Quantum-Chemical Investigations on the Structural Variability of Anion–π Interactions

<div><p>This study evaluated the effects of bone marrow-derived mesenchymal stem cells (BMSCs) or their conditioned medium (CM) on the repair and prevention of Acute Kidney Injury (AKI) induced by gentamicin (G). Animals received daily injections of G up to 20 days. On the 10^th day, injections of BMSCs, CM, CM+trypsin, CM+RNase or exosome-like microvesicles extracted from the CM were administered. In the prevention groups, the animals received the BMSCs 24 h before or on the 5^th day of G treatment. Creatinine (Cr), urea (U), FENa and cytokines were quantified. The kidneys were evaluated using hematoxylin/eosin staining and immunohystochemistry. The levels of Cr, U and FENa increased during all the periods of G treatment. The BMSC transplantation, its CM or exosome injections inhibited the increase in Cr, U, FENa, necrosis, apoptosis and also increased cell proliferation. The pro-inflammatory cytokines decreased while the anti-inflammatory cytokines increased compared to G. When the CM or its exosomes were incubated with RNase (but not trypsin), these effects were blunted. The Y chromosome was not observed in the 24-h prevention group, but it persisted in the kidney for all of the periods analyzed, suggesting that the injury is necessary for the docking and maintenance of BMSCs in the kidney. In conclusion, the BMSCs and CM minimized the G-induced renal damage through paracrine effects, most likely through the RNA carried by the exosome-like microvesicles. The use of the CM from BMSCs can be a potential therapeutic tool for this type of nephrotoxicity, allowing for the avoidance of cell transplantations.</p> </div

The biochemical results for the treatment groups transplanted with CM.

<p> <b>Data are expressed as the mean ± S.E.M. One-Way ANOVA; (p<0,05).</b></p>*<p> <b><i>vs. CTL; p<.05.</i></b></p>#<p> <b><i>vs.G_15d; p<.05.</i></b></p>+<p> <b><i>vs.G₁₅+TPS₁₀.</i></b></p

The biochemical results from the treatment groups transplanted with BMSCs.

<p>Data are expressed as the mean ± S.E.M. One-Way ANOVA; (p<0.05).</p>*<p><i>vs</i>. CTL_{11,12,15 or 20d}; <i>p<.05</i>.</p>#<p><i>vs.G_11,12,15 or _20d</i>.</p

Figure 5

<p>(A) Transmission Electron Microscopy of the exossomes-like microvesicles extracted from the BMSC conditioned medium. (B) RT-PCR for 18S RNA of exosome extracted from BMSC culture medium (EXO) untreated and treated with RNase (RNase+EXO). (C) The light micrographs of the kidney sections stained with hematoxylin eosin. (D) A graphic representation showing the quantitative analysis of the micrographs. The normal histology of the kidney tissue from control rats (CTL₁₅) and rats treated with G for 15 days (G₁₅). Some groups presented with massive tubular necrosis and unstained nuclei (G₁₅, G₁₅+RNAse+EXO₁₀), while other groups presented with intensely stained nuclei (G₁₅+EXO₁₀). Data are expressed as the mean ± S.E.M. (* p<0.05) vs. CTL15d, (# p<0.05) vs. G15.</p

The plasma cytokine levels in the control (CTL15d and CTL20d), G-treated (G15 and G20) and G-treated rats that received BMSC (G15 or 20+BMSC10) or CM (G15 or 20+CM10).

<p>The data are expressed as the mean ± S.E.M. p<0.05 (*) vs. CTL15 or 20 d; (#) vs. G15 or 20 d; (&) vs. G+BMSC20d.</p

Figure 4

<p>(A) RT-PCR for 18S RNA in culture medium (CM) from BMSC controls and culture medium treated with RNase (CM+RNAse). (B) BMSC viability after treatment with Trypsin and RNase by Trypan blue extrusion. (C) The light micrographs of the kidney sections stained with hematoxylin eosin and with immunochemistry for caspase 3 and KI67. (D) A graphic representation showing the quantitative analysis of the control rat micrographs (CTL₁₅; CTL₁₅+BMSC₁₀). The histology of the kidney tissue from rats treated with G for 15 days (G₁₅). Some groups presented with massive tubular necrosis and unstained nuclei (G₁₅, G₁₅+RNAse+CM₁₅), while other groups presented with intensely stained nuclei (G₁₅+BMSC₁₀, G₁₅+CM₁₀). Data are expressed as the mean ± S.E.M. (* p<0.05) vs. CTL15d, (# p<0.05) vs. G15.</p

Figure 1

<p>(A) FACS analysis and (B) quantification showing the monoclonal antibody expression [%] of BMSCs. Data are expressed as the mean ± S.E.M. (* p<0.05) vs. CD45, CD34 and CD11b. (C) The differentiation of BMSCs into adipocytes and osteocytes. Undifferentiated (non-induced) BMSCs were maintained in the control medium as a negative control.</p

The biochemical results for the prevention groups transplanted with BMSCs.

<p>Data are expressed as the mean ± S.E.M. One-Way ANOVA; (p<0.05).</p>*<p><i>vs</i>. CTL_10d.</p

The schematic of the experiment protocol.

<p>In the control group (CTL), the rats were treated with daily i.p. injections of a vehicle (water), while the G groups received gentamicin (40 mg/Kg BW) continuously for 10, 11, 12, 15 or 20 days. For the prevention groups, the rats received BMSC 1×106 i.v. injections 24 hours before (G₁₀+BMSC₋₁) or in the 5th day (G10d+BMSC5) of a 10-day treatment with G. For the treated groups, the rats received BMSC 1×106 i.v. injections on the 10th day of G treatment and continued receiving G for an additional 1 (G₁₁+BMSC₁₀), 2 (G₁₂+BMSC₁₀), 5 (G₁₅+BMSC₁₀) or 10 (G₂₀+BMSC₁₀) days. For the conditioned media protocol, the rats received G (40 mg/Kg/BW, i.p., daily) or water (CTL) for 15 or 20 days, and on the 10th day of the G treatment, the animals received 500 µl of a single dose of CM. In some experiments, the BMSCs were cultured for 12 h with trypsin and a concentration of 100 ug/ml (CM+TPS, 100 µg/ml) or RNase A at a concentration of 40 ug/ml, 280 Units (CM+RNAse) in DMEM without FBS.</p

PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy

<p>Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12–ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.</p