and on postoperative days 1 and 2. The animals were sedatedwith thiopental sodium (Abbot Laboratories, North Chicago, IL),intubated, and administered isofluorane (Baxter Healthcare,Deerfield, IL) general anesthesia via endotracheal tube. Anesophageal thermometer was inserted, and temperature was monitored.A dialysis catheter (Bard Access Systems, Salt Lake City, UT)was inserted via external jugular vein into the right atrium.An arterial catheter (Arrow International, Reading, PA), a venouscatheter (Diag, Minnetonka, MN), and a Swan-Ganz thermodilutioncardiac output catheter (Argon Medical, Athens, TX) were placedand transduced. Arterial, central venous, and pulmonary arterywaveforms, as well as pulmonary capillary wedge pressure (PCWP)were measured (Model Viridia 24C; Hewlett-Packard GmbH, Böblinge,Germany) before institution of continuous venovenous hemofiltration(CVVH) and at hourly intervals thereafter. Cardiac outputs weremeasured by thermodilution (Model COM-1; American Hospital Supply,Irvine, CA) before CVVH and at hourly intervals thereafter.CVVH was interrupted and the dialysis lines clamped during cardiacoutput and PCWP measurements. BP, pulse, and temperature wererecorded at 15-min intervals. Blood aliquots were obtained atvarious time interval hours for serum chemistries, blood counts,cytokine measurements, and endotoxin levels.
After all access lines were secured in the animal and baselineparameters and laboratory studies obtained, 100 ml of brothcontaining 30 x 1010E. coli/kg was instilled into the animals’peritoneal cavities by means of a small take-down of the nephrectomyincision. The E. coli strain was serotype 06:K2:H1 (ATCC, Manassas,VA). This model was chosen because it has been used as a modelof Gram-negative sepsis in other canine studies (10,11). Toensure that similar doses of bacteria were administered to eachanimal, the concentration of primary cultured bacteria was quantifiedfor each experiment by plating 2.5 x 106-fold dilutions on nutrientagar and counting visible colonies after 24-h incubation at37°C.
Within 1 h after bacterial insult, CVVH was instituted (GambroAK-10; Gambro Lundia AB, Sundsvall, Sweden) with an F-40 hollow-fiberdialyzer (Fresenius, Walnut Creek, CA). Extracorporeal bloodflow was regulated at 120 ml/min. Ultrafiltrate production wasmonitored, and a balanced electrolyte replacement solution wasinfused on a 1:1 volume replacement basis. In addition to theconventional CVVH circuit, a RAD containing porcine proximaltubule cells (cell RAD) or an otherwise identically treatedhollow-fiber dialyzer without proximal tubule cells (sham RAD)was introduced. The tubule cells seeded into the RAD had beenpassaged 3 to 4 times. Alkaline phosphatase and -glutamyltransferaseimmunostaining demonstrated that more than 95% of the cellswere positive for these proximal tubule cell markers, and CD45white blood cell marker was negative in the cell preparation.Six dogs were used in the cell RAD group and six dogs in thesham RAD group. RAD fabrication has been described in detailelsewhere (6–8). This fabrication is based on an F40 Freseniushemofiltration cartridge comprised of 4950 polysulphone hollowfibers with an inner diameter of 200 µm, a total diameterof 240 µm, and a molecular weight cut-off of 45,000 daltons.Fourteen ml/min of ultrafiltrate from the hemofilter was directedinto the luminal space of the RAD, and thus into direct contactwith cultured porcine proximal tubule cells or polysulfone hemodialysismembranes, in the cell RAD and the sham RAD, respectively. Ofthe 120 ml/min blood flow through the hemofilter, 80 ml/minof post-hemofilter blood was directed into the extraluminalspace of the RAD. Ultrafiltration rates were maintained at 14ml/min. Transmural and hydraulic pressure gradients in the RADwere adjusted to maintain a reabsorption rate of 7 ml/min ofultrafiltrate into the post-hemofilter extracapillary bloodcompartment.
Animals with sham RAD and cell RAD were resuscitated identicallyaccording to a standard protocol (10,11). To simulate humanseptic shock therapy, all dogs received intravenous ceftriaxonesodium (100 mg/kg; Roche Laboratories, Nutleg, NJ) at hour 1after bacterial administration. Volume resuscitation with 80ml/kg of crystalloid in 20 ml/kg boluses was administered whenthe animal’s mean arterial pressure declined below 70mmHg and was accompanied by a PCWP below 15 mmHg. When systemichypotension persisted after a cumulative dose of 80 ml/kg ofcrystalloid had been administered, and filling pressures asestimated by PCWP remained low, 80 ml/kg of colloid (Hespan;B. Braun, Bethlehem, PA) in divided 20 ml/kg boluses was alsoadministered. All dogs received this complete volume resuscitationregimen within the first hour after bacteria seeding. No animalsreceived vasopressor or inotropic agents. Animals were observeduntil no variation in arterial pressure waveform could be detectedor until 15 h had elapsed and were then euthanized. Blood sampleswere drawn from animals of both groups during various time intervalsof the experiment for measurement of levels of endotoxin, chemistries,cytokines, and 1,25-(OH)2 vitamin D3.
Assays
Serum chemistries were measured with an automated chemical analyzer(Synchron CX-7; Beckman Instruments, Brea, CA). IL-10 and TNF-levels in the serum samples were evaluated in triplicate byELISA. Anti-human IL-10 and anti-human TNF- antibodies, thekind gift of Dr. Robert Streiter (University of Michigan, AnnArbor, MI), showed a strong cross-species affinity with cytokinesproduced by both porcine and canine LPS-stimulated monocytesin culture. C-reactive protein levels were also measured usinga commercially available ELISA assay kit (Tri-Delta Diagnostics,Morris Plains, NJ). Levels of 1,25-(OH)2 vitamin D3 were measuredwith two commercially available kits, an ELISA assay (AmericanLaboratory Products Co., Windham, NH) and a 1,25 RIA (DiaSorin,Stillwater, MN). Endotoxin levels were determined with an assaykit (BioWhitaker, Walkersville, MD).
Statistical Analyses
Cardiovascular and cytokine data were analyzed by repeated-measuresANOVA over all time points. Endotoxin levels, plasma levelsof solutes, and survival times were compared utilizing t test,paired or nonpaired as appropriate.
The two groups of animals achieved nearly identical degreesof acute uremia after bilateral nephrectomies, as detailed inTable 1. The degree of small solute clearance achieved duringthe treatment periods with the extracorporeal CVVH circuit werealso similar between the two groups (Table 1). Both groups achievedsignificant reductions in blood urea nitrogen (BUN) and plasmacreatinine levels with hemofiltration using either cell or shamRAD.
The levels of endotoxin in blood after E. coli infusion didnot differ significantly between the two groups during the timecourse of the experiments, as displayed in Figure 1. The cardiovascularparameters in the sham RAD and cell RAD groups are summarizedin Table 2. Cardiac outputs in the cell-treated group were significantlyhigher than in the sham-treated group (Figure 2; P < 0.02).Cardiac output was substantially better maintained in the cellRAD animals compared with the sham RAD animals throughout theentire treatment period. This improved cardiac output was notassociated with differences in ventricular filling pressures,as reflected by comparable PCWP in both groups (Table 2). Withthis improved cardiac contractility, systolic BP were significantlyhigher in the cell-treated versus the sham-treated group (Figure 3;P < 0.05). The divergence of this physiologic parameterwas apparent only after hour 4 of the experimental protocol.Mean arterial pressures, diastolic BP, and systemic vascularresistances were not different between the groups (Table 2).These improved cardiovascular parameters translated into a significantsurvival advantage in the RAD group compared to the controlsham group (Figure 4, P < 0.02). The RAD animals survived13.0 ± 0.7 h versus 9.7 ± 0.5 h in the sham controls.
Figure 1. Plasma endotoxin concentrations (mean ± SE) in the sham RAD and cell renal tubule assist device (RAD) groups at various time intervals after bacterial infusion into the peritoneal cavity. The last value for the cell RAD group was measured during the last hour before death.
Figure 2. Cardiac outputs (mean ± SE) at hourly time intervals in the two groups of animals after E. coli infusion. The cardiac outputs were significantly greater in the cell RAD group compared with sham control (P < 0.02; n = 6).
Figure 3. Systolic BP (mean ± SE) at hourly time intervals after Gram-negative bacterial administration. BP was significantly better maintained (P < 0.05; n = 6) in the RAD-treated animals compared with the sham controls.
Figure 4. Survival curves of the sham and cell RAD groups. Survival time was significantly greater in the cell RAD animals than in the sham controls (P < 0.002).
The durability and viability of the cell RAD during exposureto animals in uremia and septic shock was assessed. Tubule cellcounts in the processed ultrafiltrate exiting the RAD were quantitatedhourly during the experiment. The total number of cells releasedduring the entire experimental interval averaged less than 8x 106 cells, or less than 0.1% of the total number of cellsin the device. Similar to previous studies, most of the cellloss occurred during the first hour of use of the device, whenthe less adherent cells are washed out of the device. Metabolicactivity of the cells in the BAK was assessed by measuring theplasma levels of 1,25-(OH)2 vitamin D3 prenephrectomy, baselinepre-experiment, and end (post) experiment times. The end experimentvalues were obtained during the last hour of the experiment.Table 3 displays the levels observed in both groups. As demonstrated,bilateral nephrectomy resulted in significant declines in 1,25-(OH)2D3 levels in both groups. In the sham RAD-treated animals thisvalue declined significantly further to 15.1 ± 1.5 pg/ml(P < 0.02) during the last hour of the experimental protocol,whereas RAD treatment maintained postnephrectomy pre-experimentvitamin D3 levels.
The assessment of the influence of the RAD on plasma cytokineswas limited to the pro-inflammatory cytokine (TNF-), the anti-inflammatorycytokine (IL-10), and C-reactive protein due to lack of availabilityof canine-specific reagents. Serum TNF- levels rose rapidlyafter bacterial infusion to reach peak values at hour 2 andquickly returned to near baseline levels by hour 6 (Figure 5).No significant differences were found between the values observedin the cell RAD and sham RAD groups for either the peak levelsor values during the entire treatment time course. A differentresponse was observed in the serum levels of IL-10 (Figure 6).The peak levels at 2 h after bacterial administration were similarin the two groups, but a persistently higher elevation duringthe entire treatment interval was observed in the cell RAD animalsversus the controls. The comparison of these values betweenthe two groups demonstrated significantly higher levels (P <0.01) in the cell RAD group compared with the sham RAD group.Correlations of IL-10 levels to various cardiovascular parameters,including cardiac outputs, in all animals did not reach statisticalsignificance.
Figure 5. Plasma TNF- levels at various time intervals after bacterial administration in the sham RAD and cell RAD groups. No significant differences were found between the two groups.
Figure 6. Plasma IL-10 levels at hourly intervals after Gram-negative infusion. Cell RAD values were significantly higher (P < 0.01) for the entire time course compared with sham control levels.
C-reactive protein was also measured in these animals as a generalassessment of the acute phase response to sepsis. As displayedin Figure 7, C-reactive protein levels declined acutely in thefirst hour after bacterial seeding. These levels subsequentlyincreased in both groups, with the cell RAD group having slightlybut not significantly higher values compared with the sham controlgroup at time points after 2 h.
Figure 7. C-reactive protein (CRP) plasma concentrations in the cell and sham RAD groups after E. coli peritonitis. The values between the two groups were not significantly different.
Improved treatment regimens in patients with acute renal failurein the intensive care unit (ICU) have not shared the same quantumleaps of progress that have been seen in other illnesses, suchas myocardial infarction, despite improved understanding ofthe pathophysiology of the syndrome, improvements in mechanicalventilation, detection and treatment of infection, and techniquesfor renal substitution (1,2). In this regard, the cause of deathin these patients is usually the development of a systemic inflammatoryresponse syndrome (SIRS), often secondary to sepsis, with resultingcardiovascular collapse, ischemic damage to vital organs, andmulti-organ failure (MOF) (12). The propensity of patients withARF to develop SIRS and sepsis suggests that renal function,specifically renal tubule cell function secondary to ATN, playsa critical immunomodulatory role in individuals under stressstates. The renal tubule cell’s roles in glutathione reclamation,glutathione peroxidase synthesis, other middle molecule metabolism,and activation of vitamin D3, with its important immunoregulatoryfunctions, are well recognized pathways to maintain importanttissue integrity and host defense under stress conditions (13).A less recognized role of the kidney and the renal tubule cellsis its potential immunoregulatory function. The kidney is derivedembryologically from dorsal mesoderm, a collection of cellsalso important in the development of bone marrow stem cells(14,15). The maturation of cells responsible for erythropoietinsynthesis and activation of 1,25-(OH)2 vitamin D3 in the kidneyis reflective of this embryonic origin. Phylogenetically, inbony fish and amphibians without lymph systems, the kidney isthe major antibody-producing organ (15–17). Not surprisingly,mammalian renal proximal tubule cells are immunologically active.They are antigen-presenting cells (18–20) that possessco-stimulatory molecules (21) and that synthesize and processa variety of inflammatory cytokines (22–26).
Acute renal failure and acute tubular necrosis that resultsin this loss of the kidney’s immunoregulatory functionresults in a propensity to develop SIRS, sepsis, MOF, and ahigh risk of death. A counterpart to this loss of immunologicfunction has been clearly seen in chronic renal insufficiencyand end-stage renal disease, which are clearly pro-inflammatorystates (27–29). The degree of inflammation in these patientpopulations has been highly correlated to mortality rates (28,29).The loss of renal tubular cells, rather than loss of filtrationand clearance function, may be the cause of this inflammatorydysregulation observed in these patients as well.
In the past decade, a large constellation of data has providednew insights into the inflammatory response that seems to underliethe MOF syndrome. There are now data linking patient outcometo initial plasma levels of TNF-, IL-6, and other pro-inflammatorycytokines (30–32). There is also increasing agreementthat organ dysfunction in sepsis arises from oscillating patternsof inflammation from systemic spread of mediators beyond theirusual autocrine or paracrine pathways, alternating with immunosuppressioneither as compensation for or exhaustion of the inflammatoryresponse. This futile and lethal state of affairs has been referredto as "immunologic dissonance" (33,34). This paradigm has ledto efforts to interrupt this process by targeting individualelements in the cascade or by reducing the overall burden ofinflammatory mediators by plasma replacement or adsorption.Other approaches, including plasmapheresis, plasma exchange,hemofiltration, or monoclonal antibodies directed at variouscomponents of the inflammatory cascade, have not consistentlydemonstrated clinically useful interruption of the inflammatorycascade, despite promising in vitro and animal studies (35–41).
This report provides data that support the view that the renalproximal tubule has immunomodulatory effects and influencessystemic cytokine patterns, and that therapy with renal tubulecells can ameliorate some of the hemodynamic instability seenwith septic shock. Animals treated with a cell RAD had significantlyhigher systolic BP and cardiac outputs compared with those treatedwith sham, non-cell cartridges. The higher cardiac outputs inthe dogs receiving cell therapy occurred despite left ventricularfilling pressures, as reflected in PCWP, similar to those inthe sham controls. In fact, in the hour before death, animalstreated with a sham RAD developed large declines in cardiacoutput despite rising filling pressures. Most significant ofall is that despite identical volume resuscitation protocols,animals treated with renal tubule cells in the BAK survivedsignificantly longer than animals treated with a cell-free shamdevice.
The precise mechanisms by which this enhanced survival and theimproved hemodynamics are affected are currently being evaluated.Possible mechanisms include metabolism or disposal of circulatingmyocardial depressants in sepsis, changes in inflammatory cytokinebalance, relief of oxidant stress by glutathione reclamation,and production of free-radical scavengers (3,13). To evaluatethe possible involvement of the tubule cells in the BAK in modulatingthe inflammatory response in Gram-negative septic shock, TNF-,IL-10, and CRP plasma levels were evaluated and compared inthe two animal groups during the experimental protocol. Theevaluation was limited to only these three inflammatory proteinsbecause of the lack of reagents having cross-reactivity to dogcytokine proteins. No difference in the levels of the pro-inflammatorycytokine, TNF-, was observed in these studies. CRP levels wereslightly higher in the cell-treated group after the initialdecline seen in the first hour of bacterial infusion. Of importance,IL-10 levels displayed a significantly persistent elevationduring the entire treatment interval in the RAD-treated groupcompared with sham controls. These higher IL-10 levels, however,did not correlate to the better cardiovascular parameters observedin the cell-treated animals compared with the controls.
The role of IL-10 in regulating immune response continues tobe elucidated, but data suggest that IL-10 levels have an influenceon outcome from Gram-negative sepsis. Several reports have demonstratedthat administration of recombinant IL-10 is protective againstGram-negative septic shock in murine sepsis models (42–44).Another study in a similar model demonstrated that administrationof antibodies to IL-10 was associated with higher mortality(45). The mechanism underlying the link between proximal tubulefunction and IL-10 levels remains to be detailed, but preliminarydata suggest that renal production of IL-6 induces liver productionof IL-10 (46). Further support for an immunomodulatory roleof renal tubule cells has been suggested in preliminary reportsfrom an ongoing phase I/II clinical trial in ICU patients withARF being treated with CVVH and a RAD containing human renaltubule cells (47,48).
These initial pilot experiments demonstrate the positive effectthat renal tubule cell therapy and the bioartificial kidneyhave on the devastating consequences of bacteremia and septicshock in an acutely uremic state. The use of stem or progenitorcells is being increasingly considered as a potential approachto the treatment of a variety of acute and chronic disease states(49). The potential success of this therapeutic approach liesin the growing appreciation that most disease processes arenot due to the lack of a single protein but develop due to alterationsin complex interactions of a variety of cell products. Celltherapy is dependent on cell and tissue culture methodologiesto expand specific cells to replace important differentiatedprocesses deranged or lost in various disease states. Recentapproaches have made progress by placing cells into hollow fiberbioreactors or encapsulating membranes as a means to delivercell activities to a patient. Extracorporeal liver-assist devicesand encapsulated Islet of Langerhans to treat liver failureand diabetes mellitus are the most notable examples (50,51).A reasonable extension of this approach is to add cell therapyto the current renal substitution processes of hemodialysisand hemofiltration in the acute renal failure state. These resultssuggest a potential utility of this approach in a well-establishedlarge animal model of septic shock and acute uremia.
This work was supported by the VA Research Service and NephrosTherapeutics, Inc. Special thanks to Min Wang and Jianguo Liufor assistance in this study.
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Received for publication July 2, 2002.
Accepted for publication October 7, 2002.
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Abstract
Introduction
, IL-10, and C-reactive protein (CRP) were measured during cell RAD and sham RAD treatment. IL-10 levels were significantly higher (P < 0.01) during the entire treatment interval in the RAD animals compared with sham controls. These data demonstrated in a pilot large animal experiment that the BAK with RAD altered plasma cytokine levels in acutely uremic animals with septic shock. This change was associated with improved cardiovascular performance and increased survival time. These results demonstrate that the addition of cell therapy to hemofiltration in an acutely uremic animal model with septic shock ameliorates cardiovascular dysfunction, alters systemic cytokine balance, and improves survival time. E-mail: dhumes@umich.edu 
and tumor necrosis factor production during experimental endotoxemia. Eur J Immunol 24: 1167–1171, 1994[Medline]
