13 research outputs found
Crotamine/siRNA Nanocomplexes for Functional Downregulation of Syndecan-1 in Renal Proximal Tubular Epithelial Cells
Proteinuria drives progressive tubulointerstitial fibrosis in native and transplanted kidneys, mainly through the activation of proximal tubular epithelial cells (PTECs). During proteinuria, PTEC syndecan-1 functions as a docking platform for properdin-mediated alternative complement activation. Non-viral gene delivery vectors to target PTEC syndecan-1 could be useful to slow down alternative complement activation. In this work, we characterize a PTEC-specific non-viral delivery vector composed of the cell-penetrating peptide crotamine complexed with a syndecan-1 targeting siRNA. Cell biological characterization was performed in the human PTEC HK2 cell line, using confocal microscopy, qRT-PCR, and flow cytometry. PTEC targeting in vivo was carried out in healthy mice. Crotamine/siRNA nanocomplexes are positively charged, about 100 nm in size, resistant to nuclease degradation, and showed in vitro and in vivo specificity and internalization into PTECs. The efficient suppression of syndecan-1 expression in PTECs mediated by these nanocomplexes significantly reduced properdin binding (p < 0.001), as well as the subsequent complement activation by the alternative complement pathway (p < 0.001), as observed in either normal or activated tubular conditions. To conclude, crotamine/siRNA-mediated downregulation of PTEC syndecan-1 reduced the activation of the alternative complement pathway. Therefore, we suggest that the present strategy opens new venues for targeted proximal tubular gene therapy in renal diseases.</p
Schematic representation of the sucrose splash test.
<p>Schematic representation of the sucrose splash test.</p
Effect of crotamine on contraction force of isolated diaphragm of mice.
<p>The contraction of isolated mouse diaphragms was induced by transmural electrical stimulation (0.1 Hz, 2 ms duration, under optimum voltage and supramaximal voltage) and the isometric contractions were determined by plotting the average value of contraction force of each 5 min. Crotamine (from 3 to 30 nM) increased by about 17 to 46% the amplitude of twitch-contraction of isolated muscle induced by the direct transmural electrical stimuli. The time of crotamine addition was considered as zero (vertical dashed line), and the baseline contraction amplitude was considered as 100% (horizontal dashed line). The results (in percentage) were expressed as mean ± SEM (N = 6 independent experiments). The value <i>p</i> < 0.05 for ANOVA statistical analysis compared to the control was considered significant as indicated by *, <sup>#</sup> and <sup><i>a</i></sup>, for 30, 10 and 3 nM of crotamine, respectively.</p
Sucrose splash test.
<p>The period of grooming denotes the time spent by the animal in self cleaning activity, and the grooming was quantified for periods of 5 min each, statistically significant differences between animals receiving crotamine (7.5 Όg/animal or 0.3 mg/kg BW) by intraperitoneal (<i>ip</i>) route compared to vehicle receiving control animals were observed only after the second splash (A). The animals (N = 9) were continuously monitored this experiment during 1.5 h, and two splashes were performed in this interval, as indicated by the vertical dashed lines. The significant decreased activity was observed in the animals that received crotamine (average 754 s) compared to control animals (average 1498 s) was observed (B). The results (mean ± SE) were expressed as grooming period (s). * <i>p</i> †0.05 for unpaired two-tailed t-test was used to compare total grooming observed in each group during the splash test.</p
Mean distance traveled by animals after the administration of crotamine and the voltage-dependent K<sup>+</sup> channels blocker 4-AP.
<p>The distance travelled by the animal treated with crotamine and 4-AP was quantified in timeframe periods of 10 min each (<b>A</b>), as well as the total distance travelled during 60 min was detemined (<b>B</b>), and the time of paralysis was also recorded (<b>C</b>). The values were considered significant for <i>p</i> < 0.05 for ANOVA statistical analysis. *, <sup>#</sup> and <sup><i>a</i></sup> in panels A and B refer to comparisons with control, 4-AP (30 ÎŒg/animal, which corresponds to 1.2 mg/kg BW) and crotamine (7.5 ÎŒg/animal or 0.3 mg/kg BW), respectively, while <sup><i>b</i></sup> refers to comparisons with animals receiving only crotamine.</p
Mean distance traveled by the animals after the administration of different doses of crotamine.
<p>The dose-response effect of crotamine was confirmed by the differences in the mean distance traveled by the animals after receiving 30, 15 or 7.5 ÎŒg/animal (which correspond to 0.3, 0.6 and 1.2 mg/kg BW, respectively) by intraperitoneal (<i>ip</i>) injection, quantified during 60 min <b>(A)</b> or measured in periods of each 10 min <b>(B)</b>. Total locomotor activity was continuously monitored during 60 min, and each bar in the graphic represent the average traveled distance (in meters) of at least 6â7 animals/group. The value of <i>p</i> < 0.05 for ANOVA statistical analysis was considered significant. *, <sup>#</sup> and <sup><i>a</i></sup> represent the comparison with control, 7.5 ÎŒg and 15 ÎŒg of crotamine per animal, respectively.</p
Effect of K<sup>+</sup> channel blockers on the effect of crotamine on the contraction force of isolated diaphragm of mice.
<p>The increases of muscle contraction force induced by crotamine (30 nM) were evaluated in the presence of the voltage-dependent K<sup>+</sup> channel blocker, namely 4-aminopyridine (4-AP, 1 ΌM) (panels A and B), and Ca<sup>2+</sup>-dependent K<sup>+</sup> channel blockers, namely apamin (APA, 50 nM) (panels C and D) and charibdotoxin (CBTx, 10 nM) (panels E and F), which were added into the preparation after (panels A, C and E) or before (panels B, D and F) the addition of crotamine. Crotamine or channel blockers were applied at time zero or 35 min after the first compound addition, as indicated by the vertical dashed lines. The baseline contraction amplitude was considered as 100% (horizontal dashed line). The results (mean ± SEM, N = 6 independent experiments) were expressed as a percentage. *<i>p</i> < 0.05 for ANOVA statistical analysis compared to their respective control.</p
Evaluation of paralysis time and animal behavior in the open field.
<p>Time of paralysis (<b>A</b>), number of times that the animal crossed the central area of the open field (<b>B</b>) and the external area of the open field (<b>C</b>), and amount of stools deposited by the animal in the field (<b>D</b>) were recorded and expressed in minutes (min) for time of paralysis onset (in A) and in numbers of events (N) for B, C and D. Different doses of crotamine (7.5, 15 or 30 ÎŒg/animal which correspond to 0.3, 0.6 and 1.2 mg/kg BW, respectively) was administered by intraperitoneal (<i>ip</i>) injection. The last bar in panel A, indicated by the 24 h in the open box, refers to the <i>ip</i> administration of same dose of crotamine (7.5 ÎŒg/animal or 0.3 mg/kg BW) in the same animal 24 h after first crotamine administration, showing a significant decrease in the time for paralysis onset compared to the first administration of crotamine in naĂŻve animal, a day before. *<i>p</i> < 0.05 for ANOVA statistical analysis.</p
Paralysis of mice hind limbs after intraperitoneal route administration of crotamine and/or ion channel active compounds.
<p>Paralysis of mice hind limbs after intraperitoneal route administration of crotamine and/or ion channel active compounds.</p
Schematic representation of the <i>ex vivo</i> assays.
<p>Schematic representation of the <i>ex vivo</i> assays.</p