1. Volume 83, Issue 3, Pages 392-403 (March 2013)
Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2- driven inhibin-A and microvesicle-shuttled decorin 
Fabio Sallustio, Vincenzo Costantino, Sharon N. Cox, Antonia Loverre, Chiara Divella, Marco Rizzi, Francesco P. Schena 
Kidney International 
Volume 83, Issue 3, Pages (March 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Characterization of glomerular and tubular adult renal stem/progenitor cells (ARPCs). Cytofluorimetric and immunofluorescence analysis of tubular ARPCs (tARPCs) showed the expression of CD133 (a), CD24 (b), CD44 (c), Oct-4 (d), PAX-2 (e), BMI-1 (f), and CD106 (g). Cytofluorimetric and immunofluorescence analysis of glomerular ARPCs (gARPCs) showed the expression of CD133 (h), CD24 (i), CD44 (j), Oct-4 (k), PAX-2 (l), BMI-1 (m), and CD106 (n). Original view: × 63 (d–f and k–m). FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Tubular, but not glomerular, adult renal stem/progenitor cells (ARPCs) protect renal proximal tubular epithelial cells (RPTECs) from cisplatin-induced toxicity. RPTECs were exposed to cisplatin (2.5μmol/l for 6h; RPTECs+cisp) to simulate an acute damage and then cocultured with or without tubular ARPCs (tARPCs) (a) or glomerular ARPCs (gARPCs) (b) for 1, 4, and 7 days after inducing damage. Viable RPTECs without ARPC coculture at 24h and at 4 days after cisplatin incubation significantly decreased in comparison with healthy cells. Regeneration of damaged RPTECs occurred only in coculture with tARPCs, mainly after 4 days, whereas it was absent in coculture with gARPCs. (c) Four-day coculture experiments showing that tARPCs exposed to cisplatin (2.5μmol/l for 6h; tARPCs+ cisp) did not lead to a decrease in the number of cells. On the contrary, both RPTECs and gARPCs were sensible to the drug-damaging effect. (d) Four-day coculture experiments showing the absence of repairing effect both by coculturing cisplatin-damaged RPTECs with healthy RPTECs or with healthy Human Kidney 2 (HK2) cells. However, this effect was present exclusively with tARPCs. (e) The tARPCs induced RPTEC regeneration only after cisplatin damage stimulation. No anomalous healthy RPTEC number increase was shown in 4-day tARPC cocultures, showing that the recovering effect was specific and strictly dependent on damage. Cell counts were performed with Automatic Cell Counter (Bio-Rad) and confirmed by Burker chamber assays. Histograms represent 15 independent experiments using ARPCs from 15 different subjects; *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Tubular adult renal stem/progenitor cells (tARPCs) prevent cisplatin-induced apoptosis and bring on renal proximal tubular epithelial cells (RPTEC) proliferation. Immunofluorescence and confocal microscopy assay for the detection of apoptotic marker caspase 3 and proliferative marker Ki-67. When RPTECs were exposed to cisplatin (2.5μmol/l for 6h), after 4 days from damage most of the cells became positive for caspase 3 (b, c) and their number decreased markedly compared with control (a). When cocultured with tARPCs, after 4 days from cisplatin incubation, RPTECs proliferated and all became negative for caspase 3 (d), as well as non-damaged RPTECs (a). (e) Quantification of RPTEC caspase 3 expression by calculating the percentage of positive cells in 10 different fields. (f–l) When RPTECs were damaged by cisplatin (2.5μmol/l for 6h), we showed a very low modulation of Ki-67 expression compared with healthy RPTECs (f) either after 12 or 24 or 36h of culture (g, h, and i, respectively). However, when RPTECs were cocultured with tARPCs, Ki-67 expression increased gradually with time at 12, 24, and 36h (j–l) and became more evident after 36h from damage induction (l). (m) Quantification of Ki-67 expression by calculating the percentage of positive cells in 10 different fields. RPTEC Ki-67 expression increased significantly only in coculture with tARPCs at 24 and 36h after damage induction. No. of experiments=5; **P< To-pro-3 counterstains nuclei (blue). Original view: × 40 (a, b, d, f–l); × 63 (c).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Tubular adult renal stem/progenitor cells (tARPCs)-derived supernatants induce renal proximal tubular epithelial cell (RPTEC) regeneration after cisplatin damage. (a) Effect of preconditioned tARPC supernatants, derived from 1-day and 4-day coculture experiments, respectively, on cisplatin-damaged tubular cells. Only 1-day supernatants were able to revert the cisplatin damage. (b) Experiments showing that supernatants coming from cocultures without cisplatin damage (nd-SUPR) were not able to induce RPTEC proliferation. (c) 5-bromo-2-deoxyuridine proliferation assays showing that preconditioned tARPC supernatants, coming from four different coculture experiments of 1 day, increased the proliferation rate of RPTECs surviving to the toxic damage. (d) BrdU proliferation assays showing that preconditioned tARPC supernatants diluted 1:2 with fresh medium did not lead to a significant increase of RPTEC proliferation. Diluted supernatants derived from four different 1-day coculture experiments, using four different tARPC lines. (e) Coculture experiments showing that blocking the Toll-like receptor 2 (TLR2) on tARPCs (2ng/μl TLR2 mAb for 30min; b-tARPCs) resulted in a complete inhibition of tubular cell regeneration. However, tARPCs not treated with blocking antibody were again able to increase the proliferation of tubular cells after cisplatin treatment. Cell counts were performed with Automatic Cell Counter (Bio-Rad) and confirmed by Burker chamber assay. Histograms represent 10 independent experiments using ARPCs from 10 different subjects; *P<0.05; **P<0.005.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Identification of tubular adult renal stem/progenitor cells (tARPC)-secreted molecules able to improve renal proximal tubular epithelial cell (RPTEC) repair. (a) Multiplex enzyme-linked immunosorbent assay (ELISA) on supernatants coming from cocultures to detect molecules responsible for tARPC-mediated repairing effect. Supernatants coming from cisplatin-treated RPTECs/healthy-Human Kidney 2 (HK2) cocultures were used as controls of non-repairing conditions. Among the 13 molecules tested, TRAIL, HB-EGF, INHB-A, IL-6, GRO-α, and FGF2 increased significantly only in the repairing conditions. Cisplatin-treated RPTECs/glomerular ARPCs (gARPCs) coculture conditions have not been included to simplify the graph layout. Histograms represent six independent ARPC samples from six different subjects. (b) FGF2 single ELISA validated the chemokine increase only in the repair condition. The blocking of Toll-like receptor 2 (TLR2) on tARPCs (b-tARPCs) did not decrease FGF2 levels. Supernatants coming from RPTECs/gARPCs and cisplatin-treated RPTECs/healthy RPTECs cocultures were used as controls of non-repairing conditions. (c) Inhibin-A single ELISA validated the chemokine increase only in the repairing condition. The blocking of TLR2 on tARPCs (b-tARPCs) led to a total inhibition of inhibin-A levels. Supernatants coming from RPTECs/gARPCs and cisplatin-treated RPTECs/healthy RPTECs cocultures were used as controls of non-repairing conditions. No. of experiments=10; *P<0.05; **P<0.005.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Tubular adult renal stem/progenitor cell (tARPC) paracrine factors responsible for the repairing effect. (a) Effect of FGF2 on cisplatin-damaged renal proximal tubular epithelial cells (RPTECs). Instead of tARPC coculture, 3ng/μl recombinant FGF2 was used directly on damaged cells. A small, not significant, proliferative effect was observed in 4-day coculture experiments. (b) Coculture experiments (4 days) showing that blocking of inhibin-A in the repairing condition by 1ng/μl specific antibody entirely prevented the regenerative effect of tARPCs. RPTECs+cisp+b-Inh-tARPCs is the condition with tARPCs supernatants containing inhibin-A antibody. (c) Supernatants derived from the repairing condition (SUPR) induced regeneration of cisplatin-damaged RPTECs. However, repairing condition supernatants treated with 1U/ml RNase (r-SUPR) lost their recovering effect. (d–f) Real-time PCR for, respectively, cyclin D1, decorin, and inhibin-A mRNA isolated from coculture-derived microvesicles. Cyclin D1, decorin, and inhibin-A transcripts increased in microvesicles (MVs) from repairing condition but not in MVs from damaged RPTECs without tARPCs. Their increase was inhibited by blocking the Toll-like receptor 2 (TLR2) on tARPCs (RPTEC+cisp+b-tARPC MVs). (g) Real-time PCR for Gadd45b mRNA isolated from coculture-derived microvesicles. Gadd45b transcript increased only in MVs from cisplatin-damaged RPTECs without tARPCs and in MVs from tARPC coculture in which TLR2 was blocked (RPTEC+cisp+b-tARPC MVs). Cell counts were performed with the Automatic Cell Counter (Bio-Rad) and confirmed by Burker chamber assay. Histograms represent 10 independent experiments using ARPCs from 10 different subjects; *P<0.05, **P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

8. Figure 7
Microvesicles (MVs) secreted by tubular adult renal stem/progenitor cells (tARPCs) were incorporated by renal proximal tubular epithelial cells (RPTECs). Representative micrographs showing the uptake of tARPC MVs by RPTECs after 24h of coculture with PKH26-labeled tARPCs. (a) MVs labeled with PKH26 dye and derived from PKH26-labeled tARPCs in coculture were incorporated by RPTECs in the model of cisplatin-induced cell toxicity. (b) On inducing damage on RPTECs by cisplatin and then coculturing PKH26-labeled tARPCs, the MVs incorporated by RPTECs increased notably. (c) Magnification of RPTECs incorporating MVs in the repair condition.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

9. Figure 8
Inhibin-A protein and decorin mRNA were expressed by tubular adult renal stem/progenitor cells (tARPCs) in renal tubules of patients undergoing delayed graft function (DGF). (a) Double-label immunofluorescence for CD133 (red) and inhibin-A (green) in renal tubules of transplanted patients without DGF. No inhibin-A expression was observed. (b–d) Double-label immunofluorescence showing the expression of inhibin-A (green, b, d) by CD133 tARPCs (red, c, d) in renal tubules of patients with DGF. (e–g) Double-label immunofluorescence showing coexpression of Toll-like receptor 2 (TLR2) (green, e, g) and CD133 (red, f, g) by tARPCs in renal tubules of patients with DGF. (h–j) Double-label immunofluorescence showing expression of inhibin-A (red, h, j) and TLR2 (green, i, j) by tARPCs in renal tubules of patients with DGF. (k) Fluorescence in situ hybridization (FISH) mRNA detection on a seriate tissue section of specimen in (b–d) showing decorin mRNA expressed in close proximity to tARPCs. (l) Overlay between CD133 tARPCs (red, c) and decorin mRNA (k) immunofluorescence on seriate sections showing that decorin mRNA was expressed in tARPC cytoplasm, in the extracellular space next to them, and in the tubule lumen. To-pro-3 counterstains nuclei (blue). Original view: (a) × 40; (b–l) × 63.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions