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

Epidermal Growth Factor Receptor Inhibition with Erlotinib Partially Prevents Cisplatin-Induced Nephrotoxicity in Rats

<div><p>The effects of blocking the epidermal growth factor receptor (EGFR) in acute kidney injury (AKI) are controversial. Here we investigated the renoprotective effect of erlotinib, a selective tyrosine kinase inhibitor that can block EGFR activity, on cisplatin (CP)-induced AKI. Groups of animals were given either erlotinib or vehicle from one day before up to Day 3 following induction of CP- nephrotoxicity (CP-N). In addition, we analyzed the effects of erlotinib on signaling pathways involved in CP-N by using human renal proximal tubular cells (HK-2). Compared to controls, rats treated with erlotinib exhibited significant improvement of renal function and attenuation of tubulointerstitial injury, and reduced the number of apoptotic and proliferating cells. Erlotinib-treated rats had a significant reduction of renal cortical mRNA for profibrogenic genes. The Bax/Bcl-2 mRNA and protein ratios were significantly reduced by erlotinib treatment. <i>In vitro</i>, we observed that erlotinib significantly reduced the phosphorylation of MEK1 and Akt, processes that were induced by CP in HK-2. Taken together, these data indicate that erlotinib has renoprotective properties that are likely mediated through decreases in the apoptosis and proliferation of tubular cells, effects that reflect inhibition of downstream signaling pathways of EGFR. These results suggest that erlotinib may be useful for preventing AKI in patients receiving CP chemotherapy.</p></div

Effects of erlotinib on gene expression levels for fibrogenic molecules, proinflammatory cytokines, apoptosis-regulatory molecules, and EGFR ligands in the study groups.

<p>Real-time RT-PCR for genes encoding fibrogenic molecules (a), proinflammatory cytokines (b), apoptosis-regulatory molecules (c), and EGFR ligands including proHB-EGF and TGF-α (d) in each group. The horizontal dotted lines show the expression levels of the NC rats. Data are expressed as mean ± SEM (n = 5, 14, and 14 for the NC rats, the CP+V rats, and the CP+E rats, respectively). The values were normalized to the GAPDH transcript levels and then expressed as relative quantification. Mann–Whitney test: *P<0.05, **P<0.01, NS, not significant vs. CP+V.</p

Effects of erlotinib on biochemical parameters of the study groups.

<p>Data are mean ± SEM.</p><p>Mann–Whitney test: *P<0.05, **P<0.01, vs. NC; #P <0.05, vs. CP+V.</p><p>Abbreviations:NC, normal control rats; CP+V, rats with cisplatin-induced nephrotoxicity treated with vehicle; CP+E, rats with cisplatin-induced nephrotoxicity treated with erlotinib; BW, body weight; KW, kidney weight; KW/BW ratio (%), kidney weight (g) x 100/body weight (g); Cr, creatinine; NAG, N-acetyl-β-D-glucosaminidase.</p><p>Effects of erlotinib on biochemical parameters of the study groups.</p

Light microscopic findings in the study groups.

<p>Representative images of tissues stained with (a–f) PAS or (g–i) Masson trichrome in a NC rat (a, d, g), a CP+V rat (b, e, h), and a CP+E rat (c, f, i). Original magnifications: (a–c) × 100, (d–i) × 400.</p

Immunohistochemistry for PCNA, ED1, TUNEL, and caspase-3 in the study groups.

<p>Representative pictures stained for (a–c) PCNA, (d–f) ED1, (g–i) TUNEL, and (j–l) caspase-3 in a NC rat (a, d, g, j), a CP+V rat (b, e, h, k), and a CP+E rat (c, f, i, l). Original magnifications: x 400.</p

Effects of erlotinib on pro-apoptotic and anti-apoptotic protein in the study groups.

<p>Representative western blot analysis for Bax, Bcl-2 and GAPDH (a). Densitometric analysis of western blot for Bax (b), Bcl-2 (c), and Bax/Bcl2 ratio (d) was performed using an image analyzer in each group. Data are expressed as mean ± SEM (n = 5, 14, and 14 for the NC rats, the CP+V rats, and the CP+E rats, respectively). The values were expressed after normalization to GAPDH expression and depicted as the percentage change from the average of normal controls. Mann-Whitney test: *P<0.01, **P<0.01 vs. NC, #P<0.05 vs. CP+V.</p

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