Engineering a Bioartificial Kidney Utilizing a Decellularized Matrix

Christoph Neyer, Larry Liu, Regine Labog, Seyed Bozorgi Introduction 26 million Americans have chronic kidney disease with progression of disease leading to kidney failure need for transplant to sustain life, and the number of new patients with kidney failure has averaged more than 90,000 annually [1]. Chronic kidney disease is a progressive loss of renal function which may cause: blood wastes to build to high levels and develop complications like high blood pressure, anemia (low blood count), weak bones, poor nutritional health and nerve damage [1]. Chronic kidney disease (CDK) may be caused by diabetes, high blood pressure and other disorders [1]. Current treatments for kidney's failure are dialysis and transplantation. Dialysis does not cure kidney disease, costs a lot and patients will need to have dialysis treatments for their whole life unless they are able to get a kidney transplant. Kidney transplants may come from living donors or a cadaver, and require tissue typing and blood type compatibility to reduce the risk of immune rejection of the transplanted kidney. Furthermore, the treatment requires patients to wait for a match and take anti- rejection medications, which are expensive and cause more susceptibility to diseases. To reduce the risk of post- transplantation immune rejection and provide patients relief from waiting for a tissue match, bone marrow stem cells, constructing a bioartificial kidney with the patient's own renal cells will be a potential treatment.

A paper was published in Nature Medicine magazine that claimed to obtain a functional bioartificial heart by recellularization of a decellularized cadaveric heart [3]. Although the experiment was conducted on murine and primarily on heart, it was claimed that the concept of the experiment may be applied to a human heart and other organs. Thus by mimicking the procedure of decellularizing a cadaveric kidney with no recorded medical history of dysfunction and recellularizing the obtained extra-cellular matrix (ECM) with the patient's own cells, a functional bioartificial kidney may be obtained. Background on human kidney physiology Kidneys regulate body fluid volume and composition. As an endocrine organ, kidneys synthesize renin to regulate blood pressure, erythropoietin (the main factor for red blood formation in bone marrow), and active vitamin D (enhances calcium absorption). Nephrons, as the functional unit of kidneys, filtrate through glomerular capillaries, reabsorb mostly through proximal tubule and excrete the body fluid through collecting duct. Design Ideas and Methods Decellularization of the Kidney Once a donor kidney has been obtained the kidney will then be decellularized using detergents. This can be accomplished by first perfusing the matrix with sodium doceyl sulfate (SDS), an ionic detergent commonly used in decellularization that acts as a surfactant, lysing the cells. The matrix is then perfused with Triton X-100, a non-ionic detergent. This process has been shown to remove over 96% of all DNA traces and leaves no detectable SDS residue. In the past intact

nuclei were detected using a DAPI stain and none were found [3]. Cell Source The next step in the process is to obtain kidney cells to seed on the matrix. Bone marrow-derived stem cells (BMSCs) have been reported to differentiate into renal cells, specifically mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney [8]. Mesangial cells line the vessels of the kidney, podocytes are found in the Bowman's capsule, and tubular cells are present in the renal tubule, which connects the Bowman's capsule and the collecting duct. Using dual-wavelength flow cytometric analysis [9], which is based on the differential ability of cells to efflux Hoechst 33342 (a fluorescent DNA-binding dye), enriched populations of BMSCs can be obtained from adult murine bone marrow. Differentiation of the BMSCs into mesangial cells is accomplished by seeding the BMSCs on a collagen I matrix. Nonadherent cells were then transferred to a type IV collagen matrix and subjected to a differentiation medium containing retinoic acid and platelet-derived growth factor-BB (PDGF-BB) for 24h [8]. Some of the resultant cells changed to a stellate shape and expressed Thy1 and desmin, characteristic of mesangial cells. Reseeding of the Decellularized kidney Our unique approach is in the sequential perfusion of these different cell types into the decellularized kidney matrix. First, tubular cells will be perfused in a retrograde fashion through the collecting duct of the kidney. This will result in tubular cells lining the collecting duct and renal tubule. Podocytes will then be perfused through the blood vessel system of the decellularized kidney, using an oxygenated

nutrient medium. The idea is that these cells will localize to the Bowman's capsule because of it unique matrix composition. Next, mesangial cells will be added to the perfusion solution. These cells will begin to line the blood vessels present in the kidney. Lastly, endothelial cells will be added to line the interior of the blood vessels. The kidney construct will then be cultured in a bioreactor designed to mimic renal physiology. Culturing of Bioartificial Kidney in Bioreactor While the exact design of the bioreactor is beyond the scope of this project, the bioreactor will need to perform a few key functions. First, it must supply oxygen and nutrients to the cells in the kidney. Second, it must provide an artificial outlet for waste products removed by the kidney. Lastly, it can recycle the isolated waste products back into the blood-like fluid being pumped through the blood vessels of the kidney to supply the kidney with waste to remove. This set up also allows for the easy measurement of waste removal efficiency. Once the kidney exhibits physiological waste removal capabilities, it can be transplanted into the patient. The perfusion medium will contain nephrogenic growth factors such as a combination of retinoic acid, Activin-A, and Bmp7 in order to promote kidney growth and function [5]. The time frame for culturing the bioartificial kidney within the bioreactor is unknown without actually doing the experiment, but most likely after a period of weeks to months the efficacy of the efficacy of the kidney construct could be tested by adding waste to the blood-like solution being pumped through the construct and measuring the clearance. If the construct is able to provide sufficient clearance, it can be

implanted into the patient and the patients blood will be monitored for waste levels. Product Characterization and Validation In Vitro

To characterize the contribution of bone marrow cells to the turnover rate of renal cells, male BMSCs can be transplanted into a female murine kidney and tested for Y-chromosome kidney cells. In Vivo

The BMSCs were immunostained with CD34, CD133, c-Kit, and GATA-4 to make sure they were bone marrow derived stem cells. After the BMSCs are differentiated in a tissue culture, the renal cells can be isolated using immunoselection with monoclonal antibodies (mABs) such as ST.12, which intercalated and principal cells in the renal collecting duct react to or by conjugating renal cells with cell markers and separating with FACS [12]. Cells with slow cycling time can be distinguished by retention of a nucleotide label such as bromodeoxyuridine (BrdU), which is incorporated into the DNA of cells during DNA synthesis [7]. If, after administration of a pulse of BrdU the cells are monitored for long periods of chase, only the slowly cycling cells retain a concentration of label sufficiently high to allow their staining and thus adult organspecific stem cells are often called labelretaining cells Flow cytometry of the acutely dispersed cells from the papillae of 8 rats revealed that 3% of the cells were positive for CD45, indicating that a small fraction of the isolated cells were blood cells. However, less than 0.5% of the total cells were positive for CD34, CD44, or c-Kit, indicating that if hematopoietic stem cells or bone marrow mesenchymal stem cells were isolated with the renal papillary cells, their contribution to the population of the BrdUretaining cells (about 40% of the total cells) was small

To assess kidney function, immunofluorescence will be used to determine whether the decellularized kidney is composed of differentiated renal cells. Glomerular flow rate in and out of the kidney within the bioreactor can be measured by injecting inulin into the bioreactor. Inulin is neither reabsorbed or secreted by the kidney after glomerular filtration so its rate of excretion is directly proportional to the rate of filtration of water and solutes across the glomerular filter. The normal kidney functions with a glomerural flow rate of 90mL/min/1.73m2. Renal Blood Flow tests could also be done by determining the level of creatinine, BUA (Blood Uric Acid), and BUN (Blood Urea Nitrogen) in the blood, which is normally above levels during kidney disfunction. Discussion Key Techniques and Improvements The most important technique introduced in our project is the sequential perfusion of the different cells types. Perfusion is done by pumping medium through the decellularized blood vessels of the kidney or the collecting ducts. The importance of this method is that it controls where cells end up, but eliminates the need for a tedious manual seeding process. Since the matrix content and geometry is preserved during decellularization, the differentiated cells are likely to localize to

the correct locations within the kidney construct and sequential perfusion further controls where the cells end up. If all the cells were perfused at the same time, the endothelial cells, which will tend to line the interior of the blood vessels, will prevent the other cells from migrating further into the kidney matrix. Feasibility The feasibility of this project can be separated into three parts. First, it is definitely feasible to decellularize the kidney, as the decellularization of the murine heart was successfully conducted recently in literature. Second, it is also feasible to generate the renal cells from bone marrow-derived stem cells (BM-dSC) and other forms of stem cells. However, the part that's not quite feasible is the perfusion of the bioartificial kidney because there might be various variables not known and parameters not quantified. Alternatives BMSCs were chosen because of the large number of cells that will be required to reseed the kidney matrix, there are not likely to be enough native renal papillary cells available in the patient, especially if kidney function is compromised. Advantages and Drawbacks The advantage of a bioartificial kidney is that it only takes the decellularized matrix from a donor kidney. Since the kidney does not need to be functional, it can come from a live or cadaveric human donor. An allogeneic donor is preferred to in order to avoid immunogenic matrix components. Allogeneic kidneys are also most similar in size and shape to the failing kidney, which improves the ability of the host to accept and integrate the bioartificial kidney. If no

allogeneic donor is available, a xenogenic kidney could theoretically be used. However, this brings with it issues of immunogenic matrix components. There are some important drawbacks to consider in this case. First, the decellularized kidney with implanted stem cells need to be cultured in a bioreactor. However, such a bioreactor that can grow an entire kidney has not yet been invented and its parameters are complicated. Second, during the kidney growth inside the bioreactor, it will be difficult to observe and monitor cell growth/allocation, taking into the account that there are going to be different cell types and layers interacting at same time. The third drawback as well as a major challenge is the time that's required to complete the entire process, which includes finding the best kidney match, decellularize the selected kidney, implant the stem cells, grow kidney inside bioreactor, implantation and potential post-implantation treatments. Works Cited 1. "Chronic Kidney Disease (CKD)." National Kidney Foundation. Web. 20 Apr. 2010. <http://www.kidney.org/kidneydisease/ckd/i ndex.cfm>. 2. Takahashi, K., and S. Yamanaka. "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors." Cell 126.4 (2006): 652-55. PubMed. Web. 2 May 2010. 3. Ott, HC. "Perfusion-decellularized Matrix: Using Nature's Platform to Engineer a Bioartificial Heart." Nature Medicine 14.2 (2008): 213-21. Web. 4. Peiffer, Isabelle. "Use of Xenofree Matrices and Molecularly-Defined Media to Control Human Embryonic Stem Cell

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