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Chronic hypoxia is a major contributor to Chronic Kidney Disease (CKD) after Acute Kidney Injury (AKI). However, the temporal relation between the acute insult and maladaptive renal response to hypoxia remains unclear. In this study, we analyzed the time-course of renal hemodynamics, oxidative stress, inflammation, and fibrosis, as well as epigenetic modifications, with focus on HIF1α/VEGF signaling, in the AKI to CKD transition. Sham-operated, right nephrectomy (UNx), and UNx plus renal ischemia (IR + UNx) groups of rats were included and studied at 1, 2, 3, or 4 months. The IR + UNx group developed CKD characterized by progressive proteinuria, renal dysfunction, tubular proliferation, and fibrosis. At first month post-ischemia, there was a twofold significant increase in oxidative stress and reduction in global DNA methylation that was maintained throughout the study. Hif1α and Vegfa expression were depressed in the first and second-months post-ischemia, and then Hif1α but not Vegfa expression was recovered. Interestingly, hypermethylation of the Vegfa promoter gene at the HIF1α binding site was found, since early stages of the CKD progression. Our findings suggest that renal hypoperfusion, inefficient hypoxic response, increased oxidative stress, DNA hypomethylation, and, Vegfa promoter gene hypermethylation at HIF1α binding site, are early determinants of AKI-to-CKD transition.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na + channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na + reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of αENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and αENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between 〈ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Severe cases of COVID-19 are characterized by development of acute respiratory distress syndrome (ARDS). Water accumulation in the lungs is thought to occur as consequence of an exaggerated inflammatory response. A possible mechanism could involve decreased activity of the epithelial Na.sup.+ channel, ENaC, expressed in type II pneumocytes. Reduced transepithelial Na.sup.+ reabsorption could contribute to lung edema due to reduced alveolar fluid clearance. This hypothesis is based on the observation of the presence of a novel furin cleavage site in the S protein of SARS-CoV-2 that is identical to the furin cleavage site present in the alpha subunit of ENaC. Proteolytic processing of [alpha]ENaC by furin-like proteases is essential for channel activity. Thus, competition between S protein and [alpha]ENaC for furin-mediated cleavage in SARS-CoV-2-infected cells may negatively affect channel activity. Here we present experimental evidence showing that coexpression of the S protein with ENaC in a cellular model reduces channel activity. In addition, we show that bidirectional competition for cleavage by furin-like proteases occurs between (ENaC and S protein. In transgenic mice sensitive to lethal SARS-CoV-2, however, a significant decrease in gamma ENaC expression was not observed by immunostaining of lungs infected as shown by SARS-CoV2 nucleoprotein staining.

Water homeostasis is regulated by the peptide hormone arginine vasopressin (AVP), which promotes water reabsorption in the renal collecting duct. The regulation of gene transcription is a key mechanism through which AVP modulates water transport as disruption of this mechanism leads to water balance disorders. Therefore, an important goal is to understand the regulatory processes that control gene transcription. While CREB (CREB1) has been proposed as the primary transcription factor responsible for transcription, recent evidence challenges this view, suggesting that other CREB-like transcription factors, including ATF1 and CREM, may play a role. We employed the CRISPR/Cas9 gene-editing system to delete , , and in mpkCCD cells, an immortalized mouse collecting duct cell line. These cell lines were then exposed to the vasopressin analog, dDAVP, to assess the role of these transcription factors in regulating expression. AQP2 protein levels were measured by immunoblotting and RNA-seq was used to analyze changes in mRNA abundance, as well as other transcriptomic changes. Deletion of all three transcription factors (ATF1, CREB1, and CREM) led to a significant reduction in the vasopressin-induced upregulation of AQP2 protein, confirming their role in regulating expression. RNA-seq data showed that mRNA levels mirrored changes in protein abundance, supporting the idea that these transcription factors affect transcription. Rescue experiments in triple knockout cells showed that expressing any of the three transcription factors restored the response to vasopressin. Our findings demonstrate that ATF1, CREB1, and CREM have redundant roles in regulating transcription. Based on these results and prior data, we propose that these CREB-family transcription factors may regulate gene transcription indirectly by controlling the expression of additional unidentified transcription factors.

Phosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases protein kinase A (PKA)-dependent phosphorylation of β-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. To address the role of β-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. The mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 ± 0.66 in control mice to 3.33 ± 0.82 in Ser552Ala mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between wild-type and Ser552Ala mice (both ~13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two cyclin-dependent kinase inhibitor proteins, . At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. The observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of β-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla.