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  • Although all the three Dc doses prevented

    2019-07-30

    Although all the three Dc doses prevented the Pertussis Toxin of diluted urine enriched by proteins, 1mg/kg Dc did not prevent the reduction of GFR nor BUN accumulation. Instead, because I/R+Dc1 rats presented lower GFR values compared with I/R, the FENa (the percentage of the Na+ filtered by the kidney that is excreted in the urine) was enhanced, showing the inability of these rats to conserve Na+. It seems that 1mg/kg Dc is effective for tubular damage (evaluated by urine volume and proteinuria) but not completely effective to modify glomerular filtration rate (GFR is similar to I/R and BUN is partially prevented); thus, invalidating the continued use of this dose in the study. The total beneficial effect of Dc was observed at the dose of 3 and 10mg/kg, protecting GFR, and reducing BUN and FENa. Guimarães et al. [52] demonstrated a dose-dependent effect of Dc in a renovascular hypertension model. Doxycycline treatment, at doses lower than 10mg/kg/day, attenuated hypertension but not vascular alterations found in the 2K1C hypertensive rat. However, 30mg/kg/day Dc treatment prevented vascular alterations in addition to attenuating hypertension. The authors correlated the reduced efficacy of the lower Dc doses to the lack of MMP-2 inhibition. Most previous kidney studies used 10mg/kg (oral or intraperitoneal) to prevented kidney injury by attenuating reactive oxygen species (ROS), MMPs, TGF-β1, and nephritis [14], [26], [27], [28], [29], [30]. We used intraperitoneal Dc administration at 3 different low doses (1, 3, and 10mg/kg) before inducing IRI. We observed that both 3 and 10mg/kg prevented the kidney function loss, suggesting the effectiveness of Dc in the kidney compared with the vasculature. Indeed, it has been shown that the levels of Dc measured in renal tissue averaged twice the concentration found in serum [53]. According to Prall et al. [54], 10, 50, and 100mg/kg Dc administered to mice produce a dose-dependent increase in Dc serum concentration of 2.7, 5.3, and 23.2μM, respectively. Assuming that bioavailability is similar to rats, in our study serum Dc concentration should be equal to or lower than 2.7μM and around 5.4μM in the kidney. A concentration higher than 4μM has been shown to be effective in reducing MMP activity [54]. This may partly explain the low dose effects of Dc in the kidney but not in the vasculature. The observation that low Dc doses inhibit MMP content and activity in the kidney, but not in the vasculature, reinforce this explanation. The future use of Dc treatment as a rescue therapy is unsettled. Antonio et al., [55] showed that although Dc promoted MMP inhibition associated with antioxidant and antihypertensive effects, there was no reversal of the vascular remodeling induced by hypertension. However, Kholmukhamedov et al. [56] demonstrated that minocycline or Dc were similarly protective in attenuating kidney injury when given before or after resuscitated hemorrhage. These divergent effects could be related to the ability of the kidney to accumulate Dc. In the present rat model of IRI, total MMPs activities were highly augmented as shown by the in vitro analysis. Cortical kidney extract of I/R rats incubated with both 25 and 50μM Dc blocked augmentation of total MMPs activity on its specific fluorogenic substrate. Our observation differs from in vitro studies by Guimarães et al. [52] that showed Dc concentrations below 50μM did not inhibit human recombinant MMP-2 activity, suggesting that rat MMP was more susceptible to Dc treatment. In vivo, the dual effect of MMP, whether protecting or promoting kidney injury, depends upon the temporal MMP pattern expression/activity. Among all MMPs, MMP-2 and -9 are emerging as key MMPs in kidney diseases. Kaneko et al. [17] showed that MMP-2 and -9 expression/activity are upregulated in 24h of reperfusion. MMPs levels are even higher up to 14days. MMP-2 is related with tubular repair phase after IRI and as an anti-fibrotic agent, regulating the severity of renal fibrosis [18], [19]. We observed that in I/R rats, there was an increase in MMP-2 and -9 protein content compared with control or I/R+Dc3, yet their activities did not change, suggesting that the augmented protein content sustained the enzyme activity among all three experimental groups. Indeed, the MMP-2 or -9 protein activity/protein content was reduced by 50% and 30%, respectively. Considering that MMP-2 is more related with protection and MMP-9 with tissue damage, Genetic marker may be that the loss of the protective component (MMP-2) contributes to kidney injury in I/R. Moreover, it has been shown that Dc decreases MMPs mRNA stability, thereby explaining how MMPs decreases its protein content to control levels [57], [58]. We cannot rule out the involvement of other MMP isoforms since we showed that I/R generally augmented MMPs activities. Therefore, we propose that during I/R, an imbalance exists between the MMPs isoforms that culminates in an increase of total MMP activity. Doxycycline prevents this imbalance, maintains control levels of MMP activity, including MMP-2 and -9 activities and MMP-2 and -9 protein contents.