Hedgehog blockade remodels the gut microbiota and the intestinal effector CD8+ T cells in a mouse model of mammary carcinoma.

Given the gut microbiome's rise as a potential frontier in cancer pathogenesis and therapy, leveraging microbial analyses in the study of breast tumor progression and treatment could unveil novel interactions between commensal bacteria and disease outcomes. In breast cancer, the Hedgehog (Hh) signaling pathway is a potential target for treatment due to its aberrant activation leading to poorer prognoses and drug resistance. There are limited studies that have investigated the influences of orally administered cancer therapeutics, such as Vismodegib (a pharmacological, clinically used Hh inhibitor) on the gut microbiota. Using a 4T1 mammary carcinoma mouse model and 16 S rRNA sequencing, we longitudinally mapped alterations in immunomodulating gut microbes during mammary tumor development. Next, we identified changes in the abundance of commensal microbiota in response to Vismodegib treatment of 4T1 mammary tumor-bearing mice. In addition to remodeling gut microbiota, Vismodegib treatment elicited an increase in proliferative CD8+ T cells in the colonic immune network, without any remarkable gastrointestinal-associated side effects. To our knowledge, this is the first study to assess longitudinal changes in the gut microbiome during mammary tumor development and progression. Our study also pioneers an investigation of the dynamic effects of an orally delivered Hh inhibitor on the gut microbiome and the gut-associated immune-regulatory adaptive effector CD8+ T cells. These findings inform future comprehensive studies on the consortium of altered microbes that can impact potential systemic immunomodulatory roles of Vismodegib.

Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice.

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3.

Urea has a critical role in urinary concentration. Mice lacking the inner medullary collecting duct (IMCD) urea transporter A1 (UT-A1) and urea transporter A3 (UT-A3) have very low levels of urea permeability and are unable to concentrate urine. To investigate the role of UT-A1 in the concentration of urine, we transgenically expressed UT-A1 in knockout mice lacking UT-A1 and UT-A3 using a construct with a UT-A1 gene that cannot be spliced to produce UT-A3. This construct was inserted behind the original UT-A promoter to yield a mouse expressing only UT-A1 (UT-A1(+/+)/UT-A3(-/-)). Western blot analysis demonstrated UT-A1 in the inner medulla of UT-A1(+/+)/UT-A3(-/-) and wild-type mice, but not in UT-A1/UT-A3 knockout mice, and an absence of UT-A3 in UT-A1(+/+)/UT-A3(-/-) and UT-A1/UT-A3 knockout mice. Immunohistochemistry in UT-A1(+/+)/UT-A3(-/-) mice also showed negative UT-A3 staining in kidney and other tissues and positive UT-A1 staining only in the IMCD. Urea permeability in isolated perfused IMCDs showed basal permeability in the UT-A1(+/+)/UT-A3(-/-) mice was similar to levels in wild-type mice, but vasopressin stimulation of urea permeability in wild-type mice was significantly greater (100% increase) than in UT-A1(+/+)/UT-A3(-/-) mice (8% increase). Notably, basal urine osmolalities in both wild-type and UT-A1(+/+)/UT-A3(-/-) mice increased upon overnight water restriction. We conclude that transgenic expression of UT-A1 restores basal urea permeability to the level in wild-type mice but does not restore vasopressin-stimulated levels of urea permeability. This information suggests that transgenic expression of UT-A1 alone in mice lacking UT-A1 and UT-A3 is sufficient to restore urine-concentrating ability.

Metformin, an AMPK activator, stimulates the phosphorylation of aquaporin 2 and urea transporter A1 in inner medullary collecting ducts.

Nephrogenic diabetes insipidus (NDI) is characterized by production of very large quantities of dilute urine due to an inability of the kidney to respond to vasopressin. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in ∼90% of families. These patients do not have mutations in aquaporin-2 (AQP2) or urea transporter UT-A1 (UT-A1). We tested adenosine monophosphate kinase (AMPK) since it is known to phosphorylate another vasopressin-sensitive transporter, NKCC2 (Na-K-2Cl cotransporter). We found AMPK expressed in rat inner medulla (IM). AMPK directly phosphorylated AQP2 and UT-A1 in vitro. Metformin, an AMPK activator, increased phosphorylation of both AQP2 and UT-A1 in rat inner medullary collecting ducts (IMCDs). Metformin increased the apical plasma membrane accumulation of AQP2, but not UT-A1, in rat IM. Metformin increased both osmotic water permeability and urea permeability in perfused rat terminal IMCDs. These findings suggest that metformin increases osmotic water permeability by increasing AQP2 accumulation in the apical plasma membrane but increases urea permeability by activating UT-A1 already present in the membrane. Lastly, metformin increased urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. We conclude that AMPK activation by metformin mimics many of the mechanisms by which vasopressin increases urine-concentrating ability. These findings suggest that metformin may be a novel therapeutic option for congenital NDI due to V2R mutations.

Endothelial histone deacetylase 1 activity impairs kidney microvascular NO signaling in rats fed a high-salt diet.

AIM: We aimed to test the hypothesis that a high-salt diet (HS) impairs NO signaling in kidney microvascular endothelial cells through a histone deacetylase 1 (HDAC1)-dependent mechanism. METHODS: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or HS (4% NaCl) for 2 weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. RESULTS: We found HS-induced impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS-fed rats to be sufficient to induce impaired NO signaling. This indicates the presence of a humoral factor we termed plasma-derived endothelial dysfunction mediator (PDEM). Pretreatment with the antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. CONCLUSION: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.

Endothelial Histone Deacetylase 1 Activity Impairs Kidney Microvascular NO Signaling in Rats fed a High Salt Diet.

Aim: We aimed to identify new mechanisms by which a high salt diet (HS) decreases NO production in kidney microvascular endothelial cells. Specifically, we hypothesized HS impairs NO signaling through a histone deacetylase 1 (HDAC1)-dependent mechanism. Methods: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or high salt diet (4% NaCl) for two weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. Results: We found that HS impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS fed rats to be sufficient to induce dysfunction suggesting a humoral factor, we termed Plasma Derived Endothelial-dysfunction Mediator (PDEM), mediates the endothelial dysfunction. The antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. Conclusion: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.

Early Life Stress in Mice Leads to Impaired Colonic Corticosterone Production and Prolonged Inflammation Following Induction of Colitis.

BACKGROUND: Early life stress (ELS) is an environmental trigger believed to promote increased risk of IBD. Our goal was to identify mechanisms whereby ELS in mice affects susceptibility to and/or severity of gut inflammation. METHODS: We utilized 2 published animal models of ELS. In the first model, newborn mice were separated from the dam daily for 4 to 8 hours starting on postnatal day 2 and then weaned early on postnatal day 17. Control mice were left undisturbed with the dams until weaning on postnatal day 21. In the second model, dams were fed dexamethasone or vehicle ad libitum in drinking water on postpartum days 1 to 14. Plasma and colonic corticosterone were measured in juvenile and adult mice. Colitis was induced in 4-week-old mice via intraperitoneal injection of interleukin (IL)-10 receptor blocking antibody every 5 days for 15 days. Five or 15 days later, colitis scores and transcripts for Tnf, glucocorticoid receptors, and steroidogenic enzymes were measured. RESULTS: Mice exposed to ELS displayed reduced plasma and colonic corticosterone. Control animals showed improvements in indices of inflammation following cessation of interleukin-10 receptor blockade, whereas ELS-exposed animals maintained high levels of Tnf and histological signs of colitis. In colitic animals, prior exposure to ELS was associated with significantly lower expression of genes associated with corticosterone synthesis and responsiveness. Finally, TNF stimulation of colonic crypt cells from ELS mice led to increased inhibition of corticosterone synthesis. CONCLUSIONS: Our study identifies impaired local glucocorticoid production and responsiveness as a potential mechanism whereby ELS predisposes to chronic colitis in susceptible hosts.

Using 2 distinct animal models, this study shows that in mice, early life stress leads to reduced colonic corticosterone and that induction of colitis after stress removal results in reduced transcription of glucocorticoid synthesis genes, increased Tnf, and enhanced chronicity of intestinal inflammation.

Circulating Tregs Accumulate in Omental Tumors and Acquire Adipose-Resident Features.

Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties.

Protein kinase Cα deletion causes hypotension and decreased vascular contractility.

AIM: Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. METHODS: Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. RESULTS: PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. CONCLUSION: Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip.

Vasopressin increases urine concentration through activation of aquaporin-2 (AQP2) in the collecting duct. Nonsteroidal anti-inflammatory drugs (NSAIDs) block prostaglandin E2 synthesis, and may suppress AQP2 producing a urine concentrating defect. There are four serines in AQP2 that are phosphorylated by vasopressin. To determine if chronic use of NSAIDs changes AQP2's phosphorylation at any of these residues, the effects of a non-selective NSAID, ibuprofen, and a COX-2-selective NSAID, meloxicam, were investigated. Daily ibuprofen or meloxicam increased the urine output and decreased the urine osmolality significantly by days 7 through 14. Concomitantly, meloxicam significantly reduced total AQP2 protein abundance in inner medulla (IM) tip to 64% of control and base to 63%, respectively. Ibuprofen significantly decreased total AQP2 in IM tip to 70% of control, with no change in base. Meloxicam significantly increased the ratios of p256-AQP2 and p261-AQP2 to total AQP2 in IM tip (to 44% and 40%, respectively). Ibuprofen increased the ratio of p256-AQP2 to total AQP2 in IM tip but did not affect p261-AQP2/total AQP2 in tip or base. Both ibuprofen and meloxicam increased p264-AQP2 and p269-AQP2 ratios in both tip and base. Ibuprofen increased UT-A1 levels in IM tip, but not in base. We conclude that NSAIDs reduce AQP2 abundance, contributing to decreased urine concentrating ability. They also increase some phosphorylated forms of AQP2. These changes may partially compensate for the decrease in AQP2 abundance, thereby lessening the decrease in urine osmolality.