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Engineering Renal

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  Contents 60.1 Introduction ......................... 86960.2 Basic Components .................... 86960.3 Approaches for Renal Tissue Regeneration 87060.3.1 Developmental Approaches ............. 87060.3.2 Tissue Engineering Approaches ......... 87160.4 Regeneration of Functional Tissue In Vivo 87260.5 Summary ........................... 874References .......................... 874 individuals. Although dialysis can prolong survival via ltration, other kidney functions are not replaced, leading to long-term consequences such as anemia and malnutrition [2, 3, 7]. Currently, renal transplan- tation is the only denitive treatment that can restore full kidney function. However, transplantation has several limitations, such as critical donor shortage, complications due to chronic immunosuppressive therapy and graft failure [2, 3, 7].The limitations of current therapies for renal failure have led investigators to explore the devel-opment of alternative therapeutic modalities that could improve, restore, or replace renal function. The emergence of cell-based therapies using tissue engineering and regenerative medicine strategies has  presented alternative possibilities for the manage-ment of pathologic renal conditions [1, 7–13]. The concept of kidney cell expansion followed by cell transplantation using tissue engineering and regen-erative medicine techniques has been proposed as a method to augment either isolated or total renal func-tion. Despite the fact that the kidney is considered to  be one of the more challenging organs to regenerate and/or reconstruct, investigative advances made to date have been promising [8, 10–12]. 60.2  Basic Components The unique structural and cellular heterogeneity  present within the kidney creates many challenges for tissue regeneration. The system of nephrons and 60.1  Introduction The kidney is a vital and complex organ that per-forms many critical functions [1–3]. It is responsible for ltering the body’s wastes, such as urea, from the  blood and excreting them as urine. In addition to the excretory function, the kidney maintains the body’s homeostasis by regulating acid-base balance, blood  pressure, and plasma volume. Moreover, it synthe-sizes 1, 25 vitamin D3, erythropoietin, glutathione, and free radical scavenging enzymes. It is also known that the kidney participates in the catabolism of low molecular weight proteins and in the production and regulation of cytokines [4–6].There are many conditions where the kidney functions are diminished and these lead to renal failure. End stage renal failure is a devastating con-dition which involves multiple organs in affected Regeneration of Renal Tissues  T. Aboushwareb, J. J. Yoo, A. Atala 󰀶󰀰  collecting ducts within the kidney is composed of multiple functionally and morphologically distinct segments. For this reason, appropriate conditions need to be provided for the long-term survival, dif-ferentiation, and growth of many types of cells. Re-cent efforts in the area of kidney tissue regeneration have focused on the development of a reliable cell source [14–19]. In addition, optimal growth condi-tions have been extensively investigated to provide an adequate enrichment to achieve stable renal cell expansion systems [20–24].Isolation of particular cell types that produce spe- cic factors, such as erythropoietin, may be a good approach for selective cell therapies. However, to-tal renal function would not be achieved using this approach. To create kidney tissue that would deliver full renal function, a culture containing all of the cell types comprising the functional nephron units should  be used. Optimal culture conditions to nurture renal cells have been extensively studied and the cells grown under these conditions have been reported to maintain their cellular characteristics [25]. Fur-thermore, renal cells placed in a three-dimensional culture environment are able to reconstitute renal structures.Recent investigative efforts in the search for a re-liable cell source have been expanded to stem and  progenitor cells. The use of these cells for tissue re-generation is attractive due to their ability to differen- tiate and mature into specic cell types required for regeneration. This is particularly useful in instances where primary renal cells are unavailable due to ex-tensive tissue damage. Bone marrow-derived human mesenchymal stem cells have been shown to be a po-tential source because they can differentiate into sev-eral cell lineages [14, 15, 18]. These cells have been shown to participate in kidney development when they are placed in a rat embryonic niche that allows for continued exposure to a repertoire of nephrogenic signals [19]. These cells, however, were found to contribute mainly to regeneration of damaged glom-erular endothelial cells after injury. In addition, the major cell source of kidney regeneration was found to srcinate from intrarenal cells in an ischemic renal injury model [14, 17]. Another potential cell source for kidney regeneration is circulating stem cells, which have been shown to transform into tubular and glomerular epithelial cells, podocytes, mesangial cells, and interstitial cells after renal injury [15, 16, 26–30]. These observations suggest that controlling stem and progenitor cell differentiation may lead to successful regeneration of kidney tissues.Although isolated renal cells are able to retain their phenotypic and functional characteristics in culture, transplantation of these cells in vivo may not result in structural remodeling that is appropriate for kidney tissue. In addition, cell or tissue components cannot be implanted in large volumes as diffusion of oxygen and nutrients is limited [31]. Thus, a cell-support matrix, preferably one that encourages an-giogenesis, is necessary to allow diffusion across the entire implant. A variety of synthetic and naturally derived materials have been examined in order to de-termine the ideal support structures for regeneration [9, 32–35]. Biodegradable synthetic materials, such as poly-lactic and poly-glycolic acid polymers, have  been used to provide structural support for cells. Synthetic materials can be easily fabricated and con- gured in a controlled manner, which make them at -tractive options for tissue engineering. However, nat-urally derived materials, such as collagen, laminin, and bronectin, are much more biocompatible and  provide a similar extracellular matrix environment to normal tissue. For this reason, collagen-based scaf-folds have been used in many applications [36–39]. 60.3  Approaches for Renal Tissue Regeneration 60.3.1  Developmental Approaches Transplantation of a kidney precursor, such as the metanephros, into a diseased kidney has been pro- posed as a possible method for achieving functional restoration. In an animal study, human embryonic metanephroi, transplanted into the kidneys of an im- mune decient mouse model, developed into mature kidneys [40]. The transplanted metanephroi pro- duced urine-like uid but failed to develop ureters. This study suggests that development of an in vitro system in which metanephroi could be grown may lead to transplant techniques that could produce a 870T. Aboushwareb, J. J. Yoo, A. Atala  small replacement kidney within the host. In another study, the metanephros was divided into mesenchy-mal tissue and ureteral buds, and each of the tissue segments was cultured in vitro [41]. After 8 days in culture, each portion of the mesenchymal tissues had grown to the srcinal size. A similar method was used for ureteral buds, which also propagated. These results indicate that if the mesenchyme and ureteral  buds were placed together and cultured in vitro, a metanephros-like structure would develop. This sug-gests that the metanephros could be propagated un-der optimal conditions.In another study, transplantation of metanephroi into a nonimmunosuppressed rat omentum showed that the implanted metanephroi are able to undergo differentiation and growth that is not conned by a tight organ capsule [42]. When the metanephroi with an intact ureteric bud were implanted, the metanephroi were able to enlarge and become kidney-shaped tis-sue within 3 weeks. The metanephroi transplanted into the omentum were able to develop into kidney tissue structures with a well-dened cortex and me -dulla. Mature nephrons and collecting system struc-tures are shown to be indistinguishable from those of normal kidneys by light or electron microscopy [43]. Moreover, these structures become vascularized via arteries that srcinate at the superior mesenteric ar-tery of the host [43]. It has been demonstrated that the metanephroi transplanted into the omentum sur-vive for up to 32 weeks post implantation [44]. These studies show that the developmental approach may  be a viable option for regenerating renal tissue for functional restoration. 60.3.2  Tissue Engineering Approaches The ability to grow and expand cells is one of the essential requirements in engineering tissues. The feasibility of achieving renal cell growth, expansion, and in vivo reconstitution using tissue engineering techniques was investigated [9]. Donor rabbit kid-neys were removed and perfused with a nonoxide solution which promoted iron particle entrapment in the glomeruli. Homogenization of the renal cortex and fractionation in 83 and 210 micron sieves with subsequent magnetic extraction yielded three sepa- rate puried suspensions of distal tubules, glomeruli, and proximal tubules. The cells were plated sepa-rately in vitro and after expansion, were seeded onto  biodegradable polyglycolic acid scaffolds and im- planted subcutaneously into host athymic mice. This included implants of proximal tubular cells, glomer-uli, distal tubular cells, and a mixture of all three cell types. Animals were sacriced at 1 week, 2 weeks, and 1 month after implantation and the retrieved im-  plants were analyzed. An acute inammatory phase and a chronic foreign body reaction were seen, ac-companied by vascular ingrowth by 7 days after im- plantation. Histologic examination demonstrated pro-gressive formation and organization of the nephron segments within the polymer bers with time. Renal cell proliferation in the cell-polymer scaffolds was detected by in vivo labeling of replicating cells with the thymidine analog bromodeoxyuridine [17]. BrdU incorporation into renal cell DNA was conrmed us -ing monoclonal anti-BrdU antibodies. These results demonstrated that renal specic cells can be success -fully harvested and cultured, and can subsequently attach to articial biodegradable polymers. The renal cell-polymer scaffolds can be implanted into host animals where the cells replicate and organize into nephron segments, as the polymer, which serves as a cell delivery vehicle, undergoes biodegradation.Initial experiments showed that implanted cell- polymer scaffolds gave rise to renal tubular struc-tures. However, it was unclear whether the tubular structures reconstituted de novo from dispersed renal elements, or if they merely represented frag-ments of donor tubules which survived the srcinal dissociation and culture processes intact. Further in-vestigation was conducted in order to examine the  process [45]. Mouse renal cells were harvested and expanded in culture. Subsequently, single isolated cells were seeded on biodegradable polymers and implanted into immune competent syngeneic hosts. Renal epithelial cells were observed to reconstitute into tubular structures in vivo. Sequential analyses of the retrieved implants over time demonstrated that renal epithelial cells rst organized into a cord-like structure with a solid center. Subsequent canalization into a hollow tube could be seen by 2 weeks. His- tologic examination with nephron-segment-specic lactins showed successful reconstitution of proxi-mal tubules, distal tubules, loop of Henle, collecting Chapter 60 Regeneration of Renal Tissues871  tubules, and collecting ducts. These results showed that single suspended cells are capable of reconsti-tuting into tubular structures, with homogeneous cell types within each tubule. 60.4  Regeneration of Functional Tissue In Vivo The kidneys are critical to body homeostasis because of their excretory, regulatory, and endocrine func- tions. The excretory function is initiated by ltration of blood at the glomerulus, and the regulatory func-tion is provided by the tubular segments. Although our prior studies demonstrated that renal cells seeded on biodegradable polymer scaffolds are able to form some renal structures in vivo, complete renal function could not be achieved in these studies. In a subsequent study we sought to create a functional articial renal unit which could produce urine [46]. Mouse renal cells were harvested, expanded in cul-ture, and seeded onto a tubular device constructed from polycarbonate [39]. The tubular device was connected at one end to a silastic catheter which ter-minated into a reservoir. The device was implanted subcutaneously in athymic mice. The implanted devices were retrieved and examined histologically and immunocytochemically at 1, 2, 3, 4 and 8 weeks after implantation. Fluid was collected from inside the implant, and uric acid and creatinine levels were determined.Histological examination of the implanted de-vice demonstrated extensive vascularization as well as formation of glomeruli and highly organized tubule-like structures. Immunocytochemical stain-ing with antiosteopontin antibody, which is secreted  by proximal and distal tubular cells and the cells of the thin ascending loop of Henle, stained the tubular sections. Immunohistochemical staining for alkaline  phosphatase stained proximal tubule-like structures. Uniform staining for bronectin in the extracellular matrix of newly formed tubes was observed. The uid collected from the reservoir was yellow and contained 66 mg/dl uric acid (as compared to 2 mg/dl in plasma) suggesting that these tubules are capable of unidirectional secretion and concentration of uric acid. The creatinine assay performed on the collected uid showed an 8.2-fold increase in concentration, as compared to serum. These results demonstrated that single cells from multicellular structures can become organized into functional renal units that are able to excrete high levels of solutes through a urine-like uid [46]. To determine whether renal tissue could be formed using an alternative cell source, nuclear transplanta-tion (therapeutic cloning) was performed to generate histocompatible tissues, and the feasibility of engi-neering syngeneic renal tissues in vivo using these cloned cells was investigated [25]. Nuclear material from  bovine dermal broblasts was transferred into unfertilized enucleated donor bovine eggs. Renal cells from the cloned embryos were harvested, ex- panded in vitro, and seeded onto three-dimensional renal devices (Fig. 60.1a). The devices were im- planted into the back of the same steer from which Fig. 60.1a–c Formation of functional renal tissue in vivo. a  Renal device. b  Tissue-engineered renal unit shows the accumu- lation of urine-like uid. c  There was a clear unidirectional continuity between the mature glomeruli, their tubules, and the  polycarbonate membrane 872T. Aboushwareb, J. J. Yoo, A. Atala
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