Enhanced propagation of adult human renal epithelial progenitor cells to improve cell sourcing for tissue-engineered therapeutic devices for renal diseases.

Renal cell therapy employing cells derived from adult renal epithelial cell (REC) progenitors promises to reduce the morbidity of patients with renal insufficiency due to acute renal failure and end stage renal disease. To this end, tissue engineered devices addressing the neglected biologic component of renal replacement therapy are being developed. Because human donor tissue is limited, novel enhanced progenitor cell propagation (EP) techniques have been developed and applied to adult human kidney transplant discards from six donors. Changes include more efficient digestion and the amplification of progenitors prior to terminal epithelial differentiation promoted by contact inhibition and the addition of retinoic acid. Differentiated morphology in EP populations was demonstrated by the ability to form polarized epithelium with tight junctions, apical central cilia and expression of brush border membrane enzymes. Evaluation of lipopolysaccharide stimulated interleukin-8 secretion and γ-glutamyl transpeptisade activity in EP derived cells was used to confirm therapeutic equivalence to REC obtained using published techniques, which have previously shown efficacy in large animal models and clinical trials. Yield exceeded 10(16) cells/gram cortex from the only kidney obtained due to an anatomical defect, while the average yield from diseased kidneys ranged from 1.1 × 10(9) to 8.8 × 10(11) cells/gram cortex, representing an increase of more than 10 doublings over standard methods. Application of the EP protocol to REC expansion has solved the problem of cell sourcing as the limiting factor to the manufacture of cell based therapies targeting renal diseases and may provide a method for autologous device fabrication from core kidney biopsies.

A selective cytopheretic inhibitory device for use during cardiopulmonary bypass surgery.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) can occur in association with cardiopulmonary bypass (CPB) surgery, resulting in multiple organ dysfunction (MOD). Activated neutrophils have been implicated as major inciting factors in this process. Neutrophil-depleting filters incorporated within the extracorporeal blood circuit during CPB have been developed and evaluated, with inconsistent clinical results. METHODS: A novel, biomimetic, selective cytopheretic device (SCD) was tested in vitro within a blood circuit to assess safety and interactions with blood components and further evaluated ex vivo in a bovine model of CPB surgery during ventricular assist device implantation. RESULTS: In vitro blood circuit studies demonstrated that the SCD reduces circulating neutrophils while maintaining low rates of hemolysis compared to current leukocyte-reduction filters. In the bovine CPB model, animals without SCD treatment (No SCD) demonstrated an increase in circulating white blood cell (WBC) and neutrophil counts, steadily increasing throughout CPB. SCD with only systemic heparin anticoagulation (SCD-H) acutely reduced neutrophils for the first 2 hrs of CPB, but followed with a greater than 6-fold increase in neutrophil counts. SCD treatment with regional citrate anticoagulation along the SCD circuit (SCD-C) reduced systemic neutrophil counts throughout 4 hrs of CPB despite lower amounts of eluted cells from the SCD. When analyzed for immature neutrophils, the control and SCD-H showed increasing counts at later time-points, not seen in the SCD-C group, suggesting a more complex mechanism of action than simple leukoreduction. CONCLUSIONS: These results suggest that SCD-C therapy may disrupt the systemic leukocyte response during CPB, leading to improved outcomes for CPB-mediated MOD.

The effects of a novel therapeutic device on acute kidney injury outcomes in the intensive care unit: a pilot study.

Despite decades of improvements in the provision of renal replacement therapy, the morbidity and mortality associated with acute kidney injury (AKI) in the intensive care unit (ICU) setting remains extremely high. Much of the morbidity and mortality of this disorder is the consequence of systemic cellular damage that results from immune dysregulation. This is a prospective, single-arm, single-center study designed to evaluate the safety and efficacy of treatment with a selective cytopheretic device (SCD) on clinical outcomes in AKI requiring renal replacement therapy in the ICU. The patients enrolled in the trial were compared with historical case-matched controls with respect to age and Sequential Organ Failure Assessment (SOFA) score. The mortality for the case-matched controls was 77.78%, whereas the mortality in the SCD treatment group was 22.22% (p = 0.027). Multiple regression analysis identified treatment with SCD as the only significant variable affecting mortality among age, SOFA score, average change in urine output over the first 7 days during or after treatment. Mean total urine output in the 10 subjects receiving SCD treatment increased from a baseline of approximately 500 ml/d to more than 2,000 ml/d by day 7 of treatment. The SCD represents a novel therapeutic approach to alter the acute inflammatory response seen in AKI, and further evaluation of the safety and efficacy of the device is being evaluated in a multicenter investigation in the United States under an Food and Drug Administration (FDA) approved investigational device exemption (IDE).

New insights into mechanisms of glomerular permselectivity.

Proteinuria has emerged as a key predictor of progression from renal insufficiency to end-stage renal disease, and clearly plays a pathogenic role in loss of renal function. Control of proteinuria is seen as critical to delaying disease progression, and myriad treatments which appear to reduce proteinuria have been reported and have entered clinical practice. Despite the increasing emphasis on control of proteinuria, the precise mechanism by which the kidney retains proteins in the blood remains a subject of dispute in the literature. In the past decade, mechanisms for protein retention by the kidney which transcend simple molecular sieve heuristics have been proposed. This renewed interest in renal physiology is exciting, as new insights may drive forward mechanism-based treatments for renal disease. In this review article, four schools of thought on renal protein retention are described, including three from other groups and our own hypothesis. Arguments and data supporting and refuting each paradigm are discussed without the intent or effect of supporting one to the exclusion of others.

Dialysis and nanotechnology: now, 10 years, or never?

Nanotechnology, defined as the science of material features between 10(-9) and 10(-7) of a meter, has received extensive attention in the popular press as proof-of-concept experiments in the laboratory are published. The inevitable delay between feature articles and clinical endpoints has led to unwarranted skepticism about the applicability of the technology to current medical therapy. The theoretic advantages of micro- and nanometer scale engineering to renal replacement include the manufacture of high-hydraulic permeability membranes with implanted sensing and control structures. Recent data in membrane design and testing is presented, with a review of the challenges remaining in implementation of this technology.

The future of hemodialysis membranes.

Hemodialytic treatment of patients with either acute or chronic renal failure has had a dramatic impact on the mortality rates of these patients. Unfortunately, this membrane-based therapy is still incomplete renal replacement, as the mortality and morbidity of these patients remain unacceptably high. Much progress must be made to improve the biocompatibility of hemodialysis membranes as well as their hydraulic and permselective properties to remove small solutes and 'middle molecules' in compact cartridges. The next directions of development will leverage materials and mechanical engineering technology, including microfluidics and nanofabrication, to further improve the clearance functions of the kidney to replicate glomerular permselectivity while retaining high rates of hydraulic permeability. The extension of membrane technology to biohybrid devices utilizing progenitor/stem cells will be another substantive advance for renal replacement therapy. The ability to not only replace solute and water clearance but also active reabsorptive transport and metabolic activity will add additional benefit to the therapy of patients suffering from renal failure. This area of translational research is rich in creative opportunities to improve the unmet medical needs of patients with either chronic or acute renal failure.

Cell therapy of renal failure.

The kidney is unique in that it is the first organ for which long-term ex vivo substitutive therapy has been available. The first hemodialyzer was successfully applied to a human patient with acute renal failure in 1948, and the first successful allograft transplantation was performed with a kidney in 1951. Both treatments are used today. There is ample evidence that the small solute clearance function provided by hemodialysis does not confer the same survival advantage as a functional kidney, both in acute and in chronic renal failure. To mimic the metabolic, endocrine, and immunologic functions of the kidney, our group has successfully engineered a bioartificial device that includes a conventional dialysis filter and a bioreactor containing 10(9) renal proximal tubule cells. We have demonstrated differentiated activity of these cells both in vitro and ex vivo in a large animal model. The bioreactor has been shown to confer a survival advantage in two large animal models of gram-negative sepsis, seemingly due to modulation of inflammatory mediators. This bioartificial kidney has now completed a Phase I clinical trial in acute renal failure.

The role of a bioengineered artificial kidney in renal failure.

Renal failure continues to carry substantial burden of morbidity and mortality in both acute and chronic forms, despite advances in transplantation and dialysis. There is evidence to suggest that the kidney has metabolic, endocrine, and immune effects transcending its filtration functions, even beyond secretion of renin and erythropoietin. Our laboratory has developed experience in the tissue culture of renal parenchymal cells, and has now been able to demonstrate the metabolic activity of these cells in an extracorporeal circuit recapitulating glomerulotubular anatomy. We have observed active transport of sodium, glucose, and glutathione. We describe the design and initial preclinical testing of the bioartificial kidney, as well as future directions of our research.

Bioartificial organ support for hepatic, renal, and hematologic failure.

The current strategy to the treatment of SIRS and MODS uses a multidisciplinary approach that emphasizes supportive therapy. Herein, we have presented a futuristic approach that focuses on replacing the function of failed organs using bioartificial technology (Table 1). Bioartificial organ technology may allow the intensivist to provide physiologic organ replacement either as a bridge to transplantation or as a "time-buying" element until native organs that have become acutely dysfunctional or nonfunctional in a variety of clinical settings, can recover their function or regenerate their mass. As bioartificial organ technology matures, it is conceivable as an ultimate goal that non-immunogenic bioartificial organs would be miniaturized or redesigned and acutely placed within the intracorporeal space as replacement organs.

Bioartificial kidney for full renal replacement therapy.

The rapid understanding of the cellular and molecular basis of organ function and disease processes will be translated in the next millennium 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 fields of gene therapy, cell therapy, and tissue engineering. These new technologies 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 in 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 article summarizes the current state of device development for a renal tubule assist device, a bioartificial hemofilter, and a regulatable erythropoietin cell therapy device. These individual devices have the promise to be combined to produce a wearable or implantable bioartificial kidney for full renal replacement therapy. These new approaches may result in therapeutic modalities that significantly diminish the morbidity and mortality in patients with acute renal failure or end-stage renal disease.

Design engineering of a bioartificial renal tubule cell therapy device.

The use of a bioartificial renal tubule device composed of renal proximal tubule cells grown within a hollow fiber cartridge is a first step in engineering a bioartificial kidney to provide more complete replacement therapy of renal function than is available today. In this study, the feasibility of two designs for a tubule device were investigated: one with cells grown on microcarrier beads densely packed within the extracapillary space of a hollow fiber cartridge, and the other with cells grown as a confluent monolayer within the hollow fibers themselves. First, the oxygen requirements of porcine renal proximal tubule cells were determined, both attached to microcarriers and in suspension and compared to that of proximal tubule segments. The basal rate of cell respiration was found to be 2.29 +/- 0.53 nmol O2/10(6) cells/min for our cultured proximal tubule cells in suspension and no significant difference was seen with attached cells. Proximal tubule segments displayed significantly higher respiratory rates. Cells were also found to be responsive in the presence of mitochondrial inhibitors or uncouplers, and their respiratory rates remained constant, despite multiple passaging. The resultant cell oxygen consumption parameter was used in models describing oxygen concentration profiles within the two device configurations. From these models, it was found that cells within our proposed device designs could theoretically be sustained and remain viable, with respect to oxygen limitations. Finally, flow visualization studies were performed to assess fluid flow distribution and determine optimal device configuration and geometry to decrease areas of low or stagnant flow.

Tissue engineering of a bioartificial renal tubule assist device: in vitro transport and metabolic characteristics.

BACKGROUND: Current renal substitution therapy for acute or chronic renal failure with hemodialysis or hemofiltration is life sustaining, but continues to have unacceptably high morbidity and mortality rates. This therapy is not complete renal replacement therapy because it does not provide active transport nor metabolic and endocrinologic functions of the kidney, which are located predominantly in the tubular elements of the kidney. METHODS: To optimize renal substitution therapy, a bioartificial renal tubule assist device (RAD) was developed and tested in vitro for a variety of differentiated tubular functions. High-flux hollow-fiber hemofiltration cartridges with membrane surface areas of 97 cm2 or 0. 4 m2 were used as tubular scaffolds. Porcine renal proximal tubule cells were seeded into the intraluminal spaces of the hollow fibers, which were pretreated with a synthetic extracellular matrix protein. Attached cells were expanded in the cartridge as a bioreactor system to produce confluent monolayers containing up to 1.5 x 109 cells (3. 5 x 105 cells/cm2). Near confluency was achieved along the entire membrane surface, with recovery rates for perfused inulin exceeding 97 and 95% in the smaller and larger units, respectively, compared with less than 60% recovery in noncell units. RESULTS: A single-pass perfusion system was used to assess transport characteristics of the RADs. Vectorial fluid transport from intraluminal space to antiluminal space was demonstrated and was significantly increased with the addition of albumin to the antiluminal side and inhibited by the addition of ouabain, a specific inhibitor of Na+,K+-ATPase. Other transport activities were also observed in these devices and included active bicarbonate transport, which was decreased with acetazolamide, a carbonic anhydrase inhibitor, active glucose transport, which was suppressed with phlorizin, a specific inhibitor of the sodium-dependent glucose transporters, and para-aminohippurate (PAH) secretion, which was diminished with the anion transport inhibitor probenecid. A variety of differentiated metabolic functions was also demonstrated in the RAD. Intraluminal glutathione breakdown and its constituent amino acid uptake were suppressed with the irreversible inhibitor of gamma-glutamyl transpeptidase acivicin; ammonia production was present and incremented with declines in perfusion pH. Finally, endocrinological activity with conversion of 25-hydroxy(OH)-vitamin D3 to 1,25-(OH)2 vitD3 was demonstrated in the RAD. This conversion activity was up-regulated with parathyroid hormone and down-regulated with increasing inorganic phosphate levels, which are well-defined physiological regulators of this process in vivo. CONCLUSIONS: These results clearly demonstrate the successful tissue engineering of a bioartificial RAD that possesses critical differentiated transport, and improves metabolic and endocrinological functions of the kidney. This device, when placed in series with conventional hemofiltration therapy, may provide incremental renal replacement support and potentially may decrease the high morbidity and mortality rates observed in patients with renal failure.

Replacement of renal function in uremic animals with a tissue-engineered kidney.

Current renal substitution therapy with hemodialysis or hemofiltration has been the only successful long-term ex vivo organ substitution therapy to date. Although this approach is life sustaining, it is still unacceptably suboptimal with poor clinical outcomes of patients with either chronic end-stage renal disease or acute renal failure. This current therapy utilizes synthetic membranes to substitute for the small solute clearance function of the renal glomerulus but does not replace the transport, metabolic, and endocrinologic functions of the tubular cells. The addition of tubule cell replacement therapy in a tissue-engineered bioartificial kidney comprising both biologic and synthetic components will likely optimize renal replacement to improve clinical outcomes. This report demonstrates that the combination of a synthetic hemofiltration device and a renal tubule cell therapy device containing porcine renal tubule cells in an extracorporeal perfusion circuit successfully replaces filtration, transport, metabolic, and endocrinologic functions of the kidney in acutely uremic dogs.

Limiting acute renal failure.

Although the cause may be inadequate blood flow or a toxic insult to the renal tubules--or a combination thereof--iatrogenesis often underlies acute renal failure. Awareness of the procedures and treatments most likely to precipitate renal failure can help physicians to prevent the condition, or at least to detect it at the earliest opportunity.

Insights into ototoxicity. Analogies to nephrotoxicity.

The most common ototoxic compounds in clinical practice are aminoglycoside antibiotics, cisplatin, and loop diuretics (ethacrynic acid and furosemide). These agents also have substantial renal effects in the form of nephrotoxicity or diuresis. The understanding of these renal effects may provide insight into ototoxic mechanisms. For aminoglycosides, the renal proximal tubule cell is susceptible due to high concentration achieved and slow clearance with direct effects on phosphoinositide binding and mitochondrial bioenergetics. Pathogenesis appears to involve iron-induced free-radical formation, since iron chelators prevent nephrotoxicity. Analogous effects of aminoglycosides on the inner and outer hair cells have been observed. Cisplatin is also highly concentrated in the proximal tubule cell. Less is known about the direct toxic effects of this agent on renal cells. Insights into mechanisms or renal tubule cells could be directly relevant to the inner ear. The loop diuretics are direct inhibitors of the Na(+)-K(+)-2Cl- cotransport system, which also exists in the marginal and dark cells of the stria vascularis, which are responsible for endolymph secretion. The ototoxicity of these agents may be indirect, due to changes in ionic composition and fluid volume within the endolymph.

Tissue engineering of a bioartificial renal tubule.

Development of a bioartificial renal tubule with a confluent monolayer of renal epithelial cells supported on a permeable synthetic surface may be the first step to further optimization of renal substitution therapy currently used with hemodialysis or hemofiltration. Madin-Darby canine kidney cells, a permanent renal epithelial cell line, were seeded into the lumen of single hollow fibers. Functional confluence of the cells was demonstrated by the recovery of intraluminally perfused 14C-inulin that averaged >98.9% in the cell lined units vs <7.4% in the control noncell hollow fibers during identical pressure and flow conditions. The baseline absolute fluid transport rate averaged 1.4+/-0.4 microl/30 min. To test the dependency of fluid flux with oncotic and osmotic pressure differences across the bioartificial tubule, albumin was added to the extracapillary space, followed by the addition of ouabain, an inhibitor of Na+K+ adenosine triphosphatase, the enzyme responsible for active transport across the renal epithelium. Addition of albumin resulted in a significant increase in volume transport to 4.5+/-1.0 microl/30 min. Addition of ouabain inhibited transport back to baseline levels of 2.1+/-0.4 microl/30 min. These results are the first demonstration that renal epithelial cells have been grown successfully as a confluent monolayer along a hollow fiber, and exhibit functional transport capabilities. The next steps in constructing a bioartificial renal tubule successfully are to develop a multi-fiber bioreactor with primary renal proximal tubule cells that maintain not only transport properties but also differentiated metabolic and endocrine functions, including glucose and ammonia production, and the conversion of vitamin D3 to a more active derivative. A renal tubule device may add critical renal functional components not currently substituted for, thereby improving the treatment regimens for patients with acute and chronic renal failure.

Cell therapy in kidney failure.

Current therapy for acute renal failure continues to have an exceedingly high mortality rate, exceeding 50% even with dialytic or hemofiltrative support. Current renal replacement therapy in ARF only substitutes for filtration function of the kidney but not its cellular metabolic functions. Replacing these metabolic functions may optimize current therapy for this devastating disease process. In this regard, a renal tubule assist device (RAD) has been developed to be placed in an extracorporeal continuous hemoperfusion circuit in series with a hemofilter. The RAD consists of porcine renal proximal tubule cells grown as confluent monolayers in a multifiber bioreactor with a membrane surface area from 0.4 to 1.6 m2. The cells along the inner surface of the hollow fibers are immunoprotected from the patient's blood by the hollow fiber membrane. In vitro experiments demonstrate that this device possesses differentiated renal transport, metabolic and endocrinologic properties. These properties, in fact, are responsive to normal physiological regulatory parameters. In preliminary experiments in uremic dogs, this device has also been shown to tolerate a uremic environment while providing reabsorptive, metabolic, and endocrinologic activity. Pilot human trials of the RAD are anticipated within the next year to improve current renal replacement therapy in this devastating disease process.

Acute renal failure: growth factors, cell therapy, and gene therapy.

The rapid understanding of the cellular and molecular basis of organ function and disease will be translated during the next several decades into new therapeutic approaches to a wide range of clinical disorders, including acute renal failure (ARF). The development of the biotechnology for recombinant genetic engineering has led to the prospect of using purified protein products for therapy. In this regard, the repair of ischemic and toxic ARF is critically dependent on a redundant, interactive cytokine network of growth factors to return kidney function to near normal baseline function. Recombinant growth factors are being tested both experimentally and clinically to accelerate the repair of kidney tissue in this disorder. A newer strategy in biotechnology is the development of cell therapy derivatives. Cell therapy is based on the ability to expand specific cells in tissue culture to perform differentiated tasks and to introduce these cells into the patient either in extracorporeal circuits or as implants as drug delivery vehicles of a single protein or to provide physiological functions. Cell therapy devices are being developed to replace components of renal function that are lost during ARF and chronic renal failure and are not replaced with current hemodialysis or hemofiltration. These new approaches may result in therapeutic modalities that diminish the degree of renal failure and the time needed to recover renal function in acute tubular necrosis. This article examines the future prospects of these developing therapies in the treatment of ARF.