Chronic diseases of the liver are major public health concerns worldwide. Steatosis and steatohepatitis associated with alcoholic liver disease, metabolic dysfunction-associated fatty liver disease/nonalcoholic fatty liver disease, and hepatitis B and C contribute to chronic diseases of the liver. Liver fibrosis occurs in all forms of advanced chronic diseases of the liver, the confirmation of which is typically performed by needle biopsy. Imaging approaches for liver diagnosis exist but do not provide sufficient diagnostic accuracy for defining the various stages of fibrosis or steatosis. Therefore, there is a need for improved imaging capabilities to enhance disease diagnosis. Ultrasonography-based photoacoustic imaging has recently emerged as a noninvasive, nonionizing modality, capable of capturing structural details and oxygen saturation changes during disease progression. However, its potential for detecting surrogate metabolic dysfunction-associated fatty liver disease markers, such as collagen and lipids, which are often poorly resolved by other conventional imaging techniques, has yet to be investigated in detail. The novelty of this study lies in the innovative use of spectral photoacoustic imaging for the direct detection and quantification of key biomarkers of liver disease, such as fibrosis, collagen, lipids, and oxygenated and deoxygenated hemoglobin, in a mouse model of steatotic fatty liver disease. Ultrasonography-based photoacoustic imaging, validated with magnetic resonance imaging, effectively identified increases in liver adiposity and fibrosis, enabling the noninvasive detection of changes in liver pathology associated with metabolic dysfunction.

OBJECTIVES/GOALS: Diabetic Kidney Disease (DKD) is a common diabetes complication, often linked to end-stage renal disease in the United States (US). While autophagy and miRNAs are pivotal, miR-451’s specific role remains understudied. Our study explores its renoprotective effects in an accelerated DKD mouse model. METHODS/STUDY POPULATION: We assessed the effect of miR-451 mimic treatment on Diabetic Kidney Disease (DKD) in BTBR ob/ob mice, known for their rapid DKD-like renal lesions. Mice were divided into four groups: WT (wild-type), BTBR ob/ob, WT+miR-451 (wild-type with miR-451 mimic), and BTBR ob/ob+miR-451 (BTBR ob/ob with miR-451 mimic). MiR-451 mimics were administered at 2mg/kg body weight once weekly for three consecutive weeks. We collected spot urine and monitored blood glucose levels at each time point. After the treatment period, mice were euthanized for kidney and blood samples. Western blot analysis assessed autophagy-related protein markers. Statistical analysis included Student’s t-test and ANOVA (p<0.05). RESULTS/ANTICIPATED RESULTS: The study assessed the impact of miR-451 mimic treatment in BTBR ob/ob mice. Albumin:creatinine ratio increased fourfold (p=0.01) in BTBR ob/ob mice at 5 weeks. MiR-451 mimic treatment had no impact on body weight. Blood glucose levels were notably higher in both treated and untreated BTBR ob/ob mice at 12 (425±33.1 mg/dL; p=0.04) and 13 weeks (383±25.3 mg/dL; p=0.007). However, a significant drop occurred from week 13 (554.7±10.8 mg/dL) to week 14 (289±13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis in whole kidney homogenates showed a 91% reduction (p=0.02) in YWHAZ, a predicted miR-451 target, in treated BTBR ob/ob mice and a 95% reduction (p=0.01) in WT mice. Furthermore, miR-451 mimic treatment led to a 68% increase (p=0.01) in ATG101 and a 44% increase in Beclin-1 in BTBR ob/ob mice. DISCUSSION/SIGNIFICANCE: The study uncovers miR-451-based interventions as a promising avenue to counter Diabetic Kidney Disease by modulating autophagy, potentially introducing novel therapies for at-risk individuals. However, practical DKD treatments will require further research and rigorous clinical validation to harness the full potential of these insights.

About 1 in 3 adults with either type 1 or type 2 diabetes (T2D) has some degree of renal injury or diabetic nephropathy (DN). There is a need to develop more sensitive and specific means of detecting and gauging DN progression so that therapies can be started as early as possible. We hypothesized that UE microRNAs (miRs), small, non-coding 21-25 nucleotide species, a relatively stable readout of renal metabolic health, could be utilized in this fashion. From the NIH, NIDDK Central Repository, we obtained 299 urine samples (300 ml) from African American subjects previously enrolled in the FIND study. The FIND study was initially designed to elucidate genetic determinants of DN in related patient populations using both T2D and non-diabetic control (CTRL), male (M) and female (F) subjects. Our samples included: 20 CTRL-M, 46 CTRL-F, 43 T2D-M, and 90 T2D-F). Other deidentified demographic and clinical data was obtained from the NIH. We measured urine albumin and creatinine in the samples and calculated ratios (mg albumin/g creatinine). Patients were categorized as normal (N, < 30, 120 subjects), microalbuminuric (MIC, 30-300, 33 subjects), or macroalbuminuric (MAC, >300, 46 subjects). Urine exosomes (UE) were isolated from each urine using the Total Exosome Isolation (from urine) kit (Thermofisher), which allows for precipitation of water-insoluble exosomes via centrifugation at medium speed (10,000g) for 1 hour. Small RNA was isolated followed by cDNA synthesis using commercially-available kits (Qiagen). Previously, we found miR-451a to be more highly expressed in UE from human subjects with chronic kidney disease, than in control subjects. We also found this miR to be a potential predictive biomarker for albuminuria in diabetic rats. Therefore, first we used conventional qRT-PCR to evaluate UE levels of miR-451a, as well as, the anti-sense product, miR-451b. MiR-127 was used as the internal standard, as it has been reported not to change with T2D. In MAC subjects, we found that the UE median miR-451a level increased with a fold change (FC) of 3.5 (relative to N). Similarly, miR-451b showed a 3.6-fold increase. FC in the MIC groups were 2.9 and 2.5 for miR-451a and b, respectively (relative to N). Next we conducted an unbiased screen of commonly excreted miRs in these subjects using a miR Array (miRCURY LNA miRNA Focus PCR panel, Qiagen) configured with 88 targets highly expressed in UE. We screened 19 subjects representing a range of conditions, i.e., albuminuric and normal, diabetic and non-diabetic, male and female. We found positive signals in 5 or more subjects for 16 miRs. Of those, 12, i.e., let-7b-5p, miR-16-5p, miR-23b-3p, miR-26a-5p, miR-27a-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR30e-5p, miR-141-3p, miR-203a, and miR-204-5p were expressed at mean levels 2-fold higher in MAC versus N subjects. Furthermore, 13 of these miRs were also over 2-fold higher when subjects were divided as T2D versus CTRL, i.e., the above list plus miR-200c-3p and miR-30d-5p, but not let-7b-5p in this case. When subjects were not divided by groups, miR-598-5p had the highest positive correlation with urinary albumin excretion, R = 0.53 and p = 0.035 (for slope ≠ 0). Using miRNet (McGill University), a freely available miR analysis software tool, we mined for common targets of miR-451(a & b) and miR-598. One putative target of both miR-451b and miR-598 was vascular endothelial growth factor A (VEGFA), which has been shown to play a role in DN. In sum, these miRs represent candidate targets to undergo additional scrutiny. We conclude UE miRs may hold clinical value in the non-invasive assessment of DN severity and predicting progression. NIH/NINDS U01-DK103225 (Feldman, PI); Ecelbarger Pilot. 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.

Inhibition of sodium-glucose cotransporter 2 (SGLT2) by empagliflozin (EMPA) and other "flozins" can improve glycemic control under conditions of diabetes and kidney disease. Though they act on the kidney, they also offer cardiovascular and liver protection. Previously, we found that EMPA decreased circulating triglycerides and hepatic lipid and cholesterol esters in male TallyHo mice fed a high-milk-fat diet (HMFD). The goal of this study was to determine whether the liver protection is associated with a change in metabolic function by characterizing the hepatic and circulating metabolic and lipidomic profiles using targeted LC-MS. In both male and female mice, HMFD feeding significantly altered the circulating and hepatic metabolome compared with low-fat diet (LFD). Addition of EMPA resulted in the restoration of circulating orotate (intermediate in pyrimidine biosynthesis) and hepatic dihydrofolate (intermediate in the folate and methionine cycles) levels in males and acylcarnitines in females. These changes were partially explained by altered expression of rate-limiting enzymes in these pathways. This metabolic signature was not detected when EMPA was incorporated into an LFD, suggesting that the restoration requires the metabolic shift that accompanies the HMFD. Notably, the HMFD increased expression of 18 of 20 circulating amino acids in males and 11 of 20 in females, and this pattern was reversed by EMPA. Finally, we confirmed that SGLT2 inhibition upregulates ketone bodies including β-hydroxybutyrate. Collectively, this study highlights the metabolic changes that occur with EMPA treatment, and sheds light on the possible mechanisms by which this drug offers liver and systemic protection.NEW & NOTEWORTHY Sodium-glucose cotransporter 2 (SGLT2) inhibitors, including empagliflozin, have emerged as a new treatment option for individuals with type 2 diabetes that have positive impacts on kidney and cardiovascular disease. However, less is known about their impact on other tissues, including the liver. Here, we report that empagliflozin reduces hepatic steatosis that is associated with restoring metabolic intermediates in the folate and pyrimidine biosynthesis pathways. These changes may lead to new approaches to treat nonalcoholic fatty liver disease.

Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

OBJECTIVES/GOALS: In the U.S., over 4 million people including children experience transient periods of undernutrition annually. Cardio-metabolic and renal diseases are more prevalent in this population. We are investigating therapeutic strategies to reverse the long-term risk of these diseases in a rat model of transient undernutrition followed by refeeding. METHODS/STUDY POPULATION: Thirty six female Fischer rats (3-months of age) were initially divided into 2 groups. Half were fed regular chow (CT) while the other half were severely food restricted (sFR) by 60% from 0-2 weeks (wks) followed by refeeding from 2-14 wks (sFR-Refed). These 2 groups were then subdivided and treated ± metformin (Met) from wk 7 to wk 12 (n=9/group). High precision ultrasound was conducted on live rats to assess heart and kidney function immediately after the sFR period ended (wk 2) and at the end of the study (wk 14). At the conclusion of the experiment, the rats were sacrificed and the histology of the kidney and heart tissues were analyzed in hematoxylin and eosin-stained sections. The protein to DNA ratio was also calculated in homogenates from these tissues. RESULTS/ANTICIPATED RESULTS: In sFR-Refed rats, cardiac output (CO), heart rate (HR) and renal artery blood flow (RBF) were decreased by 11 ± 1.5%#, 7.0 ± 6.0% and 22 ± 0.6%#, respectively, compared to control (CT) rats; #p<0.05. Mean glomerular diameter was reduced in the kidneys of sFR-refed rats compared to CT and this effect was attenuated by metformin treatment [(µm): CT, 406 ± 31; sFR-Refed, 383 ± 11, p<0.06; CT+Met, 393 ± 18; sFR-Refed+Met, 407 ± 18*]. Furthermore, the mean cardiomyocyte thickness was reduced in sFR-Refed rats compared to controls while metformin treatment prevented this effect [(µm): CT, 16.4 ± 3.6; sFR-Refed, 11.5 ± 2.3#; CT+Met, 16.4 ± 3.6; sFR-Refed+Met, 15.9 ± 3.2*]. #p<0.05 vs. CT, same treatment; *p<0.05 vs. Met, same diet; two-way ANOVA. DISCUSSION/SIGNIFICANCE: These findings have promising implications for metformin use to mitigate long-term impairments in heart and kidney structure and function in individuals who have experienced bouts of undernutrition earlier in life for either voluntarily (e.g., very low calorie dieting) or involuntary (e.g., very low food security) reasons.

Gluconeogenesis is an endogenous process of glucose production from non-carbohydrate carbon substrates. Both the liver and kidneys express the key enzymes necessary for endogenous glucose production and its export into circulation. We would be remiss to add that more recently gluconeogenesis has been described in the small intestine, especially under high-protein, low-carbohydrate diets. The contribution of the liver glucose release, the net glucose flux, towards systemic glucose is already well known. The liver is, in most instances, the primary bulk contributor due to the sheer size of the organ (on average, over 1 kg). The contribution of the kidney (at just over 100 g each) to endogenous glucose production is often under-appreciated, especially on a weight basis. Glucose is released from the liver through the process of glycogenolysis and gluconeogenesis. Renal glucose release is almost exclusively due to gluconeogenesis, which occurs in only a fraction of the cells in that organ (proximal tubule cells). Thus, the efficiency of glucose production from other carbon sources may be superior in the kidney relative to the liver or at least on the level. In both these tissues, gluconeogenesis regulation is under tight hormonal control and depends on the availability of substrates. Liver and renal gluconeogenesis are differentially regulated under various pathological conditions. The impact of one source vs the other changes, based on post-prandial state, acid-base balance, hormonal status, and other less understood factors. Which organ has the oar (is more influential) in driving systemic glucose homeostasis is still in-conclusive and likely changes with the daily rhythms of life. We reviewed the literature on the differences in gluconeogenesis regulation between the kidneys and the liver to gain an insight into who drives the systemic glucose levels under various physiological and pathological conditions.

The benefits of aerobic exercise on cardiorenal health in aging, insulin-resistant individuals are not fully understood. We hypothesized that chronic treadmill exercise (aerobic training) of overweight aging (10-month old) insulin-resistant TallyHo/Jng (TH) mice (prone to type 2 diabetes) would improve the comprehensive cardiovascular phenotype and metabolism. Male (M) and female (F) TH mice (n = 10/group) underwent treadmill running exercise (Ex, 10-19 meters/min) for 15-30 minutes, 4X/week or remained sedentary (Sd) for 9 weeks. Final body weights were 20 and 30% lower in M and F Ex, relative to same sex Sd groups, respectively. Ultrasound conducted a week prior to euthanizing showed renal blood flow (external velocity, pulse wave doppler) was significantly increased by Ex on average 26 and 24% in M and F, respectively (2-way ANOVA for Ex, p = 0.0089). With regard to cardiac features, while cardiac output was not significantly different between groups, stroke volume was increased in both sexes by Ex (9% in M, 5% in F). Whereas, cardiac ejection fraction and fractional shortening were significantly increased by Ex in female, but not male mice (p < 0.03 between FEx and FSd groups by Šidák's multiple comparisons testing). Left ventricular mass was not altered by Ex, but about 20% higher in M than F mice. Dual X-Ray Absorptiometry (DEXA) scanning revealed increased bone mineral content and density, in both sexes due to Ex, as well as, reduced body fat, but only in the F mice (p < 0.0001). Body fat was (%): 26.1 ± 0.4 (MSd); 26.1 ± 0.6 (MEx); 31.7 ± 1.8 (FSd); 20.8 ± 1.1 (FEx). Total lean mass was significantly higher in M, but not affected by Ex. Ex reduced final blood glucose (4-hour fasted, p = 0.0007 for Ex, mg/dl): 142 ± 5 (MSd); 128 ± 5 (MEx); 137 ± 4 (FSd); 117 ± 4 (FEx). Most fat pads (weighed at euthanasia) were reduced in mass in the F by Ex, but not necessarily in the M. Brown fat was increased by exercise in M, but reduced in F (g/40 g·bw): 0.34 ± 0.02 (MSd); 0.44 ± 0.03 (MEx); 0.37 ± 0.03 (FSd); 0.25 ± 0.04 (FEx), p = 0.0007 for interaction of exercise with sex (2-way ANOVA). Kidney, liver, and heart final wet weights were not affected by Ex, but lower in F (p < 0.0001 for sex for all 3). In conclusion, treadmill exercise to obese, aging TH M and F mice resulted in a several improved cardio-renal indices in addition to reducing weight, visceral fat mass, and increasing bone density. These studies support the prescription of vigorous cardiovascular exercise in aging, obese, insulin-resistant individuals. Funding to CE provided by the Marriott Foundation, the NIH CTSA Award pre-pilot, and internal Georgetown University sources This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

States of acute severe food restriction (sFR) can result voluntarily, e.g., anorexia nervosa (~1-4% of women in their lifetime) or involuntarily, e.g., very low food insecurity (~3.8% of U.S. households). This is often followed by periods of normal food intake, i.e., refeeding. We’ve shown increased propensity toward the development of metabolic syndrome (MetS) and cardiac injury in our sFR-Refed rat model. Rats receive 40% of their normal ad libitum grams of rat chow for 2-weeks followed by 3-months of recovery (ad libitum feeding). We hypothesized that treatment with metformin, a type 2 diabetic therapeutic, would attenuate features of MetS and potentially reverse or attenuate cardiac damage. Female Fischer rats (3-months old) were randomly assigned to one of four treatments: 1) Control (CT); 2) Metformin (MF); 3) sFR-Refed (FR); or 4) FR-MF (n = 9/group). MF (0.5% incorporated into the chow) was fed for 5-weeks (from week 8-12) of the study before euthanizing at week 14. Ultrasound was conducted to assess heart function just after food restriction ended (week 4, prior to beginning MF) and near the end of the study (week 13 after completion of MF). Cardiac output (CO) and heart rate (HR) were decreased (38 and 15%, respectively, p < 0.05) in the FR groups (week 4), relative to CT. In week 13, CO in the FR rats was still 11% lower than CT rats (p = 0.04); however, not in the FR-MF group (2% higher than CT). MF alone did not significantly alter CO or HR. There were no significant differences in final body weight; however, liver weight was increased by MF (p = 0.008, 2-way ANOVA). Fat pads were weighed and parametrial fat (visceral) was increased in the FR group and this rise attenuated by MF (g/185 g·bw): 5.7 ± 0.2 (CT); 5.5 ± 0.3 (MF); 7.0 ± 0.3 (FR); 6.1 ± 0.3 (FR-MT), (p < 0.0003 between CT and FR, unpaired t-test). To analyze the effects of MF on insulin signaling pathways in liver, hepatic cell suspensions were incubated ex vivo with insulin. Isolated proteins were biotinylated and incubated with antibody arrays (Phospho-antibody array, PIG-219, Full Moon Biosystems). Antibody spot densities were quantitated and normalized between chips. MF treatment altered the expression or phosphorylation of 24 distinct proteins (by 2-way ANOVA). FR in the presence or absence of MF altered 8. In addition, MF prevented the sFR-induced up-regulation of several proteins including the activating phosphorylation of ATP citrate lyase (ACLY), an enzyme involved in fatty acid biogenesis, and tuberous sclerosis complex (TSC2), a known inhibitor of insulin signaling. In sum, sFR-Refed rats had features of MetS including visceral adiposity, as well as, reduced cardiac function. Treatment in the post-sFR period (after body weight recovery) was beneficial in attenuating this phenotype. Thus, metformin may be therapeutic to reduce MetS and cardiac injury associated with periods of food insecurity. Funding included the Marriott Foundation fellowship to CE; and addition Georgetown University internal funding. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Severe food restriction (sFR) due to various psychological, environmental, and economical reasons can have adverse consequences to cardiovascular functioning and health. Less understood are the long-term risks for developing cardiovascular disease after the sFR period has ended. The renin-angiotensin system (RAS) is responsible for regulating blood pressure and we have found that the RAS is chronically upregulated months after the sFR is over and body weight (BW) recovered due to refeeding (sFR-Refed). AIM: Determine the role of the kidney in the chronic up-regulation of the RAS in sFR-Refed rats. Methods: We investigated the long-term consequences of sFR after refeeding on kidney structure and function in sFR-Refed rats. Female Fischer rats (3-months-old) were maintained on normal chow (Ctrl) ad libitum or a 60% caloric restricted diet for 2 weeks. Thereafter, all rats received regular chow ad libitum for 3 months. Kidney function was analyzed by precision ultrasound. ACE expression (qPCR) and activity (fluorescent assay) were measured and renal pathology was assessed by H&E staining. Results: After 2 weeks of sFR, rats lost 15% of their initial BW [DFinal/Initial): Ctrl, 1.50±0.80 vs sFR, -15.4±1.1; p<0.001; n=8]. After 3 months of refeeding, there was no detectable difference in BW, blood pressure and heart rate between Ctrl and sFR-Refed groups. However, the renal artery blood flow was reduced by 13% [(mm/s): Ctrl, 255 ± 12 vs sFR-Refed, 199 ± 7.8; p<0.02; n=4-8 Glomeruli size was reduced [(mm): Ctrl, 399 ± 6.2 vs sFR-Refed, 383 ± 3.8; p<0.05; n=9] and renal AT1R mRNA expression was increased by 1.3-fold [(fold of Ctrl): Ctrl, 1.00 ± 0.060 vs sFR-Refed, 1.29 ± 0.040; p<0.005; n=8]. Conclusion: In summary, AT1Rs in the renal cortex are up-regulated under conditions in which kidney structure and function are impaired months after the sFR period has ended and BW is restored to normal levels. These findings suggest increased renal AT1R activity contributes to the long-term renal dysfunction observed in sFR-Refed rats. Further research is needed to understand how women who are subjected either voluntarily (e.g., crash diets) or involuntarily (e.g., very low food security) to periods of inadequate caloric intake could be at increased risk for developing renal disease later in life. AHA: 940246 (AS); IH 1R01HL119380 (KS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Insulin facilitates renal sodium reabsorption and attenuates gluconeogenesis. Sex differences in this regulation have not been well characterized. Using tetracycline-inducible Cre-lox recombination, we knocked out (KO) the insulin receptor (InsR) from the renal tubule in adult male (M) and female (F) mice (C57Bl6 background) with a paired box 8 (PAX8) promoter. Body weights were not affected by the KO, but mean kidney weights were reduced in the KO mice (13 and 3%, in M and F, respectively, relative to wild-type (WT) mice). A microscopic analysis revealed 25 and 19% reductions in the proximal tubule (PT) and cortical collecting duct cell heights, respectively, in KOMs relative to WTMs. The reductions were 5 and 11% for KOFs. Western blotting of renal cortex homogenates showed decreased protein levels for the β and γ subunits of the epithelial sodium channel (ENaC) and the sodium-potassium-2-chloride cotransporter type 2 (NKCC2) in both sexes of KO mice; however, α-ENaC was upregulated in KOMs and downregulated in KOFs. Both sexes of KO mice cleared exogenously administered glucose faster than the WT mice and had lower semi-fasted, anesthetized blood glucose levels. However, KOMs (but not KOFs) demonstrated evidence of enhanced renal gluconeogenesis, including higher levels of renal glucose-6-phosphatase, the PT’s production of glucose, post-prandial blood glucose, and plasma insulin, whereas KOFs exhibited downregulation of renal high-capacity sodium glucose cotransporter (SGLT2) and upregulation of SGLT1; these changes appeared to be absent in the KOM. Overall, these findings suggest a sex-differential reliance on intact renal tubular InsR signaling which may be translationally important in type 2 diabetes, obesity, or insulin resistance when renal insulin signaling is reduced.

Sodium glucose cotransporter, type 2 inhibitors (SGLT2i) have been demonstrated to be protective of kidney by unclear mechanisms. The aim of the current study was to determine how treatment with a common SGLT2i, Empagliflozin affected kidney cortical metabolome and lipidome in high‐fat‐fed, male TallyHo/Jng (TH) mice. TH mice are prone to obesity and insulin resistance progressing to type 2 diabetes (T2D). Adult mice (n = 7 or 8/group) were fed a control high‐milk‐fat diet (60% by weight) or this same diet supplemented with empagliflozin (0.01%) for 8‐weeks, then euthanized and kidney cortex dissected and rapidly frozen. Metabolomics (targeted and untargeted) and lipidomics (targeted) were conducted on separate sections of cortex in our core facility by liquid chromatography‐followed by tandem mass‐spectroscopy. Data pre‐processing included signal drift and inter‐batch correction, as well as, removal of species with high coefficient of variability on pooled or reference analysis. Metabolites and lipids passing quality control (QC) included 207 and 467 unique species, respectively, which were further analyzed by MetaboAnalyst 5.0 and lipids, additionally by LipidSig, both publically available software platforms. Volcano plotting of metabolites and lipids revealed oppositely skewed patterns in that metabolites tended to be relatively decreased and lipid species increased with Empagliflozin. Seventy‐four metabolites met our threshold of changed > 20% change and/or p < 0.05 and were analyzed for pathway enrichment. The top three pathways down regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism. Analysis of metabolites of glycolysis/oxidative phosphorylation revealed a general 20‐45% reduction in several species including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, in general, several lipid species were increased with hierarchical clustering showing greatest effect on phosphatidylglycerol (16:1/16:1), 40% increase, p < 0.02, lysophosphatidylcholine (LPC, 20:4) 38% increase p < 0.03, and 10 additional phosphatidylcholine (PC) species. Overall, these analyses suggest a greater abundance of lipid, relative to carbohydrate and protein‐based species, in the kidney cortex in response to chronic Empagliflozin treatment. It is unclear whether these differences are due to intracellular metabolic alterations or transport reduction in substrate (glucose and peptides) for metabolic pathways. In addition, we found no evidence of elevated gluconeogenesis with Empagliflozin observing a significant reduction in one key metabolite in this particular pathway, PEP. We may speculate oxidative phosphorylation and ATP generation is maintained relatively normal by greater oxidation of lipids (as opposed to proteins and carbohydrates) in the kidney proximal tubule of Empagliflozin‐treated mice.

Persons with type 2 diabetes mellitus (T2D) are at greater risk for poor prognosis, including renal pathology, when infected with Covid-19 virus. A newer class of medications for T2D is the gliflozin class, including Empagliflozin (EMPA), which inhibits the renal proximal tubule sodium glucose cotransporter, type 2 (SGLT2) resulting in glucosuria and reduction in hyperglycemia. Whether these medications alter susceptibility and responses to Covid-19, and similar infections, is not known. The angiotensin 2 converting enzyme (ACE2) is the receptor for Covid-19, while transmembrane serine protease 2 (TMPRSS2) is a proteinase expressed on the surface of epithelial cells and facilitates virus uptake. In general, ACE2 is thought to be renoprotective, as it converts Ang I (Ang 1-10) to Ang 1-9 and Ang II (Ang 1-8) to Ang 1-7. Ang 1-8, in particular, is a potent vasoconstrictor and natriferic agent. Our aim was to determine how chronic EMPA affected ACE2 and TMPRSS2 protein levels and ACE2 activity in the kidney cortex of mice. Adult male (M) and female (F) mice of three strains (n = 8/group), i.e., C57Bl6 (C57), Swiss Webster (SW), and Tally Ho/Jng (TH) were fed high-fat control diet (C, 60% milk-fat) or EMPA-containing high-fat diet (E, 0.01%) for 12 weeks. TH mice are insulin resistant and obese, with a tendency to develop T2D over time. SW are lean and the closest genetically related background strain to TH. EMPA increased glucosuria but not body weight (p = 0.38 for treatment, 3-way ANOVA); however kidney weight (normalized to body weight) was 2-22% increased (depending on group) by EMPA (p = 0.002 for treatment). Kidney cortex protein levels of ACE2 and TMPRSS2 were determined by conventional western blotting (to observe band size and antibody specificity) and by dot blotting (to compare all samples on the same blot). Dot blotting revealed significantly higher ACE2 protein in males (p < 0.0001) relative to females and in the TH and SW strains (p < 0.0001), relative to C57. There was a significant sex-by-strain interaction in that the TH females were not significantly lower than TH males. Similarly, cortical TMPRSS2 was lower in females (p < 0.0001) and in C57 mice (p < 0.0001), but also reduced by EMPA (p = 0.030). ACE2 activity was determined in 10 μg kidney cortex by calculating the slope (over 2 hours) of the difference in fluorescence generated in the presence of the ACE2 substrate (Mca-YVADAPK(Dnp)-OH) with the addition of the ACE2-specific antagonist (MLN) minus fluorescence with addition of both captopril (ACE antagonist) plus MLN. Slopes were: (RFU/min, mean ± sem): MC57C- 19.9 ± 5.5; MC57E- 31.8 ± 5.2; MSWC- 20.2 ± 1.9; MSWE- 25.7 ± 3.6; MTHC- 18.8 ± 3.6; MTHE- 34.7 ± 11.3; FC57C- 4.7 ± 1.2; FC57E- 9.7 ± 3.4; FSWC- 4.9 ± 1.2; FSWE- 4.4 ± 1.8; FTHC- 9.0 ± 1.8; and FTHE- 16.2 ± 2.3. Three-way ANOVA revealed significant effects of both sex (p < 0.0001) and treatment (p = 0.0052) in that Empagliflozin and male sex increased the slope (activity). Overall, male sex was the greatest determinant of differences in ACE2 protein and activity and TMPRSS2 protein. EMPA increased ACE2 activity, but reduced TMPRSS2 protein levels. In general, sex differences were attenuated in the TH strain. There is the potential for these relative increases to play a role in increased sensitivity of male, obese, and/or T2D subjects to pathology associated with Covid-19; however, the beneficial effect of enhanced ACE2 to reduce Ang II may also provide countering protection.

OBJECTIVES/GOALS: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2), which underlies the current COVID-19 pandemic, among other tissues, also targets the central nervous system (CNS). The goal of this study is to investigate mechanisms of neuroinflammation in Lipopolysaccharides (LPS)-treated mouse model and SARS-CoV-2-infected hamsters. METHODS/STUDY POPULATION: In this research I will assay vascular reactivity of cerebral vessels to assess vascular dysfunction within the microcirculation. I will determine expression of proinflammatory cytokines, coagulation factors and AT1 receptors (AT1R) in isolated microvessels from the circle of Willis to assess inflammation, thrombosis and RAS activity in the microvasculature. LPS and SARS-CoV-2, are both associated with coagulopathies and because of that I will measure concentration of PAI-1, von Willebrand Factor, thrombin and D-dimer to assess the thrombotic pathway in the circulation. Histology and immunohistochemistry will assess immune cell type infiltration into the brain parenchyma, microglia activation and severity of neuroinflammation and neural injury. RESULTS/ANTICIPATED RESULTS: We hypothesize that under conditions of reduced ACE2 (e.g., SARS-CoV-2 infection), AT1R activity is upregulated in the microvasculature. In the presence of an inflammatory insult, these AT1Rs promote endothelialitis and immunothrombosis through pro-thrombotic pathways and pro-inflammatory cytokine production leading to endothelial dysfunction in the microvasculature, blood brain barrier (BBB) injury, deficits in cognition and increased anxiety. We will test this hypothesis through 2 aims: Aim 1: Determine the role of the pro-injury arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. Aim 1: Determine the role of the protective arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. DISCUSSION/SIGNIFICANCE: This study will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19. This research is a necessary first step in understanding mechanisms of brain pathogenesis that can set the groundwork for future studies of more complex models of disease.

The mechanistic target of rapamycin (mTOR), a serine-threonine-specific kinase, is a cellular energy sensor, integrating growth factor and nutrient signaling. In the collecting duct (CD) of the kidney, the epithelial sodium channel (ENaC) essential in the determination of final urine Na+ losses, has been demonstrated to be upregulated by mTOR, using cell culture and mTOR inhibition in ex vivo preparations. We tested whether CD-principal cell (PC) targeted deletion of mTOR using Cre-lox recombination would affect whole-body sodium homeostasis, blood pressure, and ENaC regulation in mice. Male and female CD-PC mTOR knockout (KO) mice and wild-type (WT) littermates (Cre-negative) were generated using aquaporin-2 (AQP2) promoter to drive Cre-recombinase. Under basal conditions, KO mice showed a reduced (∼30%) natriuretic response to benzamil (ENaC) antagonist, suggesting reduced in vivo ENaC activity. WT and KO mice were fed normal sodium (NS, 0.45% Na+) or a very low Na+ (LS, <0.02%) diet for 7-days. Switching from NS to LS resulted in significantly higher urine sodium losses (relative to WT) in the KO with adaptation occurring by day 2. Blood pressures were modestly (∼5–10 mm Hg) but significantly lower in KO mice under both diets. Western blotting showed KO mice had 20–40% reduced protein levels of all three subunits of ENaC under LS or NS diet. Immunohistochemistry (IHC) of kidney showed enhanced apical-vs.-cellular localization of all three subunits with LS, but a reduction in this ratio for γ-ENaC in the KO. Furthermore, the KO kidneys showed increased ubiquitination of α-ENaC and reduced phosphorylation of the serum and glucocorticoid regulated kinase, type 1 [serum glucocorticoid regulated kinase (SGK1)] on serine 422 (mTOR phosphorylation site). Taken together this suggests enhanced degradation as a consequence of reduced mTOR kinase activity and downstream upregulation of ubiquitination may have accounted for the reduction at least in α-ENaC. Overall, our data support a role for mTOR in ENaC activity likely via regulation of SGK1, ubiquitination, ENaC channel turnover and apical membrane residency. These data support a role for mTOR in the collecting duct in the maintenance of body sodium homeostasis.

Sodium glucose cotransporter, type 2 inhibitors, such as Empagliflozin, are protective of the kidneys by unclear mechanisms. Our aim was to determine how Empagliflozin affected kidney cortical metabolome and lipidome in mice. Adult male TALLYHO mice (prone to obesity) were treated with a high-milk-fat diet, or this diet containing Empagliflozin (0.01%), for 8 weeks. Targeted and untargeted metabolomics and lipidomics were conducted on kidney cortex by liquid chromatography followed by tandem mass-spectroscopy. Metabolites were statistically analyzed by MetaboAnalyst 5.0, LipidSig (lipid species only) and/or CEU Mass Mediator (untargeted annotation). In general, volcano plotting revealed oppositely skewed patterns for targeted metabolites (primarily hydrophilic) and lipids (hydrophobic) in that polar metabolites showed a larger number of decreased species, while non-polar (lipids) had a greater number of increased species (>20% changed and/or raw p-value < 0.05). The top three pathways regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism, with an amino acid network being highly affected, with 14 of 20 classic amino acids down-regulated. Out of 75 changed polar metabolites, only three were up-regulated, i.e., flavin mononucleotide (FMN), uridine, and ureidosuccinic acid. Both FMN and uridine have been shown to be protective of the kidney. Scrutiny of metabolites of glycolysis/gluconeogenesis/Krebs cycle revealed a 20–45% reduction in several species, including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, although overall lipid quantity was not higher, several lipid species were increased by EMPA, including those of the classes, phosphatidic acids, phosphatidylcholines, and carnitines. Overall, these analyses suggest a protection from extensive metabolic load and the corresponding oxidative stress with EMPA in kidney. This may be in response to reduced energy demands of the proximal tubule as a result of inhibition of transport and/or differences in metabolic pools available for metabolism.

By 2030, Diabetes is estimated to become the 7th leading cause of death worldwide with roughly 90‐95% of all cases attributed to Type 2 diabetes (T2D). A common comorbidity of T2D is Non‐Alcoholic Fatty Liver Disease (NAFLD), characterized by the presence of lipid accumulation with or without hepatic steatosis (NASH). While the pathogenesis of NAFLD and NASH is not entirely understood, it is associated with insulin resistance, inflammation, and hyperlipidemia. Recently, sodium‐glucose co‐transporter 2 (SGLT2) inhibitors have emerged as a treatment option for patients with T2D. This drug effectively blocks renal glucose reabsorption and in turn, improves glycemic control. It is also associated with positive renal and cardiovascular outcomes. Given the attenuation of hyperglycemia, we sought to determine if SGLT2 inhibitors also offer protection from the development of NAFLD and NASH. To test this, spontaneously diabetic male and female TallyHo mice were maintained on either a control or high milk fat diet (60%) for 24‐weeks in the continued presence or absence of Empagliflozin, one of the most well‐characterized SGLT2 inhibitors on the market. As expected, at the conclusion of 24 weeks, blood glucose values were significantly lowered in the male mice treated with Empagliflozin. We also detected a reduction in hepatic lipid accumulation by both MRI and biochemical analysis. To determine if these protective effects correlated with a reduction in inflammation, we performed a large‐scale screen of more than 60 inflammatory markers on both plasma and livers collected from the high‐fat diet‐fed male mice. The data revealed that Empagliflozin attenuates systemic inflammation; we detected a downregulation in circulating pro‐inflammatory cytokines (CD30 ligand, IL‐1α, IL‐4, IL‐9, TIMP1, TPO) and an upregulation in MIP‐1γ, an anti‐inflammatory cytokine (p<0.05 for all cytokines). In contrast, we observed indications of increased inflammation in the Empagliflozin‐treated livers. Protein expression of cleaved caspase, the active form of the inflammatory‐driven protein, was elevated when compared to non‐treated milk fat diet‐fed mice (which was already elevated compared to normal chow controls). In addition, we noted heightened levels of five pro‐inflammatory cytokines (IL‐3, IL‐6, CCL2, M‐CSF, CCL1) while there was downregulation of another three (p40/p70, TNFSF8, CCL3; p<0.05). Efforts are currently underway to confirm the expression of these common inflammatory markers and to elucidate the mechanism behind the dysregulated hepatic inflammatory pathway upon SGLT2 inhibition.