Twenty-six million Americans have chronic kidney disease with progression of the disease leading to kidney failure and the need for a kidney transplant to sustain life.21
Approximately one-third of pediatric kidney-related illnesses are due to disruption of normal kidney development, resulting in abnormalities such as aplasia, dysplasia, and obstructive uropathy.22
Current treatments such as dialysis offer temporary solutions for patients as they wait for a replacement organ; however, sustainable treatments are necessary to effectively address the problem of transplant organ supply and rejection.
Approaches to kidney tissue engineering have investigated the use of renal progenitors and precursors in three-dimensional niches to generate specific renal structures, such as renal tubules and glomeruli.23,24
The complex interactions that occur during kidney development provide both framework and recipe for engineering renal tissue. The formation of the definitive kidney (metanephros) is the result of tightly regulated and highly organized reciprocal molecular interactions between the metanephric blastema (future excretory component) and the ureteric bud (future collecting system). Signals from the blastema induce branching morphogenesis of the ureteric bud and drive mesenchymal cells surrounding the bud tips to form renal vesicles that transition into epithelial comma-shaped and then S-shaped bodies to eventually become the glomeruli. Endothelial cells infiltrate the epithelium during the S-shaped stage to form the capillaries of the glomerular tuft and lead to the maturation of the glomerular basement membrane, which is composed of ECM components, including laminin, collagen IV, entactin/nidogen, proteoglycans, and fibronectin.25–27
During development of the definitive kidney, ECM is a morphogenic modulator responsible for mediating cellular organization, regulating signal transduction pathways, and controlling cell growth and proliferation. The composition, expression pattern, and concentration of ECM macromolecules are specific to a given developmental time frame. Well-orchestrated interactions with other macromolecules and cells present during this stage of development facilitate normal nephrogenesis.28
Recapitulation of kidney ontogeny by reproducing the developmental niche with the appropriate ECM and cell populations will provide the best opportunity for the same interactions to occur in vitro
. Therefore, this study investigated the use of nature's scaffold, decellularized kidney sections, to serve as a biologically active blueprint and modulator of renal recellularization and tissue engineering. Unlike most reports of applied decellularized tissues, age-related differences in decellularization were examined. The current study has demonstrated that decellularized rhesus monkey kidneys of all ages possess well-preserved histoarchitecture and functional ECM proteins, and that fetal kidneys as tested maintain the ability to support cell attachment and migration.
The goal of this study was to develop a decellularized kidney scaffold conducive to cellular repopulation for renal tissue engineering and determine whether there are unique differences depending on age. Because this is the first study to examine decellularization of nonhuman primate kidney tissue sections, optimization of decellularization treatment was required and extensive characterization of the scaffold's physical and biomechanical properties was needed to provide insight for scaffold recellularization. Detergents such as Triton X-100 and SDS are commonly used to decellularize tissues. There is still debate over which agent achieves optimal decellularization. Results from this study demonstrated that SDS was the most effective for decellularization of kidney sections. Triton X-100 was unable to completely decellularize the tissues and caused greater disruption of the basement membrane and connective tissue ECM. A study by Cartmell and Dunn29
reported greater cell removal by SDS compared to Triton X-100 and showed that rat tendon treated with Triton X-100 resulted in the disruption of tendon collagen structure, whereas disruption was not seen in SDS-treated tendon.29
In contrast, Woods and Gratzer30
reported increased collagen denaturation of SDS-treated ligament and decreased glycosaminoglycan content. However, immunohistochemical analysis revealed only SDS treatment resulted in complete removal of the intracellular cytoskeletal protein, vimentin, compared with Triton X-100 or the organic solvent, tributyl phosphate. These studies suggest decellularization agent's effectiveness and destabilization of ECM are tissue specific. To ensure selection of optimal decellularization treatment for kidney sections, four treatment conditions were evaluated and a solution of 1% SDS at 4°C was most effective based on the following criteria: (1) minimal changes in tissue volume, (2) complete removal of cellular nuclei, and (3) preservation of functional ECM epitopes. With this treatment anuclear kidney scaffolds were produced that enabled moderate infiltration of cells from a fresh tissue explant. To our knowledge, this is the first study to examine decellularization of kidney tissue sections and in a clinically relevant nonhuman primate model.31–33
The close phylogenetic relationship and shared developmental and anatomical features of this model provide translational relevance and unique insights into future applications for regenerative medicine.
Fetal kidneys from the late second trimester are in the process of undergoing active nephrogenesis, during which time the constant remodeling of the developing kidney and presence of primitive cell types may potentiate differential susceptibility of tissue to SDS compared to more mature tissues. During nephrogenesis, induction of the metanephric mesenchyme is followed by the loss of the ECM proteins, collagen types I and III, and fibronectin, and the formation of cell aggregates and mesenchyme condensates.34
Removal of these cells and condensates with SDS treatment may leave large spaces devoid of ECM in the nephrogenic zone, resulting in collapse of the scaffold and prevention of further decellularization of the innermost cells. As the size of the nephrogenic zone decreases with increasing postnatal age, the condensing mesenchyme is replaced with mature renal structures and ECM. In older tissues, the microstructure of the scaffold experiences minimal collapse, which likely is responsible for allowing diffusion of SDS into the tissue for faster decellularization and may account for the observed increase in decellularization rate with advancing age. The current study has demonstrated age-dependent decellularization behavior of kidney sections, suggesting that critical differences in cell and ECM composition are important considerations for age-specific renal tissue engineering. These findings will aid in understanding potential age-related differences when scaffolds of different ages are decellularized and recellularized.
Biomechanical testing of engineered scaffolds and cellularized constructs is a common assessment of preservation of functional integrity and scaffold characterization.35
Unfortunately, decellularization of tissues has been associated with changes in biomechanical properties. Increased tensile stiffness was noted in decellularized ligament36
and a decrease in mechanical strength (burst pressure) for esophagus acellular matrix tissue.37
Ott et al.9
reported higher tangential modulus of decellularized heart compared to cadaveric rat ventricles; however, this group was able to successfully recellularize the matrix, suggesting that slight changes in biomechanical properties do not necessarily hinder cellular repopulation. In the study described herein, the compressive modulus of decellularized juvenile and adult kidneys decreased when compared to fresh tissue. Although biomechanical integrity is not a primary measure of functional ability for decellularized kidney scaffolds compared to engineered load bearing tissues, alteration of biomechanical properties of decellularized kidney may influence behavior of cells during repopulation; future biomechanical tests will need to be conducted after scaffold recellularization to assess how changes in the mechanical properties impact cellular repopulation.
To establish the feasibility of using a layered explant/scaffold culture for recellularization, decellularized fetal kidney scaffolds were cultured with age-matched fresh kidney explants from unrelated donors. Studies in which cell populations were seeded on SDS-decellularized tissues have demonstrated variable success in cellular repopulation. Reluctance of cells to infiltrate SDS-treated tissue was observed by Brown et al.38
as well as studies published by Cartmell and Dunn39
for patellar tendon. In contrast, Ott et al.9
demonstrated successful recellularization of SDS-decellularized rat hearts with cardiomyocytes. These studies suggest that SDS decellularization does not necessarily prevent recellularization and is likely dependent on tissue type, the decellularization protocol, and cells used for repopulation. Histological analysis of the layered explant/scaffolds in the study described here revealed that the scaffolds were capable of supporting cell attachment and migration. Select locations in which developing cortical renal structures, such as ureteric buds and renal vesicles, were in direct contact with the scaffold showed migration of Pax2-positive cells with two distinct phenotypes. The first phenotype of dispersed, individual cells double-positive for vimentin and Pax2 is likely of mesenchymal origin. The second phenotype of cell clusters triple-positive for vimentin, cytokeratin, and Pax2 may originate from the ureteric bud which has been shown to coexpress vimentin and Pax2 under some circumstances.20
Further studies are required to fully characterize the different cell population(s) that migrate into the scaffold and how these findings will relate to future kidney regeneration strategies. These results demonstrate the intrinsic capacity of decellularized kidney sections to potentiate cell–cell and cell–ECM interactions to facilitate cell-specific infiltration and begin to establish a catalog of cells that do well in decellularized tissue of a given age. These findings also establish a basis for renal recellularization and may provide a paradigm shift in how decellularized tissues of different ages are recellularized.
Taken together, these data establish foundations for in vitro
renal tissue engineering with decellularized kidney scaffolds, begin to address the impact of age in both decellularization and recellularization, and form the basis for methods that may be necessary for eventual in vivo
transplantation for patients in different age groups. More extensive recellularization experiments with a range of cell populations will be necessary to assess the full potential of these scaffolds and understand the usefulness of different cell lineages for recellularization strategies. In addition, future investigations will need to focus on the potential retention of major histocompatibility complex (MHC) class I and II antigens and other cell debris that may have the potential to elicit an immune response from the host. Findings from this study serve as initial steps toward the development of future engineered renal constructs and will set the stage for preclinical studies in nonhuman primates.18,32