Use of mouse hematopoietic stem and progenitor cells to treat acute kidney injury

Abstract

New and effective treatment for acute kidney injury remains a challenge. Here, we induced mouse hematopoietic stem and progenitor cells (HSPC) to differentiate into cells that partially resemble a renal cell phenotype and tested their therapeutic potential. We sequentially treated HSPC with a combination of protein factors for 1 wk to generate a large number of cells that expressed renal developmentally regulated genes and protein. Cell fate conversion was associated with increased histone acetylation on promoters of renal-related genes. Further treatment of the cells with a histone deacetylase inhibitor improved the efficiency of cell conversion by sixfold. Treated cells formed tubular structures in three-dimensional cultures and were integrated into tubules of embryonic kidney organ cultures. When injected under the renal capsule, they integrated into renal tubules of postischemic kidneys and expressed the epithelial marker E-cadherin. No teratoma formation was detected 2 and 6 mo after cell injection, supporting the safety of using these cells. Furthermore, intravenous injection of the cells into mice with renal ischemic injury improved kidney function and morphology by increasing endogenous renal repair and decreasing tubular cell death. The cells produced biologically effective concentrations of renotrophic factors including VEGF, IGF-1, and HGF to stimulate epithelial proliferation and tubular repair. Our study indicates that hematopoietic stem and progenitor cells can be converted to a large number of renal-like cells within a short period for potential treatment of acute kidney injury.

Fig. 1.

Lin⁻ cells can be sequentially treated to lose hematopoietic marker CD45. A: Lin⁻ cells freshly isolated from mice expressing enhanced yellow fluorescent protein (EYFP) under the kidney-specific cadherin 16 promoter showed no expression of EYFP (left). Cells obtained from whole kidney digestion showed a distinct population of EYFP-expressing cells (right, R2 area). B: nonadherent cells after 1st stage culture expressed CD45 (right) and were transferred for further treatment in cultures. Left: isotype control. C: freshly isolated Lin⁻ cells expressed CD45 (green), but CD45 expression was lost after completion of protein factor treatment in the 3rd stage cultures (red). Isotype control is shown in grey.

Fig. 2.

Treated cells resemble renal phenotypes. Lin⁻ cells isolated from mice expressing EYFP under the kidney-specific cadherin 16 promoter were given 3-stage treatments with protein factors, and the resulting cells were characterized. A: expression of a panel of renal developmentally regulated genes (arrows). No pluripotent gene Oct 4 is expressed. B: detection of Pax2 protein in treated cells. Postnatal day 1 (P1) kidneys were used as positive controls. C: expression of EYFP by flow cytometry analysis. An average of 6.3% cells expressed EYFP after treatments (right, R2 area), whereas untreated Lin⁻ cells show no EYFP expression (left). D: visualization of EYFP under fluorescent microscope. E: cells formed colonies after 3 stages of culture. F: no expression of epithelial tight junction protein zonula occludens-1 (ZO-1) by immunostaining, indicating that treated cells are not fully differentiated epithelial cells. IMCD3 cells were used as controls. Scale bar = 20 μm. 1st, 2nd, and 3rd: first, second, or third stage culture.

Fig. 3.

Treated cells have the potential for tubulogenesis. A: formation of tubular structures after 1 or 2 wk in 3-dimensional cultures. Renal epithelial cell line IMCD3 cells were used as positive controls. Inset: untreated bone marrow cells fail to grow and form tubules. B: integration of treated cells into renal tubules in embryonic kidney organ cultures. Treated cells generated from male Lin⁻ cells were cocultured with fragmented embryonic day 13.5 (E13.5) female kidneys for 1 wk. XZ and YZ images indicate that male cells [red, fluorescent in situ hybridization (FISH) signals] were integrated into female renal tubules and expressed epithelial marker E-cadherin (green, right). Control male mouse embryonic fibroblast (MEF) cells (red, FISH signals) were only detected outside the tubular structure (middle). Cultures of male embryonic kidney fragments were used as positive controls for Y-chromosome FISH analysis (left). C: integration of male treated cells (red, FISH signals) into female postischemic kidneys after injection under the renal capsule (middle). Control male MEF cells (left) failed to integrate into renal tubules. E-cadherin (green) labels epithelial cells. XZ and YZ images are shown. Right: renal tubule (dotted outline) with relatively more integration of male treated cells (red, FISH signals).

Fig. 4.

Histone acetylation enhances cell conversion. A: increased acetylation of histone H3 (Ac-H3) and H4 (Ac-H4) on promoters for cadherin 6 (Cdh6) and for kidney-specific cadherin 16 (Cdh16) during cell conversion. Chromatin immunoprecipitation (ChIP) assay was performed on cells after the 1st and 3rd stage treatments. No apparent changes in trimethylation of histone H3 (Me₃-H3) were detected. B: increased histone acetylation on promoters for renal genes after additional treatment with trichostatin A (TSA). Treated cells with and without TSA treatment were analyzed with ChIP after the 3rd stage cultures. C: increased expression of renal genes in treated cells with TSA treatment analyzed by qRT-PCR. No changes in control 18S rRNA were detected. Values are expressed as a percentage over TSA-untreated cells (means ± SE; n = 3). D: TSA treatment increased EYFP-expressing cells from 6.3 to 37.9%. Treated cells generated from wild-type mice were used as controls (n = 3). A representative flow cytometry analysis is shown. E: generation of a large number of treated cells within 1 wk of culture. Values are means ± SE (n = 3).

Fig. 5.

Transplantation of treated cells accelerates renal recovery from injury. Female mice were given an intravenous injection of 5 × 10⁶ male treated cells 2 h postinjury. A: improvement of renal function with reduction of blood urea nitrogen (BUN; left) and creatinine (right) by injection of treated cells (○, *P < 0.05; n = 5–10). No functional improvement was observed with injection of untreated bone marrow cells (BMC; ●). Sham-operated mice (▴) showed no changes in BUN or creatinine. B: improved renal morphology with treated cells injection. Brush borders of proximal tubules appear more normal (arrows) with treated cells injections. Mice with BMC injection show more casts and debris in tubular lumen (arrowheads). Scale bar = 20 μm. C: detection of male treated cells in tubules of female postischemic kidneys. Y chromosome FISH (red, arrows) indicates the integration of treated cells in tubules labeled with laminin (green). Scale bar = 20 μm. D: stimulation of cell proliferation by treated cell injection. Left: representative images of Ki-67 expression (red) in injured S3 segments of the proximal tubules (green, LTA labeling of the brush border) in kidneys of mice at 3 days after ischemic injury and treated cells or control MBC injection. Scale bars = 20 μm. Right: graph indicates significant increase in Ki-67 expression with treated cell injection at 1 and 3 days postinjury and -repair (*P < 0.05; n = 3–4). E: reduction of tubular apoptosis by treated cell injection. Left: activated caspase 3 (red) in the S3 segment of the proximal tubules labeled with LTA (green). Scale bars = 20 μm. Right: graph indicates a significant decrease in apoptosis with treated cell injection (*P < 0.05; n = 3). Kidneys were analyzed 3 days postinjury and -cell injections.

Fig. 6.

Treated cells produce renotrophic factors to reduce renal injury and increase renal recovery. A: improvement of renal function by injection of conditioned medium (CM). CM collected from treated cells was injected intraperitoneally into mice with renal ischemic injury. CM injection reduces serum BUN (left) and creatinine (right) significantly compared with control Iscove's modified Dulbecco's medium (IMDM) (*P < 0.05; n = 5). Sham-operated mice show no changes in BUN and creatinine. B: decreased tubular apoptosis by CM injection. Apoptotic proximal tubular cells were quantified by activated caspase 3 immunostaining in mice that received CM or control IMDM injections (*P < 0.05; n = 3). C: CM treatment increases bromodeoxyuridine (BrdU) incorporation in primary cultures of renal epithelial cells injured with antimycin A. Cells cultured in DMEM/F12 were used as controls (*P < 0.05; n = 3). D: VEGF, IGF-1, and HGF increase BrdU incorporation in primary cultures of renal epithelial cells injured with antimycin A. Cells cultured in DMEM/F12 were used as controls (*P < 0.05; n = 3). 1×GF, equivalent concentrations of growth factors to those detected in conditioned medium produced by treated cells; 5×GF and 10×GF, concentrations of growth factors that are 5 or 10 times higher than that in the conditioned medium.