F-Actin Distribution Changes Provoked by Acetaminophen in the Proximal Tubule in Kidney of Adult Male Rat

Acetaminophen is a drug used to treat many conditions as headache, muscle aches, arthritis, backache, toothache, and fever between others, but collateral effects of this drug are not well known yet. Here is tested its effect on proximal tubule epithelium. Acetaminophen (APAP) at doses of 200, 500, 1000 and 1500 mg/Kg i.p. caused cell damage and changes in F-actin distribution in the proximal tubule of male Wistar rats. After 48 hours of treatment, the proximal tubule epithelium showed tumefaction and necrosis. Dose of 200 mg/kg decreased the F-actin and was observed a structure in patches in the basal cytoplasm of epithelial cells of the proximal tubule. This effect was increased depending on the administered dose. Dose of 1000 mg/kg produced the highest histological damage and changes in the actin cytoskeleton. Results of this study suggested that nephrotoxic damage produced by high doses of APAP included breakdown of cytoskeleton in proximal tubule epithelium.

Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury

The mechanism of how SARS-CoV-2 causes severe multi-organ failure is largely unknown. Acute kidney injury(AKI) is one of the frequent organ damage in severe COVID-19 patients. Previous studies have shown that human renal tubule cells could be the potential host cells targeted by SARS-CoV-2. Traditional cancer cell lines or immortalized cell lines are genetically and phenotypically different from host cells. Animal models are widely used, but often fail to reflect a physiological and pathogenic status because of species tropisms. There is an unmet need for normal human epithelial cells for disease modeling. In this study, we successfully established long term cultures of normal human kidney proximal tubule epithelial cells(KPTECs) in 2 D and 3 D culture systems using conditional reprogramming(CR) and organoids techniques.These cells had the ability to differentiate and repair DNA damage, and showed no transforming property. Importantly, the CR KPTECs maintained lineage function with expression of specific transporters(SLC34 A3 and cubilin). They also expressed angiotensin-converting enzyme 2(ACE2), a receptor for SARS-CoV and SARS-CoV-2. In contrast, cancer cell line did not express endogenous SLC34 A3, cubilin and ACE2. Very interestingly, ACE2 expression was around twofold higher in 3 D organoids culture compared to that in 2 D CR culture condition. Pseudovirion assays demonstrated that SARS-CoV spike(S) protein was able to enter CR cells with luciferase reporter. This integrated 2 D CR and 3 D organoid cultures provide a physiological ex vivo model to study kidney functions, innate immune response of kidney cells to viruses, and a novel platform for drug discovery and safety evaluation.

Effect of D_(4)Dopamine Receptor on Na^(+)-K^(+)-ATPase Activity in Renal Proximal Tubule Cells

Objective:Dopamine,via its receptors,plays a vital role in the maintenance of blood pressure by modulating renal sodium transport.However,the role of the D_(4)dopamine receptor(D_(4)receptor)in renal proximal tubules(PRTs)is still unclear.This study aimed to verify the hypothesis that activation of D_(4)receptor directly inhibits the activity of the Na+-K+-ATPase(NKA)in RPT cells.Methods:NKA activity,nitric oxide(NO)and cyclic guanosine monophosphate(cGMP)levels were measured in RPT cells treated with the D_(4)receptor agonist PD168077 and/or the D_(4)receptor antagonist L745870,the NO synthase inhibitor NG-nitro-L-arginine-methyl ester(L-NAME)or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one(ODQ).Total D_(4)receptor expression and its expression in the plasma membrane were investigated by immunoblotting in RPT cells from Wistar-Kyoto(WKY)rats and spontaneously hypertensive rats(SHRs).Results:Activation of D_(4)receptors with PD168077,inhibited NKA activity in RPT cells from WKY rats in a concentration-and time-dependent manner.The inhibitory effect of PD168077 on NKA activity was prevented by the addition of the D_(4)receptor antagonist L745870,which by itself had no effect.The NO synthase inhibitor L-NAME and the soluble guanylyl cyclase inhibitor ODQ,which by themselves had no effect on NKA activity,eliminated the inhibitory effect of PD168077 on NKA activity.Activation of D_(4)receptors also increased NO levels in the culture medium and cGMP levels in RPT cells.However,the inhibitory effect of D_(4)receptors on NKA activity was absent in RPT cells from SHRs,which could be related to decreased plasma membrane expression of D_(4)receptors in SHR RPT cells.Conclusions:Activation of D_(4)receptors directly inhibits NKA activity via the NO/cGMP signaling pathway in RPT cells from WKY rats but not SHRs.Aberrant regulation of NKA activity in RPT cells may be involved in the pathogenesis of hypertension.

不同浓度尿酸和胰岛素对猪近端肾小管上皮细胞株cAMP水平及Na^+-K^+-ATP酶活性的影响

目的:观察不同浓度尿酸和胰岛素对猪近端肾小管上皮细胞株(LLCPK1)细胞内环腺苷酸(cAMP)水平和Na+K+ATP酶活性的改变,探讨代谢相关因素在细胞水平对近端肾小管上皮细胞的离子转运和细胞凋亡影响.方法:以LLCPK1为研究对象,观察尿酸浓度为0,0.1,0.2和0.4mmol/L和胰岛素浓度为0,10-9,10-8,10-7mol/L培养24h条件下,LLCPK1细胞内cAMP水平、Na+K+ATP酶活性的改变以及离子转运情况.结果:0.1,0.2和0.4mmol/L尿酸刺激24h,LLCPK1的cAMP水平、细胞Ca2+,Na+浓度升高,而Na+K+ATP酶的活性、K+浓度下降;胰岛素在高浓度时引起细胞内游离Ca2+,Na+浓度增高,而cAMP水平,Na+K+ATP酶活性及K+浓度下降.结论:尿酸和胰岛素均对LLCPK1细胞具有明显的影响,可能与代谢综合征时肾脏功能改变的发生或保护机制有关.

Toxic nephropathy

970375 Amino acid metabolism analysis and protec-tion against injury to isolated proximal tubules incu-bated with cyclosporin A. JIANG Tang(姜傥), etal. Kidney Instit, 1st Affili Hosp, Sun Yat-sen Med U-niv, Guangzhou, 510080. Chin J Nephrol 1996; 12(6):347-351. Objective: To study the mechanism of cyclosporin

Single-cell RNA sequencing reveals the transcriptomic landscape of kidneys in patients with ischemic acute kidney injury

Background:Ischemic acute kidney injury(AKI)is a common syndrome associated with considerable mortality and healthcare costs.Up to now,the underlying pathogenesis of ischemic AKI remains incompletely understood,and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking.Here,this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing(scRNA-seq)analysis in kidneys.Methods:In this study,scRNA-seq technology was applied to kidneys from two ischemic AKI patients,and three human public scRNA-seq datasets were collected as controls.Differentially expressed genes(DEGs)and cell clusters of kidneys were determined.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,as well as the ligand-receptor interaction between cells,were performed.We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion(I/R)injury induced AKI mice through immunohistochemistry staining.Results:15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control.The injured proximal tubules(PT)displayed a proapoptotic and proinflammatory phenotype.PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47,RASSF4,EBAG9,IER3,SASH1,SEPTIN7,and NUB1,which have not been reported in ischemic AKI previously.Several hub genes were validated in kidneys from human AKI and renal I/R injury mice,respectively.Furthermore,PT highly expressed DEGs enriched in endoplasmic reticulum stress,autophagy,and retinoic acid-inducible gene I(RIG-I)signaling.DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain(NOD)-like receptor signaling,estrogen signaling,interleukin(IL)-12 signaling,and IL-17 signaling.Overexpressed genes in kidney-resident immune cells including macrophages,natural killer T(NKT)cells,monocytes,and dendritic cells were associated with leukocyte activation,chemotaxis,cell adhesion,and complement activation.In addition,the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI.Conclusion:Together,this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients,altered signaling pathways,and potential cell-cell crosstalk in the development of AKI.These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.