Inhibin-A and Decorin Secreted by Human Adult Renal Stem/Progenitor Cells Through the TLR2 Engagement Induce Renal Tubular Cell Regeneration

Abstract

Acute kidney injury (AKI) is a public health problem worldwide. Several therapeutic strategies have been made to accelerate recovery and improve renal survival. Recent studies have shown that human adult renal progenitor cells (ARPCs) participate in kidney repair processes, and may be used as a possible treatment to promote regeneration in acute kidney injury. Here, we show that human tubular ARPCs (tARPCs) protect physically injured or chemically damaged renal proximal tubular epithelial cells (RPTECs) by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells. tARPCs without toll-like receptor 2 (TLR2) expression or TLR2 blocking completely abrogated this regenerative effect. Only tARPCs, and not glomerular ARPCs, were able to induce tubular cell regeneration process and it occurred only after damage detection. Moreover, we have found that ARPCs secreted inhibin-A and decorin following the RPTEC damage and that these secreted factors were directly involved in cell regeneration process. Polysaccharide synthetic vesicles containing these molecules were constructed and co-cultured with cisplatin damaged RPTECs. These synthetic vesicles were not only incorporated into the cells, but they were also able to induce a substantial increase in cell number and viability. The findings of this study increase the knowledge of renal repair processes and may be the first step in the development of new specific therapeutic strategies for renal repair.

Figure 1

tARPCs can repair RPTECs physically injured. The wound healing assay showed that tARPCs can repair physically damaged RPTECs. A scratch was performed on tubular cell monolayers to simulate a physical damage. Subsequently, tubular cells were incubated alone (A–C) or in co-culture with ARPCs on transwells (D–F). Images were captured at phase contrast microscopy at intervals of 0, 24 and 48 hours (T0, T24 and T48, respectively) to detect the gradual repair of the gap. When RPTECs were in co-culture with tARPCs, they displayed an increased capacity to fill in the damaged area (E,F), when compared to RPTECs cultured alone (B–C). After 24 hours, the scratch in co-cultured RPTECs already started to close and contact points were seen (E, black arrow). At 48 hours, many more junction points were observed between gaps in RPTECs co-cultured with ARPCs (F, black arrows) compared RPTEC cultured alone. (G) Quantization results are expressed as a ratio between area of scratches at T48 and at T24 compared to the scratch area at T0. Plots represent 3 independent experiments using ARPCs from 3 different subjects; *P < 0.05, **P < 0.005.

Figure 2

tARPCs can repair cisplatin damaged RPTECs and necrotic cells. RPTEC BrdU proliferation assays showed the capacity of ARPCs to induce the regeneration of cisplatin-damaged tubular cells. (A) RPTEC proliferation rate at 4 days after cisplatin treatment (2.5 µmol/l) significantly decreased compared with healthy cells. When damaged cells were co-cultured with tARPCs, they recovered their proliferation rate. The gARPCs did not influence RPTEC proliferation rate. (B) Necrosis was induced on RPTECs and the cells were cultured with or without ARPCs for 4 days after inducing damage. In ARPCs absence RPTECs did not recover their proliferation rate and was significantly lower compared to non-damaged cells. RPTEC proliferation rate recovered only when damaged cells were co-cultured with tARPCs, whereas with gARPCs the recovery was absent (C). tARPCs induced the repair process only after damage perception. The proliferation rate of healthy RPTECs did not change when they were co-cultured with tARPCs without cisplatin (RPTECs + tARPCs) for 4 days. (D) The repair process was induced specifically by tARPCs. No significant increase in cell proliferation was observed when damaged RPTECs were co-cultured with healthy HK2 cells or RPTECs for 4 days. (E) Cell culture experiments showed that RPTECs and gARPCs, but not tARPCs, decreased their proliferation rate after cisplatin exposition (2.5 mmol/l drug for 6 h). Plots represent 5 independent experiments using ARPCs from 5 different subjects; *P < 0.05, **P < 0.005.

Figure 3

tARPCs can abrogate cisplatin-induced apoptosis of RPTECs. After cisplatin administration, the number of cleaved-caspase 3 positive RPTECs significantly increased to 41% after 24 hours and to and 63.9% after 48 hours. Conversely, when RPTECs were co-cultured with tARPCs, apoptotic cells decreased to 24% after 24 hours, whereas after 48 hours, apoptosis was completely blocked and RPTECs did no longer express cleaved-caspase 3. Results are representative of three independent experiments.

Figure 4

The repair induced by tARPCs was mediated by the Toll-like receptor-2. (A) BrdU cell proliferation assays showed that TLR2⁺ tARPCs were able to induce the repair of cisplatin damaged RPTECs in the co-culture system. On the contrary, the TLR2⁻ tARPCs and the TLR2⁺ gARPCs did not change the proliferation rate of damaged cells. (B) Cisplatin damaged RPTECs showed a limited proliferation rate in co-culture with tARPCs when TLR2 on renal progenitors was neutralized by a specific TLR2 blocking antibody. Plots represent 5 independent experiments using ARPCs from 5 different subjects; **P < 0.005, ***P < 0.0005.

Figure 5

Difference of TLR2 expression in gARPCs and tARPCs following exposure to necrotic cell supernatants. Immunofluorescence experiments on ARPCs showing the TLR2 expression at basal conditions and after exposure to necrotic cell supernatants. (A–D) immunofluorescence showed the expression of TLR2 (green) in gARPCs, gARPCs + necrotic supernatant, tARPCs, and tARPCs + necrotic supernatans, respectively. (E–H) Immunofluorescence showed the expression of CD133 (red) in gARPCs, gARPCs + necrotic supernatant, tARPCs, and tARPCs + necrotic supernatants, respectively. (I–L) Double-label immunofluorescence showed the expression of TLR2 (green) and CD133 (red) in gARPCs, gARPCs + necrotic supernatant, tARPCs, and tARPCs + necrotic supernatant, respectively. Nuclei were stained with TO-PRO-3 (blue). Original magnification 40x. (M) Quantification of TLR2 expression by calculating the pixel ratio of positive cells in 10 different fields. TLR2 expression was significantly higher in tARPCs in basal condition and following exposure to necrotic cell supernatants. Reprinted from [Kidney International], Supplementary Figure S5, Copyright (2013), with permission from Elsevier.

Figure 6

Inhibin-A and decorin were involved in the RPTEC repair. (A) Preconditioned supernatants from co-cultures induced an increase of RPTEC proliferation rate. Supernatants treated with 1 U/ml Rnase did not influence the proliferation rate. (B–C) Representative micrographs of transmission electron microscopy showing the release of MVs from the surface of a tARPC. Micrographs show the extrusion of MVs from the surface of the tARPC. Ultrathin sections, stained with led citrate were viewed by ZEISS EM910 electron microscope. Image acquisitions were performed with magnification of ×16000. (D) MVs isolated from the medium of regenerative condition (cisplatin-damaged RPTECs in co-culture with tARPCs), carried high levels of Inhb-A mRNA, these levels are comparable to the ones found in the supernatant of non-damaged RPTECs. Moreover, Inhb-A levels sharply decreased when TLR2 on tARPCs was blocked. (E) MVs isolated from the medium of the regenerative condition (cisplatin-damaged RPTECs in co-culture with tARPCs), carried high levels of Decorin mRNA, these levels were even higher than the ones detected in the supernatant of non-damaged RPTECs. Moreover, Decorin levels sharply decreased when TLR2 on tARPCs was blocked. (F) Inhb-A protein level increased in the regenerative condition (cisplatin-damaged RPTECs in co-culture with tARPCs), and this increase was abrogated when TLR2 receptor was blocked reaching levels similar to those obtained in the damaged condition. Inhb-A did not increased in the co-cultures of gARPCs with damaged-RPTECs. (G) BrdU cell proliferation assays showed that if we treated the regenerative medium (cisplatin-damaged RPTECs in co-culture with tARPCs) with the Inhb-A blocking antibody the damaged-RPTEC failed to recover their proliferation rate. Plots represent 5 independent experiments using tARPCs from 5 different subjects; *P < 0.05, **P < 0.005.

Figure 7

Inhibin-A is expressed by damaged renal tubules that enclosed tARPCs and its exogenous administration increased damaged-RPTEC proliferation rate. (A–F) Immunofluorescence on seriate tissue sections showed a proximal tubule labeled with Lotus (green, A,D) in which there are two CD133⁺progenitor cells (red, B) co-expressing Inhb-A (magenta, E). (C) Overlay of CD133 (red, C) and lotus (A). (F) Overlay of Inhb-A (magenta, (E) and lotus (D). The expression of Inhb-A colocalized with CD133⁺progenitor cells. To-pro-3 counterstained nuclei (blue). Original view: ×63. Reprinted from [Kidney International], Supplementary Figure S7, Copyright (2013), with permission from Elsevier. (G,H) BrdU proliferation assays showing that exogenous administration of Inhb-A and decorin increased damaged-RPTEC proliferation rate. (G) Inhb-A and decorin were loaded, individually or together, within polysaccharide synthetic vesicles (SV). The addition of Inhb-A and DCN SV to cisplatin-treated RPTECs led to a substantial increase in cell proliferation after 3 days of culture. DCN-SV alone were not able to increase the RPTEC proliferation rate, whereas INHB-A-SV gave a significant increase. On the other hand, the SV loaded with DCN and Inhb-A together gave a higher increase of cell proliferation. (H) BrdU proliferation assays showed that the exogenous administration of Inhb-A and decorin in the medium increased cell proliferation as proteins included in SV.