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Extracorporeal Renal Replacement
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Extracorporeal Renal Replacement Kimberly A. Johnston*, H. David Humes*,y * Innovative Biotherapies, Ann Arbor, MI, USA y Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
INTRODUCTION The kidney is unique in that it is the first organ for which long-term ex vivo substitutive therapy has been made available and lifesaving. Renal failure prior to the era of hemodialysis and transplantation resulted in certain death, and this outcome of renal failure is still common outside the industrialized world. In the USA, 526,343 patients were listed as having end-stage renal disease (ESRD) by the 2007 US Renal Data System (USRDS) database, of whom 308,910 were receiving maintenance dialysis (US Renal Data System, 2004). The prevalence of ESRD in the USA is rising at approximately 8% per year (Neilson et al., 1997; US Renal Data System, 2004). The financial cost of dialysis is immense, estimated at $54,900 per hemodialysis patient per year and $46,121 per peritoneal dialysis patient per year. In contrast, transplant patients cost an average of $17,227 per patient per year (US Renal Data System, 2004). The higher cost of maintenance dialysis when compared with transplantation does not translate into better results; annual mortality for patients listed for transplant and awaiting a kidney is 6.3%, compared with only 3.8% for patients listed for transplant who did receive a kidney (US Renal Data System, 2004). While organ transplantation provides the best prognosis for survival, demand vastly outweighs the availability of donated organs. Current dialysis therapies include hemodialysis (HD), hemofiltration, and peritoneal dialysis (PD). Dialysis provides clearance of small molecules by diffusive flow across a semipermeable membrane and control of volume status by bulk flow of water and solutes through that membrane. These short-term effects are sufficient to abrogate the lethal acidosis, volume overload, and uremic syndromes that accompany renal failure but do not protect the patient from the increased mortality associated with dialysis-treated renal failure in either the acute or chronic form. These methodologies all address water and electrolyte balance e functional replacement of the kidney. However, they fail to provide for the lost endocrine function. Thus, the metabolic, endocrine, and immune roles of the functioning kidney are candidate mechanisms for the difference in survival noted above. The dialytic clearance of glutathione, a key tripeptide in free radical scavenging and protection against oxidant stress; the negative nitrogen balance and energy loss in the clearance of peptides and amino acids in dialysate; loss of oxidative deamination and gluconeogenesis in the tubule cell; and loss of cytokine and hormone metabolic activity by the kidney each impose substantial stress upon the dialyzed patient and as such are appropriate targets for improved renal replacement therapy. Principles of Regenerative Medicine. DOI: 10.1016/B978-0-12-381422-7.10051-3 Copyright Ó 2011 Elsevier Inc., All rights reserved.
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REQUIREMENTS OF A RENAL REPLACEMENT DEVICE Filtration is accomplished by the glomerulus, a tuft of capillaries supported by a basement membrane and specialized epithelial cells called podocytes. The renal proximal tubule, a hollow tube of cells surrounded by capillaries, receives the filtrate from the glomerulus and accomplishes the bulk of reclamation of salt, water, glucose, small proteins, amino acids, glutathione, and other substances. The tubule also performs metabolic functions, including excretion of acid as ammonia and hydroxylation of 25-hydroxy-vitamin-D3 among others. Intermittent hemodialysis is thought to replace the filtration function of the glomerulus and advances in hemodialysis and hemofiltration have focused on emulation of glomerular physiology. Recent attention has been drawn to duplicating the function of the proximal tubule. The transport of solutes and water is accomplished by ATP-driven electrolyte transporters in the luminal cell membrane. Reabsorption of small proteins and peptides in the filtrate stream is accomplished by membrane-bound proteases and specific amino acid transport proteins within the luminal membrane of the tubule cell. These amino acids are either used for protein and peptide synthesis in the tubule cell or transported into the capillaries for transport to and use by the body. The diversity and specificity of the functions of the proximal tubule cell argue against the development of an electromechanical or polymeric substitute, and so a number of years ago our research group turned its attention to the isolation and culture of renal proximal tubule cells, which research has culminated in the hollow-fiber bioreactor discussed below.
RENAL PROXIMAL TUBULE CELL SOURCING AND FABRICATION OF A BIOREACTOR 944
Critical to providing organ function replacement through cell therapy is the isolation and growth in vitro of specific cells from adult tissue. These cells must have stem cell-like characteristics, with a high capacity for self-renewal and the ability to differentiate under defined conditions into specialized cells to develop the correct structure and functional components of a physiological organ system. Methodology to isolate and grow renal proximal tubule progenitor cells from adult pig kidneys has been reported (Humes and Cielinski, 1992; Humes et al., 1996). These studies were promoted by clinical and experimental observations suggesting that progenitor cells of renal proximal tubules must exist, as tubule cells have the ability to regenerate after severe nephrotoxic or ischemic injury. Porcine cells were utilized, as the pig has been considered the best source of organs for both xenotransplantation and cell therapy devices due to its anatomic and physiological similarities to human tissue and the relative ease of breeding large numbers of pigs in closed herds. However, reports of the ability of porcine endogenous retroviruses (PERVs) to infect human cells in co-culture in vitro have raised concerns about the potential, but currently unquantifiable, risk of transmission of viral elements from porcine tissue to humans in xenotransplantation or cell therapy devices (le Tissier et al., 1997; Paradis et al., 1999). Accordingly, Humes and colleagues fabricated bioreactors containing human renal epithelial cells isolated from kidneys donated for transplantation but found unsuitable for such purpose because of anatomic or fibrotic defects. These cells performed well in studies to assess viability, durability, and physiological performance (Humes et al., 2002). The renal assist device (RAD) is a bioreactor containing proximal tubule cells grown in confluent monolayers along the inner surface of the hollow fibers of a conventional hemofiltration cartridge. Within this multifiber unit, proximal tubule cells not only maintain transport properties but also differentiated metabolic and endocrine functions. The nonbiodegradability and the pore size of the hollow fibers allow the membranes to act as both scaffolds for the cells and as an immunoprotective barrier. Completed experiments have successfully scaled up to a clinically applicable device with the use of commercially available high-flux hemofiltration hollow fiber cartridges.
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Experiments have tested the transport and metabolic functions of cells grown intraluminally within these cartridges with membrane surface areas of 97 cm2 to 0.4 m2 (Humes et al., 1999b). Starting with a hemofilter cartridge, the intraluminal surface of the hollow fibers was coated with pronectin L, a synthetic protein with multiple cell attachment sites found in the extracellular protein laminin. Renal proximal tubule progenitor cells were then seeded at a density of 105 cells/ml into the intracapillary space. The seeded cartridge was connected to the bioreactor perfusion system, in which the extracapillary space was filled with culture media and the intracapillary space perfused with similar media. After 7e14 days, light microscopy revealed a confluent monolayer of tubule cells grown on the inner surface of the fibers, and electron microscopy identified differentiated epithelial characteristics, including microvilli, endocytic vesicles, and extensive basolateral infoldings. The cells retain vectorial fluid transport properties as a result of Na,K-ATPase; differentiated active transport properties, including active electrolyte, bicarbonate, and glucose transport; differentiated metabolic activities, including intraluminal glutathione breakdown and ammonia production; and the important endocrinological conversion of 25-OH-vitD3 to 1,25-(OH)2-vitD3.
ULTRAFILTRATION MEMBRANE DEVELOPMENT In parallel with progress in stem cell work, there has been considerable interest in applying novel technology to membrane engineering. The filtration barrier in the kidney has been extensively studied. This barrier is widely considered to be trilaminate, with an endothelial cell layer, a basement membrane, and an elaborate epithelial layer bearing a specialized cell-cell junction called the glomerular slit diaphragm. Unfortunately, the cell considered responsible for the permselectivity barrier in the kidney, the glomerular podocyte, is a terminally differentiated cell with limited regenerative capacity. Damaged cells are not replaced by expansion of neighboring podocytes. Similarly, primary cultures of podocytes do not assume a differentiated phenotype in the laboratory dish, nor do they easily divide and expand in number. Despite progress with a conditionally transformed cell line derived from mouse podocytes, there seems little immediate prospect of a cell-based bioartificial glomerulus. Without a purely biological ultrafiltration unit on the horizon, advances in HD membrane development have been aimed at attempting to better reproduce the physiological process of glomerular ultrafiltration using synthetic membranes. Membrane materials are diverse, ranging from regenerated cellulose filters to metals and ceramics to modern-day polymers. Critical to the choice of materials for current dialysis membranes, biocompatibility with blood is a major concern, as well as cost and manufacturing. Synthetic polymers have become the dominant membrane materials used due a combination of these factors. The most common polymers in the manufacturing of synthetic membranes are non-degradable polymers: cellulose derivatives; nitrates; polyesters, polysulfones; polyacrilonitrile derivatives; polyamides; polyimides; polyolefins such as polyethylene, polypropylene or polyvinylchloride; and fluorinated polymers such as polytetrafluoroethylene, and polyvinylidinefluoride. Dialysis membrane clearance is based on concentration differences rather than convective separation of small solutes and low-molecular-weight proteins from large serum proteins and blood elements. In an attempt to recapitulate glomerular ultrafiltration and removal of “middle molecules,” synthetic membranes with larger pore sizes and high water permeability have been developed. These so-called “high-flux” membranes are prepared with hydrophobic base materials, including polyacrylnitrile, polysulfone, polyethersulfone, or polymide, with various hydrophilic components. Recent membrane development has focused on increasing pore size while sharpening the molecular weight cutoff of high-flux membranes to maximize removal of low-molecular-weight proteins. Removal of a distinct class of uremic toxins, such as b2-microglobulin, factor D, leptin, and adrenomedullin, while minimizing the loss of albumin, could improve treatment outcomes of patients with ESRD. This idea has spurned the
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creation of superflux or protein-leaking membranes. These membranes provide greater clearances for low-molecular-weight proteins and small protein-bound solutes, such as homocysteine and advanced glycation end products, but with significantly higher loss of albumin than high-flux dialysis membranes. The overall benefits for patients on chronic HD still require more extensive evaluation.
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Micromechanical systems (MEMS) are increasingly used to develop novel membrane technology. Current polymer membranes for renal replacement are performance limited. In general, such membranes are fairly thick or employ a multilayer scaffold for mechanical support, and they have a distribution of pore sizes rather than a regular array of uniform pores. Pores in conventional polymer membranes tend to either be roughly cylindrical, have a round orifice terminating a larger channel, or have a structure resembling an open cell sponge. These structures provide less than optimal geometries for membrane filtration for two reasons. First, a wide dispersion in pore sizes within a membrane leads to imperfect retention of molecules larger than the mean pore size of the membrane. Reducing the mean pore size of the membrane may partially solve this problem; however, it has the undesired effect of reducing the hydraulic permeability of the membrane. Second, the round shape of conventional pores dictates a dependence of hydraulic permeability on pore radius. In contrast, a pore that is slitshaped allows steric hindrance to solute passage dictated by the smallest critical dimension of the pore, while increasing hydraulic permeability based on the long dimension of the pore. Consequently, it might be predicted that filtration structures with parallel slit-shaped pores will have superior performance when compared to structures with round pores. The glomerular filtration barrier also imposes an electrostatic restriction on solute passage. This function has been variously attributed to the proteins within the slit diaphragm, the glomerular basement membrane, and the glycocalyx of the glomerular endothelial cell. In regard to artificial membranes, a double thick electrical layer related to the nanometer-scale pore size itself contributes to the rejection of charged solutes. Novel silicon nanopore membranes with 10e100 nm 45 mm slit pores, approximating the glomerular slit diaphragm, have been prototyped by an innovative process based on MEMS technology. Silicon chips bearing 1 1 mm arrays of approximately 104 slit pores were fabricated via sacrificial layer techniques. The pore structure is defined by deposition and patterning of a polysilicon film on the silicon wafer. The critical submicron pore dimension is defined by the thickness of a sacrificial SiO2 layer, which can be grown with unprecedented control to within 1 nm. Preliminary data on the transport properties of MEMS membranes are encouraging. Measured hydraulic permeabilities correlated well with theoretical predictions for flow-through slitshaped pipes, also known as Hele-Shaw flows. The observed albumin sieving coefficient data provide encouragement that protein permselectivity is also feasible with this technology. Recent laboratory data have validated the possibilities of these membranes as scaffolding for a renal tubule cell bioreactor (Ward et al., 2001).
TRANSPORT AND METABOLIC CHARACTERISTICS OF HOLLOW-FIBER BIOREACTORS As initial experiments using the single hollow-fiber model were promising, the design was scaled up to use commercially available polysulfone hollow-fiber dialysis cartridges from the manufacturers of the single hollow fibers. Single hollow-fiber measurements of transport and metabolic activity were repeated with 97 cm2 and 0.4 m2 surface area cartridges. Further exploration of the metabolic and transport characteristics of the cultured proximal tubule cells was assessed. The transport of glucose, bicarbonate, and glutathione excretion was measured in the absence and presence of a known inhibitor of an enzyme essential for the reabsorption. In each case, there was evidence of active transport and specific inhibition (Humes et al., 1999b).
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The synthesis and secretion of ammonia into the tubule is essential for renal excretion of an acid load, as it buffers secreted protons. Proximal tubule cells are able to upregulate their ammoniagenesis in response to a decline in pH, and the proximal tubule cells in the bioreactor demonstrated a stepwise increase in ammonia production with changes in pH (Humes et al., 1999b). The experiments detailed above were performed in our laboratory with porcine tubule cells that demonstrated similar results in culture, attachment, and activity with human proximal tubule cells from cadaveric organs. The final selection of cell type for use in a renal tubule device not only rests on supply and safety of cells, but also depends on the ability of cells to participate in the homeostasis of the host. The above data suggest that our laboratory has successfully isolated and cultured renal proximal tubule cells, established stable confluent monolayers within hollow-fiber bioreactors, and scaled the initial construct to a level approximating the number of proximal tubule cells in a single kidney.
PRECLINICAL CHARACTERIZATION OF THE RENAL TUBULE ASSIST DEVICE/BIOARTIFICIAL KIDNEY In keeping with its role as a metabolically active replacement for the renal proximal tubule, an extracorporeal circuit was devised that recapitulated nephron anatomy. The bioartificial kidney setup consists of a filtration device (a conventional hemofilter) followed in series by the renal tubule assist device (RAD) unit. Specifically, blood is pumped out of a patient using a peristaltic pump. The blood then enters the fibers of a hemofilter, where ultrafiltrate is formed and delivered into the fibers of the tubule lumens within the RAD downstream to the hemofilter. Processed ultrafiltrate exiting the RAD is collected and discarded as “urine.” The filtered blood exiting the hemofilter enters the RAD through the extracapillary space port and disperses among the fibers of the device. Upon exiting the RAD, the processed blood travels through a third pump and is delivered back to the patient. Heparin is delivered continuously into the blood before entering the RAD to diminish clotting within the device. The RAD is oriented horizontally and maintained at 37 C throughout its operation to ensure optimal functionality of the cells. The tubule unit is able to maintain viability because metabolic substrates and low-molecular-weight growth factors are delivered to the tubule cells from the ultrafiltration unit and the blood in the extracapillary space. Furthermore, immunoprotection of the cells grown within the hollow fiber is achieved because of the impenetrability of immunoglobulins and immunologically competent cells through the hollow fibers. Rejection of the cells, therefore, does not occur. This extracorporeal circuit containing the RAD was initially tested on uremic dogs with bilateral nephrectomies (Humes et al., 1999a). The animals were treated with either a RAD or a sham control cartridge daily for either 7 or 9 h for three successive days or for 24 h continuously. The RADs maintained viability and functionality throughout the study period. Fluid and small solutes, including blood urea nitrogen (BUN), creatinine (Cr), and electrolytes, were adequately controlled in both groups, but potassium and BUN levels were more easily controlled by RAD treatment. Furthermore, active reabsorption of Kþ, HCO3, and glucose and excretion of ammonia were accomplished only in RAD treatments. Glutathione reclamation from UF exceeded 50% in the RAD. Finally, uremic animals receiving cell therapy attained normal 1,25-(OH)2-vitD3 levels, whereas sham treatment resulted in a further decline from the already low plasma levels. Thus, these experiments clearly showed that the combination of a synthetic hemofiltration cartridge and a RAD in an extracorporeal circuit successfully replaced filtration, transport, and metabolic and endocrinological functions of the kidney in acutely uremic dogs.
CLINICAL EXPERIENCE WITH A HUMAN RENAL TUBULE ASSIST DEVICE Encouraging preclinical data led to FDA approval for an investigational new drug and phase I/II clinical trials. The first human clinical study of the bioartificial kidney containing human cells
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was carried out in 10 ICU patients with AKI receiving CVVH (Humes et al., 2004). This study demonstrated that the RAD can be used safely for up to 24 h. Cardiovascular stability was maintained, and increased native renal function, as determined by elevated urine outputs, temporally correlated with RAD treatment. All patients were critically ill with acute kidney injury (AKI) and multiple organ failure (MOF), with predicted hospital mortality rates between 80 and 95%, Six of the 10 treated patients survived past 30 days, with mortality reduced to 40%. The human renal tubule cells contained in the RAD demonstrated differentiated metabolic and endocrinological activity in this ex vivo situation, including glutathione degradation and endocrinological conversion of 25-OH-vitD3 to 1,25-(OH)2-vitD3. Plasma cytokine levels suggest that RAD therapy produces dynamic and individualized responses in patients depending on their unique pathophysiological conditions. For the subset of patients who had excessive proinflammatory levels, RAD treatment resulted in significant declines in granulocytecolony stimulating factor (G-CSF), IL-6, IL-10, and especially IL-6/IL-10 ratio, suggesting a greater decline in IL-6 relative to IL-10 levels and a less proinflammatory state. These favorable phase I/II trial results led to a randomized, controlled, open-label phase II trial conducted at 12 clinical sites in the US (Tumlin et al., 2008). Fifty-eight patients with ARF requiring CVVH in the ICU were randomized (2:1) to receive CVVH þ RAD (n ¼ 40) or CVVH alone (n ¼ 18). Despite the critical nature and life-threatening illnesses of the patients enrolled in this study, the addition of the RAD to CVVH resulted in a substantial clinical impact on survival compared with the conventional CVVH-treatment group. RAD treatment for up to 72 h promoted a statistically significant survival advantage over 180 days of follow-up in ICU patients with AKI and demonstrated an acceptable safety profile. Cox proportional hazards models suggested that the risk of death was approximately 50% of that observed in the CRRT-alone group. A follow-up phase IIb study to evaluate a commercial manufacturing process was not completed due to difficulties with the manufacturing process and clinical study design. This approach will be further evaluated when an improved scale-up manufacturing process is established. 948
CELL THERAPY OF ACUTE RENAL FAILURE DUE TO SEPSIS After a series of experiments demonstrating bioactivity, longevity, and systemic activity of the proximal tubule cells in a large animal model, a series of experiments was designed to examine the impact of cell therapy on the course of sepsis complicated by renal failure (Fissell et al., 2002a, 2003). After two initial studies supported a systemic effect and hemodynamic benefit from cell therapy in large animal models of sepsis, our laboratory pursued further evidence that cell therapy with renal proximal tubule cells alters the physiological response to sepsis (Humes et al., 2003). A porcine model of septic shock was developed from the previous work (Natanson et al., 1989; Natanson, 1990; Dinarello, 1991). Purpose-bred pigs were anesthetized and administered an intraperitoneal dose of bacteria, causing shock and renal failure. An hour later continuous venovenous hemofiltration (CVVH) was initiated with either cell or sham RAD. Urine output and mean arterial pressure declined within the first few hours after insult. Cell-treated animals survived 9.0 10.83 h versus 5.1 10.4 h (P 0.005) for shamtreated animals. Serum cytokines were similar between the two groups, with the striking exception of interleukin (IL)-6 and interferon (IFN)-g. Treatment with the cell RAD resulted in significantly lower plasma levels of both IL-6 (P 0.04) and INF-g (P 0.02) throughout the experimental time course compared to sham RAD exposure. This controlled trial of cell therapy of renal failure in a realistic animal model of sepsis has several findings not immediately expected from a priori assumptions regarding renal function. Heretofore, although renal failure has been strongly associated with poor outcome in hospitalized patients, and chronic renal failure is associated with specific defects in humoral and cellular immunity, a direct immunomodulatory effect of the kidney had not been accepted. In this trial, clear differences in survival and clear differences in a serum cytokine associated with mortality in sepsis were found between groups: The increased mortality in renal failure appears to be not attributable to inadequate solute clearance, but may arise from other bioactivity of the kidney.
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BIOARTIFICIAL KIDNEY IN END-STAGE RENAL DISEASE A bioartificial kidney for long-term use in ESRD, similar to short-term use in acute renal failure (ARF), would integrate tubular cell therapy and the filtration function of a hemofilter. As noted above, ESRD patients on conventional renal replacement therapy are at high risk for cardiovascular and infectious diseases. A recent clinical trial failed to show survival benefit from increased doses of hemodialysis above what is now standard care (Eknoyan et al., 2002), suggesting that there are important metabolic derangements not adequately treated with conventional dialytic treatment. Data from the survival of renal transplant recipients, which far exceed those from the survival of age-, sex-, and risk-matched controls awaiting transplant, also suggest that there is some metabolic function provided by the kidney that transcends this organ’s filtration function. Patients with ESRD display elevated levels of C-reactive protein (CRP), an emerging clinical marker, and pro-inflammatory cytokines, including IL-1, IL-6, and tumor necrosis factor (TNF) (Bologa et al., 1998; Kimmel et al., 1998; Zimmermann et al., 1999). All these parameters are associated with enhanced mortality in ESRD patients. Specifically, IL-6 has been identified as a single predictive factor closely correlated with mortality in hemodialysis patients (Bologa et al., 1998). Although all ESRD patients could conceivably benefit from a bioartificial kidney, patients in the inflammatory stage who display elevated levels of certain markers of chronic inflammation (most notably IL-6 and CRP) would likely benefit most and will be the target population for clinical study in the near future. For the ESRD patient population, however, there are obvious limitations in using an extracorporeal RAD connected to a hemofiltration circuit. Ideally, a bioartificial kidney suitable for long-term use in ESRD patients would be capable of performing continuously, like the native kidney, to reduce risks from fluctuations in volume status, electrolytes, and solute concentrations and to maintain acid-base and uremic toxin regulation. Such treatment requires the design and manufacture of a compact implantable or wearable dialysis apparatus and the development of miniaturized renal tubule cell devices with long service lifetimes. The ideal design of the next-generation RAD would be like that of an implantable device such as the pacemaker. Attempts have been made to develop wearable dialysis systems to improve the portability of renal replacement therapies. Gura et al. (2005) have published research into a light-weight, wearable, continuous ambulatory ultrafiltration device consisting of a hollow fiber hemofilter, a battery-operated pulsatile pump, and two micropumps to control heparin administration and ultrafiltration. This device regenerates dialysate with activated carbon, immobilized urease, zirconium hydroxide, and zirconium phosphate, similar to the once commercially available REDY dialysis system. Ronco and Fecondini (2007) have described a wearable continuous PD system consisting of a double lumen dialysate line with a peritoneal catheter, a miniaturized rotary pump, a circuit for dialysate regeneration, and a handheld computer as a remote control. These systems still rely on inconvenient dialysate and expensive dialysis regeneration devices and/or dialyzers, but they promise to improve the convenience of dialysis. In contrast to wearable dialysis systems, a hybrid bioartificial kidney integrates tubule cell and filtration functions. The first bioartificial kidney, consisting of a passive hemofilter and an active renal tubule cell bioreactor, has consistently demonstrated excellent safety and effectiveness in animal studies and FDA-approved human clinical trials, as described above (Ward et al., 2001; Humes et al., 2003, 2004; Tumlin et al., 2008). A major drawback of the current version of the bioartificial kidney is its large size, owing to the requisite extracorporeal circuit with peristaltic pumps to provide driving pressure for hemofiltration. A new smaller and more durable RAD is currently being developed by Humes and colleagues. In collaboration, Fissell and colleagues are developing a nanopore membrane to replace the filtration function of the glomerulus without the hemofilters and mechanical pumps of existing dialysis machines. A filtration device based on nanopore membrane technology
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would be implantable (Fissell et al., 2002b, 2006; Magistrelli, 2004). Further refinement of the RAD would be encouraging for ESRD patients because, in principle, such a tissue-engineered device could be free of dialysate or replacement fluid while providing functions of healthy kidney that are not offered by current dialysis strategies. The combination of cell therapy and solute clearance could be a viable renal replacement therapy, conferring dialysis independence to the patient.
IMMUNOMODULATORY EFFECT OF THE RENAL TUBULE ASSIST DEVICE
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As described earlier, RAD treatment altered systemic circulating cytokine levels in animal and human experiments. In endotoxin-challenged and gram-negative peritonitis uremic dog models, plasma levels of IL-10 were significantly higher in RAD-treated animals (Fissell et al., 2002a, 2003). The role of IL-10 in regulating immune response continues to be elucidated, but data suggest that IL-10 levels influence outcome in endotoxin shock and gram-negative sepsis. Several reports have demonstrated that administration of recombinant IL-10 is protective against gram-negative septic shock in murine sepsis models (Walley et al., 1996; Matsumoto et al., 1998). Another study in a similar model demonstrated that administration of antibodies to IL-10 was associated with higher mortality (Marchant et al., 1994). The mechanism underlying the link between proximal tubule function and IL-10 levels remains to be detailed, but preliminary data suggest that renal production of IL-6 induces liver production of IL-10 (Kielar et al., 2002). In gram-negative septic pigs without nephrectomy, RAD treatment significantly reduced plasma circulating levels of IL-6 and INF-g (Humes et al., 2003). The difference in IL-6 concentrations is especially noteworthy, since the plasma elevations of this proinflammatory cytokine have been directly correlated to outcome in patients with SIRS (Pinksy et al., 1993). The lower concentration of plasma INF-g may be important due to its central role in the inflammatory response. INF-g stimulates B-cell antibody production, enhances polymorpholeukocyte phagocytosis, and activates monocytes and macrophages to release proinflammatory cytokines (Bone, 1991; Redmond et al., 1991; Joyce et al., 1994). Excessive rates of INF-g production by NK cells have correlated with progression to lethal endotoxin shock in mice (Emoto et al., 2002). Further support for an immunomodulatory role of renal tubule cells has been suggested in the phase I/II clinical trial of the RAD containing human renal tubule cells (Humes et al., 2004). The patients treated in this study had a wide spectrum of plasma cytokine levels. The subset of patients who presented with very high plasma cytokine levels and who were treated for an adequate period showed that RAD treatment resulted in significant reductions in G-CSF, IL-6, and IL-10 levels. The greater relative reduction in IL-6/IL-10 ratio suggests renal tubule cell therapy may rebalance the excessive proinflammatory response with the concurrent anti-inflammatory response. These results are consistent with an immunomodulatory role for the RAD in patients with acute tubular necrosis and multiorgan failure. To further evaluate the RAD’s influence on local inflammation in tissue and distant organ dysfunction, especially in the lungs, a recent study compared bronchoalveolar lavage (BAL) fluid from cell-RAD-treated and non-cell, sham-treated groups in a pig model with septic shock with AKI (Humes et al., 2007). The levels of total protein in BAL were significantly higher in sham control animals than in the RAD group (143 111 compared to 78 110 mg/ml, respectively; P > 0.05). Proinflammatory cytokines, including IL-6 and IL-8, were markedly elevated in the control group. These results demonstrate an important role for renal epithelial cells in ameliorating multiorgan injury in sepsis by influencing microvascular injury and the local proinflammatory response. A more promising direction to improve outcome of AKI is to better understand and interrupt the pathophysiological processes that are activated in AKI, resulting in distant multi-organ dysfunction and eventually death. AKI results in a profound inflammatory response state resulting in microvascular dysfunction in distant organs (Okusa, 2002; Simmons et al., 2004). Leukocyte activation plays a central role in these acute inflammatory states. Disruption of the activation
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process of circulating leukocytes may limit microvascular damage and multi-organ dysfunction (Maroszynska and Fiedor, 2000). The RAD appears to influence systemic leukocyte activation and the balance of inflammatory cytokines and may alter the proinflammatory state of AKI and, ultimately, improve morbidity and mortality. Our group has recently developed a novel synthetic membrane embedded in an extracorporeal device to bind and inhibit circulating leukocytes. This “selective cytopheretic inhibitory device” (SCD) mimics immunomodulation and duplicates RAD efficacy. The SCD improved septic shock survival times in preclinical animal models and improved the survival outcome of ICU patients with multiorgan failure in a small exploratory, randomized, double-blinded, multicenter trial (Ding et al., 2008; Humes et al., 2008).
THE BIOARTIFICIAL RENAL EPITHELIAL CELL SYSTEM A next-generation RAD, the bioartificial renal epithelial cell system (BRECS), was designed by Humes and colleagues to achieve the support of 10-fold more cells in less than one-third of the volume of previous RAD designs, along with the capacity to cryopreserve the full unit in order to facilitate distribution. The polysulfone hollow fibers used as the scaffold in previous RAD designs were limited in cell attachment surface area and were prone to fracture during freezethaw. The cell-seeding scaffold for the BRECS, niobium-coated carbon disks, were selected based on their biologically inert, non-biodegradable, and favorable thermo-mechanical properties. Growth of adequate cell numbers to achieve a therapeutic impact was allowed due to disks’ high surface area. The other BRECS components, a polycarbonate housing, gasket, nuts, bolts, and access ports, were all carefully selected and thoroughly tested to withstand cryogenic temperatures, while still maintaining an uncompromised, sterile internal BRECS environment. In laboratory studies, the BRECS was able to be cryopreserved for long-term storage at 140 C, transitioned to 80 C for short-term storage, thawed, and maintained at 37 C for clinical application, accompanied by a loss of no greater than 10% of the cell dose (Buffington et al., 2009). These data demonstrate that the BRECS is the first single device that can serve as a culture vessel to maintain cells, reach cryopreservation temperatures as a full unit, and, lastly, be reconstituted to provide cell therapy. Having this storage capacity makes both emergent and acute use feasible.
CONCLUSION Despite all the advances in renal replacement therapies, a portable, continuous, dialysatefree artificial kidney remains the holy grail of renal tissue engineering. The enabling platform technologies discussed in this review advance this goal from a dream to the laboratory bench and even to the bedside. Future research in renal tissue engineering will need to focus on reproducing mechanisms of whole-body homeostasis. A high priority must be given to sensing and regulating extracellular fluid volume, even if only at the crude level of having the patient weigh him- or herself daily and adjust ultrafiltration and reabsorption by the bioartificial kidney. Chemical-field effect transistors (ChemFETs) offer the possibility of measuring electrolyte levels in a protein-free ultrafiltrate and reading out the potassium level to the patient, who could then alter diet or treat him- or herself with potassiumabsorbing resins. The critical building blocks of an autonomous bioartificial kidney are advancing rapidly with revolutionary clinical trials currently underway at multiple medical centers. The technology with which to adapt these advances to a more autonomous, dialysate-free system is under development. In addition, progress has been made in the field of cryopreservation and thus the ability to manufacture, store, and distribute bioartificial organs is advancing. The next decade, like the previous, will likely see quantum advances in renal tissue engineering.
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References Bologa, R. M., Levine, D. M., Parker, T. S., Cheigh, J. S., Serur, D., Stenzel, K. H., et al. (1998). Interleukin-6 predicts hypoalbuminemia, hypocholesterolemia, and mortality in hemodialysis patients. Am. J. Kidney Dis., 32, 107e114. Bone, R. C. (1991). The pathogenesis of sepsis. Ann. Intern. Med., 115, 457e469. Buffington, D. A., Hageman, G., Wang, M., Ding, F., Song, J., Jung, J., et al. (2009). (Abstract). Design of a compact cryopreservable bioartificial renal cell system. J. Am. Soc. Nephrol., 20, 27A. Dinarello, C. A. (1991). The proinflammatory cytokines interleukin-1 and tumor necrosis factor and the treatment of the septic shock syndrome. J. Infect. Dis., 163, 1177e1184. Ding, F., Song, J. H., Lou, L., Rojas, A., Reoma, J. L., Cook, K. E., et al. (2008). A novel selective cytopheretic inhibitory device (SCD) inhibits circulating leukocyte activation and ameliorates multiorgan dysfunction in a porcine model of septic shock. (Abstract.). J. Am. Soc. Nephrol., 19, 458A. Eknoyan, G., Beck, G. J., Cheung, A. K., Daugirdas, J. T., Greene, T., Kusek, J. W., et al., for the Hemodialysis (HEMO) Study Group. (2002). Effect of dialysis dose and membrane flux in maintenance hemodialysis. N. Engl. J. Med., 347, 2010e2019. Emoto, M., Miyamoto, M., Yoshizawa, I., Emoto, Y., Schaible, U. E., Kita, E., et al. (2002). Critical role of NK cells rather than Va14þNKT cells in lipopolysaccharide-induced lethal shock in mice. J. Immunol., 169, 1426e1432. Fissell, W. H., Dyke, D. B., Buffington, D. A., Weitzel, W. F., Westover, A. J., MacKay, S. M., et al. (2002a). Bioartificial kidney alters cytokine response and hemodynamics in endotoxin-challenged uremic animals. Blood Purif., 20, 55e60. Fissell, W. H., Humes, H. D., Roy, S., & Fleischman, A. (2002b). Initial characterization of a nanoengineered ultrafiltration membrane. J. Am. Soc. Nephrol., 13, 602A. Fissell, W. H., Lou, L., Abrishami, S., Buffington, D. A., & Humes, H. D. (2003). Bioartificial kidney ameliorates gram-negative bacteria-induced septic shock in uremic animals. J. Am. Soc. Nephrol., 14, 454e461. Fissell, W. H., Manley, S., Westover, A., Humes, H. D., Fleischman, A. J., & Roy, S. (2006). Differentiated growth of human renal tubule cells on thin-film and nanostructured materials. ASAIO J., 52, 221e227.
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Gura, V., Beizai, M., Ezon, C., & Polaschegg, H. D. (2005). Continuous renal replacement therapy for end-stage renal disease. The wearable artificial kidney (WAK). Contrib. Nephrol., 149, 325. Humes, H. D., & Cieslinski, D. A. (1992). Interaction between growth factors and retinoic acid in the induction of kidney tubulogenesis in tissue culture. Exp. Cell Res., 201, 8e15. Humes, H. D., Krauss, J. C., Cieslinski, D. A., & Funke, A. J. (1996). Tubulogenesis from isolated single cells of adult mammalian kidney: clonal analysis with a recombinant retrovirus. Am. J. Physiol., 271(1 Pt 2), F42eF49. Humes, H. D., Buffington, D. A., MacKay, S. M., Funke, A. J., & Weitzel, W. F. (1999a). Replacement of renal function in uremic animals with a tissue-engineered kidney. Nat. Biotechnol., 17, 451e455. Humes, H. D., MacKay, S. M., Funke, A. J., & Buffington, D. A. (1999b). Tissue engineering of a bioartificial renal tubule assist device: in vitro transport and metabolic characteristics. Kidney Int., 55, 2502e2514. Humes, H. D., Fissell, W. H., Weitzel, W. F., Buffington, D. A., Westover, A. J., MacKay, S. M., et al. (2002). Metabolic replacement of kidney function in uremic animals with a bioartificial kidney containing human cells. Am. J. Kidney Dis., 39, 1078e1087. Humes, H. D., Buffington, D. A., Lou, L., Abrishami, S., Wang, M., Xia, J., et al. (2003). Cell therapy with a tissueengineered reduces the multiple-organ consequences of septic shock. Crit. Care Med., 31, 2421e2428. Humes, H. D., Weitzel, W. F., Bartlett, R. H., Swaniker, F. C., Paganini, E. P., Luderer, J. R., et al. (2004). Initial clinical results of the bioartificial kidney containing human cells in ICU patients with acute renal failure. Kidney Int., 66, 1578e1588. Humes, H. D., Buffington, D. A., Lou, L., Wang, M., & Abrishami, S. (2007). Renal cell therapy ameliorates pulmonary abnormalities in a large animal model of septic shock and acute renal injury. J. Am. Soc. Nephrol., 18, A382. Humes, H. D., Dillon, J., Tolwani, A., Cremisi, H., Wali, R., Murray, P., et al. (2008). A novel selective cytopheretic inhibitory device (SCD) improves mortality in ICU patients with acute kidney injury (AKI) and multiorgan failure (MOF) in a phase II clinical study. J. Am. Soc. Nephrol., 19, 458A. Joyce, D. A., Gibbons, D. P., Green, P., Steer, J. H., Feldmann, M., & Brennan, F. M. (1994). Two inhibitors of proinflammatory cytokine release, interleukin-10 and interleukin-4, have contrasting effects on release of soluble p75 tumor necrosis factor receptor by cultured monocytes. Eur. J. Immunol., 24, 2699e2705. Kielar, M., Jeyarajah, D. R., & Lu, C. Y. (2002). The regulation of ischemic acute renal failure by extrarenal organs. Curr. Opin. Nephrol. Hypertens., 11, 451e457. Kimmel, P. L., Phillips, T. M., Simmens, S. J., Peterson, R. A., Weihs, K. L., Alleyne, S., et al. (1998). Immunologic function and survival in hemodialysis patients. Kidney Int., 54, 236e244.
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le Tissier, P., Stoye, J. P., Takeuchi, Y., Patience, C., & Weiss, R. A. (1997). Two sets of human-tropic pig retrovirus. Nature, 389, 681e682. Magistrelli, J. M. (2004). Investigating Fluid Flow Through Silicon Nanoporous Membranes. Master’s thesis. Cleveland: Case Western Reserve University. Marchant, A., Bruyns, C., Vandenabeele, P., Ducarne, M., Gerard, C., Delvaux, A., et al. (1994). Interleukin-10 controls interferon-g and tumor necrosis factor production during experimental endotoxemia. Eur. J. Immunol., 24, 1167e1171. Maroszynska, I., & Fiedor, P. (2000). Leukocytes and endothelium interaction as rate limiting step in the inflammatory response and a key factor in the ischemia-reperfusion injury. Ann. Transplant., 5, 5e11. Matsumoto, T., Tateda, K., Miyazaki, S., Furuya, N., Ohno, A., Ishii, Y., et al. (1998). Effect of interleukin-10 on gutderived sepsis caused by Pseudomonas aeruginosa in mice. Antimicrob. Agents Chemother., 42, 2853e2857. Natanson, C. (1990). Studies using a canine model to investigate the cardiovascular abnormality of and potential therapies for septic shock. Clin. Res., 38, 206e214. Natanson, C., Danner, R. L., Elin, R. J., Hosseini, J. M., Peart, K. W., Banks, S. M., et al. (1989). Role of endotoxemia in cardiovascular dysfunction and mortality: Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock. J. Clin. Invest., 83, 243e251. Neilson, E. G., Hull, A. R., Wish, J., Neylan, J. F., Sherman, D., & Suki, W. N. (1997). The Ad Hoc Committee Report on estimating the future workforce and training requirements for nephrology. J. Am. Soc. Nephrol., 8(5 Suppl. 9), S1eS4. Okusa, M. D. (2002). The inflammatory cascade in acute ischemic renal failure. Nephron, 90, 133e138. Paradis, K., Langford, G., Long, Z., Heneine, W., Sandstrom, P., Switzer, W. M., et al., The XEN 111 Study Group, Otto, E. (1999). Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science, 285, 1236e1241. Pinsky, M. R., Vincent, J. L., Deviere, J., Alegre, M., Kahn, R. J., & Dupont, E. (1993). Serum cytokine levels in human septic shock. Chest, 103, 565e576. Redmond, H. P., Chavin, K. D., Bromberg, J. S., & Daly, J. M. (1991). Inhibition of macrophase-activating cytokines is beneficial in the acute septic response. Ann. Surg., 214, 502e508. Ronco, C., & Fecondini, L. (2007). The Vicenza wearable artificial kidney for peritoneal dialysis (ViWAK PD). Blood Purif., 25, 383e388. Simmons, E. M., Himmelfarb, J., Sezer, M. T., Chertow, G. M., Mehta, R. L., Paganini, E. P., et al., for the PICARD study group. (2004). Plasma cytokine levels predict mortality in patients with acute renal failure. Kidney Int., 65, 1357e1365. Tumlin, J., Wali, R., Williams, W., Murray, P., Tolwani, A. J., Vinnikova, A. K., et al. (2008). Efficacy and safety of renal tubule cell therapy for acute renal failure. J. Am. Soc. Nephrol., 19(5), 1034e1040. US Renal Data System. (2004). USRDS 2002 Annual Data Report, Atlas of End-Stage Renal Disease in the United States. Bethesda, MD: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. Walley, K., Lukacs, N., Standiford, T., Streiter, R., & Kunkel, S. (1996). Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect. Immunol., 64, 4733e4738. Ward, R. A., Leypoldt, J. K., Clark, W. R., Ronco, C., Mishkin, G. J., & Paganini, E. P. (2001). What clinically important advances in understanding and improving dialyzer function have occurred recently? Semin. Dial., 14, 160e174. Zimmermann, J., Herrlinger, S., Pruy, A., Metzger, T., & Wanner, C. (1999). Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int., 55, 648e658.
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Extracorporeal Renal Replacement Kimberly A. Johnston*, H. David Humes*,y * Innovative Biotherapies, Ann Arbor, MI, USA y Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
INTRODUCTION The kidney is unique in that it is the first organ for which long-term ex vivo substitutive therapy has been made available and lifesaving. Renal failure prior to the era of hemodialysis and transplantation resulted in certain death, and this outcome of renal failure is still common outside the industrialized world. In the USA, 526,343 patients were listed as having end-stage renal disease (ESRD) by the 2007 US Renal Data System (USRDS) database, of whom 308,910 were receiving maintenance dialysis (US Renal Data System, 2004). The prevalence of ESRD in the USA is rising at approximately 8% per year (Neilson et al., 1997; US Renal Data System, 2004). The financial cost of dialysis is immense, estimated at $54,900 per hemodialysis patient per year and $46,121 per peritoneal dialysis patient per year. In contrast, transplant patients cost an average of $17,227 per patient per year (US Renal Data System, 2004). The higher cost of maintenance dialysis when compared with transplantation does not translate into better results; annual mortality for patients listed for transplant and awaiting a kidney is 6.3%, compared with only 3.8% for patients listed for transplant who did receive a kidney (US Renal Data System, 2004). While organ transplantation provides the best prognosis for survival, demand vastly outweighs the availability of donated organs. Current dialysis therapies include hemodialysis (HD), hemofiltration, and peritoneal dialysis (PD). Dialysis provides clearance of small molecules by diffusive flow across a semipermeable membrane and control of volume status by bulk flow of water and solutes through that membrane. These short-term effects are sufficient to abrogate the lethal acidosis, volume overload, and uremic syndromes that accompany renal failure but do not protect the patient from the increased mortality associated with dialysis-treated renal failure in either the acute or chronic form. These methodologies all address water and electrolyte balance e functional replacement of the kidney. However, they fail to provide for the lost endocrine function. Thus, the metabolic, endocrine, and immune roles of the functioning kidney are candidate mechanisms for the difference in survival noted above. The dialytic clearance of glutathione, a key tripeptide in free radical scavenging and protection against oxidant stress; the negative nitrogen balance and energy loss in the clearance of peptides and amino acids in dialysate; loss of oxidative deamination and gluconeogenesis in the tubule cell; and loss of cytokine and hormone metabolic activity by the kidney each impose substantial stress upon the dialyzed patient and as such are appropriate targets for improved renal replacement therapy. Principles of Regenerative Medicine. DOI: 10.1016/B978-0-12-381422-7.10051-3 Copyright Ó 2011 Elsevier Inc., All rights reserved.
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REQUIREMENTS OF A RENAL REPLACEMENT DEVICE Filtration is accomplished by the glomerulus, a tuft of capillaries supported by a basement membrane and specialized epithelial cells called podocytes. The renal proximal tubule, a hollow tube of cells surrounded by capillaries, receives the filtrate from the glomerulus and accomplishes the bulk of reclamation of salt, water, glucose, small proteins, amino acids, glutathione, and other substances. The tubule also performs metabolic functions, including excretion of acid as ammonia and hydroxylation of 25-hydroxy-vitamin-D3 among others. Intermittent hemodialysis is thought to replace the filtration function of the glomerulus and advances in hemodialysis and hemofiltration have focused on emulation of glomerular physiology. Recent attention has been drawn to duplicating the function of the proximal tubule. The transport of solutes and water is accomplished by ATP-driven electrolyte transporters in the luminal cell membrane. Reabsorption of small proteins and peptides in the filtrate stream is accomplished by membrane-bound proteases and specific amino acid transport proteins within the luminal membrane of the tubule cell. These amino acids are either used for protein and peptide synthesis in the tubule cell or transported into the capillaries for transport to and use by the body. The diversity and specificity of the functions of the proximal tubule cell argue against the development of an electromechanical or polymeric substitute, and so a number of years ago our research group turned its attention to the isolation and culture of renal proximal tubule cells, which research has culminated in the hollow-fiber bioreactor discussed below.
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Critical to providing organ function replacement through cell therapy is the isolation and growth in vitro of specific cells from adult tissue. These cells must have stem cell-like characteristics, with a high capacity for self-renewal and the ability to differentiate under defined conditions into specialized cells to develop the correct structure and functional components of a physiological organ system. Methodology to isolate and grow renal proximal tubule progenitor cells from adult pig kidneys has been reported (Humes and Cielinski, 1992; Humes et al., 1996). These studies were promoted by clinical and experimental observations suggesting that progenitor cells of renal proximal tubules must exist, as tubule cells have the ability to regenerate after severe nephrotoxic or ischemic injury. Porcine cells were utilized, as the pig has been considered the best source of organs for both xenotransplantation and cell therapy devices due to its anatomic and physiological similarities to human tissue and the relative ease of breeding large numbers of pigs in closed herds. However, reports of the ability of porcine endogenous retroviruses (PERVs) to infect human cells in co-culture in vitro have raised concerns about the potential, but currently unquantifiable, risk of transmission of viral elements from porcine tissue to humans in xenotransplantation or cell therapy devices (le Tissier et al., 1997; Paradis et al., 1999). Accordingly, Humes and colleagues fabricated bioreactors containing human renal epithelial cells isolated from kidneys donated for transplantation but found unsuitable for such purpose because of anatomic or fibrotic defects. These cells performed well in studies to assess viability, durability, and physiological performance (Humes et al., 2002). The renal assist device (RAD) is a bioreactor containing proximal tubule cells grown in confluent monolayers along the inner surface of the hollow fibers of a conventional hemofiltration cartridge. Within this multifiber unit, proximal tubule cells not only maintain transport properties but also differentiated metabolic and endocrine functions. The nonbiodegradability and the pore size of the hollow fibers allow the membranes to act as both scaffolds for the cells and as an immunoprotective barrier. Completed experiments have successfully scaled up to a clinically applicable device with the use of commercially available high-flux hemofiltration hollow fiber cartridges.
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Experiments have tested the transport and metabolic functions of cells grown intraluminally within these cartridges with membrane surface areas of 97 cm2 to 0.4 m2 (Humes et al., 1999b). Starting with a hemofilter cartridge, the intraluminal surface of the hollow fibers was coated with pronectin L, a synthetic protein with multiple cell attachment sites found in the extracellular protein laminin. Renal proximal tubule progenitor cells were then seeded at a density of 105 cells/ml into the intracapillary space. The seeded cartridge was connected to the bioreactor perfusion system, in which the extracapillary space was filled with culture media and the intracapillary space perfused with similar media. After 7e14 days, light microscopy revealed a confluent monolayer of tubule cells grown on the inner surface of the fibers, and electron microscopy identified differentiated epithelial characteristics, including microvilli, endocytic vesicles, and extensive basolateral infoldings. The cells retain vectorial fluid transport properties as a result of Na,K-ATPase; differentiated active transport properties, including active electrolyte, bicarbonate, and glucose transport; differentiated metabolic activities, including intraluminal glutathione breakdown and ammonia production; and the important endocrinological conversion of 25-OH-vitD3 to 1,25-(OH)2-vitD3.
ULTRAFILTRATION MEMBRANE DEVELOPMENT In parallel with progress in stem cell work, there has been considerable interest in applying novel technology to membrane engineering. The filtration barrier in the kidney has been extensively studied. This barrier is widely considered to be trilaminate, with an endothelial cell layer, a basement membrane, and an elaborate epithelial layer bearing a specialized cell-cell junction called the glomerular slit diaphragm. Unfortunately, the cell considered responsible for the permselectivity barrier in the kidney, the glomerular podocyte, is a terminally differentiated cell with limited regenerative capacity. Damaged cells are not replaced by expansion of neighboring podocytes. Similarly, primary cultures of podocytes do not assume a differentiated phenotype in the laboratory dish, nor do they easily divide and expand in number. Despite progress with a conditionally transformed cell line derived from mouse podocytes, there seems little immediate prospect of a cell-based bioartificial glomerulus. Without a purely biological ultrafiltration unit on the horizon, advances in HD membrane development have been aimed at attempting to better reproduce the physiological process of glomerular ultrafiltration using synthetic membranes. Membrane materials are diverse, ranging from regenerated cellulose filters to metals and ceramics to modern-day polymers. Critical to the choice of materials for current dialysis membranes, biocompatibility with blood is a major concern, as well as cost and manufacturing. Synthetic polymers have become the dominant membrane materials used due a combination of these factors. The most common polymers in the manufacturing of synthetic membranes are non-degradable polymers: cellulose derivatives; nitrates; polyesters, polysulfones; polyacrilonitrile derivatives; polyamides; polyimides; polyolefins such as polyethylene, polypropylene or polyvinylchloride; and fluorinated polymers such as polytetrafluoroethylene, and polyvinylidinefluoride. Dialysis membrane clearance is based on concentration differences rather than convective separation of small solutes and low-molecular-weight proteins from large serum proteins and blood elements. In an attempt to recapitulate glomerular ultrafiltration and removal of “middle molecules,” synthetic membranes with larger pore sizes and high water permeability have been developed. These so-called “high-flux” membranes are prepared with hydrophobic base materials, including polyacrylnitrile, polysulfone, polyethersulfone, or polymide, with various hydrophilic components. Recent membrane development has focused on increasing pore size while sharpening the molecular weight cutoff of high-flux membranes to maximize removal of low-molecular-weight proteins. Removal of a distinct class of uremic toxins, such as b2-microglobulin, factor D, leptin, and adrenomedullin, while minimizing the loss of albumin, could improve treatment outcomes of patients with ESRD. This idea has spurned the
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creation of superflux or protein-leaking membranes. These membranes provide greater clearances for low-molecular-weight proteins and small protein-bound solutes, such as homocysteine and advanced glycation end products, but with significantly higher loss of albumin than high-flux dialysis membranes. The overall benefits for patients on chronic HD still require more extensive evaluation.
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Micromechanical systems (MEMS) are increasingly used to develop novel membrane technology. Current polymer membranes for renal replacement are performance limited. In general, such membranes are fairly thick or employ a multilayer scaffold for mechanical support, and they have a distribution of pore sizes rather than a regular array of uniform pores. Pores in conventional polymer membranes tend to either be roughly cylindrical, have a round orifice terminating a larger channel, or have a structure resembling an open cell sponge. These structures provide less than optimal geometries for membrane filtration for two reasons. First, a wide dispersion in pore sizes within a membrane leads to imperfect retention of molecules larger than the mean pore size of the membrane. Reducing the mean pore size of the membrane may partially solve this problem; however, it has the undesired effect of reducing the hydraulic permeability of the membrane. Second, the round shape of conventional pores dictates a dependence of hydraulic permeability on pore radius. In contrast, a pore that is slitshaped allows steric hindrance to solute passage dictated by the smallest critical dimension of the pore, while increasing hydraulic permeability based on the long dimension of the pore. Consequently, it might be predicted that filtration structures with parallel slit-shaped pores will have superior performance when compared to structures with round pores. The glomerular filtration barrier also imposes an electrostatic restriction on solute passage. This function has been variously attributed to the proteins within the slit diaphragm, the glomerular basement membrane, and the glycocalyx of the glomerular endothelial cell. In regard to artificial membranes, a double thick electrical layer related to the nanometer-scale pore size itself contributes to the rejection of charged solutes. Novel silicon nanopore membranes with 10e100 nm 45 mm slit pores, approximating the glomerular slit diaphragm, have been prototyped by an innovative process based on MEMS technology. Silicon chips bearing 1 1 mm arrays of approximately 104 slit pores were fabricated via sacrificial layer techniques. The pore structure is defined by deposition and patterning of a polysilicon film on the silicon wafer. The critical submicron pore dimension is defined by the thickness of a sacrificial SiO2 layer, which can be grown with unprecedented control to within 1 nm. Preliminary data on the transport properties of MEMS membranes are encouraging. Measured hydraulic permeabilities correlated well with theoretical predictions for flow-through slitshaped pipes, also known as Hele-Shaw flows. The observed albumin sieving coefficient data provide encouragement that protein permselectivity is also feasible with this technology. Recent laboratory data have validated the possibilities of these membranes as scaffolding for a renal tubule cell bioreactor (Ward et al., 2001).
TRANSPORT AND METABOLIC CHARACTERISTICS OF HOLLOW-FIBER BIOREACTORS As initial experiments using the single hollow-fiber model were promising, the design was scaled up to use commercially available polysulfone hollow-fiber dialysis cartridges from the manufacturers of the single hollow fibers. Single hollow-fiber measurements of transport and metabolic activity were repeated with 97 cm2 and 0.4 m2 surface area cartridges. Further exploration of the metabolic and transport characteristics of the cultured proximal tubule cells was assessed. The transport of glucose, bicarbonate, and glutathione excretion was measured in the absence and presence of a known inhibitor of an enzyme essential for the reabsorption. In each case, there was evidence of active transport and specific inhibition (Humes et al., 1999b).
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The synthesis and secretion of ammonia into the tubule is essential for renal excretion of an acid load, as it buffers secreted protons. Proximal tubule cells are able to upregulate their ammoniagenesis in response to a decline in pH, and the proximal tubule cells in the bioreactor demonstrated a stepwise increase in ammonia production with changes in pH (Humes et al., 1999b). The experiments detailed above were performed in our laboratory with porcine tubule cells that demonstrated similar results in culture, attachment, and activity with human proximal tubule cells from cadaveric organs. The final selection of cell type for use in a renal tubule device not only rests on supply and safety of cells, but also depends on the ability of cells to participate in the homeostasis of the host. The above data suggest that our laboratory has successfully isolated and cultured renal proximal tubule cells, established stable confluent monolayers within hollow-fiber bioreactors, and scaled the initial construct to a level approximating the number of proximal tubule cells in a single kidney.
PRECLINICAL CHARACTERIZATION OF THE RENAL TUBULE ASSIST DEVICE/BIOARTIFICIAL KIDNEY In keeping with its role as a metabolically active replacement for the renal proximal tubule, an extracorporeal circuit was devised that recapitulated nephron anatomy. The bioartificial kidney setup consists of a filtration device (a conventional hemofilter) followed in series by the renal tubule assist device (RAD) unit. Specifically, blood is pumped out of a patient using a peristaltic pump. The blood then enters the fibers of a hemofilter, where ultrafiltrate is formed and delivered into the fibers of the tubule lumens within the RAD downstream to the hemofilter. Processed ultrafiltrate exiting the RAD is collected and discarded as “urine.” The filtered blood exiting the hemofilter enters the RAD through the extracapillary space port and disperses among the fibers of the device. Upon exiting the RAD, the processed blood travels through a third pump and is delivered back to the patient. Heparin is delivered continuously into the blood before entering the RAD to diminish clotting within the device. The RAD is oriented horizontally and maintained at 37 C throughout its operation to ensure optimal functionality of the cells. The tubule unit is able to maintain viability because metabolic substrates and low-molecular-weight growth factors are delivered to the tubule cells from the ultrafiltration unit and the blood in the extracapillary space. Furthermore, immunoprotection of the cells grown within the hollow fiber is achieved because of the impenetrability of immunoglobulins and immunologically competent cells through the hollow fibers. Rejection of the cells, therefore, does not occur. This extracorporeal circuit containing the RAD was initially tested on uremic dogs with bilateral nephrectomies (Humes et al., 1999a). The animals were treated with either a RAD or a sham control cartridge daily for either 7 or 9 h for three successive days or for 24 h continuously. The RADs maintained viability and functionality throughout the study period. Fluid and small solutes, including blood urea nitrogen (BUN), creatinine (Cr), and electrolytes, were adequately controlled in both groups, but potassium and BUN levels were more easily controlled by RAD treatment. Furthermore, active reabsorption of Kþ, HCO3, and glucose and excretion of ammonia were accomplished only in RAD treatments. Glutathione reclamation from UF exceeded 50% in the RAD. Finally, uremic animals receiving cell therapy attained normal 1,25-(OH)2-vitD3 levels, whereas sham treatment resulted in a further decline from the already low plasma levels. Thus, these experiments clearly showed that the combination of a synthetic hemofiltration cartridge and a RAD in an extracorporeal circuit successfully replaced filtration, transport, and metabolic and endocrinological functions of the kidney in acutely uremic dogs.
CLINICAL EXPERIENCE WITH A HUMAN RENAL TUBULE ASSIST DEVICE Encouraging preclinical data led to FDA approval for an investigational new drug and phase I/II clinical trials. The first human clinical study of the bioartificial kidney containing human cells
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was carried out in 10 ICU patients with AKI receiving CVVH (Humes et al., 2004). This study demonstrated that the RAD can be used safely for up to 24 h. Cardiovascular stability was maintained, and increased native renal function, as determined by elevated urine outputs, temporally correlated with RAD treatment. All patients were critically ill with acute kidney injury (AKI) and multiple organ failure (MOF), with predicted hospital mortality rates between 80 and 95%, Six of the 10 treated patients survived past 30 days, with mortality reduced to 40%. The human renal tubule cells contained in the RAD demonstrated differentiated metabolic and endocrinological activity in this ex vivo situation, including glutathione degradation and endocrinological conversion of 25-OH-vitD3 to 1,25-(OH)2-vitD3. Plasma cytokine levels suggest that RAD therapy produces dynamic and individualized responses in patients depending on their unique pathophysiological conditions. For the subset of patients who had excessive proinflammatory levels, RAD treatment resulted in significant declines in granulocytecolony stimulating factor (G-CSF), IL-6, IL-10, and especially IL-6/IL-10 ratio, suggesting a greater decline in IL-6 relative to IL-10 levels and a less proinflammatory state. These favorable phase I/II trial results led to a randomized, controlled, open-label phase II trial conducted at 12 clinical sites in the US (Tumlin et al., 2008). Fifty-eight patients with ARF requiring CVVH in the ICU were randomized (2:1) to receive CVVH þ RAD (n ¼ 40) or CVVH alone (n ¼ 18). Despite the critical nature and life-threatening illnesses of the patients enrolled in this study, the addition of the RAD to CVVH resulted in a substantial clinical impact on survival compared with the conventional CVVH-treatment group. RAD treatment for up to 72 h promoted a statistically significant survival advantage over 180 days of follow-up in ICU patients with AKI and demonstrated an acceptable safety profile. Cox proportional hazards models suggested that the risk of death was approximately 50% of that observed in the CRRT-alone group. A follow-up phase IIb study to evaluate a commercial manufacturing process was not completed due to difficulties with the manufacturing process and clinical study design. This approach will be further evaluated when an improved scale-up manufacturing process is established. 948
CELL THERAPY OF ACUTE RENAL FAILURE DUE TO SEPSIS After a series of experiments demonstrating bioactivity, longevity, and systemic activity of the proximal tubule cells in a large animal model, a series of experiments was designed to examine the impact of cell therapy on the course of sepsis complicated by renal failure (Fissell et al., 2002a, 2003). After two initial studies supported a systemic effect and hemodynamic benefit from cell therapy in large animal models of sepsis, our laboratory pursued further evidence that cell therapy with renal proximal tubule cells alters the physiological response to sepsis (Humes et al., 2003). A porcine model of septic shock was developed from the previous work (Natanson et al., 1989; Natanson, 1990; Dinarello, 1991). Purpose-bred pigs were anesthetized and administered an intraperitoneal dose of bacteria, causing shock and renal failure. An hour later continuous venovenous hemofiltration (CVVH) was initiated with either cell or sham RAD. Urine output and mean arterial pressure declined within the first few hours after insult. Cell-treated animals survived 9.0 10.83 h versus 5.1 10.4 h (P 0.005) for shamtreated animals. Serum cytokines were similar between the two groups, with the striking exception of interleukin (IL)-6 and interferon (IFN)-g. Treatment with the cell RAD resulted in significantly lower plasma levels of both IL-6 (P 0.04) and INF-g (P 0.02) throughout the experimental time course compared to sham RAD exposure. This controlled trial of cell therapy of renal failure in a realistic animal model of sepsis has several findings not immediately expected from a priori assumptions regarding renal function. Heretofore, although renal failure has been strongly associated with poor outcome in hospitalized patients, and chronic renal failure is associated with specific defects in humoral and cellular immunity, a direct immunomodulatory effect of the kidney had not been accepted. In this trial, clear differences in survival and clear differences in a serum cytokine associated with mortality in sepsis were found between groups: The increased mortality in renal failure appears to be not attributable to inadequate solute clearance, but may arise from other bioactivity of the kidney.
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BIOARTIFICIAL KIDNEY IN END-STAGE RENAL DISEASE A bioartificial kidney for long-term use in ESRD, similar to short-term use in acute renal failure (ARF), would integrate tubular cell therapy and the filtration function of a hemofilter. As noted above, ESRD patients on conventional renal replacement therapy are at high risk for cardiovascular and infectious diseases. A recent clinical trial failed to show survival benefit from increased doses of hemodialysis above what is now standard care (Eknoyan et al., 2002), suggesting that there are important metabolic derangements not adequately treated with conventional dialytic treatment. Data from the survival of renal transplant recipients, which far exceed those from the survival of age-, sex-, and risk-matched controls awaiting transplant, also suggest that there is some metabolic function provided by the kidney that transcends this organ’s filtration function. Patients with ESRD display elevated levels of C-reactive protein (CRP), an emerging clinical marker, and pro-inflammatory cytokines, including IL-1, IL-6, and tumor necrosis factor (TNF) (Bologa et al., 1998; Kimmel et al., 1998; Zimmermann et al., 1999). All these parameters are associated with enhanced mortality in ESRD patients. Specifically, IL-6 has been identified as a single predictive factor closely correlated with mortality in hemodialysis patients (Bologa et al., 1998). Although all ESRD patients could conceivably benefit from a bioartificial kidney, patients in the inflammatory stage who display elevated levels of certain markers of chronic inflammation (most notably IL-6 and CRP) would likely benefit most and will be the target population for clinical study in the near future. For the ESRD patient population, however, there are obvious limitations in using an extracorporeal RAD connected to a hemofiltration circuit. Ideally, a bioartificial kidney suitable for long-term use in ESRD patients would be capable of performing continuously, like the native kidney, to reduce risks from fluctuations in volume status, electrolytes, and solute concentrations and to maintain acid-base and uremic toxin regulation. Such treatment requires the design and manufacture of a compact implantable or wearable dialysis apparatus and the development of miniaturized renal tubule cell devices with long service lifetimes. The ideal design of the next-generation RAD would be like that of an implantable device such as the pacemaker. Attempts have been made to develop wearable dialysis systems to improve the portability of renal replacement therapies. Gura et al. (2005) have published research into a light-weight, wearable, continuous ambulatory ultrafiltration device consisting of a hollow fiber hemofilter, a battery-operated pulsatile pump, and two micropumps to control heparin administration and ultrafiltration. This device regenerates dialysate with activated carbon, immobilized urease, zirconium hydroxide, and zirconium phosphate, similar to the once commercially available REDY dialysis system. Ronco and Fecondini (2007) have described a wearable continuous PD system consisting of a double lumen dialysate line with a peritoneal catheter, a miniaturized rotary pump, a circuit for dialysate regeneration, and a handheld computer as a remote control. These systems still rely on inconvenient dialysate and expensive dialysis regeneration devices and/or dialyzers, but they promise to improve the convenience of dialysis. In contrast to wearable dialysis systems, a hybrid bioartificial kidney integrates tubule cell and filtration functions. The first bioartificial kidney, consisting of a passive hemofilter and an active renal tubule cell bioreactor, has consistently demonstrated excellent safety and effectiveness in animal studies and FDA-approved human clinical trials, as described above (Ward et al., 2001; Humes et al., 2003, 2004; Tumlin et al., 2008). A major drawback of the current version of the bioartificial kidney is its large size, owing to the requisite extracorporeal circuit with peristaltic pumps to provide driving pressure for hemofiltration. A new smaller and more durable RAD is currently being developed by Humes and colleagues. In collaboration, Fissell and colleagues are developing a nanopore membrane to replace the filtration function of the glomerulus without the hemofilters and mechanical pumps of existing dialysis machines. A filtration device based on nanopore membrane technology
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would be implantable (Fissell et al., 2002b, 2006; Magistrelli, 2004). Further refinement of the RAD would be encouraging for ESRD patients because, in principle, such a tissue-engineered device could be free of dialysate or replacement fluid while providing functions of healthy kidney that are not offered by current dialysis strategies. The combination of cell therapy and solute clearance could be a viable renal replacement therapy, conferring dialysis independence to the patient.
IMMUNOMODULATORY EFFECT OF THE RENAL TUBULE ASSIST DEVICE
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As described earlier, RAD treatment altered systemic circulating cytokine levels in animal and human experiments. In endotoxin-challenged and gram-negative peritonitis uremic dog models, plasma levels of IL-10 were significantly higher in RAD-treated animals (Fissell et al., 2002a, 2003). The role of IL-10 in regulating immune response continues to be elucidated, but data suggest that IL-10 levels influence outcome in endotoxin shock and gram-negative sepsis. Several reports have demonstrated that administration of recombinant IL-10 is protective against gram-negative septic shock in murine sepsis models (Walley et al., 1996; Matsumoto et al., 1998). Another study in a similar model demonstrated that administration of antibodies to IL-10 was associated with higher mortality (Marchant et al., 1994). The mechanism underlying the link between proximal tubule function and IL-10 levels remains to be detailed, but preliminary data suggest that renal production of IL-6 induces liver production of IL-10 (Kielar et al., 2002). In gram-negative septic pigs without nephrectomy, RAD treatment significantly reduced plasma circulating levels of IL-6 and INF-g (Humes et al., 2003). The difference in IL-6 concentrations is especially noteworthy, since the plasma elevations of this proinflammatory cytokine have been directly correlated to outcome in patients with SIRS (Pinksy et al., 1993). The lower concentration of plasma INF-g may be important due to its central role in the inflammatory response. INF-g stimulates B-cell antibody production, enhances polymorpholeukocyte phagocytosis, and activates monocytes and macrophages to release proinflammatory cytokines (Bone, 1991; Redmond et al., 1991; Joyce et al., 1994). Excessive rates of INF-g production by NK cells have correlated with progression to lethal endotoxin shock in mice (Emoto et al., 2002). Further support for an immunomodulatory role of renal tubule cells has been suggested in the phase I/II clinical trial of the RAD containing human renal tubule cells (Humes et al., 2004). The patients treated in this study had a wide spectrum of plasma cytokine levels. The subset of patients who presented with very high plasma cytokine levels and who were treated for an adequate period showed that RAD treatment resulted in significant reductions in G-CSF, IL-6, and IL-10 levels. The greater relative reduction in IL-6/IL-10 ratio suggests renal tubule cell therapy may rebalance the excessive proinflammatory response with the concurrent anti-inflammatory response. These results are consistent with an immunomodulatory role for the RAD in patients with acute tubular necrosis and multiorgan failure. To further evaluate the RAD’s influence on local inflammation in tissue and distant organ dysfunction, especially in the lungs, a recent study compared bronchoalveolar lavage (BAL) fluid from cell-RAD-treated and non-cell, sham-treated groups in a pig model with septic shock with AKI (Humes et al., 2007). The levels of total protein in BAL were significantly higher in sham control animals than in the RAD group (143 111 compared to 78 110 mg/ml, respectively; P > 0.05). Proinflammatory cytokines, including IL-6 and IL-8, were markedly elevated in the control group. These results demonstrate an important role for renal epithelial cells in ameliorating multiorgan injury in sepsis by influencing microvascular injury and the local proinflammatory response. A more promising direction to improve outcome of AKI is to better understand and interrupt the pathophysiological processes that are activated in AKI, resulting in distant multi-organ dysfunction and eventually death. AKI results in a profound inflammatory response state resulting in microvascular dysfunction in distant organs (Okusa, 2002; Simmons et al., 2004). Leukocyte activation plays a central role in these acute inflammatory states. Disruption of the activation
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process of circulating leukocytes may limit microvascular damage and multi-organ dysfunction (Maroszynska and Fiedor, 2000). The RAD appears to influence systemic leukocyte activation and the balance of inflammatory cytokines and may alter the proinflammatory state of AKI and, ultimately, improve morbidity and mortality. Our group has recently developed a novel synthetic membrane embedded in an extracorporeal device to bind and inhibit circulating leukocytes. This “selective cytopheretic inhibitory device” (SCD) mimics immunomodulation and duplicates RAD efficacy. The SCD improved septic shock survival times in preclinical animal models and improved the survival outcome of ICU patients with multiorgan failure in a small exploratory, randomized, double-blinded, multicenter trial (Ding et al., 2008; Humes et al., 2008).
THE BIOARTIFICIAL RENAL EPITHELIAL CELL SYSTEM A next-generation RAD, the bioartificial renal epithelial cell system (BRECS), was designed by Humes and colleagues to achieve the support of 10-fold more cells in less than one-third of the volume of previous RAD designs, along with the capacity to cryopreserve the full unit in order to facilitate distribution. The polysulfone hollow fibers used as the scaffold in previous RAD designs were limited in cell attachment surface area and were prone to fracture during freezethaw. The cell-seeding scaffold for the BRECS, niobium-coated carbon disks, were selected based on their biologically inert, non-biodegradable, and favorable thermo-mechanical properties. Growth of adequate cell numbers to achieve a therapeutic impact was allowed due to disks’ high surface area. The other BRECS components, a polycarbonate housing, gasket, nuts, bolts, and access ports, were all carefully selected and thoroughly tested to withstand cryogenic temperatures, while still maintaining an uncompromised, sterile internal BRECS environment. In laboratory studies, the BRECS was able to be cryopreserved for long-term storage at 140 C, transitioned to 80 C for short-term storage, thawed, and maintained at 37 C for clinical application, accompanied by a loss of no greater than 10% of the cell dose (Buffington et al., 2009). These data demonstrate that the BRECS is the first single device that can serve as a culture vessel to maintain cells, reach cryopreservation temperatures as a full unit, and, lastly, be reconstituted to provide cell therapy. Having this storage capacity makes both emergent and acute use feasible.
CONCLUSION Despite all the advances in renal replacement therapies, a portable, continuous, dialysatefree artificial kidney remains the holy grail of renal tissue engineering. The enabling platform technologies discussed in this review advance this goal from a dream to the laboratory bench and even to the bedside. Future research in renal tissue engineering will need to focus on reproducing mechanisms of whole-body homeostasis. A high priority must be given to sensing and regulating extracellular fluid volume, even if only at the crude level of having the patient weigh him- or herself daily and adjust ultrafiltration and reabsorption by the bioartificial kidney. Chemical-field effect transistors (ChemFETs) offer the possibility of measuring electrolyte levels in a protein-free ultrafiltrate and reading out the potassium level to the patient, who could then alter diet or treat him- or herself with potassiumabsorbing resins. The critical building blocks of an autonomous bioartificial kidney are advancing rapidly with revolutionary clinical trials currently underway at multiple medical centers. The technology with which to adapt these advances to a more autonomous, dialysate-free system is under development. In addition, progress has been made in the field of cryopreservation and thus the ability to manufacture, store, and distribute bioartificial organs is advancing. The next decade, like the previous, will likely see quantum advances in renal tissue engineering.
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