An arteriovenous mock circulatory loop and accompanying bond graph model for in vitro study of peripheral intravascular bioartificial organs.

BACKGROUND: Silicon nanopore membrane-based implantable bioartificial organs are dependent on arteriovenous implantation of a mechanically robust and biocompatible hemofilter. The hemofilter acts as a low-resistance, high-flow network, with blood flow physiology similar to arteriovenous shunts commonly created for hemodialysis access. A mock circulatory loop (MCL) that mimics shunt physiology is an essential tool for refinement and durability testing of arteriovenous implantable bioartificial organs and silicon blood-interfacing membranes. We sought to develop a compact and cost-effective MCL to replicate flow conditions through an arteriovenous shunt and used data from the MCL and swine to inform a bond graph mathematical model of the physical setup. METHODS: Flow physiology through bioartificial organ prototypes was obtained in the MCL and during extracorporeal attachment to swine for biologic comparison. The MCL was tested for stability overtime by measuring pressurewave variability over a 48-h period. Data obtained in vitro and extracorporeally informed creation of a bond graph model of the MCL. RESULTS: The arteriovenous MCL was a cost-effective, portable system that reproduced flow rates and pressures consistent with a pulsatile arteriovenous shunt as measured in swine. MCL performance was stable over prolonged use, providing a cost-effective simulator for enhanced testing of peripherally implanted bioartificial organ prototypes. The corresponding bond graph model recapitulates MCL and animal physiology, offering a tool for further refinement of the MCL system.

Patient Preference Trade-offs for Next-Generation Kidney Replacement Therapies.

BACKGROUND: Next-generation implantable and wearable KRTs may revolutionize the lives of patients undergoing dialysis by providing more frequent and/or prolonged therapy along with greater mobility compared with in-center hemodialysis. Medical device innovators would benefit from patient input to inform product design and development. Our objective was to determine key risk/benefit considerations for patients with kidney failure and test how these trade-offs could drive patient treatment choices. METHODS: We developed a choice-based conjoint discrete choice instrument and surveyed 498 patients with kidney failure. The choice-based conjoint instrument consisted of nine attributes of risk and benefit pertinent across KRT modalities. Attributes were derived from literature reviews, patient/clinician interviews, and pilot testing. The risk attributes were serious infection, death within 5 years, permanent device failure, surgical requirements, and follow-up requirements. The benefit attributes were fewer diet restrictions, improved mobility, pill burden, and fatigue. We created a random, full-profile, balanced overlap design with 14 choice pairs plus five fixed tasks to test validity. We used a mixed-effects regression model with attribute levels as independent predictor variables and choice decisions as dependent variables. RESULTS: All variables were significantly important to patient choice preferences, except follow-up requirements. For each 1% higher risk of death within 5 years, preference utility was lower by 2.22 ( ß =-2.22; 95% confidence interval [CI], -2.52 to -1.91), while for each 1% higher risk of serious infection, utility was lower by 1.38 ( ß =-1.46; 95% CI, -1.77 to -1.00) according to comparisons of the ß coefficients. Patients were willing to trade a 1% infection risk and 0.5% risk of death to gain complete mobility and freedom from in-center hemodialysis ( ß =1.46; 95% CI, 1.27 to 1.64). CONCLUSIONS: Despite an aversion to even a 1% higher risk of death within 5 years, serious infection, and permanent device rejection, patients with kidney failure suggested that they would trade these risks for the benefit of complete mobility.

Renal Embolization-Induced Uremic Swine Model for Assessment of Next-Generation Implantable Hemodialyzers.

Reliable models of renal failure in large animals are critical to the successful translation of the next generation of renal replacement therapies (RRT) into humans. While models exist for the induction of renal failure, none are optimized for the implantation of devices to the retroperitoneal vasculature. We successfully piloted an embolization-to-implantation protocol enabling the first implant of a silicon nanopore membrane hemodialyzer (SNMHD) in a swine renal failure model. Renal arterial embolization is a non-invasive approach to near-total nephrectomy that preserves retroperitoneal anatomy for device implants. Silicon nanopore membranes (SNM) are efficient blood-compatible membranes that enable novel approaches to RRT. Yucatan minipigs underwent staged bilateral renal arterial embolization to induce renal failure, managed by intermittent hemodialysis. A small-scale arteriovenous SNMHD prototype was implanted into the retroperitoneum. Dialysate catheters were tunneled externally for connection to a dialysate recirculation pump. SNMHD clearance was determined by intermittent sampling of recirculating dialysate. Creatinine and urea clearance through the SNMHD were 76-105 mL/min/m2 and 140-165 mL/min/m2, respectively, without albumin leakage. Normalized creatinine and urea clearance measured in the SNMHD may translate to a fully implantable clinical-scale device. This pilot study establishes a path toward therapeutic testing of the clinical-scale SNMHD and other implantable RRT devices.

Feasibility of an implantable bioreactor for renal cell therapy using silicon nanopore membranes.

The definitive treatment for end-stage renal disease is kidney transplantation, which remains limited by organ availability and post-transplant complications. Alternatively, an implantable bioartificial kidney could address both problems while enhancing the quality and length of patient life. An implantable bioartificial kidney requires a bioreactor containing renal cells to replicate key native cell functions, such as water and solute reabsorption, and metabolic and endocrinologic functions. Here, we report a proof-of-concept implantable bioreactor containing silicon nanopore membranes to offer a level of immunoprotection to human renal epithelial cells. After implantation into pigs without systemic anticoagulation or immunosuppression therapy for 7 days, we show that cells maintain >90% viability and functionality, with normal or elevated transporter gene expression and vitamin D activation. Despite implantation into a xenograft model, we find that cells exhibit minimal damage, and recipient cytokine levels are not suggestive of hyperacute rejection. These initial data confirm the potential feasibility of an implantable bioreactor for renal cell therapy utilizing silicon nanopore membranes.

Characterization of human islet function in a convection-driven intravascular bioartificial pancreas.

Clinical islet transplantation for treatment of type 1 diabetes (T1D) is limited by the shortage of pancreas donors and need for lifelong immunosuppressive therapy. A convection-driven intravascular bioartificial pancreas (iBAP) based on highly permeable, yet immunologically protective, silicon nanopore membranes (SNM) holds promise to sustain islet function without the need for immunosuppressants. Here, we investigate short-term functionality of encapsulated human islets in an iBAP prototype. Using the finite element method (FEM), we calculated predicted oxygen profiles within islet scaffolds at normalized perifusion rates of 14-200 nl/min/IEQ. The modeling showed the need for minimum in vitro and in vivo islet perifusion rates of 28 and 100 nl/min/IEQ, respectively to support metabolic insulin production requirements in the iBAP. In vitro glucose-stimulated insulin secretion (GSIS) profiles revealed a first-phase response time of <15 min and comparable insulin production rates to standard perifusion systems (~10 pg/min/IEQ) for perifusion rates of 100-200 nl/min/IEQ. An intravenous glucose tolerance test (IVGTT), performed at a perifusion rate of 100-170 nl/min/IEQ in a non-diabetic pig, demonstrated a clinically relevant C-peptide production rate (1.0-2.8 pg/min/IEQ) with a response time of <5 min.

Inhibition of Transforming Growth Factor-ß Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-ß (TGF-ß) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-ß receptors possibly responsible for the improved differentiation, evaluate whether TGF-ß inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-ß signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-ß inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-ß signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-ß by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-ß signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Wearable and implantable artificial kidney devices for end-stage kidney disease treatment: Current status and review.

BACKGROUND: Chronic kidney disease (CKD) is a major cause of early death worldwide. By 2030, 14.5 million people will have end-stage kidney disease (ESKD, or CKD stage 5), yet only 5.4 million will receive kidney replacement therapy (KRT) due to economic, social, and political factors. Even for those who are offered KRT by various means of dialysis, the life expectancy remains far too low. OBSERVATION: Researchers from different fields of artificial organs collaborate to overcome the challenges of creating products such as Wearable and/or Implantable Artificial Kidneys capable of providing long-term effective physiologic kidney functions such as removal of uremic toxins, electrolyte homeostasis, and fluid regulation. A focus should be to develop easily accessible, safe, and inexpensive KRT options that enable a good quality of life and will also be available for patients in less-developed regions of the world. CONCLUSIONS: Hence, it is required to discuss some of the limits and burdens of transplantation and different techniques of dialysis, including those performed at home. Furthermore, hurdles must be considered and overcome to develop wearable and implantable artificial kidney devices that can help to improve the quality of life and life expectancy of patients with CKD.

Functionalizing Polyacrylamide Hydrogels for Renal Cell Culture Under Fluid Shear Stress.

A functional renal tubule bioreactor needs to reproduce the reabsorption and barrier functions of the renal tubule. Our prior work has demonstrated that primary human renal tubule cells respond favorably when cultured on substrates with elasticity similar to healthy tissue and when subjected to fluid shear stress. Polyacrylamide (PA) is widely used in industrial processes such as water purification because it is electrically neutral and chemically inert. PA is a versatile tool as the concentration and mechanical properties of the gel are easily adjusted by varying the proportions of monomer and crosslinker. Control of mechanical properties is attractive for preparing cell culture substrates with tunable stiffness, but PA's inert chemical properties require additional steps to prepare PA for cell attachment, such as chemical reactions to bind extracellular matrix proteins. Methods based on protein functionalization for cell attachment work well in the short term but fail to provide sufficient attachment to withstand the mechanical traction of fluid shear stress. In our present work, we tested the effects of subjecting primary renal tubule cells to fluid shear stress on an elastic substrate by developing a simple method of incorporating N-(3-Aminopropyl) methacrylamide hydrochloride (APMA) into PA hydrogels. Integration of APMA into the PA hydrogel formed a nondegradable elastic substrate promoting excellent long-term cell attachment despite the forces of fluid shear stress. Impact statement Cell culture on artificial materials requires the presence of ligands on the surface to which extracellular matrix receptors on the cell can bind. Simple nonspecific adsorption or covalent linkage of plasma or extracellular matrix proteins only suffices for short-term static culture. Prolonged culture may result in degradation of the original protein such that linkage is severed but new proteins secreted by the cell are blocked from adsorbing to the artificial scaffold. This results in detachment and loss of cell mass, as well as defects in monolayers. We present a simple technique to integrate amine moeities into a polyacrylamide hydrogel that resist degradation and support long-term culture.

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Superporous agarose scaffolds for encapsulation of adult human islets and human stem-cell-derived ß cells for intravascular bioartificial pancreas applications.

Type 1 diabetic patients with severe hypoglycemia unawareness have benefitted from cellular therapies, such as pancreas or islet transplantation; however, donor shortage and the need for immunosuppression limits widespread clinical application. We previously developed an intravascular bioartificial pancreas (iBAP) using silicon nanopore membranes (SNM) for immunoprotection. To ensure ample nutrient delivery, the iBAP will need a cell scaffold with high hydraulic permeability to provide mechanical support and maintain islet viability and function. Here, we examine the feasibility of superporous agarose (SPA) as a potential cell scaffold in the iBAP. SPA exhibits 66-fold greater hydraulic permeability than the SNM along with a short (<10 µm) diffusion distance to the nearest islet. SPA also supports short-term functionality of both encapsulated human islets and stem-cell-derived enriched ß-clusters in a convection-based system, demonstrated by high viability (>95%) and biphasic insulin responses to dynamic glucose stimulus. These findings suggest that the SPA scaffold will not limit nutrient delivery in a convection-based bioartificial pancreas and merits continued investigation.

Reconsidering Garth Robinson: fluid flow and the glomerular filtration barrier.

PURPOSE OF REVIEW: The goal of this review is to present recent models of the filtration barrier that may suggest mechanism-based treatments for proteinuric renal disease. The vast majority of renal failure occurs in diseases of glomerular proteinuria. The physiology of the filtration barrier remains incompletely understood, preventing invention of mechanism-based therapies. Research is currently dominated by molecular biology approaches to the kidney instead of engineering-based filtration and transport models. RECENT FINDINGS: Reexamination of two older paradigms (basement membrane and slit diaphragm) and critical analysis of newer models may provide mechanistic insight to guide further research. We expand on our theory of podocyte-basement membrane mechanical interactions and speculate on mechanisms of action of the leading treatment for proteinuria, angiotensin blockade. SUMMARY: Treatment of proteinuria remains largely empiric and based on inhibition of the renin-angiotensin-aldosterone system, with additional benefit from statins and vitamin D. Improved definition of transport phenomena in the capillary wall may suggest rational design of new interventions.

Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

INTRODUCTION: Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. METHODS: We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. RESULTS: piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. CONCLUSIONS: Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

Ambulatory Hemodialysis-Technology Landscape and Potential for Patient-Centered Treatment.

CKD is a worldwide health problem and the number of patients requiring kidney replacement therapy is rising. In the United States, most patients with ESKD rely on in-center hemodialysis, which is burdensome and does not provide the same long-term benefits as kidney transplantation. Intensive hemodialysis treatments have demonstrated improved clinical outcomes, but its wider adoption is limited by equipment complexity and patient apprehension. Ambulatory devices for hemodialysis offer the potential for self-care treatment outside the clinical setting as well as frequent and prolonged sessions. This article explains the motivation for ambulatory hemodialysis and provides an overview of the necessary features of key technologies that will be the basis for new wearable and implantable devices. Early work by pioneers of hemodialysis is described followed by recent experience using a wearable unit on patients. Finally, ongoing efforts to develop an implantable device for kidney replacement and its potential for implantable hemodialysis are presented.

Application of physiological shear stress to renal tubular epithelial cells.

Renal tubular epithelial cells are consistently exposed to flow of glomerular filtrate that creates fluid shear stress at the apical cell surface. This biophysical stimulus regulates several critical renal epithelial cell functions, including transport, protein uptake, and barrier function. Defining the in vivo mechanical conditions in the kidney tubule is important for accurately recapitulating these conditions in vitro. Here we provide a summary of the fluid flow conditions in the kidney and how this translates into different levels of fluid shear stress down the length of the nephron. A detailed method is provided for measuring fluid flow in the proximal tubule by intravital microscopy. Devices to mimic in vivo fluid shear stress for in vitro studies are discussed, and we present two methods for culture and analysis of renal tubule epithelial cells exposed physiological levels of fluid shear stress. The first is a microfluidic device that permits application of controlled shear stress to cells cultured on porous membranes. The second is culture of renal tubule cells on an orbital shaker. Each method has advantages and disadvantages that should be considered in the context of the specific experimental objectives.

Reassessment of Exosome Composition.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.