OBJECTIVETo demonstrate the molecular expression of carbonic anhydrase IV (CA IV) in rabbit corneal endothelium.

METHODSReverse transcriptase polymerase chain reaction (RT-PCR) was performed using cultured and fresh rabbit corneal endothelial total RNA and specific primers for CA IV. The RT-PCR product was subcloned and sequenced. Immunoblotting and indirect immunofluorescence staining were performed to detect protein expression and distribution of CA IV using fresh and cultured rabbit corneal endothelium and rat anti-CA IV polyclonal antibody.

RESULTSRT-PCR screening gave positive bands at the predicted size for CA IV from fresh and cultured rabbit corneal endothelium. Sequencing further confirmed the identity of CA IV in corneal endothelium. Immunoblotting analysis showed a single band at 52 kDa for freshly isolated and cultured endothelial cells. Indirect immunofluorescence staining revealed an apparent positive staining in cultured endothelial cells.

CONCLUSIONCarbonic anhydrase IV is expressed in rabbit corneal endothelium, which could contribute to the transendothelial HCO(3)(-) flux that is necessary to maintain corneal hydration and transparency.

Animals ; Base Sequence ; Bicarbonates ; metabolism ; Carbonic Anhydrase IV ; analysis ; genetics ; Cells, Cultured ; Endothelium, Corneal ; cytology ; enzymology ; Fluorescent Antibody Technique, Indirect ; Molecular Sequence Data ; RNA, Messenger ; analysis ; Rabbits

Objective To investigate the role and mechanism of circular RNA SNRK (circSNRK) in ischemia-reperfusion injury (IRI). Methods A hypoxia-reoxygenation (IRI) cell model was established. The expression level of circSNRK after IRI treatment and the effect of overexpression of circSNRK on cell proliferation and apoptosis were detected. The targets of circSNRK were identified. HK2 cells were divided into the blank group (Mock group), IRI group, control plasmid+IRI group (IRI+NC group), human circSNRK overexpression+IRI group (IRI+circSNRK group), human circSNRK overexpression+IRI+protein kinase B (Akt) inhibitor group (IRI+circSNRK+MK2206 group) and control plasmid group (NC group). Cell proliferation and apoptosis were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the target of circSNRK were carried out. The expression levels of CDKN1A, Akt, B-cell lymphoma (Bcl)-2, cysteinyl aspartate specific proteinase (Caspase)-9 messenger RNA (mRNA), and those of p21, Bcl-2, Caspase-9, Akt and p-Akt proteins were detected in the Mock, IRI, IRI+NC and IRI+circSNRK groups, respectively. Cell proliferation and apoptosis were determined in the NC, IRI+NC, IRI+circSNRK and IRI+circSNRK+MK2206 groups. Results Compared with the Mock group, the expression level of circSNRK was lower, and cell proliferation capability of HK2 cells was decreased and cell apoptosis was increased in the IRI group. In the IRI+circSNRK group, cell proliferation capability was higher, whereas cell apoptosis was lower than those in the IRI+NC group. circSNRK could act on 648 targets through 51 microRNAs (miRNAs). GO enrichment analysis revealed that the targets of circSNRK were mainly enriched in biological processes (such as cell process and biological regulation), cell components (such as cell parts, cells and extracellular parts), and molecular functions (such as binding, binding proteins and enzymes). KEGG enrichment analysis showed that the targets of circSNRK were mainly enriched in cancer signaling pathway, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, miRNA in cancer and other related signaling pathways. Compared with the Mock group, the relative expression levels of CDKN1A and Caspase-9 mRNA were higher, the expression level of miR-99a-5p RNA was higher and the relative expression levels of Akt and Bcl-2 mRNA were lower in the IRI group. Compared with the IRI+NC group, the relative expression levels of CDKN1A and Caspase-9 mRNA were lower, those of Akt and Bcl-2 mRNA were higher, and the expression level of miR-99a-5p RNA was lower in the IRI+circSNRK group, and the differences were statistically significant (all P < 0.05). Compared with the Mock group, the expression levels of p21 and Caspase-9 proteins were higher, while those of p-Akt, Akt and Bcl-2 proteins were lower in the IRI group. Compared with the IRI+NC group, the expression levels of p21 and Caspase-9 proteins were lower, whereas those of p-Akt, Akt and Bcl-2 proteins were higher in the IRI+circSNRK group. The miR-99a-5p binding sites were observed in circSNRK and Akt. Compared with the NC group, cell proliferation capability was declined in the IRI+NC group. Compared with the IRI+NC group, cell proliferation capability was elevated in the IRI+circSNRK group. Compared with the IRI+circSNRK group, cell proliferation capability was declined in the IRI+circSNRK+MK2206 group (all P < 0.05). The cell apoptosis level in the IRI+NC group was higher than that in the NC group. The cell apoptosis level in the IRI+circSNRK group was lower compared with that in the IRI+NC group. The cell apoptosis level in the IRI+circSNRK+MK2206 group was higher than that in the IRI+circSNRK group. Conclusions Under IRI conditions, circSNRK may affect the proliferation and apoptosis of HK2 cells probably via the Akt signaling pathway.

Objective To evaluate the clinical efficacy of percutaneous transluminal angioplasty (PTA) combined with stent implantation in the treatment of transplant renal artery stenosis (TRAS) after renal transplantation. Methods Clinical data of 21 patients with TRAS after renal transplantation undergoing PTA combined with stent implantation were retrospectively analyzed. The incidence of TRAS in renal transplant recipients was summarized. The changes of relevant indexes in patients with TRAS were statistically compared before and after interventional treatment. Clinical prognosis of patients with TRAS was evaluated. Results The incidence of TRAS in renal transplant recipients was 4.1%(21/507). TRAS was diagnosed at postoperative 5 (4, 7) months, and 67% (14/21) of patients developed TRAS within postoperative 6 months. Compared with the values before interventional therapy, the serum creatinine level, systolic and diastolic blood pressure and peak flow velocity of transplant renal artery of patients with TRAS were significantly decreased, and the estimated glomerular filtration rate (eGFR) and interlobar arterial resistance index were significantly increased at 1 week and 1 month after interventional therapy (all P < 0.05). During postoperative follow-up after PTA combined with stent implantation, 1 patient suffered re-stenosis of the transplant renal artery, which was improved after simple balloon dilatation. One patient developed pseudoaneurysm formation at the puncture site of the right femoral artery. One patient presented with renal atrophy and loss of function due to atresia of the transplant renal artery. All the remaining 18 patients were well recovered after surgery. Conclusions PTA combined with stent implantation is the optimal treatment of TRAS after renal transplantation, which can significantly improve the function of transplant kidney and considerably prolong the survival time of transplant kidney.