AbstractHypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

Micro-RNAs (miRNAs) are regulators of gene expression and play an important role in physiological homeostasis and disease. In biofluids, miRNAs can be found in protein complexes or in extracellular vesicles (EVs). Altered urinary miRNAs are reported as potential biomarkers for chronic kidney disease (CKD). In this context, we compared established urinary protein biomarkers for kidney injury with urinary miRNA profiles in obese ZSF1 and hypertensive renin transgenic rats. Additionally, the benefit of urinary EV enrichment was investigated in vivo and the potential association of urinary miRNAs with renal fibrosis in vitro. Kidney damage in both rat models was confirmed by histopathology, proteinuria, and increased levels of urinary protein biomarkers. In total, 290 miRNAs were elevated in obese ZSF1 rats compared with lean controls, whereas 38 miRNAs were altered in obese ZSF1 rats during 14-26 weeks of age. These 38 miRNAs correlated better with disease progression than established urinary protein biomarkers. MiRNAs increased in obese ZSF1 rats were associated with renal inflammation, fibrosis, and glomerular injury. Eight miRNAs were also changed in urinary EVs of renin transgenic rats, including one which might play a role in endothelial dysfunction. EV enrichment increased the number and detection level of several miRNAs implicated in renal fibrosis in vitro and in vivo. Our results show the benefit of EV enrichment for miRNA detection and the potential of total urine and urinary EV-associated miRNAs as biomarkers of altered kidney physiology, renal fibrosis and glomerular injury, and disease progression in hypertension and obesity-induced CKD.

Abstract Background The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and SGLT2 inhibitors have demonstrated clinical benefits in HFrEF and CKD patients with T2D. Cardiovascular protection with finerenone and the SGLT2 inhibitor empagliflozin in combination in hypertensive cardiorenal disease is unknown. Purpose To test the hypothesis that the combination of finerenone with empagliflozin provides cardiovascular protection in preclinical hypertension-induced end-organ damage. Methods Cardiovascular morbidity and mortality was studied in hypertensive L-NAME (20 mg/L) treated renin-transgenic (mRen2)27 rats. Rats (10–11 weeks old female, n=13–17/group) were treated once daily orally for up to 7 weeks with placebo, finerenone (1 and 3 mg/kg), empagliflozin (3 and 10 mg/kg), or a combination of the respective low doses. Blood pressure (week 1, 3 and 5), urinary (week 2 and 6) and plasma parameters (week 6 and at the end of the study) were determined during the course of the study, while cardiac histology and left ventricular gene expression analysis were performed after study end. Results Empagliflozin induced a strong and dose-dependent increase in urinary glucose excretion which was not influenced by finerenone co-administration in the combination arm. Treatment with 3 mg/kg finerenone and the low dose combination significantly decreased systolic blood pressure (SBP) after 3 and 5 weeks as well as plasma uric acid after 6 weeks. SBP was significantly more reduced in the combination arm vs. the individual monotherapies after 3 weeks. Plasma NT-proBNP was reduced by empagliflozin, finerenone and the combination with similar efficacy. There was a dose-dependent protection from cardiac vasculopathy, cardiac and vascular fibrosis with both agents while low dose combination therapy was more efficient than the respective monotherapy dosages on these cardiac histology parameters. Placebo-treated rats demonstrated a ca. 50% survival rate over the course of 7 weeks while low dose combination provided the most prominent survival benefit (93%). Conclusion Non-steroidal MR antagonism by finerenone and SGLT2 inhibition by empagliflozin confer cardiovascular protection in preclinical hypertensive-induced cardiorenal disease. Combination of these two modes of action at low dosages revealed efficacious reduction in blood pressure, cardiac lesions and mortality indicating a strong potential for combined clinical use in cardiorenal patient populations. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): BAYER AG

The pathogenesis of hyperglycemia-dependent diabetic longterm complications involves the accumulation of reactive metabolites which were recently shown to affect cation channels such as Nav1.8 and TRPA1 channels as targets and essential effector molecules in signaling pathways involved in the disease process.

The publication of this meeting abstract has been retracted at the request of the authors because of prior publication of the full report in Circulation Research.

TRPM4 have an extensive role in regulating intracellular calcium dependent cell functions in many cells including mast cells, chromaffin cells and recently in neurons. TRPM4 proteins constitute calcium activated, but calcium impermeable, non-selective cation channels and are expressed both in atrial and ventricular cardiomyocytes. The Trpm4 gene has recently been associated with several disorders, including cardiac conduction diseases such as Progressive Familial Heart Block Type 1, Right Bundle Branch Block and Brugada Syndrome. The physiological function of TRPM4 in the heart remains however incompletely understood. The objective of this study was to establish the role of TRPM4 in left ventricular muscle function. We used TRPM4 knockout mice and performed patch-clamp experiments, membrane potential measurements, microfluorometry, contractility measurements and in vivo pressure-volume loop analysis. We demonstrate that TRPM4 proteins are functionally present in mouse ventricular myocytes and are activated upon calcium induced calcium release. Measurements of action potential duration show that TRPM4 is active during the repolarization of the ventricular action potential. We show a significantly decreased time for 50 % and 90 % repolarization in Trpm4 knockout ventricular myocytes. We also provide evidence that this change in action potential shape leads to an increased driving force for the L-type calcium current during the action potential. Moreover, in vivo pressure-volume analysis and in vitro contraction strength measurement on isolated papillary muscles displays an increased β-adrenergic inotropic response in TRPM4 knockout mice. Taking together these data, our results show that functional TRPM4 proteins are novel determinants of the inotropic effect of β-adrenergic stimulation on the ventricular heart muscle.

Recently we showed that transient receptor potential TRPM4 proteins act as Ca2+‐activated nonselective cation channels in mast cells that critically limit the driving force for Ca2+ influx, release of inflammatory mediators and anaphylactic responses. In addition to mast cells we detected TRPM4 proteins in heart, aortic endothelial cells, kidney and adrenal gland from mice. Earley et al suggested that TRPM4 is expressed in vascular smooth muscle cells and it is supposed to act as a mechanosensor regulating vascular myogenic tone.Telemetric blood pressure measurements reveal that mean arterial blood pressure (MAP) is elevated in TRPM4‐/‐ mice by 11mmHg under basal conditions. Blood pressure elevation is observed during the complete circadian period and most notable under resting conditions. Plasma aldosterone levels, renin mRNA expression in the kidney and renin plasma concentrations are not different in TRPM4‐/‐ mice and there are no differences in MAP in response to application of phenylephrine (PE), norepinephrine and L‐NAME. PE induced contraction and acetylcholine induced relaxation of aortic rings is not altered. Currently we analyse the contractility of resistance vessels with respect to smooth muscle‐dependent contraction and endothelium‐dependent relaxation. Inhibition of ganglionic transmission with hexamethonium abolishes the difference in MAP between both genotypes similarly like application of prazosin. Analysis of plasma catecholamine level revealed a significant elevation of epinephrine in TRPM4‐/‐ mice. These results suggest that an increased neurogenic tone contributes to the development of the hypertension.