Tolvaptan, a vasopressin type 2 receptor antagonist initially developed to increase free-water diuresis, has been approved for the treatment of autosomal dominant polycystic kidney disease in multiple countries. Furthermore, tolvaptan has been shown to improve the renal functions in rodent models of chronic kidney disease (CKD); however, the underlying molecular mechanisms remain unknown. CKD is characterized by increased levels of oxidative stress, and an antioxidant transcription factor—nuclear factor erythroid 2-related factor 2 (Nrf2)—has been gaining attention as a therapeutic target. Therefore, we investigated the effects of tolvaptan and a well-known Nrf2 activator, bardoxolone methyl (BARD) on Nrf2. To determine the role of tolvaptan, we used a renal cortical collecting duct (mpkCCD) cell line and mouse kidneys. Tolvaptan activated Nrf2 and increased mRNA and protein expression of antioxidant enzyme heme oxygenase-1 (HO-1) in mpkCCD cells and the outer medulla of mouse kidneys. In contrast to BARD, tolvaptan regulated the antioxidant systems via a unique mechanism. Tolvaptan activated the Nrf2/HO-1 antioxidant pathway through phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, tolvaptan and BARD could successfully generate synergistic activating effects on Nrf2/HO-1 antioxidant pathway, suggesting that this combination therapy can contribute to the treatment of CKD.
Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA–PKA interaction, rather than other AKAP–PKA interactions, was robustly dissociated by PKA activation. AKAP–PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA–PKA interaction is a promising drug target for the development of anti-aquaretics.
BACKGROUND: Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. METHODS AND RESULTS: We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix ( Sp7 ), osteocalcin ( Bglap ), and osteopontin ( Spp1 ) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p , miR-17~92 cluster-originated miR-17-5p / miR-20a-5p , and miR-106b-5p . Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2 -targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p , miR-17-5p , 20a-5p , and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. CONCLUSIONS: The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.
A 59-year-old Japanese man was admitted to our hospital with leg edema, fatigue and fever. He had a past medical history of duodenal ulcer, fatty liver, hyperuricemia and positive rheumatoid factor, but was not receiving any medication. His son had died 2 years prior to this admission after following a clinical course that suggested TAFRO syndrome.The patient showed mild fever (about 37-38 °C), polypnea and severe leg edema on admission. Chest examination revealed decreased breath sounds in the right lung. No superficial lymph nodes were palpable. Laboratory tests on admission revealed mild thrombocytopenia, hypoproteinemia, elevation of C-reactive protein (CRP), alkaline phosphatase (ALP), creatinine (Cr), and urine N-acetyl-β-d-glucosaminidase (NAG) ( Table 1). Urine testing showed granular casts without proteinuria or hematuria. Concentrations of aspartate aminotransferase (AST) and alanine transaminase (ALT) were elevated, but unchanged compared with the results of a medical check 6 months before admission, and so were attributed to fatty liver. Chest X-ray and computed tomography (CT) showed mild lymphadenopathy in the mediastinum, bilateral pleural effusion and ascites (Fig. 1).Blood, sputum and urine culture tests were all negative and no other infectious diseases were present. Negative results were obtained for all autoantibodies. Among the tumor markers, only soluble interleukin-2 receptor (sIL-2R) was elevated. Bone marrow biopsy was performed to rule out malignant lymphoma, revealing normocellular Abstract Successful use of tocilizumab (TCZ) to treat TAFRO syndrome has recently been reported. In those cases, TCZ was used with steroid. We present herein the case of a 59-year-old man with very severe TAFRO syndrome who was successfully treated using TCZ without steroid. He showed rapidly progressive anasarca, acute renal failure and very severe thrombocytopenia. We initially used steroid, but its efficacy was limited. Moreover, steroid use had to be stopped as soon as possible, because hemorrhagic shock developed due to severe duodenal ulcer. After overcoming infections (about 40 days after stopping steroid), administration of TCZ was started and the patient was discharged in clinical remission.
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