Dataset Information

Connexin32 plays a crucial role in ROS-mediated endoplasmic reticulum stress apoptosis signaling pathway in ischemia reperfusion-induced acute kidney injury.

ABSTRACT:

Background

Ischemia-reperfusion (I/R)-induced acute kidney injury (AKI) not only prolongs the length of hospital stay, but also seriously affects the patient's survival rate. Although our previous investigation has verified that reactive oxygen species (ROS) transferred through gap junction composed of connexin32 (Cx32) contributed to AKI, its underlying mechanisms were not fully understood and viable preventive or therapeutic regimens were still lacking. Among various mechanisms involved in organs I/R-induced injuries, endoplasmic reticulum stress (ERS)-related apoptosis is currently considered to be an important participant. Thus, in present study, we focused on the underlying mechanisms of I/R-induced AKI, and postulated that Cx32 mediated ROS/ERS/apoptosis signal pathway activation played an important part in I/R-induced AKI.

Methods

We established renal I/R models with Cx32^+/+ and Cx32^-/- mice, which underwent double kidneys clamping and recanalization. ROS scavenger (N-acetylcysteine, NAC) and ERS inhibitors (4-phenyl butyric acid, 4-PBA, and tauroursodeoxycholic acid, TUDCA) were used to decrease the content of ROS and attenuate ERS activation, respectively.

Results

Renal damage was progressively exacerbated in a time-dependent manner at the reperfusion stage, that was consistent with the alternation of ERS activation, including glucose regulated protein 78 (BiP/GRP78), X box-binding protein1, and C/EBP homologous protein expression. TUDCA or 4-PBA application attenuated I/R-induced ERS activation and protected against renal tubular epithelial cells apoptosis and renal damage. Cx32 deficiency decreased ROS generation and distribution between the neighboring cells, which attenuated I/R-induced ERS activation, and improved cell apoptosis and renal damage.

Conclusion

Cx32 mediated ROS/ERS/apoptosis signal pathway activation played an important part in I/R-induced AKI. Cx32 deficiency, ROS elimination, and ERS inhibition all could protect against I/R-induced AKI.

SUBMITTER: Gu Y

PROVIDER: S-EPMC5935959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aims: Perioperative acute kidney injury (AKI) resulting from renal ischemia reperfusion (IR) is not conducive to the postoperative surgical recovery. Our previous study demonstrated that reactive oxygen species (ROS) transmitted by gap junction (GJ) composed of connexin32 (Cx32) contributed to AKI. However, the precise underlying pathophysiologic mechanisms were largely unknown. This study focuses on the underlying mechanisms related to ROS transmitted by Cx32 responsible for AKI aggravation. Results: In a set of in vivo studies, renal IR was found to cause severe impairment in renal tissues with massive ROS generation, which occurred contemporaneously with activation of NF-κB/p53/p53 upregulated modulator of apoptosis (PUMA)-mediated mitochondrial apoptosis pathways. Cx32 deficiency alleviated renal IR-induced AKI, and simultaneously attenuated ROS generation and distribution in renal tissues, which further inhibited NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathways. Correspondingly, in a set of in vitro studies, hypoxia reoxygenation (HR)-induced cellular injury, and cell apoptosis in both human kidney tubular epithelial cells (HK-2s) and rat kidney tubular epithelial cells (NRK52Es) were significantly attenuated by Cx32 inhibitors or Cx32 gene knockdown. More importantly, Cx32 inhibition not only decreased ROS generation and distribution in human or rat kidney tubular epithelial cells but also inhibited its downstream NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathway activation. Innovation and Conclusion: This is the first identification of the underlying mechanisms of IR-induced renal injury integrally which demonstrates the critical role played by Cx32 in IR-induced AKI. Moreover, GJ composed of Cx32 manipulates ROS generation and distribution between neighboring cells, and alters activation of NF-κB/p53/PUMA-mediated mitochondrial apoptotic pathways. Both inhibiting Cx32 function and scavenging ROS effectively reduce mitochondrial apoptosis and subsequently attenuate AKI, providing effective strategies for kidney protection.

Project description:Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion (I/R) injury. In this study, three key proteins in the endoplasmic reticulum stress pathway (glucose-regulated protein 78, caspase-12, and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor. Female Sprague-Dawley rats received ovariectomy (OVX), and then cerebral I/R rat models (OVX + I/R) were established by middle cerebral artery occlusion. Immediately after I/R, rat models were injected with 100 μg/kg E2 (OVX + I/R + E2), or 100 μg/kg G protein-coupled estrogen receptor agonist G1 (OVX + I/R + G1) in the lateral ventricle. Longa scoring was used to detect neurobehavioral changes in each group. Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining. Morphological changes in neurons were observed by Nissl staining. Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group, neurological function was remarkably improved, infarct volume was reduced, number of normal Nissl bodies was dramatically increased, and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention. To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum, caspase-12 distribution and expression were detected by immunofluorescence, and mRNA and protein levels of glucose-regulated protein 78, caspase-12, and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay. The results showed that compared with the OVX + I/R group, E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78, C/EBP homologous protein, and caspase-12. However, the G protein-coupled estrogen receptor antagonist G15 (OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury. These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus, thereby improving dysfunction caused by cerebral I/R injury. Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. SHZ A2017-171) on February 27, 2017.

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