William H. Fissell

Bioartificial Kidney Ameliorates Gram-Negative Bacteria-Induced Septic Shock in Uremic Animals

The bioartificial kidney (BAK) consists of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 10(9) porcine renal proximal tubule cells. BAK replaces filtration, transport, and metabolic and endocrinologic activities of a kidney. Previous work in an acutely uremic dog model 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 intraperitoneally with 30 x 10(10) bacteria/kg of E. coli. One hour after bacterial administration, animals were placed in a continuous venovenous hemofiltration circuit with either a sham RAD without cells (n = 6) or a RAD with cells (n = 6). BP, cardiac output, heart rate, pulmonary capillary wedge pressure, and systemic vascular resistance were measured throughout the study. All animals tested were in renal failure, with blood urea nitrogen and serum creatinine concentrations greater than 60 and 6 mg/dl, respectively. RAD treatment 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 to 9 h after bacterial administration, whereas all RAD-treated animals survived more than 10 h. Plasma levels of TNF-alpha, 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.

A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells.

We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell–cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO‐1 tight junction protein and acetylated α‐tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F‐actin and exhibited disassembly of cytosolic F‐actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca2+ medium. The transport rate of a fluorescently labeled tracer molecule (FITC‐inulin) was measured before and after Ca2+ switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes. Biotechnol. Bioeng. 2010;107:707–716.

Differentiated Growth of Human Renal Tubule Cells on Thin-film and Nanostructured Materials

Over 300,000 Americans are dependent on hemodialysis as treatment for renal failure, and kidney transplantation is limited by scarcity of donor organs. This shortage has prompted research into tissue engineering of renal replacement therapy. Existing bioartificial kidneys are large and their use labor intensive, but they have shown improved survival compared to conventional therapy in preclinical studies and an US Food and Drug Administration–approved phase 2 clinical trial. This hybrid technology will require miniaturization of hemofilters, cell culture substrates, sensors, and integration of control electronics. Using the same harvesting and isolation techniques used in preparing bioartificial kidneys for clinical use, we characterized human renal tubule cell growth on a variety of silicon and related thin-film material substrates commonly used in the construction of microelectromechanical systems (MEMS), as well as novel silicon nanopore membranes (SNMs). Human cortical tubular epithelial cells (HCTC) were seeded onto samples of single-crystal silicon, polycrystalline silicon, silicon dioxide, silicon nitride, SU-8 photoresist, SNMs, and polyester tissue culture inserts, and grown to confluence. The cells formed confluent monolayers with tight junctions and central cilia. Transepithelial resistances were similar between SNMs and polyester membranes. The differentiated growth of human tubular epithelial cells on MEMS materials strongly suggests that miniaturization of the existing bioartificial kidney will be feasible, paving the way for widespread application of this novel technology.

Cell therapy with a tissue-engineered kidney reduces the multiple-organ consequences of septic shock.

ObjectiveGram-negative septic shock has a clinical mortality rate approaching 50%. The cause of death is secondary to a systemic inflammatory response syndrome with resulting cardiovascular collapse, ischemic damage to vital organs, and multiple-organ systems failure. Renal tubule cell injury occurs early in septic shock but is not clinically appreciated. Since renal tubule cells appear to play a critical role in the immunoregulation of stress states, renal cell therapy during septic shock may alter the detrimental multiple-organ consequences of systemic Gram-negative infection. The development of a tissue-engineered bioartificial kidney consisting of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 109 renal proximal tubule cells may be a new therapeutic approach to this clinical disorder. DesignLaboratory study. SettingUniversity medical school. SubjectsPigs weighing 30–35 kg. InterventionsTo assess the effect of the bioartificial kidney and the RAD in septic shock, pigs were administered 30 × 1010 bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in a continuous venovenous hemofiltration extracorporeal circuit with either a sham RAD without cells or a RAD with cells. Measurements and Main ResultsIn this animal model, septic shock resulted within hours in acute tubule necrosis in the kidneys of all animals. Renal cell therapy resulted in significantly higher cardiac outputs and renal blood flow rates in treated animals compared with sham controls. RAD treatment also was associated with significantly lower plasma circulating concentrations of interleukin-6 and interferon-&ggr; compared with sham-treated animals. IL-6 release rates from peripheral blood mononuclear cells isolated from RAD-treated animals were significantly higher after endotoxin stimulation than those isolated from control animals. These physiologic and molecular alterations were associated with nearly a doubling of the average survival time in the RAD-treated group compared with the sham control group. ConclusionThese results demonstrate that renal cell therapy ameliorates cardiac and vascular dysfunction, alters systemic cytokine abnormalities, and improves survival time in a large animal model of Gram-negative septic shock. A cell therapeutic approach with a tissue-engineered bioartificial kidney may be a new treatment modality for this current unmet medical need.

Hypophosphatemia during continuous hemodialysis is associated with prolonged respiratory failure in patients with acute kidney injury

BACKGROUND Hypophosphatemia is common in critically ill patients and has been associated with generalized muscle weakness, ventilatory failure and myocardial dysfunction. Continuous renal replacement therapy causes phosphate depletion, particularly with prolonged and intensive therapy. In a prospective observational cohort of critically ill patients with acute kidney injury (AKI), we examined the incidence of hypophosphatemia during dialysis, associated risk factors and its relationship with prolonged respiratory failure and 28-day mortality. METHODS This is a single-center prospective observational study. Included in the study were 321 patients with AKI on continuous dialysis as initial treatment modality. RESULTS Four per cent of the patients had a phosphate level <2 mg/dL at initiation and 27% during dialysis. Low baseline phosphate was associated with older age, female gender, parenteral nutrition, vasopressor support, low calcium, and high urea, bilirubin and creatinine, whereas hypophosphatemia during dialysis correlated with the ischemic acute tubular necrosis etiology of renal failure, intensive dose and longer therapy. Serum phosphate decline during dialysis was associated with higher incidence of prolonged respiratory failure requiring tracheostomy [odds ratio (OR) = 1.81; 95% confidence interval (CI) = 1.07-3.08], but not 28-day mortality (OR = 1.16; 95% CI = 0.76-1.77) in multivariable analysis. CONCLUSIONS Hypophosphatemia occurs frequently during dialysis, particularly with long and intensive treatment. Decline in serum phosphate levels during dialysis is associated with higher incidence of prolonged respiratory failure requiring tracheostomy, but not 28-day mortality.

Bioartificial Kidney Alters Cytokine Response and Hemodynamics in Endotoxin-Challenged Uremic Animals

The mortality from sepsis complicated by renal failure remains extremely high despite the application of modern renal replacement therapy. This study investigated whether treatment with a bioartificial kidney consisting of a hemofilter in a continuous venovenous hemofiltration circuit (CVVH) with a cartridge containing renal proximal tubule cells, also called the Renal Tubule Assist Device (RAD), would alter the course of sepsis in an animal model. The RAD has been previously characterized in vitro and ex vivo and provides transport, metabolic and endocrine activity. Mongrel dogs (n = 10) underwent surgical nephrectomy and 48 h later were treated with CVVH and either a RAD containing cells (n = 5) or an identically prepared sham cartridge (n = 5). After 4 h of therapy, intravenous endotoxin 2 mg/kg was infused over 1 h to simulate gram-negative septic shock. Data on blood pressure, cardiac output and systemic markers of inflammation were collected. Mean peak levels of an anti- inflammatory cytokine, IL-10, were significantly higher in cell-treated animals (15.25 vs. 6.29 ng/ml; p = 0.037), and mean arterial pressures were higher in cell-treated versus sham-treated animals (p < 0.04). We have demonstrated that treatment of an animal model of endotoxin shock and renal failure with a bioartificial kidney has measurable effects on circulating mediators of inflammation and on hemodynamic stability of the challenged animal.

The bioartificial kidney: current status and future promise

The rapid understanding of the cellular and molecular bases of organ function and disease processes will be translated in the next decade into new therapeutic approaches to a wide range of clinical disorders, including acute and chronic renal failure. Central to these new therapies are the developing technologies of cell therapy and tissue engineering, which are based on the ability to expand stem or progenitor cells in tissue culture to perform differentiated tasks and to introduce these cells into the patient either via extracorporeal circuits or as implantable constructs. Cell therapy devices are currently being developed to replace the filtrative, metabolic, and endocrinologic functions of the kidney lost in both acute and chronic renal failure. This review summarizes the current state of development of a wearable or implantable bioartificial kidney. These devices have the promise to be combined to produce a wearable or implantable bioartificial kidney for full renal replacement therapy that may significantly diminish morbidity and mortality in patients with acute or chronic kidney disease.

Pharmacokinetics and Pharmacodynamics of Piperacillin-Tazobactam in 42 Patients Treated with Concomitant CRRT

BACKGROUND AND OBJECTIVES Current recommendations for piperacillin-tazobactam dosing in patients receiving continuous renal replacement therapy originate from studies with relatively few patients and lower continuous renal replacement therapy doses than commonly used today. This study measured the pharmacokinetic and pharmacodynamic characteristics of piperacillin-tazobactam in patients treated with continuous renal replacement therapy using contemporary equipment and prescriptions. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS A multicenter prospective observational study in the intensive care units of two academic medical centers was performed, enrolling patients with AKI or ESRD receiving piperacillin-tazobactam while being treated with continuous renal replacement therapy. Pregnant women, children, and patients with end stage liver disease were excluded from enrollment. Plasma and continuous renal replacement therapy effluent samples were analyzed for piperacillin and tazobactam levels using HPLC. Pharmacokinetic and pharmacodynamic parameters were calculated using standard equations. Multivariate analyses were used to examine the association of patient and continuous renal replacement therapy characteristics with piperacillin pharmacokinetic parameters. RESULTS Forty-two of fifty-five subjects enrolled had complete sampling. Volume of distribution (median=0.38 L/kg, intraquartile range=0.20 L/kg) and elimination rate constants (median=0.104 h(-1), intraquartile range=0.052 h(-1)) were highly variable, and clinical parameters could explain only a small fraction of the large variability in pharmacokinetic parameters. Probability of target attainment for piperacillin was 83% for total drug but only 77% when the unbound fraction was considered. CONCLUSIONS There is significant patient to patient variability in pharmacokinetic/pharmacodynamic parameters in patients receiving continuous renal replacement therapy. Many patients did not achieve pharmacodynamic targets, suggesting that therapeutic drug monitoring might optimize therapy.

Hemocompatibility of Silicon-Based Substrates for Biomedical Implant Applications

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories—coagulation (thrombin–antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems.

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor.

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS–PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS–PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS–PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process. Biotechnol. Bioeng. 2012; 109:797–803.