Chloroquine augments radiation-induced apoptosis in NPC cells.

<p>A) Cell cycle analysis. Radiation (6 Gy) induces a G2M arrest in all 5 NPC cell lines and the immortalized nasoepithelial cell line NP69. Radiation also induces apoptosis measured by an increase in subG1 in all NPC cell lines except of C666-1. Treatment with chloroquine (20 μM) significantly augments apoptosis in radiation-sensitive NPC cell lines compared to cells radiated only (CNE-2 and HNE-1: P<0.001; CNE-1: P<0.01; HONE-1: P<0.05; Student’s t-test). The data represent the means of three independent experiments and the corresponding standard error. (B) Combined treatment with chloroquine and radiation increases the number of cells with activated caspase-3 in radiation-sensitive NPC-cell lines. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001). (C) Hoechst 33258 staining. Pretreatment with chloroquine before radiation increases the percentage of cells with morphological signs of apoptosis (condensed and fragmented nuclei) in radiation-sensitive NPC cell lines CNE-2 and HNE-1, but not in the radioresistant cell line C666-1 and the immortalized nasoepithelial cell line NP69. Morphologic changes were examined under a fluorescence microscope at 200x magnification, phase contrast images are shown for cell density comparison. Data of all experiments were shown at 72h after radiation.</p

Knock-down of ATG3, ATG5, ATG6 or ATG7 by siRNA substitutes for the enhancing effect of chloroquine on radiation-induced apoptosis in NPC cells.

<p>Silencing of ATG3, ATG5, ATG6 or ATG7 by specific siRNA enhances radiation-induced apoptosis to a similar extent as chloroquine in radiation-sensitive NPC cell lines treated with scrambled siRNA as shown by an increase in subG1-DNA-content (A) or increase of active caspase-3 (B) both measured by flow cytometry. After transfection the cells were treated as described before. Quantitative data are reported as means ± S.E.M. (triplicate samples) (Student ‘s t- test; <b>*</b> = P<0.05).</p

Chloroquine blocks radiation-induced autophagy in NPC cells.

<p>(A) Immunoblot for LC-I and LC-II. Preincubation with chloroquine before radiation increases expression of LC-II in radiation-sensitive NPC cells 8h post treatment (B) Immunofluorescence for autophagic vacuoles. Combined treatment of chloroquine and radiation increases the formation of autophagic vacuoles 8h post treatment in cell lines CNE-2 and HNE-1. Autophagic vacuoles were examined under a fluorescence microscope at 200x and 400x magnification. (C) Flow cytometric analysis of autophagic vacuoles. Combined treatment of chloroquine and radiation increases the number of cells with autophagic vacuoles 8h post treatment in radiation-sensitive NPC cell lines. (Student’s t- test; <b>*</b> = P<0.05; <b>**</b> = P<0.01; <b>***</b> = P<0.001) (D) Transmission electron microscopy. Photomicrographs show normal nuclear and mitochondrial morphologies in untreated cells and cells treated with chloroquine. Especially, in irradiated CNE-2 and HNE-1 cells the number of autophagosomes is significantly increased 8h following radiotherapy and further augmented by pretreatment with chloroquine.</p

Mesenchymal Stem Cells from Rats with Chronic Kidney Disease Exhibit Premature Senescence and Loss of Regenerative Potential

<div><p>Mesenchymal stem cell (MSC) transplantation has the potential for organ repair. Nevertheless, some factors might lessen the regenerative potential of MSCs, e.g. donor age or systemic disease. It is thus important to carefully assess the patient's suitability for autologous MSC transplantation. Here we investigated the effects of chronic kidney disease (CKD) on MSC function. We isolated bone marrow MSCs from remnant kidney rats (RK) with CKD (CKD-RK-MSC) and found signs of premature senescence: spontaneous adipogenesis, reduced proliferation capacity, active senescence-associated-β-galactosidase, accumulation of actin and a modulated secretion profile. The functionality of CKD-RK-MSCs <i>in vivo</i> was tested in rats with acute anti-Thy1.1-nephritis, where healthy MSCs have been shown to be beneficial. Rats received healthy MSCs, CKD-RK-MSC or medium by injection into the left renal artery. Kidneys receiving healthy MSCs exhibited accelerated healing of glomerular lesions, whereas CKD-RK-MSC or medium exerted no benefit. The negative influence of advanced CKD/uremia on MSCs was confirmed in a second model of CKD, adenine nephropathy (AD). MSCs from rats with adenine nephropathy (CKD-AD-MSC) also exhibited cellular modifications and functional deficits <i>in vivo</i>. We conclude that CKD leads to a sustained loss of <i>in vitro</i> and <i>in vivo</i> functionality in MSCs, possibly due to premature cellular senescence. Considering autologous MSC therapy in human renal disease, studies identifying uremia-associated mechanisms that account for altered MSC function are urgently needed.</p></div

Classification of stages of CKD in rats.

<p>All remnant kidney rats (“RK”) were sacrificed after a renal disease duration >17 weeks (mean life expectancy of a healthy lab rat ≈75 weeks). All animals had elevated serum urea and serum creatinine levels at the time of sacrifice. We chose s-urea as a marker for overall uremia and calculated creatinine-clearance to divide the animals into two groups: rats with serum urea >20 mmol/l+creatinine-clearance <1.0 l/24 h (CKDsev-RK) and rats with serum urea ≤20 mmol/l and creatinine-clearance >1.0 l/24 h (CKDmod-RK). Rats with adenine nephropathy (4-week diet containing 0.75% adenine, “AD”) also showed a markedly decreased renal function (CKDsev-AD).</p

Analysis of renal function and histology on Day 4 and Day 6 of anti-Thy1.1-nephritis.

<p>(A) Experimental design. (B–D) Comparison of rats that had anti-Thy1.1-nephritis and received H-MSC (“Healthy”, n = 7), TG-MSC (“TG”, n = 8), CKDmod-RK-MSC (“CKDmod-RK”, n = 6) or control DMEM (“Medium”, n = 10) injected into the left renal artery on Day 2 after disease induction and were analysed on Day 4. (E) Experimental design. (F–H) Comparison of rats that had anti-Thy1.1-nephritis and received H-MSC (“Healthy”, n = 7), TG-MSC (“TG”, n = 7), CKDmod-RK-MSC (“CKDmod-RK”, n = 6) or control DMEM (“Medium”, n = 9) injected into the left renal artery on Day 2 after disease induction and were analysed on Day 6. * p<0.05; ** p<0.01; *** p<0.001. All data: mean ± SD.</p

Analysis of renal function and histology on Day 6 of anti-Thy1.1-nephritis, 4 days after treatment with CKDsev-AD-MSC.

<p>Left kidneys of rats with acute Thy1.1 nephritis that were treated with CKDsev-AD-MSC did not show significant differences in mesangiolysis (E), glomerular collagen I (F) or α-SMA- positive glomerular area (G) compared to untreated right control kidneys on Day 6. * p<0.05; ** p<0.01; *** p<0.001. All data: mean ± SD.</p

Premature senescence in MSCs from remnant kidney rats.

<p>(A) Quantification of enzymatic staining for SA-β-gal in H-MSC (n = 7), CKDmod-RK-MSC (n = 7), CKDsev-RK-MSC (n = 8) and MSCs from healthy old donors (n = 4). Significantly more CKDsev-RK-MSC contain active SA-β-gal compared to H-MSC or MSCs from old donors (p = 0.002 and p = 0.036, respectively). Representative pictures of SA-β-gal staining in H-MSC and CKDsev-RK-MSC are shown (magnification 200×). (B) SA-β-gal activity is also significantly higher in CKDsev-RK-MSC (n = 5) than in H-MSC (n = 6) or MSCs from old donors (n = 6). (C) Expression of Gas7 mRNA (growth-arrest-specific protein 7) in H-MSC (n = 5), CKDmod-RK-MSC (n = 5) and CKDsev-RK-MSC (n = 5). CKDmod-RK-MSC produce significantly more Gas7 mRNA compared to H-MSC (p = 0.01). (D) Cell population doubling time (Passage 2) is significantly higher in all CKD-MSCs (CKDmod-RK (n = 15), CKDsev (n = 4)) than in H-MSC (n = 6), TG-MSC (n = 7) or MSCs from old donors (n = 4). (E) Western blots demonstrate that CKDmod-RK-MSC contain significantly more actin than H-MSC or MSCs from old donors (H-MSC n = 7, TG-MSC n = 4, CKDmod-RK-MSC n = 6, CKDsev-RK-MSC n = 6, healthy old controls (>9 months) n = 5). (F) CKDsev-RK-MSC in Passage 3 spontaneously differentiate into adipocytes (native cell culture image, magnification 200×). Lipid vacuoles are visualized by oil red O staining. Magnification 200×. (G) RT-qPCR for markers of adipogenesis (adiponectin, peroxisome proliferator-activated receptor γ (PPARγ), lipoprotein lipase (LLIPA)) in H-MSC (n = 11), CKDmod-RK-MSC (n = 6), CKDsev-RK-MSC (n = 4) and healthy MSCs from old donors (n = 6). mRNA expression of PPARγ and LLIPA is significantly increased in CKDsev-RK-MSC vs. H-MSC (p = 0.008 and p = 0.03, respectively). All MSCs in Passage 3. * p<0.05; ** p<0.01; *** p<0.001. All data: mean ± SD.</p

In vitro characterization of MSCs from rats with adenine nephropathy.

<p>(A) CKDsev-AD-MSC have a decreased proliferation capacity (cell population doubling time 116.1±57.7 h (n = 6) vs. 43±8.2 h in H-MSC (n = 5); p = 0.02). (B) CKD-sev-AD-MSC (n = 8) express significantly more PDGF-A and PDGF-C than H-MSC (n = 9) (p = 0.008 and p = 0.005 respectively). CKDsev-AD-MSC contained active SA-β-gal (C) and in some cases lipid vacuoles (D). * p<0.05; ** p<0.01; *** p<0.001. All data: mean ± SD.</p

Secretory phenotype of MSCs from healthy and CKD donors.

<p>(A) ELISA for activated TGF-β in conditioned medium from MSC in Passage 2 or 3. Supernatants of CKDmod-RK-MSC (n = 8) contained less TGF-β compared to healthy wildtype (healthy, n = 6) or healthy transgenic (TG, n = 4) MSC. Culture medium was used as control (n = 2) (CKDsev-RK-MSC, n = 3). (B) PDGF- and PDGF-receptor expression in H-MSC (n = 9), CKDmod-RK-MSC (n = 19) and CKDsev-RK-MSC (n = 11): PDGF-A and PDGF-C expression is significantly higher in CKDsev-RK-MSC compared to H-MSC. CKDmod-RK-MSC also express significantly more PDGF-A than H-MSC. (C) RT-qPCR for collagen types I and III in NRK49-F fibroblasts stimulated with conditioned medium from healthy MSC (H-MSC) or CKD-MSC for 24 h (n = 3, each). Supernatants from CKDsev-RK-MSC induced a significant increase of collagen type I production in NRK cells. * p<0.05. All data: mean ± SD.</p