Brenner and Rector's The Kidney, 8th ed.

CHAPTER 68. Tissue Engineering and Regeneration

Marc R. Hammerman

  

 

Tissue Engineering a Kidney: End-Stage Disease, 2199

  

 

Integration of New Nephrons into the Kidney, 2199

  

 

Antigen Presentation, 2200

  

 

Major Histocompatibility Complex Expression, 2200

  

 

Immune Response to Transplanted Fetal Renal Tissue, 2200

  

 

Means by Which Renal Anlagen are Vascularized, 2201

  

 

Growing New Kidneys in Situ: Organogenesis, 2202

  

 

Isotransplantation/Allotransplantation of Renal Anlagen, 2202

  

 

Availability of Renal Anlagen, 2207

  

 

Xenotransplantation of Renal Anlagen, 2207

  

 

Use of Embryonic Stem Cells, Renal Precursor Cells, or Other Cells to Engineer New Nephrons and Repair Damaged Nephrons, 2209

  

 

Embryonic Stem Cells, 2209

  

 

Renal Precursor Cells, 2209

  

 

Nonrenal Precursor Cells, 2211

  

 

Generation of Histocompatible Tissues Using Nuclear Transplantation: Therapeutic Cloning, 2211

  

 

Tissue Engineering a Kidney: Enhancing Recovery from Acute Kidney Injury, Glomerulonephritis, and Congenital Kidney Disease, 2211

  

 

Bone Marrow-Derived Stem Cells, 2211

  

 

Endothelial Cells, 2212

  

 

Renal Tubule Cell Assist Device, 2212

  

 

Conclusions, 2212

At the present time, dialysis and renal allotransplantation are standard treatments to replace kidney function in the setting of end-stage disease. Dialysis is life-preserving, but it replaces only a small fraction of normal kidney function and has considerable morbidity rates.[1] Transplantation is limited by the number of human organs available. [2] [3] It is likely that these modalities will eventually be supplanted by one or more alternatives currently under development. Several alternatives, now at the frontier of nephrology, employ the technologies of tissue engineering and regeneration.[4]

The major goal of tissue engineering is to replace, repair, or enhance the biologic function of damaged tissues or organs. [4] [5] [6] In the case of the end-stage kidney, the tissue engineer has a formidable task. This is because the mature kidney is a remarkably complex structure, the function of which is dependent on the growth and differentiation of its precursor cells into a mature organ consisting of many different cell types. Al Awqati and Oliver[7] have estimated that there are at least 26 terminally differentiated cell types in the kidney of a newborn mouse that arise from at least four cell types present in the undifferentiated metanephric blastema when renal development begins.

Delineation of 26 terminally differentiated nephron cell types takes into account cell morphology, location, and function.[7] In order for glomerular filtration, reabsorption, and secretion of fluid and electrolytes to take place in a manner that will sustain life, individual nephrons must be integrated in three dimensions with one another and with a collecting system, the origin of which is yet another separate structure, the ureteric bud.[8] Concomitantly, vascularization must occur in a unique organ-specific manner from endothelial precursors that may originate from both inside and outside of the developing renal anlage.[9] In addition to its filtration reabsorptive and secretory functions, the kidney is an endocrine/metabolic organ. It is a major site of erythropoetin,[10] renin[11] synthesis, 1-α hydroxylation of 25(OH)D3,[12] and 5″ deiodination of thyroid hormone.[13] In an ideal tissue-engineered kidney, these functions will be recapitulated.

During the past decades, a number of different approaches have been applied toward tissue engineering a new kidney to treat end-stage disease. The goals of each approach are to replace some, if not all, of the functions described earlier. This chapter summarizes the progress to date recorded for four approaches that are directed toward recapitulating kidney filtration, reabsorption, and secretion: integration of new nephrons into the kidney; growing new kidneys in situ or organogenesis; use of embryonic stem (ES) cells, renal precursor cells, or nonrenal precursors to engineer new nephrons or repair damaged nephrons; and generation of histocompatible kidneys using nuclear transplantation or therapeutic cloning. Some approaches recapitulate endocrine/metabolic function as well. In that nephron structure/function is so complex, most strategies employ a self-assembly component, linking them inextricably with generation from embryonic precursors already programmed to differentiate into a kidney. In this chapter, the precursors are referred to as metanephroi, renal anlagen, or primordia.

In the setting of acute kidney injury (AKI), the task of the kidney tissue engineer is less formidable than is the case for end-stage disease. Here, the need is to enhance regeneration of damaged parts of the nephron in an otherwise intact organ. This chapter reviews evidence that bone marrow-derived stem cells participate in recovery following acute injury and delineates strategies advanced for the therapeutic use of bone marrow-derived cells, endothelial cells, or renal cells incorporated into a renal tubule cell assist device to enhance recovery from AKI.

TISSUE ENGINEERING A KIDNEY: END-STAGE DISEASE

Integration of New Nephrons into the Kidney

The methodology for studies directed toward integrating new nephrons into the kidney derives from a literature describing the transplantation of embryonic renal metanephric primordia/anlagen. Renal primordia have been transplanted successfully to the chorioallantoic membrane of developing birds,[14] the anterior eye chamber, [15] [16] beneath the renal capsule, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] into the renal cortex of recipients, [26] [27] and into the abdominal cavity. [23] [24] [25] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] Most studies that employed renal subcapsular transplantation, and placement into the anterior chamber of the eye and onto the chorioallantoic membrane were conducted to define the immune response to fetal kidney transplants or to delineate the means by which renal anlagen are vascularized. However, information emerged from these studies leading to approaches that employ transplantation to enhance renal function by growing new nephrons.

Perusal of the literature provides four theoretical reasons why the use of developing metanephroi for transplantation might be advantageous relative to developed kidneys. First, if developing renal anlagen are obtained at a sufficiently early stage, antigen-presenting cells (APCs) that mediate direct host recognition of alloantigen or xenoantigen are absent. [17] [19] [23] [29] Second, donor antigens such as major histocompatibility complex (MHC) class I and II may not be expressed on renal anlagen. [18] [21] Third, the immune response to transplanted fetal renal tissue differs from that to adult tissue. [17] [20] [21] [22] [23] [25] Fourth, one might expect a transplanted anlage to be supplied by host blood vessels [24] [31] [34] [38] and, as such, be less susceptible to humoral rejection post-transplantation across a discordant xenogeneic barrier.[3]

Antigen Presentation

In order that a host T cell mediated response directed toward antigens from transplanted tissue can be mounted, transplant antigens must first be presented to host T cells. Transplant antigens can be presented by APCs originating from within the donor organ (direct presentation) or by host APCs (indirect presentation). One theoretical advantage gained through the use of embryonic tissue for transplantation is that functional APCs may be absent from the transplants either because APCs have yet to mature in the embryos or migrate into the tissue.[43]

The metanephric kidneys originate in the rat on day 12.5 of a 21-day gestation period.[25] Foglia and colleagues[17] transplanted kidneys from adult rats or metanephroi from outbred Sprague Dawley rat embryos aged embryonic day (E)15-E21, beneath the renal capsule of nonimmunosuppressed adult Sprague Dawley hosts. Under these conditions, adult kidney transplants undergo acute rejection within 7 days. [17] [25] In contrast, growth and survival of embryonic transplants was age dependent in that enlargement and differentiation in situ over 15 to 30 days, was best for metanephroi obtained from E15 embryos, and worsened progressively for those obtained on E16-E21. Anlagen from E15 embryos showed maturation of renal elements when examined 10 days post-transplantation without rejection, whereas those obtained on E20 had a poor architecture and dense lymphocytic infiltrate, whereas liver harvested on E15 transplanted beneath the renal capsule underwent little growth and prompt rejection.

Xu and colleagues[41] and I[30] found a similar age dependence for successful allotransplantation of rat metanephroi into the omentum.

Velasco and Hegre[19] transplanted metanephroi or liver tissue from E15, E17, E18, or E19 inbred Fisher rat embryos with rat major histocompatibility complex (RT1) RT1lvl, beneath the renal capsule of RT1—incompatible Wistar Furth adult rats (RT1u). All embryonic hepatic grafts were rejected within 10 days. In contrast, the degree of rejection of the metanephroi was age-dependent, those from E15 embryos showing minimal or moderate rejection and those from older embryos showing more. If liver and metanephroi from E15 embryos were cotransplanted at different sites, metanephroi underwent a more severe rejection than if they were implanted without liver. APCs populate liver well before E15 in rats but are not present in circulation until days later.[43] It was speculated that the absence of APCs in metanephroi from E15 embryos, together with their presence in liver obtained concurrently, explains the differential fate of metanephroi transplanted with or without liver. Under the former conditions but not the latter, direct presentation of donor antigens to host T cells takes place.[19]

Major Histocompatibility Complex Expression

In the mouse, metanephroi arise on day 11.5 of a 20-day gestation period.[31] Statter and co-workers[18] transplanted metanephroi originating from E14-to-adult C57Bl/6 mice (H-2b) beneath the renal capsule of adult congenic B10.A hosts (H-2a). Expression of donor and host-specific class I (H2Kb) and class II (Aβb) transcripts in E14 donor tissue was low and increased progressively in renal tissue from older mice. After transplantation, surviving kidney grafts showed enhanced expression of class I and II transcripts. However, neither class I nor II protein could be detected in transplanted renal primordia.

In human embryos, the metanephric kidneys arise during the first trimester. [20] [21] [22] Dekel and co-workers [20] [21] [22] carried out a series of investigations in which human adult or embryonic kidney tissue is transplanted beneath the kidney capsule of immunodeficient rats (severe combined immunodeficiency [SCID/Lewis and SCID/nude chimeric rats]). Human adult kidney fragments transplanted beneath the renal capsule of such rats survive for as long as 2 months. Architecture of the transplanted tissue and the normal structure of glomeruli are preserved. Intraperitoneal infusion post-transplantation of allogeneic human peripheral blood mononuclear cells (PBMC) results in rejection of adult grafts.

Human fetal kidney fragments transplanted beneath the renal capsule of immunodeficient rats display rapid growth and development. Glomeruli and tubular structures are maintained for as long as 4 months post-transplantation. In contrast to the case for transplanted adult human kidney fragments, infusion of allogeneic human PBMC into hosts results in either minimal human T cell infiltration or infiltrates that do not result in rejection or interfere with the continued growth of the human fetal renal tissue. Fetal human kidney grafts have reduced expression of tissue human leukocyte antigen (HLA) class I and II relative to the adult grafts, consistent with reduced effectiveness in inducing an alloantigen-primed T cell response.[20]

Immune Response to Transplanted Fetal Renal Tissue

Dekel and colleagues have shown that transcript levels for interferon-γ and interleukin-2 in fetal human kidneys grafted under the renal capsule of immunodeficient rats are markedly reduced post-transplantation relative to levels in adult human kidney tissue grafted to the same site. Peak levels of these cytokines appear late after PBMC infusion. Concomitant with these findings, interleukin-4 mRNA is up-regulated during the early-phase post-PBMC infusion, and interleukin 10 mRNA is expressed throughout the post-PBMC infusion interval. In addition, levels of mRNA coding for chemokines RANTES and macrophage inflammatory protein 1 (MIP1) beta, their receptor chemokine (C-C motif) receptor 5 (CCR5), and the cytolytic effector molecule Fas ligand are suppressed in the fetal grafts relative to levels in adult grafts. Thus, fetal kidney induces the down-regulation of Th1 cytokines, chemokines, and fas ligand, and the sparing of Th2 cytokines in the grafts. The findings suggest that the human immune response of kidney rejection is dependent on whether the target organ is of fetal or adult origin. An allogeneic immune system appears to mount a T helper 2–biased response when the target organ is fetal, resulting in enhanced survival of transplanted tissue relative to adult tissue against which a T helper 1–biased response is mounted.[22]

Subsequently, this group showed that developing human kidneys had restricted expression of multiple factors that determine immune recognition. Thirteen of 57 genes that were significantly up-regulated in adult versus fetal human kidney tissue belonged to the HLA class I and II systems. In addition, molecules that mediate trafficking of leukocytes into the graft such as chemokines RANTES and MCP-1, adhesion molecule E-selectin, proinflammatory cytokines such as osteopontin and complement genes had reduced expressions in embryonic relative to adult kidneys. Reduced immunogenicity of embryonic human or pig kidneys transplanted into immunodeficient mice was confirmed by the absence of cellular rejection following infusion of human PBMCs.[23]

Means by Which Renal Anlagen are Vascularized

The major arterial vessels supplying the kidney originate from lateral branches of the abdominal aorta that terminates in a plexus of arteries in close proximity to the renal pelvis, the renal artery rete.[44] It is a matter of controversy whether the renal microvasculature (smaller vessels and glomerular capillaries) arises exclusively by means of this angiogenic process, or also in part from endothelial cells resident in the developing metanephros. However, it is clear that during its development, the renal anlage is able to attract at least its major arterial vessels, from the developing aorta.[9] In that its blood supply originates, at least in part, from outside of the developing renal anlage, the kidney may be regarded as a chimeric organ. Its ability to attract its own vasculature in situ establishes the renal anlagen as cellular transplants, capable of attracting a blood supply from an appropriate vascular bed.[3]

Insight into the origin of the renal microvasculature supply is provided by experiments in which developing kidneys are transplanted to ectopic sites. However, the results of these experiments are somewhat contradictory. One explanation for the differences may be that the means of vascularization is site specific. For mouse or chick metanephroi obtained from E11.5 embryos grafted onto the chorioallantoic membrane of the quail, the vasculature is derived entirely from the host.[14] In the case of metanephroi from E11-12 mouse embryos grafted into the anterior chamber of the eye in genetically identical mice, the glomerular endothelium derives from both donor and host. [15] [16] For metanephroi from E15 rat embryos transplanted into the abdominal cavity of mice,[31] or from E28 pig embryos transplanted into the abdominal cavity of rats, [24] [38] [40] or mice[34] the microvasculature is largely or entirely host. In all cases, large external vessels derive from the host.

Host immune responses directed against antigens located on the endothelium of a transplanted vascularized organ such as a kidney, or mediated by transplant endothelial cells are reduced in proportion to the extent that an organ can be transplanted in cellular form[3] and be supplied by host vessels as it develops in situ. Such reduction would be beneficial in the case of allotransplantation. However, for xenotransplantation, it could provide a way to ameliorate hyperacute and acute vascular rejection that represent two major obstacles to the use of nonprimate vascularized organs, such as kidneys from adult pigs, for transplantation into humans.[3]

In that humans and pigs are of comparable size, and share a similar renal physiology, and because pigs are plentiful and can be bred to be pathogen free, pigs represent an ideal kidney donor for humans. [45] [46] Unfortunately, the transplantation of whole vascularized organs, such as the kidney originating from pigs into the group of primates that includes humans, the great apes, and Old World monkeys, is rendered problematic because of the processes of humoral rejection (hyperacute and acute vascular rejection) that occur across this xenogeneic barrier. [3] [46]

Hyperacute rejection occurs as a result of the binding of preformed or natural xenoreactive antibodies present in the circulation of hosts to cells of the donor species followed by activation of the hosts's complement system. Approximately 85% of the natural antibodies in humans that bind to pig cells are directed against galactose-a-1, 3-galactose (a-gal), a sugar expressed on the vascular endothelium of cells in most mammals but not in humans, great apes, and Old World monkeys.[46] The etiology of acute vascular rejection is multifactorial and incompletely understood. Several of the processes implicated as causative reflect a fundamental incompatibility between host proteins/protein systems, and the vascular endothelium of the donor. Factors that are thought to contribute include circulating xenoreactive antibodies that trigger adverse reactions in transplant endothelium, the failure of primate natural killer cells to recognize porcine MHC I molecules, and incompatibilities between porcine proteins/receptors and circulating primate/human proteins such as clotting factors.[46]

Humoral rejection following the transplantation of pig kidneys into nonhuman primates can be ameliorated or overcome through the use of genetically altered organs originating from pigs transgenic for the human complement activator, decay accelerating factor (hDAF),[47] or the use of organs from transgenics that do not express α-gal.[48] Unfortunately, neither the immunosuppressive regimens nor the outcomes used for pig-to-primate kidney transplantation would be acceptable in humans. Transplantation of kidneys from pigs transgenic for hDAF in combination with host immunosuppression and splenectomy enabled survival for up to 78 days in otherwise anephric cynomolgous monkeys. However, there was a high incidence of adverse events such as development of edema, or ascites, vomiting, diarrhea, or lymphoproliferative disorders. All recipients had to be euthanized because of renal failure, gastrointestinal hemorrhage, or pancreatitis.[47] Survival for as long as 83 days was achieved posttransplantation of kidneys from α-gal-deficient donors into immunosuppressed, thymectomized, and splenectomized baboons, but only if vascularized pig thymic tissue was cotransplanted.[48] Baboons died from serum sickness, infection, or myocardial infarction.

In contrast to xenotransplantation of whole vascularized organs from pig to primates, cellular transplants such as pancreatic islets from pigs can be transplanted into humans without triggering hyperacute or acute vascular rejection.[3] As delineated previously, the renal metanephric anlage is a candidate for cell transplantation. It had been speculated that developing nephrons implanted beneath the renal capsule[25] or into tunnels fashioned in the cortices of host kidneys [26] [27] might become incorporated into the collecting system of the host, and thereby increase host renal function. Woolf and co-workers [26] [27] implanted pieces of sectioned renal anlagen originating from embryonic day (E)13-E16 mice into tunnels fashioned in the cortex of kidneys of newborn outbred mice. Differentiation and growth of donor nephrons occurred in the host kidney. Glomeruli were vascularized, mature proximal tubules were formed, and extension of metanephric tubules into the renal medulla was observed. However, incorporation of donor nephrons into the collecting system of hosts was not demonstrated.

We performed experiments similar to those of Woolf and colleagues[25] in which metanephroi from E15 Sprague Dawley rat embryos were implanted beneath the renal capsule of adult Sprague Dawley hosts. Hosts received no immunosuppression. E15 renal anlagen contained segments of ureteric bud and condensing metanephric blastema but no glomeruli. To determine whether subcapsularly transplanted rat renal anlagen became integrated into host kidneys, we examined kidneys of host rats 6 weeks postsubcapsular transplantation. To clear blood from the organ, kidneys were back perfused. This results in a blanching of the kidney as blood is replaced by perfusate. Normally, the entire kidney blanches. However, following perfusion of kidneys that contained a transplanted renal anlagen, blood remained in the transplanted structure relative to the host kidney ( Fig. 68-1A , cortex arrows). Most likely, this reflects a reduced perfusion in chimeric blood vessels (derived from transplant and host kidneys) that have been shown to supply subrenal capsularly transplanted anlagen relative to perfusion in those supplying only the host kidney. Blood could be traced into the papilla of the host kidney (see Fig. 68-1A , medulla, arrows). Histologic examination of kidneys showed that glomeruli (g) in the transplanted kidney (see Fig. 68-1B ) had been poorly perfused relative to glomeruli present in the host kidney (see Fig. 68-1C ), in that they contained more red blood cells. Also, glomeruli in the transplanted kidneys (g) were smaller than those in the host kidney (G).

FIGURE 68-1  (A) Photograph of a midsagittal section obtained following perfusion of a kidney originating from a rat 6 weeks post-transplantation. Arrows show portions of unperfused transplanted embryonic kidney; (B-C) Photomicrographs of hematoxylin and eosin-stained kidneys: (B) Glomerulus (g) within transplanted embryonic kidney, (C) glomerulus (G) within host kidney.  (Reprinted with permission by Rogers SA, Lowell JA, Hammerman NA, Hammerman MR: Transplantation of developing metanephroi into adult rats. Kidney Int 54:27–77, 1998.)

 

 

Collecting ducts from transplanted renal anlagen migrated toward the papilla of host kidneys in parallel with the vasculature.[25] However, like Woolf and associates, [26] [27] I was unable to determine that any connection between the collecting systems of donor and host kidneys was made.[25] In addition, I found that the growth of transplanted renal anlagen was constrained by their placement beneath the host kidney capsule.

The studies summarized previously show that it is possible to integrate new filtering nephrons into kidneys. If integration were accompanied by incorporation into the collecting system of hosts, subrenal capsular transplantation of renal primordia would represent a strategy for increasing renal function. However, to be applicable to humans with end-stage renal disease, primordia must incorporate into host nephrons in end-stage kidneys that are small and fibrotic. Such conditions are not duplicated by subcapsular transplantations performed thus far.

GROWING NEW KIDNEYS IN SITU: ORGANOGENESIS

Isotransplantation/Allotransplantation of Renal Anlagen

Renal anlagen transplanted into a host rodent's fold of mesentery, undergo differentiation and growth in hosts that is not confined by a tight organ capsule. [25] [41] [42] Growth is enhanced if one of the hosts kidneys is removed at the time of implantation [25] [41] [42] or if the host is pregnant.[42] A renal anlage in a retroperitoneal dissection from an E15 rat embryo is shown in Figure 68-2A . The ureteric bud is delineated by an arrowhead. If transplanted into an adult rat with its ureteric bud attached, the renal anlage enlarges and becomes kidney shaped within 3 weeks (see Fig. 68-2B ). The ureteric bud differentiates into a ureter (see Fig. 68-2B , arrowhead). In contrast to transplanted developed kidneys that undergo acute rejection,[25] renal anlagen transplanted into nonimmunosuppressed hosts have a normal kidney structure and ultrastructure postdevelopment in situ and become vascularized through arteries that originate from the superior mesenteric artery of hosts and veins that originate from the host omentum.[32] Figure 68-3A is a hematoxylin and eosin (H & E)–stained section of a renal anlagen from an E15 rat embryo consisting of branched utereric bud (ub) and undifferentiated metanephric blastema (mb). Figure 68-3B shows a renal anlagen or metanephros (M), 3 weeks postallotransplantation. An artery (a) and vein (v) originating from the host are delineated. Figure 68-3C shows a radiocontrast study that demonstrates the metanephros is supplied by the host's superior mesenteric artery (SMA). A ureteroureterostomy (arrow) between the ureter originating from the transplanted renal anlage (M) and host ureter is shown in Figure 68-3D .

FIGURE 68-2  (A) Photograph of retroperitoneal dissection from an E15 rat embryo showing renal anlage or metanephros (m) and ureteric bud (arrowhead). (B) Photograph of a developed renal anlage (m) in the omentum of an adult host rat 3 weeks posttransplantation. Arrowhead shows developed ureter. Magnification is shown.  (Reproduced with permission from Hammerman MR: Transplantation of developing kidneys. Transplant Rev 16:62–71, 2002.)

 

 

FIGURE 68-3  (A) H & E stained E15 rat metanephros consisting of undifferentiated metanephric blastema (mb) and ureteric bud (ub). Arrowheads show branched ureteric bud. (B) Artery (a) and vein (v) originating from the host's peritoneum supplying the developed renal anlage or metanephros (M). (C) Radiocontrast image of kidney (K) and developed renal anlage (M), 6 weeks posttransplantation into the peritoneum of a host rat. SMA, superior mesenteric artery. (D) Ureteroureterostomy (arrow). Magnifications are shown for A and for B and D (in B).  (Reproduced with permission by Hammerman MR: Transplantation of developing kidneys. Transplant Rev 16:62–71, 2002, and Rogers SA, Hammerman MR: Prolongation of life in anephric rats following de novo renal organogenesis. Organogenesis 1:22–25, 2004.)

 

 

Differentiated structures at 20 weeks' postimplantation are illustrated in Figure 68-4 that shows H & E-stained sections of a developed renal anlage. The cross sectional diameter of the developed renal anlage shown in Figure 68-4A(approximately 1.2 cm) is about one half the diameter of a normal rat kidney.[25] Its ureter (u) is labeled. Figure 68-4B shows a glomerulus (g) proximal tubule (pt), distal tubule (dt) and collecting duct (cd) in the cortex. A glomerulus (g) and collecting duct (arrow) are labeled in Figure 68-4C . A glomerulus (g) proximal tubule (pt), and distal tubule (dt) are labeled in Figure 68-4D . A collecting duct (cd) is shown in Figure 68-4E . Electron microscopy of a developed renal anlage[33] reveals normal renal structures ( Fig. 68-5 ). Developed renal anlagen transplanted onto the omentum produce urine that is excreted in the normal manner following ureteroureterostomy between transplant and host (see Fig. 68-3D ), a procedure that can be readily carried out at if renal anlagen are implanted in close proximity to the host ureter. [25] [30] [36]

FIGURE 68-4  H & E-stained sections of a developed metanephros 20 weeks' post-transplantation. (A) The ureter (u) is shown; (B) a glomerulus (g), proximal tubule (pt), distal tubule (dt), and collecting duct (cd) in the cortex. (C) A glomerulus (g) and collecting duct (arrow) are labeled; (D) a glomerulus (g) proximal tubule (pt), and distal tubule (dt) are labeled; (E) a collecting duct (cd) is labeled. Magnifications are shown in A-D (for D and E).  (Reproduced with permission by Rogers SA, Hammerman MR: Prolongation of life in anephric rats following de novo renal organogenesis. Organogenesis 1:22–25, 2004.)

 

 

FIGURE 68-5  Electron micrographs of transplanted rat renal anlagen. Glomerular capillary loops show labeled: (A) mesangial cell (m); (B) endothelial cell (en); and (C) epithelial cell (ep), endothelial cell (en), podocytes (pd), and a basement membrane (arrows). (D) A proximal tubule (pt) with a brush border membrane (arrowhead); (E) proximal tubule (pt), distal tubule (dt), and collecting duct (cd). Magnifications shown for C and E.  (Reproduced with permission by Hammerman MR: Implantation of renal rudiments. In Polak J, Hench L, Kemp P (eds): Future Strategies for Organ Replacement. London, Imperial College Press, 2002, pp 199-211; and Hammerman MR: Transplantation of embryonic kidneys. Clin Sci 103:599–612, 2001.)

 

 

Levels of renal function in transplanted renal anlagen (glomerular filtration rate [GFR]) were determined by measuring inulin clearance in otherwise anephric rats. In initial experiments, GFRs were very low.[25] However, as shown in Table 68-1 incubation of renal anlagen with growth factors before implantation increased GFRs more than 100-fold compared with those in rats with nongrowth factor-incubated renal anlagen implanted concurrently.[32] GFRs in growth factor-treated renal anlagen are about 6% of normal. Others have reported even higher levels of GFR in rat-to-rat transplants.[39] Renal plasma flow, another parameter of renal function, was measured in transplanted renal anlagen by calculating P-aminohippurate (PAH) clearances. The ratio of GFR/PAH clearance (filtration fraction) was 0.6, comparable with filtration fractions measured in rats with reduced renal function.[32] Urine flow rates in transplanted rats are about 12% of the inulin clearance (GFR) measured in growth factor-treated renal anlagen (see Table 68-1 ). The urine volume (UV)/GFR of 0.12 demonstrates that transplanted renal anlagen can concentrate urine.[32]


TABLE 68-1   -- Urine Volumes and Inulin Clearances[*][†]

 

Vehicle

Gr Factors

Urine volume (UV) (uL/hr)

31 ± 9.1

621 ± 62

Inulin clearance (GFR) uL/min/100 g

0.24 ± 0.06

27 ± 8.2

uL/hr

38

5313

UV/GFR

0.8

0.12

Reproduced from Hammerman MR: Transplantation of developing kidneys. Transplant Rev 16:62–71, 2002.

GFR, glomerular filtration rate.

 

*

Some data are expressed as mean ± SEM.

UV/GFR is calculated using values expressed as uL/hour.

 

 

Hemodialysis provides renal failure patients with GFRs that are about 10% of normal.[6] Therefore, 6% of normal approximates a level of renal function that would be expected to preserve life. Indeed, life can be prolonged in otherwise anephric rat hosts by prior transplantation and ureteroureterostomy of a renal primordia. [36] [39] Survival as a function of time postremoval of all native renal mass (all renal function from the implant) is shown in Figure 68-6 . Control rats (no transplanted renal primordia) lived 67 ± 2.7 hours (range 48 to 78 hours) postremoval of all native host renal mass. Rats in the TX group (transplanted primordium, but with the ureterouretersostomy severed such that urine was discharged into the peritoneal cavity) lived 65 ± 6.0 hours (range 55 to 76 hours), no longer than controls. Rats in the group with transplanted primordium with intact uteretoureterostomy that permitted excretion of urine (TX-EXCR) lived 125 ± 12 hours (range 108 to 170 hours), significantly greater than control or TX rats.[36]

FIGURE 68-6  Survival of rats as a function of time after removal of both kidneys. Control rats (n = 13) had no transplanted renal primordia. Rats in the TX group (n = 4) had a transplanted renal primordium, but with the ureterouretersostomy severed such that urine was discharged into the peritoneal cavity. Rats in the group with transplanted primordium with intact uteretoureterostomy that permitted excretion of urine (TX-EXCR) (n = 5), had a transplanted renal primordium with an intact uteretoureterostomy that permitted excretion of urine.  (Reproduced with permission by Rogers SA, Hammerman MR: Prolongation of life in anephric rats following de novo renal organogenesis. Organogenesis 1:22–25, 2004.)

 

 

Using inbred congenic rats (PVG-RT1C and PVG-RT1avl) we have shown that renal anlagen can be transplanted across the RT1 locus into non immune-suppressed hosts. A state of peripheral immune tolerance secondary to T cell ‘ignorance’ permits the survival of transplanted renal anlagen. Most likely, the “ignorance” results from the absence of APCs originating from the donor in the embryonic renal tissue, and the consequent absence of direct presentation of transplant antigen to host T cells (presentation by donor dendritic cells to host T cells)[29] as was shown previously for subrenal capsular transplants. [17] [19]

Metanephroi arise in embryonic pigs between E20-E28. [24] [35] Transplantation of renal anlagen from E28 pigs to adult pigs can be carried out without host immunosuppression.[35]

Availability of Renal Anlagen

In the case of human renal allotransplantation, there is an unavoidable delay between the time of harvest from donors and the time of implantation into recipients. Before removal from the donor, human renal allografts are flushed with a preservation solution, often University of Wisconsin (UW) solution, and stored subsequently in ice cold UW solution. Theoretically, renal anlagen could be harvested immediately before implantation into humans. However, practically it would be best if anlagen could be stored in vitro for a period of time prior to transplantation. The ability to store anlagen would permit distribution to sites for transplantation, distant from the site of harvesting and would allow time to plan the transplant procedure.

To determine whether renal anlagen can be stored in vitro before transplantation, we transplanted renal anlagen from E15 rat embryos into the omentum of nonimmunosuppressed uninephrectomized (host) rats either directly or suspended in ice cold UW preservation solution for 3 days prior to implantation. The size and extent of tissue differentiation preimplantation of E15 renal anlagen implanted directly is not distinguishable from the size and differentiation of renal anlagen preserved for 3 days. By 4 weeks after transplantation, renal anlagen that had been preserved for 3 days had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts with normal structure had developed. At 12 weeks' post-transplantation, GFRs of preserved renal anlagen are comparable to those of anlagen implanted directly, consistent with the viability of preserved renal anlagen.[30]

Xenotransplantation of Renal Anlagen

I transplanted renal anlagen from an E15 Lewis rat embryo across a concordant xenogeneic barrier into the peritoneum of 10-week-old C57Bl/6J mice. In mice that receive immunosuppression, but not in its absence, the transplanted rat renal anlage undergoes differentiation and growth in situ ( Fig. 68-7 ).[31] To gain insight into the origin of the vasculature (donor versus host) of renal anlagen transplanted in the omentum using our rat-to-mouse model, I stained developing rat renal anlagen using mouse specific antibodies directed against the endothelial antigen CD31. The vasculature of the transplanted developed rat kidney transplanted into the mouse is largely of mouse origin including glomerular capillary loops. In contrast, the capillary loops in rat renal anlagen transplanted into rats do not stain for mouse CD31.[31]

FIGURE 68-7  (A) Photograph and (B-D) photomicrographs of H & E-stained sections of rat renal anlagen 2 weeks' post-transplantation into a mouse omentum showing: (A) the developed renal anlage or metanephros (m); (B) a nephrogenic zone (NZ), cortex (c), and medulla (M); (C) developing nephron (arrowhead) in the NZ; and (D) developed glomerulus (g) deeper within the cortex. Magnifications shown; A, B, and D (for C and D).  (Reproduced with permission by Rogers SA, Hammerman MR: Transplantation of rat metanephroi into mice. Am J Physiol 280:R1865–R1869, 2001.)

 

 

Using a highly disparate model (pig to rodent), we transplanted E28 pig renal anlagen consisting of undifferentiated stroma , branched ureteric bud and primitive developing nephrons into the peritoneum of Lewis rats [37] [38] [40] or C57Bl/6J mice. [34] [35] Two to seven weeks' post-transplantation, no trace of the renal anlagen could be found in hosts that received no immunosuppression. Figure 68-8 illustrates E28 pig renal anlagen before transplantation (seeFigs. 68-8A, B ) and 6 to 7 weeks' post-transplantation into immunosuppressed rats (see Figs. 68-8C to 68-8F ).[37]

FIGURE 68-8  Photographs (A, C) and photomicrographs (B, D-F) of pig renal anlagen. (A, B) Anlagen from E28 embryo (s, stroma; ub, ureteric bud). (C-F) Pig renal anlagen 6 to 7 weeks post-transplantation in a rat mesentery. (C) Anlage after remo-val from the omentum (u, ureter). (D) Cortex and medulla. Nephrogenic zone (arrowheads). Arrow points toward medulla; (E) Cortex with a glomerulus (g) proximal tubule (pt) and distal tubule (dt) labeled. (F) Medulla with collecting ducts (cd) labeled. Magnifications are shown for A and B (in A); for C; for D and for E and F (in E).  (Reproduced with permission by Rogers SA, Liapis H, Hammerman MR: Normalization of glucose post-transplantation of pig pancreatic anlagen into non-immunosuppressed diabetic rats depends on obtaining anlagen prior to embryonic day 35. Transplant Immunol 14:67–75, 2005.)

 

 

Shown in Figure 68-9 are glomeruli from rat kidneys, and pig kidneys and glomeruli within pig renal anlagen transplanted into rats 8 weeks previously, stained with anti-rat endothelial antigen 1 (RECA-1) that is specific for rat endothelium, or anti-CD31 that is specific for pig endothelium. The origin of the glomerular vasculature in transplants is rat (host).[38] Nonglomerular renal vasculature is also of host origin. [34] [38]

FIGURE 68-9  Photomicrographs of stained sections of: (A, B) rat kidney; or (C, D) pig kidney and (E, F) a pig renal anlagen from an E28 embryo 8 weeks' post-transplantation into a rat omentum, stained with RECA-1 (A, B, E) or CD31 (C, D, F). Glomerular capillaries in transplants stain positive for RECA-1 that is specific for rat endothelium (E) and negative for CD31 that is specific for pig endothelium (F). Magnifications are shown for A-D (in A) and E and F in E.  (Reproduced with permission by Takeda S, Rogers SA, Hammerman MR: Differential origin for endothelial and mesangial cells after transplantation of pig fetal renal primordia into rat. Transplant Immunology 15:211–215, 2006.)

 

 

Dekel and colleagues[24] successfully transplanted renal anlagen originating from pig embryos aged E20-21 to E27-28 beneath the renal capsule of immunodeficient mice. Most transplants from the E20-25 donors fail to develop or evolve into growths containing few glomeruli and tubules, but other differentiated derivatives such as blood vessels, cartilage, and bone. In contrast, the transplants originating from E27-28 pig embryos all exhibited significant growth and full differentiation into mature glomeruli and tubules. Dekel and colleagues[24] found mouse CD31 expression in external vessels as well as developing glomeruli and small capillaries of pig renal anlage xenografts, consistent with a host origin for the vasculature of the developed renal anlage cellular transplants. In addition, Dekel and co-workers transplanted adult pig kidney tissue or E27-28 pig renal anlagen beneath the renal capsule or onto the testicular fat of immunocompetent Balb/c mice. Some hosts were treated with CTLA4-Ig. Evaluation of adult or E27-28 embryonic tissues 2 weeks postimplantation into non CTLA4-Ig-treated hosts showed rejection of tissues. In CTLA4-Ig-treated hosts, most E27-28 renal anlagen underwent growth and differentiation. In contrast, all adult kidney grafts had a disturbed morphology, necrotic tissue, and a high degree of lymphocyte infiltration. The authors interpreted these data as being consistent with an immune advantage of the developing precursor transplants over developed adult kidney transplants in fully immunocompetent hosts.[24]

Dekel and co-workers[23] implanted metanephroi from E70 human embryos intraperitoneally into immunodeficient (SCID) mice. Transplanted kidneys survived for more than 2 months post-transplantation. Hybridization to cDNA arrays of RNA derived from normal human renal anlagen at 8, 12, 16, or 20 weeks of gestation demonstrated a subset of 240 genes the expressions of which changed substantially with time. Clustering analysis of global gene expression in transplants post-transplantation revealed a temporal profile of gene expression similar to that observed in the normal human kidneys during development, consistent with recapitulation of a renal developmental program.[23] Comparison of the expression profiles of developing metanephroi to a Wilms tumor specimen revealed no similarity consistent with no threat of malignant transformation after transplantation of human kidney precursors.[23]

USE OF EMBRYONIC STEM CELLS, RENAL PRECURSOR CELLS, OR OTHER CELLS TO ENGINEER NEW NEPHRONS AND REPAIR DAMAGED NEPHRONS

Embryonic Stem Cells

Human ES cells transplanted into immunodeficient mice differentiate into teratomas containing structures from all three germ layers. [3] [49] [50] [51] Differentiated structures include glomerulus- and renal tubule-like elements,[50]consistent with a potential role for ES cells to bioengineer new kidneys. However, glomerulus- and tubule-like structures are not glomeruli and tubules, and for reasons delineated in the introduction of this chapter, the structural complexity of kidney makes it unlikely that a functional replacement organ can be generated from ES cells alone.

A novel step towards use of ES cells in renal engineering was employed by Streenhard and co-workers,[52] who microinjected mouse ES cells that were tagged with a lineage marker into E12-13 mouse metanephroi. ES cell derivatives differentiated into lumenized tubules with apical junctional complexes and primary cilia and assembled a basement membrane around their basal aspect following 3 to 5 days in organ culture. Some of the structures expressed proximal tubule markers. In contrast, there was only rare evidence for integration of ES cells into the glomeruli that form in organ culture. These observations suggest that the developing kidney microenvironment provides cues for entry of ES cells into a nephrogenic program in vitro.

Renal Precursor Cells

With the exception of its nerve supply, the kidney is a mesodermal derivative.[8] Therefore, it is conceivable that each of its original cell types, plus the ureteric bud, could originate from a renal-specific descendent of a ES cell. Al Awqati and Oliver[7] have speculated that, if it is present at all, such a renal precursor cell might be located somewhere within the intermediate mesoderm at a given stage of embryogenesis. Oliver and co-workers[53] have shown that cultures of metanephic blastema can generate not only renal epithelial cells but also cells that are positive for α-smooth muscle actin, indicating that they are myofibroblast precursors, as well as cells that express receptors for vascular endothelial growth factor, consistent with an endothelial lineage. Their observations suggest metanephric blastema may contain embryonic renal pre-cursor cells.

If renal-precursor cells exist and can be identified, it might be possible to induce them to divide, and have the progenitors organize themselves into a kidney. Maeshima and associates[54] have shown that mouse kidneys contain cells that retain a bromodeoxyuridine (BrdU) label for extended periods of time as a result of slow turnover. Such cells, termed label-retaining cells (LRCs) are believed to represent the stem cell compartment in a number of tissues, but had not previously been identified in the kidney. During recovery after renal ischemic injury, LRCs in mouse kidneys underwent cell division and became positive for proliferating cell nuclear antigen (PCNA). In contrast, PCNA-positive but BrdU-negative tubular cells were rarely observed, suggesting that the cells proliferating during the process of tubular regeneration are derived from LRCs. At an early stage of regeneration, descendants of LRC expressed a mesenchymal marker, but became positive for an epithelial marker after several cell divisions, consistent with proliferation and differentiation into epithelial cells during repair postischemic injury.

Oliver and colleagues[55] found that low-cycling BrdU-retaining cells were sparse in adult rodent kidneys except in the renal papilla.[55] During repair following transient renal ischemia in rats, the BrdU-retaining cells entered the cell cycle and disappeared from the papilla. Single-cell clones of the papillary cells coexpressed mesenchymal and epithelial proteins, and gave rise to myofibroblasts and cells that expressed neuronal markers consistent with a stem cell identity. Following isolation and injection into the renal cortex the papillary stem cells incorporated into renal parenchyma. If LRCs are renal precursors and participate in recovery postischemic injury, identification of agents that enhance their differentiation and their use as therapeutic modalities could represent new approaches to kidney regeneration in other settings. [54] [55]

CD133 is a glycoprotein expressed on primitive cell populations such as CD34+ hematopoetic stem and progenitor cells. Bussolati and colleagues[56] isolated a population of CD133+ cells from normal adult human kidneys that expressed PAX-2 an embryonic renal marker. The cells were capable of expansion and limited self-renewal in vitro. On subcutaneous implantation in SCID mice, undifferentiated CD133+ cells formed tubular structures expressing renal epithelial markers. Following intravenous injection into SCID, mice with glycerol-induced tubular necrosis, expanded CD133+ cells homed to the kidney and integrated into tubules.[56]

Elger and associates[57] have characterized a nephrogenic zone in kidneys from the adult skate in which renal precursor-like cells reside. The tissue responds to partial reduction of renal mass with the formation of new nephrons. This morphogenetic process that the authors term neonephrogenesis appears to be an important mechanism for renal growth as well as repair of injured kidney in the elasmobranch. Shown in Figure 68-10A is the nephogenic zone from a control animal. Figure 68-10B shows neonephrogenesis in the nephrogenic zone from the contralateral kidney of an animal that had undergone unilateral nephrectomy 10 weeks previously. Neonephrogenesis has not been demonstrated in mammals. However, if the processes that mediate new nephron formation in adult skates can be recapitulated in higher mammals, it may represent way to grow new nephrons integrated into diseased kidneys.

FIGURE 68-10  Nephrogenic zone from control animal (A) and of contralateral nonoperated kidney from an animal that underwent unilateral nephrectomy (B)(A) In control animals, few developmental nephron stages are present. A stage I (early stage) developing nephron is located adjacent to the lateral bundle (LB) of a stage III to IV (later stage) developing nephron. Three cross-sections of the same bundle (consisting of five tubular profiles each) are seen. A young glomerulus (GL) of stage IV is located nearby in the mesial tissue. (B) Growth rate and formation of new nephrons is enhanced in the nephrectomized animal. The mass of aggregated mesenchymal cells (MES) is enhanced, and several stages II (II) and at least one stage III (III) are present. The mesial tissue (MT, delineated by a bracket) is irrigated by venous sinusoids (VS), whereas the zone containing the mesenchymal aggregates is virtually devoid of vasculature. Paraffin sections, Masson-Goldner staining, LM.  (Reproduced with permission by Elger M, Hentschel H, Litteral J, et al: Nephrogenesis is induced by partial nephrectomy in the elasmobranch Leucoraja erinacrea. J Am Soc Nephrol 14:1506–1518, 2003.)

 

 

Advances in understanding the molecular biology of rodent renal development have led to the ability to culture the components of the developing rat kidney (the ureteric bud and metanephric blastema) in isolation from one another. Steer and co-workers[58] have described a method for subculturing and propagating either component in isolation. Ureteric buds can be combined with freshly isolated metanephric blastema to form a large number of rat neokidneys derived from a single progenitor in vitro. The authors speculate that neokidneys could be grown from component parts. A colony of neokidneys derived from a single renal component could lead to a large supply of genetically identical renal tissue. In addition, isolated mesenchyme or blastema could be transfected with constructs designed to regulate the growth characteristics of the neokidney or enhance the function of one or more cellular components. Finally, it might be possible to create a chimeric structure using ureteric bud as a scaffold that could be recombined with allo- or xenoderived nonrenal mesenchymal cells. Such cells could be engineered to differentiate into a renal phenotype when exposed to kidney-specific signals from the scaffold.[58]

Nonrenal Precursor Cells

Yokoo and associates[59] injected human mesenchymal stem cells (hMSC) labeled with LacZ into E9.5 mouse embryos or E11.5 rat embryos at the site of early renal organogenesis, and subjected the whole embryos to culture. After 48 hours of whole culture, metanephroi were dissected from whole embryos and cultured in vitro for 6 days. hMSC-derived LacZ-labeled cells contribute to renal structures in organ-cultured metanephroi. Subsequently, the investigators implanted LacZ-labeled hMSC that had been transfected with glial cell line-derived neurotrophic factor into the nephrogenic site of E11.5 rat embryos. Following 48 hours of whole embryo culture, metanephroi containing hMSC were dissected out and transplanted into the omentum of uninephrectomized rats. Transplants enlarged over 2 weeks in nonimmunosuppressed rats, became vascularized by host vessels, and contained hMSC-derived LacZ-positive cells that were morphologically identical to resident renal cells. These findings suggest that self-organs from autologous MSC can be generated using inherent developmental and angiogenic systems.[60]

GENERATION OF HISTOCOMPATIBLE TISSUES USING NUCLEAR TRANSPLANTATION: THERAPEUTIC CLONING

Lanza and colleagues[61] have created bioengineered tissues from cardiac, skeletal and renal cells cloned from adult bovine fibroblasts. They transplanted subcutaneously into an adult bovine host, cultured dispersed kidney cells from its E56 cloned embryo, seeded on collagen-coated cylindrical polycarbonate membranes. Cloned and passaged kidney cells expressed renal specific proteins in vitro including synaptopodin, aquaporin-1, aquaporin 2, and Tamm-Horsfall protein. After expansion, the cells produced both 1, 25(OH)D3 and erythropoietin.

Straw-colored fluid was produced by renal units ( Fig. 68-11 ) that had differentiated in situ into glomerulus-like and tubule-like structures and undergone vascularization. Chemical analysis of collected fluid suggested unidirectional secretion and concentration of urea nitrogen and creatinine. Cells within renal units produced synaptopodin, aquaporins-1 and 2, and Tamm-Horsfall protein. No rejection response was detected in hosts to the cloned renal cells.[61]

FIGURE 68-11  (A) Illustration of renal unit and units retrieved 3 months after implantation. (B) Unseeded control. (C) Seeded with allogeneic control cells. (D) Seeded with cloned cells, showing the accumulation of urine-like fluid.  (Reproduced with permission by Yokoo T, Fukui A, Ohashi T, et al: Xenobiotic kidney organogenesis from human mesencymal stem cells using a growing rodent embryo. J Am Soc Nephrol 17:1026–1034, 2006.)

 

 

TISSUE ENGINEERING A KIDNEY: ENHANCING RECOVERY FROM ACUTE KIDNEY INJURY, GLOMERULONEPHRITIS, AND CONGENITAL KIDNEY DISEASE

Bone Marrow-Derived Stem Cells

The discovery of bone marrow-derived stem cells that possess the ability to differentiate into multiple cell lineages has led to the hypothesis that such cells become incorporated into a number of organs as part of normal processes of cell turnover or organ repair. In support of bone marrow participating in kidney cell turnover, it was demonstrated that Y chromosome-containing cells are among renal interstitial and tubular epithelial cells of female mice into which male-derived bone marrow was transplanted, [62] [63] and that green fluorescent protein (GFP)–labeled cells can be found among mesangial cells in kidneys of mice with GFP-tagged marrow.[64]

Lin and colleagues [65] [66] and Kale and co-workers[67] showed in mice that cells from adult bone marrow are mobilized into the circulation by transient renal ischemia and home specifically to injured regions of the renal tubule. Loss of stem cells following bone marrow ablation resulted in more renal dysfunction postischemia, whereas stem cell infusion reversed this effect. In female mice that received bone marrow from male donors, it was estimated that 11% of proliferating renal epithelial cells postischemic injury were derived from bone marrow, whereas 89% originated from host cells.[66] Morigi and colleagues[68] showed that injection of mesenchymal stem cells of male bone marrow origin protected cisplatin-treated syngeneic female mice from renal functional impairment and injury. Mesenchymal stem cells were shown to engraft in the damaged kidney, differentiate into tubular epithelial cells, and accelerate tubular proliferation. In contrast, bone marrow-derived hematopoetic stem cells failed to exert comparable effects. Ikarashi and associates[69] demonstrated that bone marrow-derived endothelial precursor cells participate in glomerular endothelial cell turnover in rats rendered glomerulosclerotic by injection of anti-Thy-1.1 antibody followed by unilateral nephrectomy. Subsequently, this group showed that infused bone marrow cells contribute to regeneration of endothelial and mesangial cells and ameliorate progressive glomerulosclerosis in the anti-Thy 1.1 unilateral nephrectomy model.[70]

It was shown in male human recipients of kidneys from female donors that following an episode of acute tubular necrosis (ATN), 1% of renal tubules contained cells with Y chromosomes. In contrast, no Y chromosome containing cells were seen in transplanted kidneys of similarly sex-mismatched men who did not develop ATN. This suggests that recipient-derived cells do not routinely repopulate transplanted kidney but may participate in repair.[71]

The studies cited earlier are consistent with a bone marrow source for renal stem cells in adults. However, the results of these studies must be interpreted with caution. As noted by Poulsom,[63] partial repopulation of the kidney with cells that have come to resemble their neighbors is not the same as showing that the organ has acquired new resident stem cells with functional competence as diverse and broad as that expected of an indigenous population. Apropos of the above, Duffield and co-workers[72] failed to find a significant contribution of bone marrow-derived cells to the restoration of epithelial integrity after a renal ischemic insult in mice. Also, Dekel and colleagues[73] failed to demonstrate incorporation of human CD34+CD133+ hematopoetic stem cells into tubular or stromal elements postinjection into ischemic kidneys, or after cotransplantation with embryonic pig kidneys beneath the kidney capsule of immunodeficient mice. In contrast, a few cells engrafted in peritubular capillaries and expressed human CD31, an endothelial marker. In addition, Szczypka and associates[74] failed to find bone marrow-derived cell incorporation into mouse renal tubules after folic acid-induced injury, although rare cells could be detected in glomeruli.

Endothelial Cells

Brodsky and associates[75] demonstrated that injection through the aorta of adult human endothelial cells into athymic nude rats subjected to renal ischemia results in a dramatic protection of the kidney against injury and dysfunction. Morphologic studies demonstrated the engraftment of injected human cells into the rat renal vasculature and suggested that the infusion/engraftment ameliorated the endothelial dysfunction that occurred postischemia. Infusion of skeletal muscle-derived stem cells from adult mice that had been differentiated in vitro along the endothelial lineage has a similar salutary effect on the course of acute ischemic injury in mice.[76]

Mesenchymal Stem Cells

Kutner and associates[77] showed that fluorescently labeled rat mesenchymal stem cells (MSCs) were detected in glomeuli of kidneys from rats with anti-Thy1.1 glomerulonephritis after injection into the renal artery. MSCs accelerated glomerular healing probably related to paracrine growth factor release and not to differentiation into resident glomerular cells. MSCs injected into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close sililarities to human Alport disease, prevented loss of peritubular capillaries and reduced renal fibrosis but did not have an impact on renal function. MSCs localized to kidneys of COL4A3 mice after injection and expressed growth factors, but did not differentiate into renal cells.[78]

Renal Tubule Cell Assist Device

Hemodialysis replaces some filtration functions of the kidney but does not recapitulate endocrine/metabolic activities of renal cells. To address this deficiency, Humes and co-workers[79] have developed a synthetic hemofiltration cartridge and a renal tubule cell assist device (RAD) containing porcine or human[80] cells in an extracorporeal circuit. The RAD replaces both renal filtration and endocrine/metabolic activity. Cells are grown as confluent monolayers along the inner surface of hollow fibers within a standard hemofiltration cartridge. The nonbiodegradability and the pore size of the hollow fibers permit the membranes to act as both scaffolds for the cells and as an immunoprotective barrier. The isolated/expanded renal cells show differentiated renal transport, metabolic and endocrine activity.

The RAD placed in series to a standard hollow fiber hemofiltration cartridge with ultrafiltrate and postfiltered blood connections can reproduce functional relationship between the glomerulus and tubule. Its use in the treatment of acutely uremic dogs increases excretion of ammonia, and enhances glutathione metabolism and 1,25(OH)2D3 production during 24 hours of treatment, and ameliorates gram-negative bacteria-induced septic shock.[81] The evaluation of this device in the treatment of severely ill patients with acute renal failure has been initiated.[82]

CONCLUSIONS

Evidence for participation of renal- or nonrenal-derived cells in the regeneration of damaged nephron components that occurs in the setting of renal injury has prompted studies demonstrating the utility for such cells to enhance recovery following experimental renal injury. [54] [55] [56] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] Notes of caution have been sounded in terms of the interpretation of studies that employ bone marrow cells, [63] [72] [73] [74] and further work is warranted.

There is a need for a tissue-engineered kidney to replace renal function in humans with end-stage disease. However, kidney structure is complex, its embryologic origin is diverse, and glomerular filtration, reabsorption, and secretion depend on precise anatomic relationships between thousands to millions of nephrons and a collecting system. [4] [6] In all probability, a tissue engineering solution for end-stage disease will require growing entirely new nephrons or an entirely new kidney from cells already programmed to differentiate into these structures. Four approaches toward tissue engineering of a new kidney have been reviewed in this chapter.

In terms of their incorporation into a tissue-engineered organ to replace renal function long-term, endocrine/metabolic functions of kidneys are probably less important than filtration reabsorption, and secretion. Active forms vitamin D and erythropoetin, routinely administered to patients with end-stage renal failure, can correct endocrine deficiencies.[6]

The feasibility of integrating new nephrons into a diseased kidney [25] [26] [27] using existing approaches is probably low. Even if a way were found to connect the new nephron units to the host's collecting system, the diseased and fibrotic state of the host kidney would limit the utility of this technique. On the other hand, the generation of new nephrons in response to loss of functional renal mass that occurs in the skate[57] shows that adult vertebrate kidneys are capable of neonephrogenesis. If the conditions under which this process occurs can be understood, perhaps they can be recapitulated in mammals.

For reasons outlined earlier, the use of ES cells to grow a kidney appears to be a technology that will not soon come to fruition. However, the feasibility for use of nuclear transfer-generated cells and tissue as transplants has been demonstrated in a large animal model, the cow.[61] Although such a strategy could not be employed in humans, because ethical considerations require that preimplantation embryos not be developed in vitro beyond the blastocyst stage, these findings may be applicable to engineered adult native cells or human ES cells.[61]

The intra-abdominal transplantation of intact organ precursors preprogrammed to differentiate into a kidney and attract a vasculature (metanephroi) has been employed toward the goal of growing a kidney. Successful transplantation in immunocompetent hosts across defined allogenic barriers is possible without immunosuppression, [18] [19] [29] in part because direct presentation of transplant antigens to host T cells does not occur, [17] [19] [29] MHC antigens are not expressed on transplanted tissues, [18] [21] and the host immune system mounts a T helper 2–biased response when the target organ is of fetal origin.[22]

Unlike developed kidneys, metanephroi are nonvascularized or minimally so. The predominant host origin of the vasculature that does develop in situ under many allogeneic and xenogeneic transplant conditions [24] [31] [34] [38] [60]could serve to ameliorate rejection responses that occur as a result of an incompatibilities between hosts and foreign endothelium and currently represent significant obstacles to the use in humans of organs from other species such as pigs, which in many other ways, represent an ideal kidney donor for humans.[45]

The technologies described in this chapter are not mutually exclusive. One could envision culturing components of the developing kidney derived from cloned animals,[61] separately from one another[58] with or without stem cells,[52] [60] or noncellular biomaterials, [42] [61] and combining them in isolated embryos [59] [60] before implantation into humans to create an unlimited supply of donor organs. No matter how it is accomplished, the availability of such donor organs would result in a paradigm shift in how the world thinks about renal replacement/transplantation: There would be no need to transport kidneys across long distances; transplantation could be done electively at a convenient time; transplantation could be offered to high-risk individuals and could be repeated as needed. Thus, there is considerable therapeutic potential for renal tissue engineering and regeneration.

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