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Regeneration and experimental orthotopic transplantation of a bioengineered kidney

Journal name:
Nature Medicine
Volume:
19,
Pages:
646–651
Year published:
DOI:
doi:10.1038/nm.3154
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Accepted
Published online
Corrected online

Abstract

Approximately 100,000 individuals in the United States currently await kidney transplantation, and 400,000 individuals live with end-stage kidney disease requiring hemodialysis. The creation of a transplantable graft to permanently replace kidney function would address donor organ shortage and the morbidity associated with immunosuppression. Such a bioengineered graft must have the kidney's architecture and function and permit perfusion, filtration, secretion, absorption and drainage of urine. We decellularized rat, porcine and human kidneys by detergent perfusion, yielding acellular scaffolds with vascular, cortical and medullary architecture, a collecting system and ureters. To regenerate functional tissue, we seeded rat kidney scaffolds with epithelial and endothelial cells and perfused these cell-seeded constructs in a whole-organ bioreactor. The resulting grafts produced rudimentary urine in vitro when perfused through their intrinsic vascular bed. When transplanted in an orthotopic position in rat, the grafts were perfused by the recipient's circulation and produced urine through the ureteral conduit in vivo.

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  1. Perfusion decellularization of whole rat kidneys.
    Figure 1: Perfusion decellularization of whole rat kidneys.

    (a) Time-lapse photographs of a cadaveric rat kidney undergoing antegrade renal arterial perfusion decellularization. Shown are a freshly isolated kidney (left) and the same kidney after 6 h (middle) and 12 h (right) of SDS perfusion. Ra, renal artery; Rv, renal vein; U, ureter. (b) Representative corresponding Movat's pentachrome–stained sections of rat kidney during perfusion decellularization (black arrowheads indicate the Bowman's capsule). Scale bars, 250 μm (main images); 50 μm (insets). (c) Representative immunohistochemical stains of cadaveric rat kidney sections showing the distribution of elastin (black arrowheads indicate elastic fibers in the tunica media of cortical vessels), collagen IV and laminin (black arrowheads indicate the glomerular basement membranes). Scale bars, 250 μm (main images); 50 μm (insets). (d) Corresponding sections of decellularized rat kidney tissue after immunohistochemical staining for elastin, collagen IV and laminin confirming the preservation of extracellular matrix proteins in the absence of cells. Black arrowheads indicate the preserved vascular and glomerular basement membranes. Scale bars, 250 μm (main images); 50 μm (insets). (e) Transmission electron micrograph (TEM) of a cadaveric rat glomerulus showing capillaries (C), the mesangial matrix (M) and podocytes (P) surrounded by Bowman's capsule (BC). Scale bar, 10 μm. (f) TEM of decellularized rat glomerulus showing acellularity in decellularized kidneys with preserved capillaries, mesangial matrix and Bowman's space encapsulated by Bowman's capsule. Scale bar, 10 μm. (g,h) Biochemical quantification of DNA and total collagen in cadaveric and decellularized rat kidney tissue showing a reduction of DNA content and a preservation of collagen after perfusion decellularization. Data are shown as the mean ± s.d. NS, not significant. Statistical significance was determined by Student's t test.

  2. Perfusion decellularization of porcine and human kidneys.
    Figure 2: Perfusion decellularization of porcine and human kidneys.

    (a) Photograph of cadaveric (left) and decellularized (right) human kidneys suggesting that perfusion decellularization of rat kidneys can be upscaled to generate acellular kidney ECMs of clinically relevant size. Ra, renal artery; Rv, renal vein; U, ureter. (b) Corresponding pentachrome staining for decellularized human kidneys (black arrowheads indicate acellular glomeruli). Scale bar, 250 μm; insets are ×40 magnification. (c) Photograph of cadaveric (left) and decellularized (right) porcine kidneys. (d) Corresponding pentachrome staining for decellularized porcine kidneys (black arrowheads indicate acellular glomeruli). Scale bar, 250 μm; insets are ×40 magnification.

  3. Cell seeding and whole-organ culture of decellularized rat kidneys.
    Figure 3: Cell seeding and whole-organ culture of decellularized rat kidneys.

    (a) Schematic of a cell-seeding apparatus enabling endothelial cell seeding through port A attached to the renal artery (Ra) and epithelial cell seeding through port B attached to the ureter (U) while negative pressure in the organ chamber is applied to port C, thereby generating a transrenal pressure gradient. (b) Schematic of a whole-organ culture in a bioreactor enabling tissue perfusion through port A attached to the renal artery and drainage to a reservoir through port B. K, kidney. (c) Cell-seeded decellularized rat kidney in whole-organ culture. (d) Fluorescence micrographs of a re-endothelialized kidney constructs. CD31-positive (red) and DAPI-positive HUVECs line the vascular tree across the entire graft cross section (image reconstruction, left) and form a monolayer to glomerular capillaries (right; white arrowheads indicate endothelial cells). Scale bar, 500 μm (left); 50 μm (right). (e) Fluorescence micrographs of re-endothelialized and re-epithelialized kidney constructs showing engraftment of podocin-expressing cells (green) and endothelial cells (CD31 positive; red) in a glomerulus (left; white arrowheads indicate Bowman's capsule and the asterisk indicates the vascular pole); engraftment of Na/K-ATPase–expressing cells (green) in a basolateral distribution in tubuli resembling proximal tubular structures with the appropriate nuclear polarity (left middle); engraftment of E-cadherin–expressing cells in tubuli resembling distal tubular structures (right middle); and a three-dimensional reconstruction of a re-endothelialized vessel leading into a glomerulus (white arrowheads indicate Bowman's capsule, and the asterisk indicates the vascular pole). T, tubule; Ptc, peritubular capillary. Scale bar, 25 μm (left); 10 μm (middle and right). (f) Image reconstruction of an entire graft cross section confirming engraftment of podocin-expressing epithelial cells (left) and representative immunohistochemical staining of a reseeded glomerulus showing podocin expression (right). Scale bar, 500 μm (left); 50 μm (right). (g) Nephrin expression in regenerated glomeruli. Scale bar, 50 μm. (h) Aquaporin-1 expression in regenerated proximal tubular structures (left); Na/K-ATPase expression in regenerated proximal tubular epithelium (middle left); E-cadherin expression in regenerated distal tubular epithelium (middle right); and β-1 integrin expression in a regenerated glomerulus (right). Scale bars, 50 μm. (i) Representative TEM of a regenerated glomerulus showing a capillary with red blood cells (R) and foot processes along the glomerular basement membrane (black arrowheads; left) and TEM of a podocyte (P) adherent to the glomerular basement membrane (black arrowheads; right). BC, Bowman's capsule. Scale bars, 2 μm. (j) Scanning electron micrograph of a glomerulus (white arrowheads) in a regenerated kidney graft cross section. The asterisk indicates a vascular pedicle. Scale bar, 10 μm.

  4. In vitro function of bioengineered kidney grafts and orthotopic transplantation.
    Figure 4: In vitro function of bioengineered kidney grafts and orthotopic transplantation.

    (a) Photograph of a bioengineered rat kidney construct undergoing in vitro testing. The kidney is perfused through the cannulated renal artery (Ra) and renal vein (Rv), while urine is drained through the ureter (U). The white arrowhead indicates the urine-air interface in the drainage tubing. (b) Bar graph summarizing average urine flow rate (ml min−1) for decellularized, cadaveric and regenerated kidneys perfused at 80 mm Hg and regenerated kidneys perfused at 120 mm Hg (regenerated*). Decellularized kidneys showed a polyuric state while regenerated constructs were relatively oliguric compared to cadaveric kidneys. (c) Bar graph showing the average creatinine clearance in cadaveric, decellularized and regenerated kidneys perfused at 80 mm Hg and regenerated kidneys perfused at 120 mm Hg (regenerated*).With increased perfusion pressure creatinine clearance in regenerated kidneys improved. (d) Bar graph showing vascular resistance of cadaveric decellularized and regenerated kidneys showing an increase in vascular resistance with decellularization and partial recovery in regenerated kidneys. Error bars, s.d. (b–d). (e) Photograph of rat peritoneum after laparotomy, left nephrectomy and orthotopic transplantation of a regenerated left kidney construct. The recipient left renal artery and left renal vein are connected to the regenerated kidney's renal artery and vein. The regenerated kidney's ureter remained cannulated for collection of urine production after implantation (left). Right, photograph of the transplanted regenerated kidney construct after unclamping of left renal artery and renal vein showing homogeneous perfusion of the graft without signs of bleeding. (f) Composite histologic image of a transplanted regenerated kidney confirming perfusion across the entire kidney cross section and the absence of parenchymal bleeding. Scale bar, 500 μm.

Change history

Corrected online 18 October 2013

In the version of this article initially published, the human kidneys in Figure 2a,b were incorrectly described as porcine and the porcine kidneys in Figure 2c,d were incorrectly described as human in the figure legend. The errors have been corrected in the HTML and PDF versions of the article.

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Author information

Affiliations

  1. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Jeremy J Song,
    • Jacques P Guyette,
    • Sarah E Gilpin,
    • Gabriel Gonzalez,
    • Joseph P Vacanti &
    • Harald C Ott

  2. Harvard Medical School, Boston, Massachusetts, USA.

    • Jeremy J Song,
    • Jacques P Guyette,
    • Sarah E Gilpin,
    • Gabriel Gonzalez,
    • Joseph P Vacanti &
    • Harald C Ott

  3. Department of Surgery, Division of Pediatric Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Joseph P Vacanti

  4. Department of Surgery, Division of Thoracic Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Harald C Ott

Contributions

H.C.O. conceived, designed and oversaw all of the studies, collection of results, interpretation of the data and writing of the manuscript and was also responsible for the primary undertaking, completion and supervision of all experiments. J.J.S. and J.P.G. performed animal surgeries, conducted decellularization and whole-organ culture experiments and performed in vitro and in vivo testing. S.E.G. was responsible for cell culture, preparation of cell suspensions and matrix characterization. G.G. characterized fetal lung cells and scaffolds and regenerated constructs using various imaging techniques. J.P.V. provided input on tissue engineering aspects and reviewed the manuscript.

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The authors declare no competing financial interests.

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