Phosphatidylserine eversion regulated by phospholipid scramblase activated by TGF-β1/Smad signaling in the early stage of kidney stone formation

The mechanism underlying phosphatidylserine eversion in renal tubule cells following calcium oxalate-mediated damage remains unclear; therefore, we investigated the effects of TGF-β1/Smad signaling on phosphatidylserine eversion in the renal tubule cell membrane during the early stage of kidney stone development. In a rat model of early stage of calcium oxalate stone formation, phosphatidylserine eversion on the renal tubular cell membrane was detected by flow cytometry, and the expression of TGF-β1 (transforming growth factor-β1), Smad7, and phospholipid scramblase in the renal tubular cell membrane was measured by western blotting. We observed that the TGF-β1/Smad signaling pathway increased phosphatidylserine eversion at the organism level. The results of in vitro studies demonstrated that oxalate exposure to renal tubule cells induced TGF-β1 expression, increasing phospholipid scramblase activity and phosphatidylserine eversion in the renal tubule cell membrane. These results indicate that TGF-β1 stimulates phosphatidylserine eversion by increasing the phospholipid scramblase activity in the renal tubule cell membrane during the early stage of kidney stone development. The results of this study form a basis for further detailed research on the development of therapeutic agents that specifically treat urolithiasis and exert fewer adverse effects.

PM2.5 Induces Early Epithelial Mesenchymal Transition in Human Proximal Tubular Epithelial Cells through Activation of IL-6/STAT3 Pathway

Particulate matter exposure has been known as a potential risk for the global burden of disease, such as respiratory and cardiovascular diseases. Accumulating evidence suggests that PM2.5 (particulate matter with a diameter less than 2.5 μm) is associated with increased risk of kidney disease, but the mechanisms underlying the renal injury caused by PM2.5 remain to be elucidated. This study investigated the effects of PM2.5 on human proximal tubular epithelial (HK-2) cells by monolayer and 3D spheroid cultures and explored the potential mechanisms. The typical morphology of HK-2 cells showed epithelial–mesenchymal transition (EMT), resulting in reduced adhesion and enhanced migration after PM2.5 exposure, and was accompanied by decreased E-cadherin expression and increased vimentin and α-SMA expressions. Exposure to PM2.5 in the HK-2 cells could lead to an increase in interleukin-6 (IL-6) levels and cause the activation of signal transducer and activator of transcription 3 (STAT3), which is involved in EMT features of HK-2 cells. Furthermore, blocking IL-6/STAT3 signaling by an IL-6 neutralizing antibody or STAT3 inhibitor was sufficient to reverse PM2.5-induced EMT characteristics of the HK-2 cells. Our study suggests that PM2.5 could induce early renal tubule cell injury, contributing to EMT change, and the induction of IL-6/STAT3 pathway may play an important role in this process.

Gastrin, via activation of PPARα, protects the kidney against hypertensive injury

Hypertensive nephropathy (HN) is a common cause of end-stage renal disease with renal fibrosis; chronic kidney disease is associated with elevated serum gastrin. However, the relationship between gastrin and renal fibrosis in HN is still unknown. We, now, report that mice with angiotensin II (Ang II)-induced HN had increased renal cholecystokinin receptor B (CCKBR) expression. Knockout of CCKBR in mice aggravated, while long-term subcutaneous infusion of gastrin ameliorated the renal injury and interstitial fibrosis in HN and unilateral ureteral obstruction (UUO). The protective effects of gastrin on renal fibrosis can be independent of its regulation of blood pressure, because in UUO, gastrin decreased renal fibrosis without affecting blood pressure. Gastrin treatment decreased Ang II-induced renal tubule cell apoptosis, reversed Ang II-mediated inhibition of macrophage efferocytosis, and reduced renal inflammation. A screening of the regulatory factors of efferocytosis showed involvement of peroxisome proliferator-activated receptor α (PPAR-α). Knockdown of PPAR-α by shRNA blocked the anti-fibrotic effect of gastrin in vitro in mouse renal proximal tubule cells and macrophages. Immunofluorescence microscopy, Western blotting, luciferase reporter, and Cut&tag-qPCR analyses showed that CCKBR may be a transcription factor of PPAR-α, because gastrin treatment induced CCKBR translocation from cytosol to nucleus, binding to the PPAR-α promoter region, and increasing PPAR-α gene transcription. In conclusion, gastrin protects against HN by normalizing blood pressure, decreasing renal tubule cell apoptosis, and increasing macrophage efferocytosis. Gastrin-mediated CCKBR nuclear translocation may make it act as a transcription factor of PPAR-α, which is a novel signaling pathway. Gastrin may be a new potential drug for HN therapy.

Brincidofovir (CMX001) Inhibits BK Polyomavirus Replication in Primary Human Urothelial Cells

ABSTRACTBK polyomavirus (BKPyV)-associated hemorrhagic cystitis (PyVHC) complicates 5 to 15% of allogeneic hematopoietic stem cell transplantations. Targeted antivirals are still unavailable. Brincidofovir (BCV; previously CMX001) has shown inhibitory activity against diverse viruses, including BKPyV in a primary human renal tubule cell culture model of polyomavirus-associated nephropathy. We investigated the effects of BCV in BKPyV-infected and uninfected primary human urothelial cells (HUCs), the target cells of BKPyV in PyVHC. The BCV concentrations causing 50 and 90% reductions (EC50and EC90) in the number of intracellular BKPyV genome equivalents per cell (icBKPyV) were 0.27 μM and 0.59 μM, respectively. At 0.63 μM, BCV reduced viral late gene expression by 90% and halted progeny release. Preinfection treatment for only 24 h reduced icBKPyV similarly to treatment from 2 to 72 h postinfection, while combined pre- and postinfection treatment suppressed icBKPyV completely. After investigating BCV's effects on HUC viability, mean selectivity indices at 50 and 90% inhibition (SI50and SI90) calculated for cellular DNA replication were 2.7 and 2.9, respectively, those for mitochondrial activity were 8.9 and 10.4, those for total ATP were 8.6 and 8.2, and those for membrane integrity were 25.9 and 16.7. The antiviral and cytostatic effects, but less so the cytotoxic effects, were inversely related to cell density. The cytotoxic effects at concentrations of ≥10 μM were rapid and likely related to BCV's lipid moiety. After carefully defining the antiviral, cytostatic, and cytotoxic properties of BCV in HUCs, we conclude that a preemptive or prophylactic approach in PyVHC is likely to give the best results.

Final Amended Safety Assessment of Hydroquinone as Used in Cosmetics

Hydroquinone is an aromatic compound that functions in cosmetics as an antioxidant, fragrance, reducing agent, or polymerization inhibitor. Hydroquinone is also used as a skin bleaching agent. Safety and toxicity information indicate that hydroquinone is dermally absorbed in humans from both aqueous and alcoholic formulations and is excreted mainly as the glucuronide or sulfate conjugates. Hydroquinone is associated with altered immune function in vitro and in vivo in animals and an increased incidence of renal tubule cell tumors and leukemia in F344 rats, but the relevance to humans is uncertain. Quantitatively, however, the use of hydroquinone in cosmetics is unlikely to result in renal neoplasia through this mode of action. Thus, hydroquinone is safe at concentrations of ≤1% in hair dyes and is safe for use in nail adhesives. Hydroquinone should not be used in other leave-on cosmetics.

1α,25(OH)2D3-Induced Transrepression by Vitamin D Receptor through E-Box-Type Elements in the Human Parathyroid Hormone Gene Promoter

Although transactivation by the liganded vitamin D receptor (VDR) is well described at the molecular level, the precise molecular mechanism of negative regulation by the liganded VDR remains to be elucidated. We have previously reported a novel class of negative vitamin D response element (nVDRE) called 1αnVDRE in the human 25(OH)D31α-hydroxylase [1α(OH)ase] gene by 1α,25(OH)2D3-bound VDR. This element was composed of two E-box-type motifs that bound to VDIR for transactivation, which was attenuated by liganded VDR. Here, we explore the possible functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters. Functional mapping of the hPTH and hPTHrP promoters identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE; −87 to −60 bp) and in the hPTHrP promoter (hPTHrPnVDRE; −850 to −600 bp; −463 to −104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. These results suggest that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression.