INTRODUCTION AND AIMS
Acute kidney injury (AKI) is emerging as a public health problem worldwide. Acute tubular apoptosis and necrosis play a central role in the process underlying kidney failure after ischemic or nephrotoxic challenge. Depending on the severity and the duration of the insult, tubular damage may recover, although a critical number of surviving cells is required to reconstitute structural integrity. Recent studies showed that contribution of extra-renal stem cells is less important than resident ones. Adult renal progenitor cells (ARPCs) can participate in kidney repair processes and might therefore be used in the clinic to improve regeneration in acute and progressive kidney disease. The precise mechanisms by which ARPCs bring to renal proximal tubular repair and regeneration remain largely unknown. Therefore, understanding RPTEC repair and regeneration mechanisms may uncover new therapeutic targets that promote renal recovery and decrease the severity of ARF. We studied the influence of ARPC on the regenerative process of cisplatin-injured renal proximal tubular epithelial cells (RPTEC) and Human Kidney 2 (HK2) cells and the mechanisms underlying the ARPC regenerative processes. Moreover, we investigated the nature of the regenerative factors secreted by ARPCs.
We set up an in vitro model of cisplatin-induced toxicity, in which RPTECs were co-coltured with ARPCs for 1, 4 and 7 days. CD133-positive ARPCs were isolated by magnetic sorting, starting from healthy sections of kidney removed during resection for renal carcinoma. Caspase 3 and Ki-67 expressions were studied to evaluate apoptosis and proliferation by immunofluorescence experiments. Cell proliferation induced by ARPCs was evaluated by BrdU proliferation assay. By bioinformatic analyses on microarray data and by multiplex cytokine assays, we identified some specific cytokines and growth factors secreted specifically by ARPCs following the damage perception.
Exposure of RPTECs or HK2 cells to cisplatin markedly reduced cell number and their viability, but co-culture with tubular ARPCs (tARPCs) provided a protective effect by promoting tubular cell proliferation and inhibiting apoptosis. Tubular cell regeneration process is specific of tARPCs and occurs only following the sensing of a damage. On the contrary, when glomerular ARPCs were co-cultured with damaged tubular cells, any effect of regeneration was observed (Figure 1). We showed that tARPCs protect RPTECs and HK2 cells from cisplatin toxicity by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells (Figure 2). Surprisingly, regenerative effect was completely cancelled blocking the Toll-like receptor 2 (TLR-2), expressed by tARPCs (Figure 3). We identified some specific cytokines, growth factors and microvescicle-shuttled mRNAs, secreted by ARPCs and dependent from TLR2 activation, that worked synergistically and were essential in the regenerative process (Figure 4). Among chemokines useful for regenerative processes we identified Inhibin A and FGF2, that are involved in the renal regeneration and tubular cell proliferation (Figure 5 and 6).
In conclusion, we demonstrated, for the first time, that tARPCs could have a regenerative effect on damaged RPTEC, by both preventing apoptosis and enhancing proliferation of surviving cells, principally by means of their secretoma, through the TLR2 engagement, and identified factors that were indispensable for the regenerative processes.