Therapeutic effects of human mesenchymal stem cells in Wistar-Kyoto rats with anti-glomerular basement membrane glomerulonephritis.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) have become a promising therapeutic approach in many clinical conditions. The hypothesis that MSCs can provide a potential therapy for human anti-glomerular basement membrane (GBM) glomerulonephritis (GN) was tested. METHODS: Nephrotoxic serum nephritis was induced in Wistar-Kyoto rats on day 0. Groups of animals were given either human MSCs (hMSCs, 3×10(6)) or vehicle by intravenous injection on day 4; all rats were sacrificed at either day 7 or day 13. RESULTS: Fluorescently labeled hMSCs were localized in glomeruli and tubulointerstitium 5 h after hMSC administration and persisted until 48 h, but hMSCs were barely detectable after 7 days. hMSC-treated rats had decreased kidney weight, proteinuria, and glomerular tuft area at each time point. The serum creatinine level and degree of glomerular crescent formation were decreased by hMSC treatment on day 13. ED1-positive macrophages, CD8-positive cells, and TUNEL-positive apoptotic cells in glomeruli were reduced by hMSC treatment on day 7, and this trend in apoptotic cells persisted to day 13. Renal cortical mRNA for TNF-α, IL-1β, and IL-17, and the serum IL-17A level were decreased, whereas renal cortical mRNA for IL-4 and Foxp3 and the serum IL-10 level were increased in the MSC-treated group on day 7. Collagen types I and III and TGF-β mRNA were decreased by hMSC treatment on day 13. CONCLUSION: The present results demonstrated that anti-inflammatory and immunomodulatory effects were involved in the mechanism of attenuating established experimental anti-GBM GN by hMSCs. These results suggest that hMSCs are a promising therapeutic candidate for the treatment of anti-GBM GN

Double immunostaining for ED1 or RECA-1 with IL-1β in the study groups.

<p>Kidney sections were stained using two-color immunohistochemistry with ED1 or RECA-1 stained red and IL-1β stained brown in an HBSS-treated rat with nephritis (<b>a,c</b>) and an MSC-treated rat with nephritis (<b>b,d</b>). A large number of ED1+ macrophages shows double staining for IL-1β in the WKY-HBSS rats (circles) (<b>a</b>). RECA-1 is partially double-stained with IL-1β in both the WKY-HBSS rats and the WKY-MSC rats (squares) (<b>c,d</b>). Original magnifications, x1000.</p

Detection of CM-DiI-labeled MSCs in lungs, liver, and spleen.

<p>Representative pictures of frozen tissue sections of lungs, liver, and spleen of an MSC-treated rat at 5 h after CM-DiI-labeled hMSC administration. The presence of CM-DiI-labeled hMSCs (red stained cells; arrows) was examined by counterstaining with actin antibody (green staining). Original magnifications, x200 (a–f).</p

Detection of CM-DiI-labeled MSCs in kidneys.

<p>Representative pictures of frozen tissue sections of kidney of an MSC-treated rat at 5 h (a–f) or 24 h (g-i) after CM-DiI-labeled hMSC administration. The presence of CM-DiI-labeled hMSCs (red stained cells; arrows) was examined by counterstaining with actin antibody (green staining). Original magnifications, x400 (a–f), x200 (g–i). Asterisk (*) represents glomerulus.</p

Immunohistochemistry for TUNEL, CD8, and ED-1 in the study groups.

<p>Representative pictures stained with immunohistochemistry for TUNEL (a, d), CD8 (b, e), and ED-1 (c, f) of an HBSS-treated rat (a–c) and an MSC-treated rat (d–f) on day 7. Kidney sections were stained using two-color immunohistochemistry with ED1 or podocin stained red and TUNEL stained brown in an HBSS-treated rat (g,h). Some portion of the TUNEL+ cells is stained with ED1+ (arrows) and podocin + (arrowhead), Original magnifications, x1000.</p

Results of immunohistochemistry.

<p>Data are means ± SEM.</p><p>Mann-Whitney test: *P<0.05, ****P<0.0001 vs. WKY rats with the same duration of HBSS treatment.</p>a<p>The rats were treated with either HBSS or MSC at day 4, and sacrificed at day 7.</p>b<p>The rats were treated with either HBSS or MSC at day 4, and sacrificed at day 13.</p><p>Semiquantitative assessment of the ED1 score and quantitative assessment of TUNEL+ and CD8+ cells per glomerular cross-section in each group. Each group contained 10–14 rats, and 50 glomeruli per rat were evaluated in a blind fashion.</p

Effects of MSCs on urinary collagen levels in the study groups.

<p>Urinary collagen levels of each group measured as described in materials and methods. Data are expressed as means ± SEM. Mann-Whitney test: *p<0.05; HBSS-treated rats with nephritis vs. MSC-treated rats with nephritis.</p

Light microscopic findings in the study groups.

<p>Representative pictures stained with silver on day 7 (<b>a</b>–<b>d</b>) and day 13 (<b>e</b>–<b>h</b>) in an HBSS-treated rat with nephritis (<b>a, b, e, f</b>) and an MSC-treated rat with nephritis (<b>c, d, g, h</b>). The glomeruli on day 7 show severe fibrinoid necrosis and cellular crescent formation (arrows) (<b>a, b</b>). The crescentic glomeruli have started to transform from cellular to fibrocellular (arrows), and there is some segmental glomerulosclerosis (arrowheads) on day 13 (<b>e, f</b>). hMSC treatment reduces glomerular hypertrophy on day 7 (<b>c, d</b>) and day 13 (<b>g, h</b>). hMSC treatment also reduces crescent formation on day 13 (<b>g, h</b>). Original magnifications, x200 (a, c, e, g), x1000 (b, d, f, h).</p

Results of real-time RT-PCR for profibrogenic genes in renal cortex on day 13.

<p>Real-time RT-PCR for profibrogenic genes. Data are means±SEM.</p><p>Mann-Whitney test: *<i>P</i><0.05, **<i>P</i><0.01 <i>vs.</i> WKY rats with HBSS treatment.</p><p>The values were normalized to the GAPDH values and then expressed as relative quantification.</p