GTS-21 attenuates cisplatin-induced renal inflammation.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. COX-2 (<i>Ptgs2</i>) mRNA expression in the renal cortical tissues was measured by qPCR (A). Data are shown as mean (±SD) fold-change (vs. <i>Gapdh</i> housekeeping gene). Kidney homogenates were also analyzed for levels of inflammatory marker proteins (IL-6, B; IL-1β, C; and CXCL1, D) by Meso Scale Discovery (MSD) platform. Data are shown as mean (pg) per g of protein (±SD). *** = p<0.001 vs. CTRL, ** = p<0.01 vs. CTRL, * = p<0.05 vs. CTRL, ⁺⁺ = p<0.01 vs. CIS, ⁺ = p<0.05 vs. CIS.</p

Cisplatin-induced renal neutrophil infiltration is blunted by GTS-21 pretreatment.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Fixed kidney tissues were evaluated for neutrophils by Leder staining. Representative images for each group are shown in (A) at 400x magnification (scale bar, 50 μm) and the mean number of neutrophils per high power field, HPF (±SEM) is shown in (B). Frozen renal tissues were analyzed for myeloperoxidase (MPO) (C). Data are shown as mean MPO levels (pg) per mg protein (±SD). *** = p<0.001 vs. CTRL, ** = p<0.01 vs. CTRL, ⁺⁺⁺ = p<0.001 vs. CIS, ⁺ = p<0.05 vs. CIS.</p

Cisplatin induces renal damage and GTS-21 exerts renoprotective effect.

<p>C57BL/6 mice (males, n = 8 mice per group) were injected daily for 4 days, prior to cisplatin (20 mg/kg, i.p.), with either saline (cisplatin only group, CIS) or GTS-21 (GTS-21 pretreatment group, GTS+CIS, 4mg/kg, i.p, twice) and until euthanasia 72hrs post-cisplatin. The control group (CTRL) received saline injections in place of GTS-21 or cisplatin. Kidney damage was assessed by measuring blood urea nitrogen (BUN, A) and plasma creatinine (B) levels. For tubular injury, fixed kidney tissues were stained with Periodic Acid Schiff reagent (PAS, C), scale bar, 50 μm; representative images for each group (x400) are shown and were scored for histological damage (0–4) (D). The scores were based on the percentage of tubules affected (0: <10%; 1: 10–25%; 2: 26–50%; 3: 51–75%; 4: >75%). Data are shown as mean ± SD. *** = p<0.001 vs. CTRL, * = p<0.05 vs. CTRL, ⁺⁺ = p<0.01 vs. CIS, ⁺ = p<0.05 vs. CIS.</p

Proposed mechanisms by which GTS-21 attenuates cisplatin-induced AKI.

<p>Cisplatin treatment promotes renal inflammation and oxidative stress, renal cell apoptosis/necrosis, leading renal tissue damage and ultimately, AKI. GTS-21 treatment, acting through the cholinergic anti-inflammatory pathway, protects against cisplatin-induced AKI by reducing influx transporter expression while increasing CIS efflux transporter expression, and by modulating renal inflammation and oxidative stress pathways that lead to kidney cell death and AKI.</p

GTS-21 attenuates cisplatin-induced renal cell apoptosis.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Renal cell apoptosis was measured by TUNEL staining and representative photomicrographs are shown (at 400x magnification; scale bar, 50 μm) in (A). Apoptosis was determined by counting the number of TUNEL positive cells per high power field (HPF) using random sections and the mean apoptosis scores (±SEM) are shown (B). *** = p<0.001 vs. CTRL, ⁺⁺ = p<0.01 vs. CIS.</p

Downregulation of renal cisplatin influx transporter CTR1 expression by GTS-21.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Representative western blots showing renal CTR1 and GAPDH protein expression is shown in (A). GAPDH was used as loading control for normalization. Quantitation of band ratios: (B) CTR1/GAPDH (mean band density (±SD)) is shown. ⁺⁺ = p<0.01 vs. CIS.</p

GTS-21 administration blocks cisplatin-induced renal ERK activation.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. ERK1/2 (ERK) protein expression and its phosphorylation (p-ERK) in the renal cortical tissues were measured by western blotting. GAPDH was used as loading control for normalization. Representative blots are shown in (A) and quantitation of the band densities are shown in (B, ERK/GAPDH) and (C, p-ERK/GAPDH). All data are expressed as mean band density (±SD). * = p<0.05 vs. CTRL, ⁺⁺⁺ = p<0.001 vs. CIS.</p

Cisplatin-induced renal ATP depletion and oxidative stress are improved by GTS-21.

<p>(A) ATP levels: Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Kidney homogenates were analyzed for renal ATP levels (shown as mean ATP concentration (nmol/mg, mean±SD), corrected for the protein levels. *** = p<0.001 vs. CTRL, ⁺⁺ = p<0.01 vs. CIS. (B) GSH levels (reduced glutathione): LLC-PK₁ cells were treated with GTS-21 (GTS, 1μM) for 1hr followed by cisplatin (CIS, 30μM) treatment or vehicle treatment (CTRL). After 20hrs, cells were harvested and the amount of GSH were assayed in the cell lysates to measure oxidative stress and corrected for the protein amount (nmol/mg, mean ±SD). *** = p<0.001 vs. CTRL, ⁺ = p<0.05 vs. CIS. (C) DCFH-DA assay: LLC-PK₁ cells were treated with GTS-21 (GTS, 1μM) or NAC (2mM) or vehicle (UNTD). After 16hrs, cells were harvested and treated with either cisplatin (CIS, 50μg/mL) or vehicle (0μg/mL). After 3.5hrs, cells were labeled with DCFH-DA (20μM) for 20 min. Labeled cells were analyzed for oxidative stress by the conversion of the non-fluorescent DCFH-DA into a fluorescent compound DCF measured 30min later. Data (from 8–12 wells per condition) are shown as mean oxidative stress (or fluorescence) (±SD). *** = p<0.001 vs. vehicle, untreated (UNTD), ⁺⁺⁺ = p<0.001 vs. vehicle NAC, ^‡‡‡ = p<0.001 vs. vehicle GTS, ^$$$ = p<0.001 vs. CIS (UNTD), ^### = p<0.001 vs. CIS (UNTD).</p

GTS-21 does not compromise cisplatin-induced tumor cell killing <i>in vitro</i>.

<p>Cancer cells (H460-lung; MCF7-breast, and CT26-colon) were treated with GTS-21 (at concentrations indicated) for 1hr followed by vehicle or cisplatin (0–230μM final) addition, and were assayed for cytotoxicity using the MTT assay 24 hrs later (n = 8–12 wells per condition). The IC₅₀ values (mean±SD) for the tumor cell killing by cisplatin were calculated as described in the methods section.</p

GTS-21 upregulates the renal expression of cisplatin efflux transporters.

<p>Mice (CTRL, CIS and GTS+CIS groups, n = 8 per group) were treated and euthanized as described in the methods section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188797#pone.0188797.g001" target="_blank">Fig 1</a>. Representative western blots for renal MRP2 (ABCC2), MRP4 (ABCC4) and MRP6 (ABCC6) protein expression are shown in (A). ABCC and MRP stand for ATP-binding cassette subfamily C and multidrug resistance-associated protein respectively. GAPDH was used as loading control for normalization. Quantitation of band ratios: (B) MRP2/GAPDH, (C) MRP4/GAPDH and (D) MRP6/GAPDH (mean band density (±SD) are shown. (E) Renal platinum (¹⁹⁵Pt) accumulation, measured by ICP-MS, is shown as mean±SD (ng/g kidney tissue). ** = p<0.01 vs. CTRL, * = p<0.05 vs. CTRL, ⁺⁺⁺ = p<0.001 vs. CIS, ⁺⁺ = p<0.01 vs. CIS.</p