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