Female Mice Research Articles

Impact of lymphangiogenesis on renal phosphate handling following kidney injury

Overconsumption of inorganic phosphate (Pi) causes tubular cell damage and kidney dysfunction, and hyperphosphatemia can lead to Ca2+ retention, vascular calcification, and cardiovascular disease. Conversely, kidney injury and chronic kidney disease (CKD) cause hyperphosphatemia and Pi retention. We hypothesized that the damage caused by chronic Pi consumption likely induces kidney lymphangiogenesis and that changes to renal lymphatic density may alter kidney mineral handling. We identified significant lymphangiogenesis and increased expression of the lymphatic growth factors VEGF-C/D in kidneys of mice fed a 2% Pi diet for 2 months and as early as three weeks of diet. We then utilized “KidVD” mice, a mouse model of kidney-specific inducible expression of VEGF-D, to expand renal lymphatic density and test Pi handling. Mineral handling was largely normal in male and female KidVD mice compared to littermates on Pi diet. In a Pi-driven CKD model mice were injured with 10 mg/kg cisplatin dose, allowed to recover for 2 weeks, then challenged with chow or 2% Pi diet for 3 weeks with VEGF-D induction. This resulted in Pi retention in all mice. Pi handling in both male and female KidVD mice were normal to controls. Female KidVD, however, demonstrated significantly increased Ca2+ excretion compared to controls. Circulating FGF23 levels were significantly elevated in male and female with VEGF-D overexpression. Gene expression of Fgfr1 was also significantly increased in male and female KidVD mice. Renal lymphangiogenesis thus appears to specifically impact Ca2+ retention through the FGF23-Fgfr1 axis. National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK119497). This is the full abstract presented at the American Physiology Summit 2024 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.

Identifying the time course and contributors for Western diet-induced increase in glycocalyx barrier function

The endothelial glycocalyx is a dynamic gel-like structure bound to the vascular endothelium composed of glycosaminoglycans, such as hyaluronan. The glycocalyx plays a critical role in cardiovascular health, acting as a physical barrier to the contents of flowing blood, including red blood cells. Interestingly, we have shown that 12 weeks of Western diet (WD), characterized by excess saturated fat and sugar content, results in greater glycocalyx barrier function in the mesenteric microcirculation that is strongly related to blood glucose values. Currently, the time course that WD induces an increase in glycocalyx barrier function is unknown, as are the contributors to that increase. Thus, we first examined glycocalyx barrier function 1, 2, 4, and 12 weeks after initiating WD in non-fasted male and female UM-HET3 mice (n=9-14/group). Glycocalyx barrier function was assessed in the mesenteric microcirculation by determining perfused boundary region (PBR), which represents red blood cell penetration into the glycocalyx. A higher PBR indicates worse glycocalyx barrier function. Compared to age-matched normal chow (NC) control mice, PBR was lower in WD at week 1 (NC: 2.35±0.05 vs. WD: 1.94±0.04 μm, p<0.05), 2 (NC: 2.36±0.04 vs. WD: 2.01±0.07 μm, p<0.05), 4 (NC: 2.38±0.04 vs. WD: 1.91±0.1 μm, p<0.05), and 12 (NC: 2.41±0.03 vs. WD 1.99±0.04 μm, p<0.05). While blood glucose was higher in WD compared to NC mice at week 12 (NC: 166±3 vs. WD 179±4 mg/dL, p<0.05), it was similar between NC and WD at week 1, 2, and 4 (P>0.05). These data indicate that the WD-induced increase in glycocalyx barrier function occurs as early as 1 week after initiating WD and appears to be independent of blood glucose. Because glycocalyx barrier function was assessed in the mesenteric microcirculation in non-fasted mice, we then sought to determine if the WD-induced increase in glycocalyx barrier function was due to postprandial state. Thus, we determined PBR in non-fasted and fasted (6h) male and female UM-HET3 mice 1 week after initiating WD (n=14-16/group). Fasting status did not affect PBR in age-matched NC (non-fasted 2.32±0.05 vs. fasted: 2.25±0.04 μm, P>0.05) or WD mice (non-fasted 1.94±0.04 vs. fasted: 2.06±0.05 μm, P>0.05), though WD had lower PBR than NC mice in non-fasted and fasted states (p<0.05). These data indicate that glycocalyx barrier function is unaffected by fasting status. Given the role of hyaluronan in the glycocalyx, we then sought to determine if the WD-induced increase in glycocalyx barrier function was due to higher hyaluronan content in the glycocalyx. Thus, we examined PBR pre- and 30 min post-hyaluronidase administration (35 U) in male and female UM-HET3 mice 1 week after initiating WD (n=5/group). There was a tendency for hyaluronidase administration to increase PBR in age-matched NC mice (Pre: 2.37±0.07 vs. Post: 2.65±0.12 μm, P=0.11), whereas PBR increased in WD mice after hyaluronidase administration (Pre: 2.13±0.04 vs. Post: 2.53±0.09 μm, p<0.05). Additionally, the difference in PBR between NC and WD mice was no longer present post-hyaluronidase administration (P>0.05). Taken together, these data indicate that glycocalyx barrier function is increased as early as 1 week after initiating WD and appears to be independent of blood glucose or fasting status but is at least partially due to increased hyaluronan content in the glycocalyx. This study was partly funded by a grant from the National Institutes of Health (R00 AT010017). This is the full abstract presented at the American Physiology Summit 2024 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.

Deletion of SGK1 in Smooth Muscle Cells Induces Sex-specific Differences in Obesity-mediated Metabolic Dysfunction and Adipose Tissue Remodeling

Objectives: Obesity, characterized by an excess of adipose tissue, is an important risk factor for the development of metabolic diseases such as type 2 diabetes. In obesity, poor metabolic health is partly due to glucose intolerance, a condition in which whole-body glucose regulation is impaired. Adipose tissue is an endocrine organ central for optimal metabolic health and its expansion by excessive fat intake induces metabolic dysfunction. The molecular mechanisms that promote metabolic dysfunction in obesity are not fully understood. Adipose tissue function is dependent on the presence of new vascular networks capable of supplying nutrients and oxygen during adipose tissue expansion. Thus, pathological vascular remodeling may promote adipose tissue expansion and dysfunction in obesity. In this regard, our previous studies have shown that serum and glucocorticoid-inducible kinase 1 (SGK1) in vascular smooth muscle cells (vSMCs) mediates pathological vascular remodeling in obesity. SGK1 is a serine/threonine kinase that becomes elevated in the vasculature during obesity and is implicated in the development of metabolic syndrome. Therefore, we hypothesize that smooth muscle cell derived-SGK1 promotes pathological adipose tissue remodeling and metabolic dysfunction during obesity by perturbing adipose tissue vascularization. Methods: To test this hypothesis, these studies utilized our novel SMC-specific SGK1 knockout (smSGK1KO) mouse model to study the influence of SMC-derived SGK1 on adipose tissue remodeling and overall metabolic health in vivo. Male and female 8-week-old smSGK1WT and smSGK1KO mice were subjected to either a 10% low-fat diet (LF) or a high-fat diet (HF) for 8 weeks with weekly body weight measurements. Upon completion of respective diets, mice were subjected to glucose tolerance tests (GTT) to assess glucose tolerance and metabolic health. Adipose tissues from the subcutaneous (inguinal) and intra-abdominal (epididymal/gonadal) white adipose tissue (WAT) depots were harvested to assess overall adiposity and vascularization. Results: Our data show sexual dimorphism exists in the way smSGK1KO mice respond to a chronic high-fat diet. When fed a HF diet, female smSGK1KO mice gained approximately 60% more weight than their female smSGK1WT counterparts. In contrast, weight gain was about 50% lower in male smSGK1KO compared to male smSGK1WT mice on the same HF diet. Despite the differences in weight gain, inguinal and epididymal/gonadal WAT were reduced in male and female smSGK1KO mice fed a HF diet. Surprisingly, GTT was similar between female smSGK1KO and smSGK1WT mice, despite the increased weight gain, while male smSGK1KO mice displayed improvement in glucose tolerance in comparison to their wildtype counterparts. Moreover, male smSGK1KO mice appear to have enhanced vascularization in WAT depots as evidenced by increased vascular branching in fat pads. Conclusions: These studies suggest that SGK1 in smooth muscle cells contributes to adipose tissue remodeling and metabolic dysfunction under obesogenic conditions. Additionally, these studies indicate a need to understand the mechanisms by which crosstalk between the vasculature and adipose tissue modulates obesity and overall metabolic health. This research is supported in part by grant number 5U24DK132733-02 from the National Institute of Health, NIH. This research is supported in part by grant number 5T32HL007609-36 from the National Institute of Health, NIH. This research is supported in part by grant number 2100832 from the National Science Foundation, NSF. This is the full abstract presented at the American Physiology Summit 2024 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.

Urothelial Piezo1 channels do not alter voiding or afferent nerve sensitivity in the mouse

The mechanosensitive piezo ion channels, Piezo1 and Piezo2, are expressed in tissues of the lower urinary tract, including the urothelium and sensory nerves. Interest in a role of piezo mechanosensitive ion channels in mediating sensory physiology in the lower urinary tract has been highlighted by the recent reports of putative voiding dysfunction in mice lacking Piezo2 channels in the lower extremities and sensory nerves (Marshall et al 2020) and Piezo1/2 in urothelium (Dalghi et al 2022). To investigate the potential role of urothelial Piezo1 channels in mediating normal function in the lower urinary tract, we generated a mouse line in which the Piezo1 gene can be conditionally knocked out in the urothelium (Piezo1UPKcKO mice). We examined spontaneous voiding behavior in male and female Piezo1UPKcKO and control mice during the light and dark phases of the day/night cycle. There were no gender differences in voiding parameters in our study (p=0.659, 3-way ANOVA), thus we did not isolate gender as a variable for further analysis. Piezo1UPKcKO mice (n=9) voided 6 ± 1 times per 24 hours, while the control mice (n=11) voided 7 ± 1 times per 24 hours (p = 0.7281). A clear nocturnal voiding pattern was observed with both Piezo1UPKcKO and control mice exhibiting more frequent voids during the dark cycle, but there were no differences in the number of voids, intermicturition intervals, or void volumes in any cycle between Piezo1UPKcKO and control mice. These results indicate that spontaneous voiding behavior remains fully intact in both male and female Piezo1UPKcKO mice during the light and dark phases of the day/night cycle. Furthermore, in an ex vivo bladder model where we measure afferent nerve output from the bladder during filling cycles, sensory nerve activity in the Piezo1UPKcKO mice was similar to that in bladders from control animals. The maximum frequency of afferent nerve activity during bladder filling reached 295 ± 28 Hz in control mice (n=6) and 336 ± 34 Hz in Piezo1UPKcKO mice (n=6; p = 0.368). Functional Piezo1 activity could be demonstrated in bladders from both Piezo1UPKcKO and control mice, as the pharmacological activator for Piezo1 channels, Yoda1 (5 μM) increased maximum afferent nerve activity during bladder filling by 60 ± 15 Hz in control mice (P = 0.0029) and 33 ± 15 Hz in Piezo1UPKcKO mice (P = 0.0567). In Ca2+ imaging experiments, we found that Yoda1 induced an increase in Ca2+ signaling events in urothelial tissue from control mice. Thus, it appears that urothelial cells express functional Piezo1 ion channels, but activity of these channels is not required for maintaining normal sensory nerve activity and voiding behavior. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2024 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.

Role of CFTR in Polycystic Kidney Disease

Introduction: Polycystic kidney disease (PKD) causes numerous cysts to grow in the kidneys, liver and pancreas. These cysts are filled with fluid. In the United States about 600,000 people have PKD, which is the fourth leading cause of kidney failure. The two main types of PKD are autosomal dominant PKD (ADPKD), which is usually diagnosed in adulthood and autosomal recessive PKD (ARPKD), which can be diagnosed in the womb or shortly after a baby is born. ADPKD is associated with the slow but relentless formation of multiple renal cysts. CFTR is known to be involved in secreting fluid in ADPKD through its localization in the apical membrane. Thus, the plasma membrane is the expected site where CFTR functions. Here, we show that CFTR unexpectedly is located in primary cilia and plays a role in cilia length. ARPKD is associated with systemic and portal hypertension, fibrosis of the liver and kidney and enlarged kidneys, with fusiform dilation of the collecting ducts. Although it has been postulated that CFTR plays a role in fluid secretion in ADPKD, the opposite is true in ARPKD. For example, a double mutant of CFTR and polycystin/polyductin mice develop massively enlarged kidneys and die from renal failure at ~24 days after birth (Nakanishi et. al (2001). J. Am. Soc. Nephrol. 12, 719-725). Thus, knocking down CFTR makes the disease worse. Methods: We used for ADPKD a mouse model bearing the R3277C mutation in PC1 and ARPKD mouse model bearing pkhd1Pkhd1del3-4/del3-4 deletion. We injected male and female mice with 30 mg/kg of VX-809 every other day. Also, we injected ADPKD and ARPKD mice once with AAV1 del27-264CFTR at 30 days old and sacrificed at 90 days. Results: Cyst areas are significantly reduced in kidney and liver after treatment with VX809 or with AAV1 del27-264CFTR. Conclusion: These data indicate that VX-809 reduces proliferation and the presence of CFTR at the apical membrane while increasing CFTR at the basolateral membrane in kidney in ADPKD model and liver in ARPKD model. Demonstration of kidney and liver cyst reduction increases the therapeutic potential of VX-809 as a treatment of ADPKD and ARPKD. Also we are reporting direct evidence of involvement of CFTR in cyst reduction in liver and in kidney by using gene therapy method treating ADPKD and ARPKD mice with AAV1 del27-264CFTR which would bind to WTCFTR and correct the traffcking of CFTR in ADPKD and ARPKD mice. NIH and PKD Foundation. This is the full abstract presented at the American Physiology Summit 2024 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.

The consequences of environment-induced heat stress on the kidney in female mice

Environment-induced heat stress (EIHS) is caused by a sustained elevation in body temperature, due to prolonged exposure to excess heat and humidity. Given the present and increasing frequency of EIHS and the gap in knowledge regarding EIHS-mediated impacts to the kidney, the purpose of this investigation was to determine the extent to which EIHS alters kidney health. We hypothesized EIHS would cause kidney injury and cellular dysfunction. To test this hypothesis, 7 wk old C57 female mice were assigned to either EIHS (n=14; 36.7 ± 0.16 °C, 36.3 ± 0.5 % humidity) or thermoneutral (TN) conditions (n=12, 31.2 ± 1.01 °C, 35 ± 0.7% humidity) for 24 h, kidneys were removed, weighed, and were either fixed for histology or frozen for biochemical measures. Environment-induced HS increased rectal temperature by 2.1 °C (p< 0.001), decreased body weight by 10% (p=0.03), and decreased absolute kidney weight (10%; p=0.02) compared to TN. Environment-induced HS increased relative protein abundance of heat shock proteins 70 (48%, p<0.01), 90 (37%, p<0.01), and 60 (36%, p=0.02), as well as heat shock factor 1 (2-fold, p=0.02) compared to TN. Histological inspection revealed EIHS animals had increased vacuolation in the proximal convoluted tubules (p<0.01) compared to TN animals. Using a western blot approach, we measured biomarkers of kidney tubular health and discovered EIHS increased protein abundance of NGAL (88%, p<0.01), IL-10 (36%, p=0.05), IL-6 (46%, p<0.01), MCP-1 (74%, p=0.04), and L-FABP (99%, p<0.01), which are suggestive of kidney injury. Environment-induced HS increased markers of inflammatory signaling including TLR4 (44%, p=0.04), phosphorylated (p-) NFκB (93%, p<0.01), p-JNK (54 kD; 51%, p=0.02), p-JNK (46 kD, 35%, p=0.02), AP-1 (33%, p=0.02), and TNFα (39%, p=0.05). Endoplasmic reticulum stress was increased by EIHS as Bip, p-IRE1α, Perk, XBP1s, ATF4, and CHOP were increased 27-190% (p<0.05-0.01) compared to TN animals. Markers of mitochondrial abundance remained similar between groups; however, EIHS may alter mitochondrial fusion and fission as Opa1 (71%, p=0.01), MFN2 (29%, p=0.04) and DRP1 (59%, p=0.04) were increased compared to TN; however, MFN1, FIS1, and p-DRP1 were similar between groups. Collectively, these data suggest that 24 h of EIHS causes kidney injury, but may also be suffcient to initiate changes that contribute to acclimation. This study was supported by USDA 2020-02716. This is the full abstract presented at the American Physiology Summit 2024 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.

Early-life inflammation increases ethanol consumption in adolescent male mice

Recent studies have demonstrated that stress during the critical windows of development can evoke a cascade of neurological changes that can result in neuropsychiatric disorders later in life. In this study, we examined the effect of early-life inflammation on ethanol consumption in adolescent mice. C57BL/6J mice were assigned to either the control or Lipopolysaccharide (LPS) group on postnatal day 14 (P14). In the latter group, LPS at a dose of 50 μg/kg was injected intraperitoneally. The mice were weaned at P21, and behavior tests were performed at P45. Ethanol consumption was assessed using a two-bottle choice drinking paradigm. Anxiety-like behaviors were assessed by marble burying test (MBT), open field (OF), and elevated plus maze (EPM). Ethanol-induced loss of righting reflex (LORR), hypothermia and ethanol metabolism were assessed to evaluate ethanol intoxication. P14 LPS-injected adolescent male mice exhibited significantly increased ethanol preference and consumption, with a similar taste preference for saccharin and avoidance of quinine. The adolescent male mice showed increased anxiety-like behaviors in the OF and EPM tests, and an increased duration of LORR, without affecting the hypothermic effects of ethanol and ethanol metabolism. Interestingly, these behavioral changes were not obvious in female mice. In conclusion, our data indicate that early-life inflammation may be a risk factor for ethanol consumption in adolescents with greater changes observed in male mice. Significance StatementOur study is the first preclinical model to report the enhancement effect of early-life inflammation on ethanol consumption in adolescent male mice and our findings provide a valuable mouse model to examine the neurobiological mechanisms mediating the long-lasting effects of early-life inflammation on alcohol use disorders vulnerability.

Gut microbes regulate glomerular filtration rate in health and in a model of chronic kidney disease

Gut microbes have been implicated in varied physiological and pathophysiological processes. In this study, we hypothesized that gut microbes modulate glomerular filtration rate (GFR). Gut microbiota was depleted using a modified antibiotic (ABX) treatment protocol with a mixture of 1g/L ampicillin, 1g/L neomycin, and 0.5 g/L vancomycin (modified from Rakoff-Nahoum et al. 2004). C57BL/6J male and female mice (6-weeks-old) were treated with ABX in drinking water for five weeks. Bacteria reduction with ABX treatment was confirmed by qPCR of fecal samples using pan-bacterial primers (Ct value for Females, F: ctrl 13±0.18 vs ABX 21±0.41; Males, M: 13±0.11 vs ABX 20±0.44, n=8). GFR measurements were performed in conscious and unrestrained mice by transcutaneous detection of fluorescent FITC-sinistrin. The GFR was significantly increased in ABX-treated mice (F: ctrl 1190±14 vs ABX 1638±21, p=0.03; M: ctrl 1020±25 vs ABX 1381±29, p=0.005, n=8). To investigate if the increased GFR was due to the absence of gut microbiota, we measured GFR in germ-free (GF) mice (born and raised without gut microbes), conventional mice (Ctrl, born with gut microbes), and conventionalized GF mice (CGF, born without gut microbes but given a fecal slurry containing gut microbes by oral gavage). GFR was increased in GF mice (F: 1853±32, p=0.003; M: 1308±16, p=0.02) versus both conventional (F: 1217±17; M: 1087±18) and CGF (F: 1326±31, p=0.02; M: 1101±10, p=0.03, n=7-8). GFR was similar in Ctrl and CGF mice. To further investigate if ABX can increase GFR in chronic kidney disease (CKD) when GFR is impaired, GFR was measured in adenine-induced CKD. Adenine (in food pellets) and ABX (in drinking water) treatments were begun simultaneously; adenine decreased GFR in females at weeks 4 and 6, and in males at weeks 2, 4 and 6. In females, mice co-treated with ABX and adenine had increased GFR on week 4 (chow: 1298±11 vs adenine: 853±8, p<0.0001; adenine vs adenine+ABX: 1040±9, p=0.04) but not week 6 (chow: 1238±21 vs adenine: 618±9, p=0.0001; adenine vs adenine+ABX: 881±14, p=0.09, n=7-9). Of note, GFR exhibited no difference between chow vs adenine (data not shown) on week 2. In males, ABX increased GFR on week 2 (chow: 1116±31 vs adenine: 913±47, p=0.002; adenine vs adenine+ABX: 1150±49, p=0.0003) but not week 4 (chow: 1154±38 vs adenine: 732±58, p<0.0001; adenine vs adenine+ABX: 877±81, p=0.37) or 6 (chow: 1157±43 vs adenine: 513±51, p<0.0001; adenine vs adenine+ABX: 591±50, p=0.56, n=7-9). To explore the mechanism by which ABX increase GFR we tested the hypothesis that, in the absence of gut microbes, proximal tubular Na+ reabsorption increases, leading to a reduced Na+ delivery to the macula densa and altering tubuloglomerular feedback (TGF) to increase GFR. To test this, we used the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) to impair Na+ reabsorption and increase Na+ delivery to the macula densa. As seen previously, treatment with ABX increased GFR on week 3 (F: 1430±13, p=0.04; M:1341±9, p=0.01) and week 5 (F: 1857±21, p=0.002, M: 1443±10, p=0.0001) compared to before treatment (F: 1140±10, M: 1090±11, n=8-9). Co-treatment of EMPA and ABX impaired GFR increases on week 3 (F: 1281±18, p=0.9, M: 1201±11, p=0.9) but not week 5 (F: 1650±21, p=0.01; M: 1281±20, p=0.003) when comparing to before the treatment (F: 1199±15; M: 1109±8, n=8-9). In this study, both the absence (GF) and suppression (ABX) of gut microbes increased GFR in healthy females and males. Moreover, ABX partially restored GFR in adenine-induced CKD females and males. These data suggest that gut microbes influence GFR in both health and disease, and that the mechanism may partially involve TGF. AHA Career Development Award (JX) and NIDDK Diabetic Complications Consortium (RRID:SCR_001415m www.diacomp.org, grants DK076169 and DK 115255 (subaward to JLP)) and American Heart Association Established Investigator Award (to JLP). This is the full abstract presented at the American Physiology Summit 2024 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.

Myeloid AMPK signaling restricts fibrosis but is not required for metformin improvements during CDAHFD-induced NASH in mice

Metabolic programming underpins inflammatory processes of immune cells. In the context of chronic liver disease, liver macrophage activation and response to hepatocellular damage is dependent on profound metabolic changes. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a first line therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1fl/fl/Prkaa2fl/fl (Flox) control and Flox-LysM-Cre+ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, decrease expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Particulate Matter Exposure Induces Adverse Effects on Endometrium and Fertility via Aberrant Inflammatory and Apoptotic Pathways In Vitro and In Vivo

This study aimed to evaluate the adverse effects of particulate matter (PM) exposure on endometrial cells and fertility and to identify possible underlying mechanisms. Thirteen women (aged 15–52 years) were included in this study. Enrolled patients underwent laparoscopic surgery at Gangnam Severance Hospital between 1 January and 31 December, 2021. For in vivo experiments, 36 female and nine male C57BL/6 mice were randomly divided into control(vehicle), low-dose(10 mg/kg/d), and high-dose exposure groups(20 mg/kg/d). PM was inhaled nasally for four weeks and natural mating was performed. NIST® SRM® 1648a was used for PM exposure. qRT-PCR, western blotting and Masson’s trichrome staining were performed. PM treatment in human endometrial stromal cells induced inflammation with significant upregulation of IL-1β, p-NF-kB, and p-c-Jun compared to those of controls. Additionally, PM treatment significantly increased apoptosis in human endometrial stromal cells by downregulating p-AKT and upregulating p-p53/p53, Cas-3, BAX/Bcl-2, p-AMPK, and p-ERK. After PM treatment, the relative expression of IL-1β, IL-6, TNF-α, p-NF-κB, p-c-Jun, and p-Nrf2/Nrf2 significantly increased in murine endometrium compared to those of the controls. Expression of apoptotic proteins p53, p27, and Cas-3, was also significantly elevated in murine endometrium of the PM exposure group compared to that of the controls. A significant increase in expression of procollagen Ⅰ, and Masson’s trichrome staining scores in the murine endometrium was noted after PM treatment. PM treatment significantly decreased ERα expression. After natural mating, all 3 female mice in the control group gave birth to 25 offspring (mean 8.1), whereas in the low-dose PM treatment group, two of three female mice gave birth to nine offspring (mean 4.5). No pregnant mice or offspring was present in the high-dose PM treatment group. PM exposure induces adverse effects on the endometrium through aberrant activation of inflammatory and apoptotic pathways and is associated with detrimental effects on murine fertility.

Alteration of the bile acid pool attenuates neuroinflammation in the EAE mouse model of Multiple Sclerosis

Bile acids are critical signaling molecules that mediate gut crosstalk with other organs, including the brain. The primary bile acids, such as chenodeoxycholic acid (CDCA), are synthesized in the liver and modified by gut bacteria to generate secondary bile acids like the 12α-hydroxylated deoxycholic acid (DCA). Bile acids are effciently absorbed in the distal ileum, and recent studies have implicated them in neuroinflammation and the pathophysiology of several neurological diseases such as Multiple Sclerosis (MS). However, the specific effects of different types of bile acids on MS have not been thoroughly investigated. We hypothesize that bile acids differentially affect neuroinflammation and that treatment with cholestyramine (bile acid sequestrant) modulates astrocyte activation in a mouse model of MS. Methods: Primary astrocyte cultures were prepared from 6-10-week-old C57BL/6 mice and incubated with cytokines in the presence and absence of bile acids. The expression of inflammatory and astrocyte activation markers was assessed by real-time qPCR. The effects of blocking bile acid absorption on MS were examined using the established Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS in 8-week-old female mice (C57BL/6). Mice were fed either a standard chow diet or a standard chow diet supplemented with 2% cholestyramine, a bile acid sequestrant, to block bile acid absorption. Statistical significance between groups was determined by an unpaired T-test. Results: Astrocytes treated with cytokines (TNF-α, 10 ng/mL, IL1-β, 10 ng/mL, 24 h) significantly increased the mRNA expression of TNF-α and IL1-β and markers of astrocyte activation, lipocalin 2 (LCN2), and Serpina3n. Co-treatment with cytokines and CDCA but not DCA (50 μM, 24 h) significantly decreased TNF-α (~56%, p<0.01), IL1-β (~40%, p<0.01), LCN2 (~26%, p<.001), and Serpina3n (~44%, p<.001), vs cytokines alone. The taurine-conjugate, TCDCA, also resulted in significant decreases in the expression of TNF-α (~72%, p<0.001), IL1-β (~53%, p<.001), LCN2 (~30%, p<0.05), and Serpina3n (~16%, p<0.01), suggesting an anti-inflammatory effect specific to CDCA and its taurine conjugate on astrocytes. Cholestyramine treatment in EAE mice was associated with significant reductions in the mRNA expression of TNF-α (~40%, p<0.05) and Serpina3n (~30%, p= 0.217) in the frontal cortex of the brain. Together, these data imply that altering the composition of the bile acid pool may be a viable therapeutic strategy for MS patients by attenuating neuroinflammation. University of Illinois at Chicago, JBVMC, Chicago, IL. This is the full abstract presented at the American Physiology Summit 2024 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.

Testing of acute and sub-acute toxicity profile of novel naproxen sodium nanoformulation in male and female mice

Nanodrugs offer promising alternatives to conventionally used over the counter drugs. Compared to its free form, therapeutic benefits, and gastric tissue safety of naproxen sodium nanoformulation (NpNF) were recently demonstrated. Essential regulatory safety data for this formulation are, however, not available. To address this, male and female BALB/c mice were subjected to acute and 14-day repeated-oral dose assessments. Our data indicate that NpNF was well tolerated up to 2000 mg/kg b.w. A 14-day subacute toxicity testing revealed that the oral administration of low dose (30 mg/kg) NpNF did not produce any adverse effects on blood profile and serum biochemical parameters. Levels of oxidative stress markers and antioxidant enzymes neared normal. Histology of selected tissues also showed no evidence of toxicity. In contrast, a ten-fold increase in NpNF dosage (300 mg/kg), demonstrated, irrespective of gender, mild to moderate toxicity (p<0.05) in the brain, stomach, and heart tissues, while ROS, LPO, CAT, SOD, POD, and GSH levels remained unaffected in the liver, kidney, spleen, testis, and seminal vesicles. No effect on serum biochemical parameters, overall indicated a no-observed-adverse-effect level (NOAEL) is 300 mg/kg. Further increase in dosage (1000 mg/kg) significantly altered all parameters demonstrating that high dose is toxic.

APOE and sex impacts functional recovery after cervical spinal cord injury

Genetic factors are key to understanding recovery following spinal cord injury (SCI). Prior work conducted in our laboratory demonstrated that APOE genotype impacts respiratory plasticity after cervical SCI. Specifically, female mice expressing the APOE4 allele had impairments in their ability to generate and maintain robust diaphragmatic activity following cervical SCI and intermittent hypoxia treatment relative to APOE3 counterparts. Subsequently we showed that different APOE alleles leads to altered neural respiratory control even without a spinal cord injury and that the effect is sex dependent. To extend these findings, we are now investigating the impact of APOE genotype and sex on locomotor and respiratory motor recovery in a humanized APOE knock-in mouse model homozygous for either the APOE3 and E4 alleles. Further, we examined how genotype and sex effected plasticity and serotonin levels below the lesion. We hypothesized that animals with the APOE4 allele will demonstrate impaired recovery following SCI across all measures. We performed a left C2 hemisection injury on male and female mice of both APOE3 and APOE4 genotype (n = 8/group). We recorded whole body plethysmography and CatWalk gait analysis weekly beginning at pre-injury through three weeks post injury. Consistent with our previous findings male APOE3 mice responded better than APOE4 counterparts during respiratory challenge even following SCI. Specifically APOE3 male mice showed greater improvements in respiratory rate during both normoxia and hypoxia challenge at early timepoints following injury. Interestingly during catwalk gait analysis APOE4 male mice recovered function faster than APOE3 counterparts in the weeks following SCI. This work is critical because it lays the foundation for understanding how diverse genotypes in the clinical SCI population may indicate the need to personalize treatment for individuals following injury. AG065220 AG060056 Nielsen Foundation. This is the full abstract presented at the American Physiology Summit 2024 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.

Incomplete reverse remodeling in aged mice exposed to pulmonary hypertension-induced right ventricular failure

Right ventricle (RV) function is the strongest predictor of survival in pulmonary hypertension (PH) and age-related HF; however no therapies exist to improve RV function. Understanding mechanisms by which the RV undoes pathological remodeling (reverse remodeling) might aid in identifying therapies for this unmet need. Our objective was to determine whether the aged RV can undergo reverse remodeling following pathological afterload (PH). We hypothesized that return to normoxia after hypoxia-induced PH would result in attenuation of RV mass and improved RV function. We exposed male and female aged (~18 months) C57Bl6 mice to hypobaric hypoxia (HH) for 4 weeks before returning them to normoxia for 3 (WK3RR) or 6 (WK6RR) weeks. HH induced RV hypertrophy by RV myocyte area which was attenuated at Wk3RR and WK6RR in both male (Con: 425 μm2 ± 104, HH: 740 μm2 ± 125, WK3RR: 447 μm2 ± 129, WK6RR: 530 μm2 ± 187) and female mice (Con: 340 μm2 ± 116, HH: 770 μm2 ± 35, WK3RR: 516 μm2 ± 186, WK6RR: 655 μm2 ± 224). HH stimulated fibrosis by collagen deposition that was also reversed during normoxia re-exposure in male mice only (Con: 4.1% ± 1.0, HH: 10.8% ± 0.8, WK3RR: 4.2% ± 0.8, WK6RR: 2.5% ± 0.7). However, HH decreased RV systolic function by fractional area change (FAC) that was also not rescued by normoxia re-exposure. The aged RV can undergo morphological reverse remodeling in response to HH; however RV function does not improve with resolution of afterload. Further investigation into the mechanisms of reverse remodeling may identify potential drug therapies for maladaptive RV remodeling. AHA-Predoctoral Fellowship 897622 (BDM). This is the full abstract presented at the American Physiology Summit 2024 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 Proportion of The Neonatal Iron Pool is Acquired from the Gestational Diet in a Murine Model

BackgroundIron is crucial for growth and development, but excess iron is harmful. Neonatal mice have elevated levels of circulating iron, but the source of this iron is unclear. This lack of understanding makes it difficult to optimize early life iron balance. ObjectiveIdentify the origins of neonatal tissue-specific iron pools using dietary manipulation and cross-fostering murine models. MethodsTo determine whether tissue-specific neonatal iron was primarily acquired during gestation or after birth, pups born to iron sufficient or iron deficient dams were cross-fostered and tissues were harvested at postnatal day 3-5 (P3-5) to measure iron content. A separate set of female mice were fed a diet enriched with the stable iron isotope iron57 (57Fe) for four generations to replace naturally abundant liver iron56 (56Fe) stores with 57Fe. To quantify the proportions of neonatal iron acquired during gestation, pups born to dams with 56Fe or 57Fe stores were cross-fostered and tissues were harvested at P3-5 to determine 56Fe:57Fe ratios by inductively coupled plasma mass spectrometry (ICP-MS). Finally, to quantify the proportion of neonatal iron acquired from the maternal diet, female mice with 56Fe or 57Fe stores switched diets upon mating and pup tissues were harvested on P0 to determine 56Fe:57Fe ratios by ICP-MS. ResultsPerinatal iron deficiency resulted in smaller pups and gestational iron deficiency resulted in lower neonatal serum and liver iron. Cross-fostering between dams with 56Fe and 57Fe stores demonstrated that up to 70% of neonatal serum, liver and brain iron were acquired during gestation. Dietary manipulation experiments using dams with 56Fe and 57Fe stores showed that over half of neonatal serum, liver and brain iron were from the dam’s gestational diet rather than pre-conception iron stores. ConclusionsThis study provides quantitative values for the sources of neonatal iron, which may inform approaches to optimize neonatal iron status.

Paneth Cell-Specific TRPV6 Channel Plays a Crucial Role in Alcohol-Associated Dysbiosis of Gut Microbiota and Tissue Injury at the Gut-Liver Axis

Introduction: Endotoxemia plays an essential role in alcohol-associated tissue injury. Two factors contributing to endotoxemia are gut microbiota dysbiosis and epithelial barrier dysfunction. Previous studies indicated that TRPV6 channel-mediated Ca2+ influx plays a crucial role in alcohol-induced epithelial tight junction (TJ) disruption, mucosal barrier dysfunction, and endotoxemia. Trpv6 knockout mice were resistant to alcohol-induced intestinal barrier dysfunction and modulated microbiota dysbiosis, raising the question of how TRPV6 regulates microbiota composition. In this study, we investigated the role of Paneth cell-specific TRPV6 in alcohol-induced microbiota dysbiosis and barrier dysfunction. Methods: Adult Trpv6fl/fl (WT) and Trpv6fl/fl-Defa6Cre (KO; Paneth cell-specific TRPV6 knockout) mice were fed a Lieber-DiCarli liquid diet with EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, &amp; 6% 1wk). Control groups were pair-fed an isocaloric EtOH-free diet. Intestinal permeability was measured by vascular-to-luminal flux of FITC-inulin in vivo, and mucosal inflammation was assessed by RT-PCR for cytokines/chemokines. Plasma lipopolysaccharide (LPS) was measured to evaluate endotoxemia. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing. Lipidome in colonic flushing was analyzed using nontargeted liquid chromatography-mass spectrometry. Microbiome and lipidome data were processed by bioinformatic analyses. Results and conclusion: Data show that colon length was reduced, and colonic mucosal permeability in vivo was elevated by EtOH in WT mice. The increase in mucosal permeability was associated with increased plasma LPS levels. EtOH feeding elevated the expression of IL-6, IL-1β, and TNFα in the colon. EtOH-induced changes in colon length, mucosal permeability, cytokine expression, and plasma LPS were absent in KO mice. 16S rRNA sequencing data indicated that the microbiota composition in KO mice differed from that of WT mice. The relative abundance of Prevotellaceae, Xanthomonadaceae, and Streptococaceae were higher, and Psuedomonadaceae and Verrucomicrobiaceae abundance were lower in KO mice compared to WT mice. EtOH-induced increase in the abundance of Norcardiaceae and Psuedomonadaceae was low or absent in KO mice. EtOH increased Rhodococcus, Odoribacter, and Pseudomonas abundance at the genus level in WT but not KO mice. On the other hand, Clostridium, Turicibacter, and Helicobacter abundance were increased by EtOH in KO but not WT mice. Lipidomic analysis of colonic flushing showed that phospholipid contents were higher in KO mice than WT mice, irrespective of sex and treatments. A cluster of sphingolipids was dramatically high in KO female mice, which was reduced by EtOH feeding. Short-chain fatty acid esters of hydroxy fatty acids (AAHFA 3:0/24:0;O) were elevated by EtOH in male WT mice but not in female WT and male or female KO mice. In conclusion, these data indicate that Paneth cell-specific deletion of TRPV6 attenuates alcohol-induced changes in gut microbiota, colonic luminal lipids, mucosal barrier function, and endotoxemia in mice. NIH/NIAAA, RO1-AA029270; Veterans Administration IO1-BX003014. This is the full abstract presented at the American Physiology Summit 2024 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.

Lysine Acetylation of Aquaporin-3 in Renal Principal Cell Mediates Water Homeostasis

The kidneys function to maintain water homeostasis. High plasma osmolality activates the release of the antidiuretic hormone vasopressin (AVP), which acts on the kidneys through its receptor AVPR2 on the principal cell (PC) to increase apical aquaporin-2 (AQP2) and basolateral AQP3 abundance for enhanced water reabsorption. However, unlike AQP2, the regulation mechanism(s) of AQP3 is unclear. We previously identified a novel AQP3 post-translational modification: acetylation of lysine 282 (K282), which we hypothesize to positively regulate PC water permeability. We developed mouse models of acetylated AQP3 by mutating K282 to glutamine (K282Q; Q mutant) and deacetylated AQP3 by mutating K282 to arginine (K282R; R mutant), on C57BL/6J background to determine the functional consequences of lysine acetylated AQP3. Male and female mice (10-15 weeks old) received a standard chow diet and free access to water. We first investigated the responses to endogenous AVPR2 activation by subjecting the mice to 18-hr overnight water deprivation and compared to mice with ad libitum water access (ad lib). All dehydrated mice (males WT 3087 ± 291 mOsm/kg, n = 8; Q 3688 ± 113 mOsm/kg, n = 6; R 3447 ± 171 mOsm/kg, n = 11; females WT 3393 ± 266 mOsm/kg, n = 8; Q 3586 ± 206 mOsm/kg, n = 9; R 3749 ± 126 mOsm/kg, n = 10) had higher urine osmolality than ad lib mice (males WT 2409 ± 165 mOsm/kg, n = 8; Q 2428 ± 316 mOsm/kg, n = 7; R 1960 ± 196 mOsm/kg, n = 7; females WT 2281 ± 96 mOsm/kg, n = 8; Q 1935 ± 190 mOsm/kg, n = 11; R 2279 ± 249, n = 7, two-way ANOVA p[treatment] &lt;0.0001 for both sexes). However, there were no significant differences among the genotypes in both sexes [two-way ANOVA p[sex] &gt; 0.05). For plasma osmolality, all dehydrated mice in both sexes (males WT 332 ± 2 mOsm/kg, n = 10; Q 324 ± 2 mOsm/kg, n = 8; R 323 ± 2 mOsm/kg n = 11; females WT 316 ± 3 mOsm/kg, n =10; Q 320 ± 2 mOsm/kg, n = 10; R 308 ± 3 mOsm/kg, n = 11) had higher values than ad lib mice (males WT 314 ± 3 mOsm/kg, n = 10; Q 309 ± 1 mOsm/kg, n = 9; R 313 ± 3 mOsm/kg n = 10; females WT 312 ± 2 mOsm/kg, n =11; Q 305 ± 2 mOsm/kg, n = 11; R 301 ± 4 mOsm/kg, n = 10, 2-way ANOVA p[treatment] &lt; 0.0001 for males and = 0.0006 for females). Female R mutants had lower plasma osmolality than WTs and Q in both ad lib and dehydrated groups (2-way ANOVA p[genotype] = 0.0045). In males, compared to WTs, Q and R mutants had similar plasma osmolality in the ad lib but lower in dehydrated group (2-way ANOVA p[genotype] = 0.0359). This suggests that deacetylated AQP3 may result in a reset osmostat, although this remains to be explored further. We next compared the responses of WT and Q mutants to 4-hour exogenous AVPR2 activation by dDAVP (10 ng, i.p). Q females exhibited lower plasma osmolality than WT females (286 ± 4 vs. 305 ± 2 mOsm/kg, n = 11 for both, Student’s t-test p = 0.0005) suggesting lysine acetylation of AQP3 leads to acute enhancement of water permeability. However, there were no significant differences between Q and WT males (300 ± 5 (n = 7) vs. 303 ± 2 mOsm/kg (n = 8), Student’s t-test p &gt; 0.05). Therefore, we conclude that lysine acetylation of AQP3 contributes to renal PC water permeability and this may be sex-dependent. Thus, acetylated AQP3 is a novel mechanism to maintain water homeostasis. R01DK128001. This is the full abstract presented at the American Physiology Summit 2024 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.

2-Methoxyestradiol protects from angiotensin II-induced renal CD3+/FOXP3+ T cell-dysregulation via 12/15 lipoxygenase inhibition

We previously showed that 2-methoxyestradiol (2-ME), an active metabolite of 17β-estradiol (E2) and lox (12/15-lipoxygenase) gene disruption, protects against angiotensin (Ang) II-induced hypertension. Moreover, 2-ME reduces Ang II-induced increases in plasma levels of 12(S)-hydroxyeicosatetraenoic acid (HETE), a major arachidonic acid-LOX-generated metabolite in ovariectomized (OVX, endogenous E2 depleted) wild-type (WT) mice. 2-ME also exerts anti-inflammatory effects, and regulatory T cells (Tregs) have been reported to impact Ang II-induced hypertension in rodent models, with premenopausal females being protected relative to males. This study tested the hypothesis that 2-ME protects against the inflammatory effects of Ang II by upregulating Tregs in the kidney via LOX inhibition in female mice. Intact (ovaries present) or OVX WT and OVX-LOX knock-out (KO) mice (8-10 weeks, n=3-4/group) were infused with Ang II (700 ng/kg/min) or saline (vehicle) via osmotic minipumps (S.C.) with or without 2-ME (15 μg/g, I.P., every third day) treatment for 2 weeks. Renal protein expressions of CD3+ T cells and FOXP3+ Tregs were evaluated by immunofluorescence staining and mRNA expressions of tnf-α and il-10 by RT-PCR using TaqMan assay (relative to saline-infused intact WT) in the whole kidney. Results represented as mean ± SEM were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The CD3+, tnf-α and il-10 expressions were comparable between saline-infused intact WT, OVX-WT, and OVX-KO (CD3+: 8.3±0.3, 7.0±0.5, 6.3±0.3; tnf-α: 1.0±0.0, 0.9±0.0, 0.8±0.2; il-10: 1.1±0.1, 0.9±0.1, 1.0±0.0). However, saline-infused intact WT and OVX-KO expressed higher Tregs (p&lt;0.05) as compared to OVX-WT (7.6±0.3, 7.3±0.3 vs 4.0±0.5). Ang II increased CD3+, Tregs, and il-10 (p&lt;0.05) but not tnf-α expressions as compared to its vehicle in intact WT (12.6±0.3, 15.3±0.3, 3.7±0.3,0.8±0.1). Ang II also increased CD3+ (11.0±0.6) and Tregs (11.0±0.6) in OVX-KO (p&lt;0.05), without altering mRNA expressions of tnf-α (0.9±0.3) and il-10 (1.5±0.1) . In contrast, Ang II resulted in a greater increase in CD3+ (25.0±1.1) in OVX-WT than OVX-KO and intact WT, besides increasing tnf-α expression (2.5±0.4) (p&lt;0.05). 2-ME reduced CD3+ (16.7±0.9) and tnf-α (1.4±0.4) expressions, and increased Tregs (10.0±1.0) and il-10 (2.0±0.0) expressions in Ang II-infused OVX-WT mice (p&lt;0.05). 2-ME also reduced CD3+ (6.7±0.3, p&lt;0.05) and showed a trend to increase Tregs (14.0±1.1, P=0.056), and il-10 (2.3±0.2, P=0.058) expressions without altering tnf-α (0.7±0.1) expression in Ang II-infused OVX-KO mice. In conclusion, Ang II produces a specific effect on the renal T cell profile, greater increases in proinflammatory T cells in OVX mice, and anti-inflammatory Tregs in intact female mice, most likely mediated by E2 through metabolite 2-ME via inhibition of LOX. R01HL19134-47, AHA Award#940538. This is the full abstract presented at the American Physiology Summit 2024 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.

The role of gut microbiota and innate immune response in an autoimmune pancreatitis model.

Although the involvement of intestinal microbiota in innate immunity has been reported recently, the pathogenicity of autoimmune pancreatitis (AIP) remains unclear. This study aimed to investigate whether probiotics ameliorate inflammation in AIP through interactions with innate immunity. The AIP mouse model was generated by intraperitoneal administration of E. coli to C56BL/6 female mice. Alterations in the intestinal microbiota in the AIP group were evaluated using high-throughput sequencing. Peritoneal macrophages (PMs) were collected and cocultured in vitro with Lactobacillus gasseri (LG) or ligands of toll-like receptors (TLRs). LG was administered intraperitoneally to AIP model mice, and pancreatitis activity was evaluated to examine the ameliorative effects of LG. In the AIP model mice, inflammation was significantly induced in the pancreas, and the intestinal microbiota was altered with decreased LG. Antimicrobial treatment suppressed pancreatitis. In vitro, E. coli stimulation increased inflammatory cytokine expression, which was significantly decreased when the LG or TLR7 ligand was cocultured with PMs. Intraperitoneal administration of LG to AIP model mice significantly suppressed pancreatitis. The mouse model demonstrated the involvement of intestinal microbiota in pancreatitis, and LG administration suppressed pancreatitis, possibly through TLR7 signaling in PMs. LG may be a helpful probiotic for treating AIP.

Deletion of ADAM17 on microglia attenuates salt-sensitive hypertension

Previous work from our group identified ADAM17-mediated shedding as a major mechanism responsible for ACE2 shedding, thus impairing ACE2 compensatory activity and contributing to the development of salt sensitive hypertension. While microglia have been shown to contribute to salt sensitive hypertension, the mechanisms responsible are unknown. Accordingly, we hypothesize that ADAM17 promotes microglia priming, sympatho-excitation and neuro-inflammation in salt sensitive hypertension, and thus targeting ADAM17 or its maturation, via iRhom2, provides a multi-pronged strategy to normalize BP. Here, we aimed to evaluate whether deletion of ADAM17 on microglia attenuates hypertension. Uninephrectomized male and female mice (14–16-week-old, n=10/group) with selective deletion of ADAM17 in microglia (Cx3CR1-ADAM17tom) or with global deletion of iRhom2 (iRhom2-KO) and littermate controls (Cx3CR1-Cretom) mice were used to monitor blood pressure (BP telemetry, DSI) at baseline and following the initiation of salt sensitive hypertension (DOCA 1mg/g body weight sc + 1% saline po for 3 weeks). Following the establishment of hypertension (7 days post DOCA implantation) mice were injected with tamoxifen (200 nl ICV) and monitored for 2 more weeks. At the end of the treatment, mice were euthanized, and their brains were collected. Baseline mean arterial pressure (MAP) was not different in Cx3CR1-ADAM17tom, iRhom2-KO compared to controls (102±1, 106±1 and 104±1 mmHg, respectively). Surprisingly, all mice developed hypertension following DOCA implantation, including iRhom2 KO (Cx3CR1-ADAM17tom: 131±1, iRhom2 KO: 131±1, control: 126±1 mmHg, respectively. p&lt;0.05). Interestingly, following tamoxifen injection Cx3CR1-ADAM17tom mice showed a blunted hypertension compared to the control group (MAP: 122±0.2 vs. 146±0.5 mmHg, one way ANOVA, p&lt;0.05) suggesting that ADAM17 in microglia contributes to salt sensitive hypertension. In vitro, human microglia cells (iCell microglia — hIPSC) were shown to express lower ACE2 protein (0.03 ± 0.1 vs. 1.18 ± 0.3, Student’s t test, p&lt;0.05) when exposed to Ang-II (100 ng) with a trend for ADAM17 upregulation (1.5 ± 0.3 vs. 1.1 ± 0.1, p&lt;0.05). Together, our data suggest that overactivity of the renin-angiotensin system is associated with overexpression of ADAM17 in microglia and a simultaneous downregulation of ACE2. In addition, knockdown of ADAM17 specifically on microglia blunted the development of hypertension. We conclude that ADAM17 expression in microglia plays a pivotal role in salt sensitive hypertension. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 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.