Mitochondrial DNA-enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity

Over the last several years, one of the major advances in the field of alcoholic liver disease research was the discovery that binge alcohol consumption induced neutrophilia and hepatic neutrophil infiltration in chronically ethanol-fed mice and human subjects with excessive alcohol use (EAU); however, the underlying mechanisms remain obscure. Here, we demonstrated that chronic EAU patients with a history of recent excessive drinking (EAU + RD) had higher serum levels of mitochondrial DNA (mtDNA)-enriched microparticles (MPs) than EAU without recent drinking (EAU - RD) and healthy controls, which correlated positively with circulating neutrophils. Similarly, mice with chronic-plus-binge (E10d + 1B) ethanol feeding also had markedly elevated serum levels of mtDNA-enriched MPs, with activation of hepatic ER stress and inflammatory responses. Inhibition of ER stress by gene KO or inhibitors attenuated ethanol-induced elevation of mtDNA-enriched MPs, neutrophilia, and liver injury. The data from the study of hepatocyte-specific deletion of the protein kinase RNA-like ER kinase (Perk) gene in mice and of cultured hepatocytes demonstrated that hepatocytes were the main source of mtDNA-enriched MPs after ethanol feeding. Finally, administration of mtDNA-enriched MPs isolated from E10d+1B-fed mice caused neutrophilia in mice. In conclusion, E10d + 1B ethanol consumption activates hepatic ER stress-dependent mtDNA-enriched MP release, leading to neutrophilia and liver injury

HIV-1 Vpr suppresses expression of the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule

HIV-associated nephropathy (HIVAN) impairs functions of both glomeruli and tubules. Attention has been previously focused on the HIVAN glomerulopathy. Tubular injury has drawn increased attention because sodium wasting is common in hospitalized HIV/AIDS patients. We used viral protein R (Vpr)-transgenic mice to investigate the mechanisms whereby Vpr contributes to urinary sodium wasting. In phosphoenolpyruvate carboxykinase promoter-driven Vpr-transgenic mice, in situ hybridization showed that Vpr mRNA was expressed in all nephron segments, including the distal convoluted tubule. Vpr-transgenic mice, compared with wild-type littermates, markedly increased urinary sodium excretion, despite similar plasma renin activity and aldosterone levels. Kidneys from Vpr-transgenic mice also markedly reduced protein abundance of the Na+-Cl- cotransporter (NCC), while mineralocorticoid receptor (MR) protein expression level was unchanged. In African green monkey kidney cells, Vpr abrogated the aldosterone-mediated stimulation of MR transcriptional activity. Gene expression of Slc12a3 (NCC) in Vpr-transgenic mice was significantly lower compared with wild-type mice, assessed by both qRT-PCR and RNAScope in situ hybridization analysis. Chromatin immunoprecipitation assays identified multiple MR response elements (MRE), located from 5 kb upstream of the transcription start site and extending to the third exon of the SLC12A3 gene. Mutation of MRE and SP1 sites in the SLC12A3 promoter region abrogated the transcriptional responses to aldosterone and Vpr, indicating that functional MRE and SP1 are required for the SLC12A3 gene suppression in response to Vpr. Thus, Vpr attenuates MR transcriptional activity and inhibits Slc12a3 transcription in the distal convoluted tubule and contributes to salt wasting in Vpr-transgenic mice

NCC protein expression was expressed in the immortalized human renal distal convoluted tubule cells.

The immortalized human renal distal convoluted tubule cells were previously characterized [8]. Here, additional immunofluorescence staining was performed. Green, anti-NCC antibody (Cat. No.: AB3553, 1:500 dilution); Blue, nucleus. Scale bar, 100 μm. (TIF)</p

Suppression of MR transcriptional activity by Vpr in human DCT cells.

(A) Human DCT cells were treated with or without soluble Vpr (sVpr, 100ng/ml) for 24-hr followed by aldosterone treatment for the time indicated. Protein expression of NCC and MR were determined by immunoblotting. (B) The density of NCC was quantified by densitometry. The aldosterone-enhanced NCC protein expression, with a peak at 3-hr treatment, was abolished by sVpr (100ng/ml). n = 3. *P P C) Human DCT cells were treated with sVpr, aldosterone, or eplerenone (10μM), individually or in combination as indicated. Expression of SLC12A3 mRNA was determined by qRT-PCR and normalized to β-actin mRNA. The aldosterone-induced increase in SLC12A3 mRNA expression was attenuated by Vpr, but eplerenone had no further effect on SLC12A3 mRNA expression. n = 4–6. **P P (TIF)</p

Primers for qRT-PCR in mouse kidneys and hDCT cells.

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Primary antibodies used in this study.

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Body weight and urine volume of WT and Vpr Tg mice.

(A) Four days after the low salt diet the mice were weighed. n = 7–10. n.s., not significant, Student’s t test. (B) Mouse were placed in metabolic cages and overnight urine was collected before and after the mice were placed on low salt diet. n = 9. n.s., not significant, ANOVA, Bonferroni correction. (TIF)</p