Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.
WNT ligands induce Ca2+ signaling on target cells. PKD1 (Polycystin 1) is considered an orphan, atypical G protein coupled receptor complexed with TRPP2 (Polycystin 2 or PKD2), a Ca2+-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole cell currents and Ca2+ influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2−/− fibroblasts lack WNT-induced Ca2+ currents and are unable to polarize during directed cell migration. In Xenopus embryos, PKD1, Dishevelled 2 (DVL2), and WNT9A act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca2+ signaling as one of the causes of ADPKD.
Primary cilia start forming within the G1 phase of the cell cycle and continue to grow as cells exit the cell cycle (G0). They start resorbing when cells re-enter the cell cycle (S phase) and are practically invisible in mitosis. The mechanisms by which cilium biogenesis and disassembly are coupled to the cell cycle are complex and not well understood. We previously identified the centrosomal phosphoprotein NDE1 as a negative regulator of ciliary length and showed that its levels inversely correlate with ciliogenesis. Here, we identify the tumor suppressor FBW7 (also known as FBXW7, CDC4, AGO, or SEL-10) as the E3 ligase that mediates the destruction of NDE1 upon entry into G1. CDK5, a kinase active in G1/G0, primes NDE1 for FBW7-mediated recognition. Cells depleted of FBW7 or CDK5 show enhanced levels of NDE1 and a reduction in ciliary length, which is corrected in cells depleted of both FBW7 or CDK5 and NDE1. These data show that cell cycle-dependent mechanisms can control ciliary length through a CDK5-FBW7-NDE1 pathway.
Cilia and flagella are evolutionarily conserved, membrane-bound, microtubule-based organelles on the surface of most eukaryotic cells. They play important roles in coordinating a variety of signaling pathways during growth, development, cell mobility, and tissue homeostasis. Defects in ciliary structure or function are associated with multiple human disorders called ciliopathies. These diseases affect diverse tissues, including, but not limited to the eyes, kidneys, brain, and lungs. Many processes must be coordinated simultaneously in order to initiate ciliogenesis. These include cell cycle, vesicular trafficking, and axonemal extension. Centrioles play a central role in both cell cycle progression and ciliogenesis, making the transition between basal bodies and mitotic spindle organizers integral to both processes. The maturation of centrioles involves a functional shift from cell division toward cilium nucleation which takes place concurrently with its migration and fusion to the plasma membrane. Several proteinaceous structures of the distal appendages in mother centrioles are required for this docking process. Ciliary assembly and maintenance requires a precise balance between two indispensable processes; so called assembly and disassembly. The interplay between them determines the length of the resulting cilia. These processes require a highly conserved transport system to provide the necessary substances at the tips of the cilia and to recycle ciliary turnover products to the base using a based microtubule intraflagellar transport (IFT) system. In this review; we discuss the stages of ciliogenesis as well as mechanisms controlling the lengths of assembled cilia.
BackgroundAcute kidney injury (AKI) is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here we hypothesize that quantification of SARS-CoV-2 viral load in urine sediment from COVID-19 patients correlates with occurrence of AKI and mortality. MethodsSARS-CoV-2 viral load in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, hospitalized between March 15th and June 8th, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsies and urine sediments. Viral infectivity assays were performed from 32 urine sediments. ResultsTwenty COVID-19 patients (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load 4-times higher than non-AKI COVID-19 patients. U-viral load was highest (7.7-fold) within two weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsies in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. ConclusionOur results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from COVID-19 patients correlated with increased incidence of AKI and mortality. Urinary viral detection could inform medical care of COVID-19 patients with kidney injury to improve prognosis.
Mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by progressive metabolic alterations that include obesity, hypertension, and chronic kidney disease (CKD). SNPs in the ALMS1 gene in the general population are associated with lower GFR, hypertension and metabolic syndrome. ALMS1 is widely expressed where it localizes to endosomes, centrosomes and the base of cilia. Our lab has previously shown that ALMS1 is expressed in renal tubules of the Thick Ascending Limb (TAL) where NKCC2 is the primary apical NaCl transporter. Whole animal gene deletion of ALMS1 increases blood pressure, and inhibits NKCC2 endocytosis, increasing its surface expression and renal NaCl reabsorption. However, whole animal ALMS1 KO rats or mice develop obesity, affecting the interpretation of organ-specific data. We hypothesized that ALMS1 is a part of protein complex that binds apical NKCC2 in TALs and promotes its endocytosis. To study the role of ALMS1 in the nephron without influence of obesity we generated a loxP flanked exon 7 ALMS1 transgenic mouse line, that we crossed with nephron specific inducible Pax8-rtTA Cre mice (Pax8-ALMS1 KO). 4 weeks after inducing Cre expression with doxycycline (2mg/ml in 2% sucrose), ALMS1 expression in TAL suspensions decreased by 75% (p<0.01). To study the role of ALMS1 in TAL function, we measured surface to total NKCC2 ratio, which was increased by 53±15% in TALs from Pax8-ALMS1 KO compared to control floxed ALMS1 (p<0.01). Recombinant ALMS1 (GST-Carboxyl terminus-ALMS1), or immunoprecipitation of ALMS1, pulled down NKCC2 from WT TAL lysates (n=3), suggesting that the mechanism by which ALMS1 regulates surface NKCC2 involves protein-protein interactions. 4 weeks after inducing Cre expression with doxycycline we measured bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was higher in Pax8-ALMS1 KO, compared to control (control: 115±19 vs Pax8-ALMS1 KO: 146±30 μmols Na/4h, p<0.05). Since whole animal ALMS1 deletion induced hypertension, we measured systolic blood pressure (SBP) by tail cuff one month after inducible deletion of ALMS1 or in floxed ALMS1 mice treated with doxycycline as controls. Baseline SBP in normal Na diet (0.4%) was similar between strains. However, SBP increased by 12±3 mmHg in Pax8-ALMS1 KO, but not in control floxed ALMS1 littermates after 9 days of high Na diet (WT: 100±10, Pax8-ALMS1 KO: 115±11 mmHg, p<0.05 from baseline, n=4). We conclude that ALMS1, binds NKCC2 to regulate its surface expression. Depletion of ALMS1 in the nephron enhances surface NKCC2, TAL NaCl reabsorption, and induces salt-sensitivity of blood pressure. Our data indicate that regulation of the ALMS1-NKCC2 interaction in the TAL is important for renal NaCl reabsorption and regulation of blood pressure. Funding: NIH R56DK131114, Henry Ford Fund This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
A large fraction of patients with diabetes mellitus develop diabetic nephropathy and are hypertensive. The Akita mice is one of the few mouse models of diabetes mellitus with features of the human disease including hypertension and kidney damage. Previous data showed increased levels of reactive oxygen species in the kidneys of Akita mice. We found that hydrogen peroxide (H 2 O 2 ) is a potent stimulus for renin release from the juxtaglomerular (JG) cells in vitro and in vivo . Plasma pro-renin levels are elevated in diabetic patients and thought to contribute to hypertension and renal damage. Thus, we hypothesized that H 2 O 2 levels are higher in JG cells from Akita mice leading to increased renin and pro-renin release in diabetes. We measured H 2 O 2 by transducing primary cultures of JG cells with the fluorescent protein H 2 O 2 sensor Hyper, under control of the renin1 promoter. JG cells isolated from Akita mice had higher baseline levels of H 2 O 2 (C57= 100, Akita=222.5±79%, p <0.05). We also found that pro-renin expression was higher in JG cells from Akita by 79±21% ( p <0.05). In addition, JG cells from Akita mice have higher pro-renin (n=4; p <0.05) and renin (C57= 100, Akita=219.2±26%; p <0.04, n=6) release to the media when compared to JG cells from C57. Western Blots showed that the NADPH oxidase isoform NOX1 is upregulated in JG cells from Akita mice (C57=100%; Akita=147±10%; n=4; p <0.05), whereas NOX2 or NOX4 were not different. NOX1 co-localized with renin-containing granules in kidney sections analyzed by confocal microscopy (n=4). Silencing NOX1 in JG cells from Akita mice decreased renin release to C57 control levels; (n=6; p <0.01) while silencing NOX4 had no effect (n=6). Expression of Catalase, which scavenges H 2 O 2 , was reduced in JG cells from Akita mice (C57=100%; Akita= 36±9.2%; n=4; p <0.05) and overexpression of Catalase in JG cells from Akita decreased baseline renin release by 35±8% ( p <0.05). We conclude that renin cells from diabetic Akita mice have higher endogenous H 2 O 2 leading to increased renin and pro-renin release via enhanced NOX1 expression and decreased catalase. Enhanced H 2 O 2 -dependent renin release in Akita JG cells could be involved in renal damage and hypertension in diabetes mellitus.
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