Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells

Abstract

Ischemia causes kidney tubular cell damage and abnormal renal function. The kidney is capable of morphological restoration of tubules and recovery of function. Recently, it has been suggested that cells repopulating the ischemically injured tubule derive from bone marrow stem cells. We studied kidney repair in chimeric mice expressing GFP or bacterial β-gal or harboring the male Y chromosome exclusively in bone marrow-derived cells. In GFP chimeras, some interstitial cells but not tubular cells expressed GFP after ischemic injury. More than 99% of those GFP interstitial cells were leukocytes. In female mice with male bone marrow, occasional tubular cells (0.06%) appeared to be positive for the Y chromosome, but deconvolution microscopy revealed these to be artifactual. In β-gal chimeras, some tubular cells also appeared to express β-gal as assessed by X-gal staining, but following suppression of endogenous (mammalian) β-gal, no tubular cells could be found that stained with X-gal after ischemic injury. Whereas there was an absence of bone marrow-derived tubular cells, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. Upon i.v. injection of bone marrow mesenchymal stromal cells, postischemic functional renal impairment was reduced, but there was no evidence of differentiation of these cells into tubular cells of the kidney. Thus, our data indicate that bone marrow-derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult. It is likely that intrinsic tubular cell proliferation accounts for functionally significant replenishment of the tubular epithelium after ischemia.

Pericytes and Perivascular Fibroblasts Are the Primary Source of Collagen-Producing Cells in Obstructive Fibrosis of the Kidney

“…9 In brief, ten million BM cells from male donors in 200 l of PBS were injected into the lateral tail vein of lethally irradiated (1000 Rads over 30 minutes) isogenic female recipients and chimerism was confirmed after 6 weeks by fluorescent in situ hybridization of buffy coat cells according to the method described previously. 9 Briefly, methanol-acetic acid fixed buffy coat cells were incubated with 1 mol/L sodium thiocyanate (80°C, 10 minutes), then digested with proteinase K (37°C, 15 minutes), incubated with 0.1 mol/L HCl (37°C, 10 minutes), fixed with 4% paraformaldehyde, dehydrated, and airdried. Fluorescent in situ hybridization mouse Y chromosome-specific probe Star*Fish (Cambio, Cambridge, UK) was added in the manufacturer's buffer.…”

“…9,10 Wounded skin was dissected and cut transversely to expose the re-epithelialization of epidermis and the subepidermal granulation tissue and fixed as above. Primary antibodies against the following proteins were used for immunolabeling: ␣SMA-Cy3 (1:200, clone 1A4, Sigma), CD14, CD11b, CD11c, CD45, CD16/32, MHC class II (I-A/I-E) (1:200, eBioscience), CD34, Ly6C, (Pharmingen) 7/4, (ABD-Serotec), Ki-67 (1:200, clone SP6, Fisher), laminin (1:100, Sigma), NG2 (1:500), platelet-derived growth factor (PDGF) receptor beta (PDGFR␤ ͓1:500͔, podocin ͓1:200͔; S100A4 ͓1:200͔, DAKO, and also Abcam), and WT1 (1:100, Santa Cruz).…”

“…Fluorescent conjugated affinity purified secondary antibody labeling (1:400 to 1:800, Jackson), mounting with Vectashield/4,6-diamidino-2-phenylindole (DAPI), image capture and processing were performed as previously described. 9,10 To study proliferation of coll1a1-GFP cells, 5-bromo-2Ј-deoxyuridine (BrdU), 50 g/g was injected i.p. 2 hours before sacrifice.…”

“…Quantification of specific cells in tissue sections was performed as previously described. 9 In brief, sections were colabeled with DAPI, and Coll1a1-GFPϩ cells were identified by blue and green nuclear colocalization; ␣SMAϩ, NG2ϩ, or PDGFR␤ ϩ cells were identified by greater than 75% of the cell area immediately surrounding nuclei (detected by DAPI) staining positive with Cy3 fluorescence indicative of the antigen expression; Ki-67ϩ or BrdUϩ cells were identified by positive nuclear staining for Cy3 fluorescence. Specific cells were counted in 10 cortical interstitial fields randomly selected at original magnification ϫ400 per mouse.…”

TMIGD1 Is a Novel Adhesion Molecule That Protects Epithelial Cells from Oxidative Cell Injury

“…15 Cell adhesion plays a prominent role in these regenerative processes. 16 Recently, we identified immunoglobulin (Ig) and proline-rich receptor-1 (IGPR-1) as a novel cell adhesion molecule encoded by transmembrane and Ig domain-containing 2 (TMIGD2).…”