/pdf/applications-and-tuning-strategies-for-transcription-factor-1p16n070.pdf

Applications and Tuning Strategies for Transcription Factor-Based Metabolite Biosensors

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/β hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands. 

https://doi.org/10.1016/j.crmeth.2023.100394

A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands

Background The renal mechanisms involved in the maintenance of human hypertension and resistance to treatment are not well understood. Animal studies suggest that chronic renal inflammation contributes to hypertension. We studied cells shed in first-morning urine samples from individuals who were hypertensive who exhibited difficult-to-control blood pressure (BP). We performed bulk RNA sequencing of these shed cells to develop transcriptome-wide associations with BP. We also analyzed nephron-specific genes and used an unbiased bioinformatic approach to find signaling pathways activated in difficult-to-control hypertension. Methods and Results Participants who completed the SPRINT (Systolic Blood Pressure Intervention Trial) at a single trial site were recruited, and cells shed in first-morning urine samples collected. A total of 47 participants were divided into 2 groups based on hypertension control. The BP-difficult group (n=29) had systolic BP>140 mm Hg, >120 mm Hg after intensive treatment for hypertension, or required more than the median number of antihypertensive drugs used in SPRINT. The easy-to-control BP group (n=18) comprised the remainder of the participants. A total of 60 differentially expressed genes were identified with a >2-fold change in the BP-difficult group. In BP-difficult participants, 2 of the most upregulated genes were associated with inflammation: Tumor Necrosis Factor Alpha Induced Protien 6 (fold change, 7.76; P=0.006) and Serpin Family B Member 9 (fold change, 5.10; P=0.007). Biological pathway analysis revealed an overrepresentation of inflammatory networks, including interferon signaling, granulocyte adhesion and diapedesis, and Janus Kinase family kinases in the BP-difficult group (P<0.001). Conclusions We conclude that transcriptomes from cells shed in first-morning urine identify a gene expression profile in difficult-to-control hypertension that associates with renal inflammation.

Urine Cell Transcriptomes Implicate Specific Renal Inflammatory Pathways Associated With Difficult-to-Control Hypertension.

Multiphoton (MP) microscopy of live animals is a useful tool for visualizing the structure and function of the kidney cortex with intact regulatory systems. It has also been applied to monitor ion fluxes (chloride and pH) in the live mouse brain. However, measuring and quantifying dynamic changes in urine concentration capacity and luminal Na in distal tubules have not been done by intravital MP microscopy. We hypothesize that intravital MP microscopy of the kidney can be used to perform dynamic measurements of sodium and urine concentrating capacity in the nephron in vivo. Male C57BL/6 mice were anesthetized with isoflurane and canulated through the left carotid artery. Tetramethylrhodamine-Dextran (TMR-Dextran 10,000 M.W., which filters freely and is not reabsorbed) and CoroNa Green, a Na ion indicator, were injected as a bolus. We exposed the left kidney and imaged proximal and distal tubules using MP microscopy at 850 nm excitation while acquiring emission at >580 nm (TMR-Dextran) and 525/25 nm (CoroNa Green). As expected, dextran concentration was higher in distal tubules after concentration in the loop of Henle and are easily visualized. After obtaining baseline measurements, we injected a timed bolus of furosemide (0.5 mg/kg) to inhibit the Na-K-2Cl cotransporter (NKCC2) and rapidly decrease urine concentration. Given that NKCC2-mediated salt reabsorption is necessary to establish the interstitial concentration gradient, we predicted that furosemide would cause a rapid dilution of dextran and increase sodium concentration in distal tubules. 5 minutes after furosemide treatment, we observed a 33±7% (p<0.05) decrease in TMR-Dextran fluorescence intensity (FI) in distal tubules, which further decreased by 55±19% and reached a plateau after 20 min. There were no significant changes in dextran FI in proximal tubules (n=10 mice). Changes in flow rate and concentrating capacity affect the CoroNa Green FI in distal tubules, independently of Na concentration. Therefore, we ratioed changes in CoroNa Green FI over those of TMR-Dextran. We found that furosemide increased the CoroNa Green/dextran ratio FI in the distal tubules by 42±18% at 5 minutes, which further increased to 131±25% after 20 minutes (p<0.05). There was a minor but significant increase in the CoroNa Green/dextran ratio 3 minutes after furosemide in proximal tubules (n=8), which later returned to baseline. Time controls after saline injection showed no effect on TMR-dextran nor CoroNa Green/dextran FI (n=5). These results indicate that we were able to measure the changes in sodium and urine concentration capacity after furosemide treatment. Our data show that intravital MP microscopy is an effective tool for dynamically measuring urine concentrating capacity in vivo. Future experiments will focus on the effect of other diuretics, salt diets, and hydration status on renal tubular dynamics. NIH-NIDDK, AHA 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.

Measuring urine concentration capacity and luminal Na in live mice by intravital multi-photon microscopy

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACEN). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling. 

https://doi.org/10.1016/j.jmccpl.2022.100024

Pablo A. Ortiz

Papers

A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands

Urine Cell Transcriptomes Implicate Specific Renal Inflammatory Pathways Associated With Difficult-to-Control Hypertension.

Measuring urine concentration capacity and luminal Na in live mice by intravital multi-photon microscopy

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction