In Silico Analysis of Adjuvant Head and Neck Online Adaptive Radiation Therapy.

Purpose: Recently developed online adaptive radiation therapy (OnART) systems enable frequent treatment plan adaptation, but data supporting a dosimetric benefit in postoperative head and neck radiation therapy (RT) are sparse. We performed an in silico dosimetric study to assess the potential benefits of a single versus weekly OnART in the treatment of patients with head and neck squamous cell carcinoma in the adjuvant setting. Methods and Materials: Twelve patients receiving conventionally fractionated RT over 6 weeks and 12 patients receiving hypofractionated RT over 3 weeks on a clinical trial were analyzed. The OnART emulator was used to virtually adapt either once midtreatment or weekly based on the patient's routinely performed cone beam computed tomography. The planning target volume (PTV) coverage, dose heterogeneity, and cumulative dose to the organs at risk for these 2 adaptive approaches were compared with the nonadapted plan. Results: In total, 13, 8, and 3 patients had oral cavity, oropharynx, and larynx primaries, respectively. In the conventionally fractionated RT cohort, weekly OnART led to a significant improvement in PTV V100% coverage (6.2%), hot spot (-1.2 Gy), and maximum cord dose (-3.1 Gy), whereas the mean ipsilateral parotid dose increased modestly (1.8 Gy) versus the nonadapted plan. When adapting once midtreatment, PTV coverage improved with a smaller magnitude (0.2%-2.5%), whereas dose increased to the ipsilateral parotid (1.0-1.1 Gy) and mandible (0.2-0.7 Gy). For the hypofractionated RT cohort, similar benefit was observed with weekly OnART, including significant improvement in PTV coverage, hot spot, and maximum cord dose, whereas no consistent dosimetric advantage was seen when adapting once midtreatment. Conclusions: For head and neck squamous cell carcinoma adjuvant RT, there was a limited benefit of single OnART, but weekly adaptations meaningfully improved the dosimetric criteria, predominantly PTV coverage and dose heterogeneity. A prospective study is ongoing to determine the clinical benefit of OnART in this setting.

Treatment of Oligometastatic Non-Small Cell Lung Cancer: An ASTRO/ESTRO Clinical Practice Guideline.

PURPOSE: This joint guideline by American Society for Radiation Oncology (ASTRO) and the European Society for Radiotherapy and Oncology (ESTRO) was initiated to review evidence and provide recommendations regarding the use of local therapy in the management of extracranial oligometastatic non-small cell lung cancer (NSCLC). Local therapy is defined as the comprehensive treatment of all known cancer-primary tumor, regional nodal metastases, and metastases-with definitive intent. METHODS: ASTRO and ESTRO convened a task force to address 5 key questions focused on the use of local (radiation, surgery, other ablative methods) and systemic therapy in the management of oligometastatic NSCLC. The questions address clinical scenarios for using local therapy, sequencing and timing when integrating local with systemic therapies, radiation techniques critical for oligometastatic disease targeting and treatment delivery, and the role of local therapy for oligoprogression or recurrent disease. Recommendations were based on a systematic literature review and created using ASTRO guidelines methodology. RESULTS: Based on the lack of significant randomized phase 3 trials, a patient-centered, multidisciplinary approach was strongly recommended for all decision-making regarding potential treatment. Integration of definitive local therapy was only relevant if technically feasible and clinically safe to all disease sites, defined as 5 or fewer distinct sites. Conditional recommendations were given for definitive local therapies in synchronous, metachronous, oligopersistent, and oligoprogressive conditions for extracranial disease. Radiation and surgery were the only primary definitive local therapy modalities recommended for use in the management of patients with oligometastatic disease, with indications provided for choosing one over the other. Sequencing recommendations were provided for systemic and local therapy integration. Finally, multiple recommendations were provided for the optimal technical use of hypofractionated radiation or stereotactic body radiation therapy as definitive local therapy, including dose and fractionation. CONCLUSIONS: Presently, data regarding clinical benefits of local therapy on overall and other survival outcomes is still sparse for oligometastatic NSCLC. However, with rapidly evolving data being generated supporting local therapy in oligometastatic NSCLC, this guideline attempted to frame recommendations as a function of the quality of data available to make decisions in a multidisciplinary approach incorporating patient goals and tolerances.

Aspirin use is associated with improvement in distant metastases outcome in patients with residual disease after neoadjuvant chemotherapy.

PURPOSE: Aspirin (ASA) use has been correlated with improved outcomes in high-risk patients at risk for distant metastases. Breast cancer (BC) patients with residual disease, particularly nodal disease (ypN +) after neoadjuvant chemotherapy (NAC), are high-risk patients portending worse outcomes. We hypothesized that ASA use can reduce distant metastases and improve outcomes in these patients. METHODS: Patients at our institutions from 2005 to 2018, with BC who did not achieve complete response (pCR) after NAC were reviewed (IRB protocol STU- 052012-019). Data, including evidence of ASA use, and clinico-pathologic parameters were analyzed. Survival outcomes were obtained (Kaplan Meier analysis) and univariate (UVA) and multivariable (MVA) Cox proportional hazards regression analyses were performed. RESULTS: 637 did not achieve pCR (ypN+ = 422). 138 were ASA users. Median follow-up for the control and ASA group were 3.8 (IQR 2.2-6.3) and 3.8 (IQR 2.5-6.4) years, respectively. Majority were stage II/III. 387 were hormone receptor positive, 191 HER2 +, and 157 triple negative. On UVA, ASA use, PR status, pathologic and clinical stage showed significance for DMFS, and disease-free survival (DFS). On MVA, ASA use associated with improved 5-year DFS (p = .01, 87.0% vs 79.6%, adjusted HR = 0.48) and improved 5-year DMFS (p = .04, 92.8% vs 89.2%, adjusted HR = 0.57). In the ypN + patients, ASA use associated with improved 5-year DMFS (p = .008, 85.7% vs 70.7%, adjusted HR = 0.43) and DFS (p = .02, 86.8% vs 74.3%, adjusted HR = 0.48). CONCLUSION: For non-responders, particularly ypN + patients, ASA use associated with improved outcome. These hypotheses-generating results suggest for development of prospective clinical trials of augmented ASA use in selected very high-risk BC patients.

Feasibility and efficacy of active breathing coordinator assisted deep inspiration breath hold technique for treatment of locally advanced breast cancer.

BACKGROUND: Active breathing coordinator (ABC)-assisted deep inspiration breath hold (DIBH) is an important organ sparing radiation therapy (RT) technique for left-sided breast cancer patients. Patients with advanced breast cancer undergoing chest wall and regional nodal irradiation often require a field matching technique. While field matching has been demonstrated to be safe and effective in free breathing patients, its safety and accuracy in DIBH/ABC use has not been previously reported. PURPOSE: To report the accuracy, feasibility, and safety of field matching with ABC/DIBH for patients receiving breast/chest wall irradiation with nodal irradiation using a three-field technique. METHODS: From December 2012 to May 2018, breast cancer patients undergoing ABC/DIBH-based RT at a single institution were reviewed. For each fraction, the amount of overlap/gap between the supraclavicular and the tangential field were measured and recorded. Patient characteristics, including acute and delayed skin toxicities, were analyzed. RESULTS: A total of 202 patients utilized ABC/DIBH and 4973 fractions had gap/overlap measurements available for analysis. The average gap/overlap measured at junction was 0.28 mm ± 0.99 mm. A total of 72% of fractions had no measurable gap/overlap (0 mm), while 5.6% had an overlap and 22.7% a gap. There was no significant trend for worsening or improvement of gap/overlap measurements with increasing fraction number per patient. OSLD measurements were compared to the planned dose. The median dose 1 cm above the junction was 106% ± 7% of planned dose (range 94%-116%). One centimeter below the junction, the median dose was 114% ± 11% of planned dose (range 95%-131%). At the junction, the median dose was 106% ± 16.3% of planned dose (range 86%-131%). Acute skin toxicity was similar to historically reported values (grade 3, 5.4%, grade 4, 0%). CONCLUSION: ABC-assisted DIBH is a safe and technically feasible method of delivering RT in the setting of complex matching field technique for breast and regional nodal treatments.

Stereotactic Ablative Radiation (SAbR) for Oligometastatic RCC.

A significant proportion of metastatic renal cell carcinoma (mRCC) patients present with oligometastatic disease. Retrospective and limited prospective data suggests that a subgroup of patients with oligometastatic mRCC benefits from aggressive local therapy. With the emerging data of high local control efficacy with low toxicity of stereotactic ablative radiation (SAbR) for both CNS and extra-cranial mRCC, SAbR may play a critical role in the multi-modality management of mRCC patients with oligometastatic disease. In addition to local control benefit, the benefit of SAbR in this patient population can range from longitudinal disease control, maintaining quality of life, deferring systemic therapy, immune-modulation and even improving survival. A review of the retrospective data suggests that SAbR benefits oligometastatic mRCC patients with metachronous metastases, and perhaps those with indolent biology. Large prospective trials are indicated to successfully integrate SAbR of oligometastatic mRCC with the available systemic therapies to harness the optimal benefit of SAbR for this patient population.

Incorporating Routine Magnetic Resonance Imaging-based Planning for the Delivery of High-dose-rate Brachytherapy for Prostate Cancer: An Evaluation of Clinical Feasibility and Dosimetric Outcomes.

Introduction To evaluate the implementation and dosimetric outcomes of magnetic resonance imaging (MRI) planning for improved target and normal tissue definition for the treatment of prostate cancer with high-dose-rate brachytherapy (HDRBT). Methods From August 2015 to October 2017, 137 unique patients with newly diagnosed localized prostate cancer underwent a total of 174 outpatient brachytherapy procedures using MRI-based treatment planning. Patients receiving brachytherapy as monotherapy underwent two separate procedures while those receiving brachytherapy as a boost after external beam radiation therapy underwent a single procedure. The target volume was defined as the prostate +/- seminal vesicles as clinically appropriate without any additional margin. Pre-treatment dose-volume histogram (DVH) goals to the target were: D90≥95%, V90≥95%, V100≥90%, V150≤30%, V200≤15%. DVH goals to organs-at-risk (OARs): urethra D.01cc ≤115%, bladder D1cc ≤75%, rectum D1cc ≤75%, neurovascular bundle D0.1cc ≤100%, penile bulb D1cc ≤100%. Procedure times were recorded at each step of the procedure, from catheter insertion to removal. Results The median target volume was 45.9 cc, the median volume receiving the prescription dose was 53.0 cc, and the median selectivity index was 0.9. The median values for target dosimetry were as follows: D90=99.9%, V90=95.7%, V100=90.1%, V150=28.1%, V200=10.5%. The median values for OAR dosimetry were: urethra D.01cc=114.3%, bladder D1cc=68.3%, rectum D1cc=51.8%, left neurovascular bundle D0.1cc=86.8%, right neurovascular bundle D0.1cc=88.5%, penile bulb D1cc=31.7%. The median time from catheter insertion to end of HDRBT delivery was four hours 14 minutes (range 2:56-9:08); total treatment package time was five hours 32 minutes (range 3:31-9:45). Conclusion Routine MRI-based treatment planning is feasible for the delivery of HDRBT for prostate cancer. We met stringent dosimetric criteria despite more objective target and normal tissue definition with MRI imaging. Treatment package time remains reasonable. We have adopted MRI as our standard imaging modality for HDRBT for prostate cancer.

GammaKnife versus VMAT radiosurgery plan quality for many brain metastases.

The purpose of this work was to compare dose distributions between two radiosurgery modalities, single-isocenter volumetric modulated arc therapy (VMAT), and GammaKnife Perfexion (GK), in the treatment of a large number (≥7) of brain metastases. Twelve patients with 103 brain metastases were analyzed. The median number of targets per patient was 8 (range: 7-14). GK plans were compared to noncoplanar VMAT plans using both 6-MV flattening filter-free (FFF) and 10-MV FFF modes. Parameters analyzed included radiation therapy oncology group conformity index (CI), 12, 6, and 3 Gy isodose volumes (V12 Gy, V6 Gy, V3 Gy), mean and maximum hippocampal dose, and maximum skin dose. There were statistically significant differences in CI (2.5 ± 1.6 vs 1.6 ± 0.8 and 1.7 ± 0.9, P < 0.001, P < 0.001), V12 Gy (2.8 ± 6.1 cc vs 3.0 ± 5.2 cc and 3.1 ± 5.4 cc, P = 0.003, P < 0.001), and V3 Gy (323.0 ± 294.8 cc vs, 880.1 ± 369.1 cc and 937.9 ±  vs 361.9 cc, P = 0.005, P = 0.001) between GK versus both 6-MV FFF and 10-MV FFF. No significant differences existed for maximum hippocampal or skin doses. In conclusion, highly optimized VMAT produced improved conformity at the expense of a higher V12 Gy and V3 Gy volume when compared with highly optimized GK.

Acute toxicity of craniospinal irradiation with volumetric-modulated arc therapy in children with solid tumors.

BACKGROUND: Craniospinal irradiation (CSI) is an important part of curative radiation therapy (RT) for many types of pediatric brain or solid tumors. After conventional CSI, long term survivors may experience sequelae due to unintended dose to normal tissue. Volumetric modulated arc therapy (VMAT) CSI reduces off-target doses at the cost of greater complexity and error risk, and we describe our initial experience in a group of pediatric patients with solid tumors presenting with disseminated or recurrent disease. PROCEDURE: Pediatric patients with brain tumors were identified at Children's Hospital Los Angeles from 2013 to 2015. Clinical characteristics, acute toxicity, and radiotherapy data were abstracted from their medical records. We identified 19 patients who received VMAT CSI. Quality assurance was performed with a cylindrical detector array and ion chamber measurements at the arc junctions. RESULTS: Patients had medulloblastoma or supratentorial primitive neuro-ectodermal tumor (n = 14, 11 high risk), germ cell tumors (two), relapsed neuroblastoma (two), and atypical teratoid/rhabdoid tumor (one). The most common acute toxicity was hematologic, including leukopenia (11% grade [Gr] 2, 26% Gr 3, and 63% Gr 4), anemia (89% Gr 2), and thrombocytopenia (16% Gr 1-2, 26% Gr 3, and 37% Gr 4). Despite leukopenia, we encountered only two Gr 3 infections (urinary tract and lung). The majority required blood products (89% red blood cells and 68% platelets). Weight loss was also common (47% Gr 1 and 26% Gr 2). CONCLUSIONS: VMAT CSI, along with chemotherapy and anesthesia, is feasible with supportive care. Daily image-guided RT improves accuracy and reduces the risk of spinal cord overdose without increasing treatment time. Further research is needed to determine whether reducing doses to organs, such as thyroid, heart, or hippocampus, offsets the risk of increased volume of low-dose irradiation.

A Case of Streptococcus anginosus Brain Abscess Caused by Contiguous Spread from Sinusitis in an Immunocompetent Patient.

Brain abscesses are infections of the brain parenchyma that can arise from either contiguous spread from local infection or by hematogenous spread from a distant site. Streptococcus anginosus of the Streptococcus anginosus group (SAG) is a commensal microbe of the mucosae of the oral cavity, gastrointestinal tract, and urogenital tract. We present a case of mono-microbial brain abscess caused by contiguous spread from relatively asymptomatic sinusitis that initially presented as a subdural hemorrhage on computed tomography. A 70-year-old male presented, obtunded, with a Glasgow Coma Score of eight. The patient seized on arrival. A computed tomography scan was read as a subdural hemorrhage, and magnetic resonance imaging showed a heterogeneous area at the anterior tip of the left frontal lobe interpreted as a frontoparietal abscess, along with pansinusitis. Craniotomy revealed a loculated abscess. Culture grew only Streptococcus anginosus. The patient did well postoperatively, was extubated by day five with rapidly improving neurological function, and was discharged to inpatient rehab by hospital-day eight for the continuation of intravenous antibiotics. This case represents a frontal lobe abscess caused by the contiguous spread of Streptococcus anginosus from a frontal sinus infection. This is a relatively rare presentation of SAG infection in an immunocompetent patient. The case outlines the importance of imaging modality choice in the various stages of brain abscess formation, and the necessity of maintaining an index of suspicion for brain abscess in patients with few traditional risk factors and little to no history on presentation.

Radiotherapy after high-dose chemotherapy with autologous hematopoietic cell rescue: Quality assessment of Head Start III.

BACKGROUND: The use of high-dose chemotherapy with autologous hematopoietic cell rescue (AuHCR) in Head Start III is a potentially curative approach for the management of young children with central nervous system neoplasms. We report the potential influence of quality and timing of radiation therapy on the survival of patients treated on the study. PROCEDURE: Between 2003 and 2009, 220 children with newly diagnosed central nervous system neoplasms were enrolled on the study. Radiation therapy was indicated following AuHCR for children between 6 and 10 years old or those younger than 6 years with residual tumor preconsolidation. Records were received for 42 patients and reviewed to determine adherence to protocol treatment volume and dose guidelines. Of these patients, seven were irradiated prior to consolidation, and additional four patients who initially avoided radiation therapy after AuHCR were subsequently treated at relapse. RESULTS: Of the 31 patients who were fully evaluable, 2 refused radiation therapy until recurrence and 4 progressed between recovery from AuHCR and radiation therapy. Of the remaining 25 patients, 8 had violations in their indication, dose, or treatment volume. All violations occurred in patients under 6 years of age. Two patients could have avoided radiation therapy. There were 6 violations in the 23 patients who received radiation therapy for guideline indications. CONCLUSION: All protocol violations occurred in patients under 6 years of age and were associated with decreased overall survival as was the time to start radiotherapy of greater than 11 weeks. When indicated, starting radiation therapy soon after neutrophil and platelet recovery may improve the outcome for these high-risk children.

Re-irradiation of Recurrent Pineal Germ Cell Tumors with Radiosurgery: Report of Two Cases and Review of Literature.

Primary intracranial germ cell tumors are rare, representing less than 5% of all central nervous system tumors. Overall, the majority of germ cell tumors are germinomas and approximately one-third are non-germinomatous germ cell tumors (NGGCT), which include teratoma, embryonal carcinoma, yolk sac tumor (endodermal sinus tumor), choriocarcinoma, or mixed malignant germ cell tumor. Germ cell tumors may secrete detectable levels of proteins into the blood and/or cerebrospinal fluid, and these proteins can be used for diagnostic purposes or to monitor tumor recurrence. Germinomas have long been known to be highly curable with radiation therapy alone. However, many late effects of whole brain or craniospinal irradiation have been well documented. Strategies have been developed to reduce the dose and volume of radiation therapy, often in combination with chemotherapy. In contrast, patients with NGGCT have a poorer prognosis, with about 60% cured with multimodality chemoradiation. There are no standard approaches for relapsed germ cell tumors. Options may be limited by prior treatment. Radiation therapy has been utilized alone or in combination with chemotherapy or high-dose chemotherapy and transplant. We discuss two cases and review options for frameless radiosurgery or fractionated radiotherapy.

Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells.

We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry 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.

Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1.

AIMS: The present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues. MAIN METHODS: Tissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR. KEY FINDINGS: The effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart. SIGNIFICANCE: Per1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.

A role for the circadian clock protein Per1 in the regulation of the NaCl co-transporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells.

It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention.

The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na(+) transport genes in the collecting duct, including the α-subunit of the renal epithelial Na(+) channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3ß-dehydrogenase isomerase (3ß-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na(+) retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3ß-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3ß-HSD. We postulated that mice with reduced Per1 would have a renal Na(+)-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na(+) compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na(+) retention.

Role of Per1 and the mineralocorticoid receptor in the coordinate regulation of αENaC in renal cortical collecting duct cells.

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney.

Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1-3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.