[PubMed] [Google Scholar] 15. detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na+-K+-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney. polymerase, and 35 amplification cycles were performed using the following parameters: 95C for 30 s, 55C for 30 s, and 72C for 1 min followed by a final 10-min extension at 72C. Table 1. Sequences and exon numbers = 3 or more. Statistical analyses were performed using Graphpad Prism (version 6). All graphs/plots were made with Graphpad Prism (version 6). An unpaired Student’s values of 0.05 were considered significant. RESULTS Pharmacological blockade of Per1 nuclear entry in vivo results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. Per1 must be phosphorylated by CK1/ to enter the nucleus (22). Our laboratory has previously shown that pharmacological inhibition of CK1/ recapitulates the effects of Per1 knockdown, including decreased ENaC mRNA levels, protein levels, and ENaC activity (33, 35). To determine if Per1 regulates NHE3, SGLT1, and SGLT2 in vivo, WT mice were treated with vehicle or the CK1/ inhibitor PF670462 as previously described (34). Kidneys were harvested, and the cortex was dissected. mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. PF670462 treatment resulted in significantly decreased levels of NHE3 (Fig. 1= 4. * 0.05 compared with WT mice. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear entry in vitro results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. To further investigate our in vivo results, the human proximal tubule cell line HK-2 was used for subsequent experiments (19, 49). Per1 was knocked down using siRNA in HK-2 cells, and mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. As expected, Per1 knockdown resulted in significantly decreased mRNA expression of Per1 (Fig. 2= 3. * 0.05; ** 0.01. To further explore the potential role of Per1 in the regulation of NHE3 and SGLT1, HK-2 cells were treated with PF670462, and mRNA expression of NHE3, SGLT1, and SGLT2 was determined by quantitative real-time PCR. After 24 h, treatment with 10 M PF670462 resulted in a significant reduction of NHE3 and SGLT1 mRNA (Fig. 3, and and = 3. * 0.05. Open in a separate window Fig. 4. Pharmacological blockade of Per1 nuclear entry results in decreased nuclear Per1 in vitro. = 3. ** 0.01. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear entry results in decreased transcription of NHE3 and SGLT1. Measurement of short-lived hnRNA is a measure of transcriptional activity (10, 23). To assess if the effect of CK1/ inhibition or Per1 knockdown on NHE3 and SGLT1 DO34 analog was transcriptional, hnRNA levels were assessed by PCR amplification of intron-exon junctions using cDNA templates from HK-2 cells treated with either PF670462 for 24 h or Per1 siRNA for 48 h. Per1 siRNA-mediated knockdown or blockade of Per1 nuclear entry led to significantly decreased hnRNA expression of both NHE3 (Fig. 5= 3. * 0.05. ** 0.01. Pharmacological inhibition of Per1.[PubMed] [Google Scholar] 43. of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na+-K+-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney. polymerase, and 35 amplification cycles were performed using the following parameters: 95C for 30 s, 55C for 30 s, and 72C for 1 min followed by a final 10-min extension at 72C. Table 1. Sequences and exon numbers = 3 or more. Statistical analyses were performed using Graphpad Prism (version 6). All graphs/plots were made with Graphpad Prism (version 6). An unpaired Student’s values of 0.05 were considered significant. RESULTS Pharmacological blockade of Per1 nuclear entry DO34 analog in vivo results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. Per1 must be phosphorylated by CK1/ to enter the nucleus (22). Our laboratory has previously shown that pharmacological inhibition of CK1/ recapitulates the effects of Per1 knockdown, including decreased ENaC mRNA levels, protein levels, and ENaC activity (33, 35). To determine if Per1 regulates NHE3, SGLT1, and SGLT2 in vivo, WT mice were treated with vehicle or the CK1/ inhibitor PF670462 as previously described (34). Kidneys were harvested, and the cortex was dissected. mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. PF670462 treatment resulted in significantly decreased levels of NHE3 (Fig. 1= 4. * 0.05 compared with WT mice. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear entry in vitro results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. To further investigate our in vivo results, the human proximal tubule cell line HK-2 was used for subsequent experiments (19, 49). Per1 was knocked down using siRNA in HK-2 cells, and mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. As expected, Per1 knockdown resulted in significantly decreased mRNA expression of Per1 (Fig. 2= 3. * 0.05; ** 0.01. To further explore the potential role of Per1 in the regulation of NHE3 and SGLT1, HK-2 cells were treated with PF670462, and mRNA expression of NHE3, SGLT1, and SGLT2 was determined by quantitative real-time PCR. After 24 h, treatment with 10 M PF670462 resulted in a significant reduction of NHE3 and SGLT1 mRNA (Fig. 3, and and = 3. * 0.05. Open in a separate window Fig. 4. Pharmacological blockade of Per1 nuclear entry results in decreased nuclear Per1 in vitro. = 3. ** 0.01. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear entry results in decreased transcription of NHE3 and SGLT1. Measurement of short-lived hnRNA is a measure of transcriptional activity (10, 23). To assess if the effect of CK1/ inhibition or Per1 knockdown on NHE3 and SGLT1 was transcriptional, hnRNA levels were assessed by PCR amplification of intron-exon junctions using cDNA templates from HK-2 cells treated with either PF670462 for 24 h or Per1 siRNA for 48 h. Per1 siRNA-mediated knockdown or blockade of Per1 nuclear entry led to significantly decreased hnRNA expression of both NHE3 (Fig. 5= 3..Acta Physiol Scand 173: 59C66, 2001. resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na+-K+-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney. polymerase, and 35 amplification cycles were performed using the following parameters: 95C for 30 s, 55C for 30 s, and 72C for 1 min followed by a final 10-min extension at 72C. Table 1. Sequences and exon numbers = 3 or more. Statistical analyses were performed using Graphpad Prism (version 6). All graphs/plots were made with Graphpad Prism (version 6). An unpaired Student’s values of 0.05 were considered significant. RESULTS Pharmacological blockade of Per1 nuclear entry in vivo results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. Per1 must be phosphorylated by CK1/ to enter the nucleus (22). Our laboratory has previously shown that pharmacological inhibition of CK1/ recapitulates the effects of Per1 knockdown, including decreased ENaC mRNA levels, protein levels, and ENaC activity (33, 35). To determine if Per1 regulates NHE3, SGLT1, and SGLT2 in vivo, WT mice were treated with vehicle or the CK1/ inhibitor PF670462 as previously described (34). Kidneys were harvested, and the cortex was dissected. mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. PF670462 treatment resulted in significantly decreased levels of NHE3 (Fig. 1= 4. * 0.05 compared with WT mice. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear entry in vitro results in decreased mRNA expression of NHE3 and SGLT1 but not SGLT2. To further investigate our in vivo results, the human proximal tubule cell line HK-2 was used for subsequent experiments (19, 49). Per1 was knocked down using siRNA in HK-2 cells, and mRNA levels of NHE3, SGLT1, and SGLT2 were measured by quantitative real-time PCR. As expected, Per1 knockdown resulted in significantly decreased mRNA expression of Per1 (Fig. 2= 3. * 0.05; ** 0.01. To further explore the potential role of Per1 in the regulation of NHE3 and SGLT1, HK-2 cells were treated with PF670462, and mRNA expression of NHE3, SGLT1, and SGLT2 was dependant on quantitative real-time PCR. After 24 h, treatment with 10 M PF670462 led to a significant reduced amount of NHE3 and SGLT1 mRNA (Fig. 3, and and = 3. * 0.05. Open up in another windowpane Rabbit polyclonal to ANKRD1 Fig. 4. Pharmacological blockade of Per1 nuclear admittance results in reduced nuclear Per1 in vitro. = 3. ** 0.01. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear admittance results in reduced transcription of NHE3 and SGLT1. Dimension of short-lived hnRNA can be a way of measuring transcriptional activity (10, 23). To assess if the result of CK1/ inhibition or Per1 knockdown on NHE3 and SGLT1 was transcriptional, hnRNA amounts had been evaluated by PCR amplification of intron-exon junctions using cDNA web templates from HK-2 cells treated with either PF670462 for 24 h or Per1 siRNA for 48 h. Per1 siRNA-mediated knockdown or blockade of Per1 nuclear admittance led to considerably decreased hnRNA manifestation of both NHE3 (Fig. 5= 3. * 0.05. ** 0.01. Pharmacological inhibition of Per1 nuclear admittance results in reduced relationships of Per1 and CLOCK with promoters of NHE3 and SGLT1. As referred to above, rules.Curr Opin Nephrol Hypertens 22: 439C444, 2013. renal cortex of mice. Per1 little interfering RNA and pharmacological blockade of Per1 nuclear admittance in human being proximal tubule HK-2 cells yielded the same outcomes. Study of heterogeneous nuclear RNA recommended that the consequences of Per1 on NHE3 and SGLT1 manifestation occurred at the amount of transcription. Per1 as well as the circadian proteins CLOCK had been recognized at promoters of NHE3 and SGLT1. Significantly, both membrane and intracellular proteins degrees of NHE3 and SGLT1 had been reduced after blockade of nuclear Per1 admittance. This impact was connected with decreased activity of Na+-K+-ATPase. These data show a job for Per1 in the transcriptional rules of NHE3 and SGLT1 in the kidney. polymerase, and 35 amplification cycles had been performed using the next guidelines: 95C for 30 s, 55C for 30 s, and 72C for 1 min accompanied by your final 10-min expansion at 72C. Desk 1. Sequences and exon amounts = 3 or even more. Statistical analyses had been performed using Graphpad Prism (edition 6). All graphs/plots had been made out of Graphpad Prism (edition 6). An unpaired Student’s ideals of 0.05 were considered significant. Outcomes Pharmacological blockade of Per1 nuclear admittance in vivo leads to decreased mRNA manifestation of NHE3 and SGLT1 however, not SGLT2. Per1 should be phosphorylated by CK1/ to enter the nucleus (22). Our lab has previously demonstrated that pharmacological inhibition of CK1/ recapitulates the consequences of Per1 knockdown, including reduced ENaC mRNA amounts, proteins amounts, and ENaC activity (33, 35). To see whether Per1 regulates NHE3, SGLT1, and SGLT2 in vivo, WT mice had been treated with automobile or the CK1/ inhibitor PF670462 as previously referred to (34). Kidneys had been harvested, as well as the cortex was dissected. mRNA degrees of NHE3, SGLT1, and SGLT2 had been assessed by quantitative real-time PCR. PF670462 treatment led to significantly decreased degrees of NHE3 (Fig. 1= 4. * 0.05 weighed against WT mice. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear admittance in vitro leads to decreased mRNA manifestation of NHE3 and SGLT1 however, not SGLT2. To help expand check out our in vivo outcomes, the human being proximal tubule cell range HK-2 was useful for following tests (19, 49). Per1 was knocked down using siRNA in HK-2 cells, and mRNA degrees of NHE3, SGLT1, and SGLT2 had DO34 analog been assessed by quantitative real-time PCR. Needlessly to say, Per1 knockdown led to significantly reduced mRNA manifestation of Per1 (Fig. 2= 3. * 0.05; ** 0.01. To help expand explore the part of Per1 in the rules of NHE3 and SGLT1, HK-2 cells had been treated with PF670462, and mRNA manifestation of NHE3, SGLT1, and SGLT2 was dependant on quantitative real-time PCR. After 24 h, treatment with 10 M PF670462 led to a significant reduced amount of NHE3 and SGLT1 mRNA (Fig. 3, and and = 3. * 0.05. Open up in another windowpane Fig. 4. Pharmacological blockade of Per1 nuclear admittance results in reduced nuclear Per1 in vitro. = 3. ** 0.01. Per1 siRNA-mediated knockdown or pharmacological inhibition of Per1 nuclear admittance results in reduced transcription of NHE3 and SGLT1. Dimension of short-lived hnRNA can be a way of measuring transcriptional activity (10, 23). To assess if the result of CK1/ inhibition or Per1 knockdown on NHE3 and SGLT1 was transcriptional, hnRNA amounts had been evaluated by PCR amplification of intron-exon junctions using cDNA web templates from HK-2 cells treated with either PF670462 for 24 h or Per1 siRNA for 48 h. Per1 siRNA-mediated knockdown or blockade of Per1 nuclear admittance led to considerably decreased hnRNA manifestation of both NHE3 (Fig. 5= 3. * 0.05. ** 0.01. Pharmacological inhibition of Per1.
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