Activation of the renal GLP‐1R leads to expression of Ren1 in the renal vascular tree

Abstract The GLP‐1 receptor (GLP‐1R) in the kidney is expressed exclusively in vascular smooth muscle cells in arteries and arterioles. Downstream effects of the activation of the renal vascular GLP‐1R are elusive but may involve regulation of the renin‐angiotensin‐aldosterone system (RAAS). The expression of Ren1 in the mouse renal vasculature was investigated by in situ hybridization after a single subcutaneous dose of liraglutide, semaglutide and after repeated injections of liraglutide. Single and repeated exposure to GLP‐1R agonists induced expression of Ren1 in the renal vascular smooth muscle cell compartment compared with vehicle injected controls (p < .0001) for both semaglutide and liraglutide. The present data show a robust induction of Ren1 expression in the vascular smooth muscle cells of the kidney after single and repeated GLP‐1R activation and this renin recruitment may be involved in the effects of GLP‐1R agonist treatment on kidney disease.

tubular cells. Stimulation of the angiotensin II receptor type-1 (AT 1 ) leads to sodium reabsorption, vasoconstriction, aldosterone secretion and sympathetic nerve activation, ultimately restoring blood pressure and kidney perfusion. 13 A second angiotensin II receptor is present in renal endothelial cells leading to a nitric oxide dependent vasodilation opposing the effects of AT 1 receptor stimulation. 14 In humans, renin is coded for by REN, whereas in some mouse strains two genes code for Renin: Ren1 and Ren2. 15 The C57BL/6 strain only have one gene for renin: Ren1, which holds 76% homology to human REN. 16 The Ren1 gene codes for the renin precursor, the pro-renin complex, which is cleaved into active renin by association with a soluble or membrane bound pro-renin receptor. 17 Besides activation of the pro-renin complex, the membranous pro-renin receptor is involved in intracellular signalling including regulation of cell survival, cell cycle, growth and intracellular acidosis. 18 In rodents, transgenic manipulation of renin expression levels can lead to renal disease, hypertension and inflammation 19 indicating that tight regulation of renin transcription and bioactivation could play an important role as a physiological regulator of kidney function. This study describes the effect of GLP-1RAs on the expression of Ren1 mRNA in the mouse kidney after acute and chronic exposure.

| In vivo
The studies were approved by the Danish Animal Experimentation Inspectorate under the Ministry of Environment and Food and carried out by trained and licensed personnel. All animals were housed in cages of eight animals per cage under 12-h light dark cycle at 22 ± 2°C and 50 ± 20% humidity with ad libitum access to food and water. The mice were acclimatized for two weeks prior to study initiation. Animal caretakers inspected animals daily.

| Experimental protocol-26 days exposure to liraglutide in NTN mice
Eighteen C57BL/6J male mice, 8-10 weeks (Taconic, Lille Skensved Denmark), were divided into three groups. Two groups were anaesthetized in isoflurane and pre-sensitized by two subcutaneous injections of 100 µg sheep IgG (P130-100, lot#1128-7, Bethyl Laboratories Inc, Denmark) in Complete Freund's adjuvant (5 mg/mL mycobacterium butyricum in mineral oil, MP Biomedicals, Eschwege, Germany) each injection. Four days later, 100µL nephrotoxic serum (NTS) from sheep was given intravenously in the tail vein (Probetex, San Antonio, USA), Batch PTX-001S). One group received once daily subcutaneous injection with vehicle (pH = 7.4; 50 mmol/L phosphate; 70 mmol/L sodium chloride; 0.05% polysorbate 80) or liraglutide (270 nmol/kg, Novo Nordisk, Måløv, Denmark) after a dose titration scheme of 0.3, 0.6 and 1 mg/mL each day such that the animals received 1 mg/mL on the day of the NTS injection. The last group (n = 6) did not get NTS and was left untreated, thus serving as a naïve control. Following 26 days of treatment, animals were euthanized approximately 24 h after last dosing and the right kidney was collected.

| In situ hybridization of REN1 and immunohistochemical staining of GLP1R
All kidneys were dissected free of surrounding tissue and placed in

| Quantification of histological staining
Slides were scanned using the Olympus VS120 Virtual Slide  with the vehicle treated animals (p < .0001 for both compounds, Figure 1M).

| Ren1 expression in kidneys from 26 days liraglutide treated NTN mice
ISH for Ren1 after 26 days dosing with liraglutide showed a significant increase of Ren1 mRNA expression as compared with naïve mice (p = .0004) and NTN vehicle treated mice (p = .0005, Figure 2A).
Comparison of arteries from vehicle treated NTN mice and naïve mice showed that NTN induction per se did not affect Ren1 mRNA expression in arteries (Figure 2A, C, D). A clear signal of positive Ren1 hybridization was detected in the arteries of liraglutide treated animals ( Figure 2E).

| DISCUSS ION
In the present study, we show that GLP-1R activation by liraglutide contractile to renin producing cells is a well-described phenomenon known as renin recruitment. 20 The Ren1 gene (in humans the REN gene) codes for the precursor pro-renin. The level of active renin in plasma is dependent on bioactivation by proteolytic and non-proteolytic cleavage of the propart from renin by binding to a soluble or membranous pro-renin receptor. 21,22 The expression of renin is regulated by the levels of angiotensin II and renal perfusion pressure 23 and may be affected by physiological and pathological conditions including gender and diabetes mellitus. 24 Consequently, the levels of renin mRNA expression cannot be directly translated into the amount of active renin in plasma. 25 Published results from human studies of plasma renin levels after treatment with either native GLP-1 or GLP-1RA are inconsistent. In randomized placebo controlled blinded cross-over studies of native GLP-1 infusion in healthy men, one study showed that there was no effect of native GLP-1 infusion on renin plasma levels in healthy volunteers, 10 whereas another showed significantly decreased renin plasma levels. 26 In a similar setup, there were no acute effects on renin levels in men with T2DM, 11,27 however, in a placebo controlled experimental setup in healthy subjects with high sodium intake, native GLP-1 infusion resulted in unchanged renin plasma levels, but showed significantly reduced angiotensin II levels by 20%. 28 29 and forskolin, a cAMP activator, is known to induce renin recruitment. [30][31][32] Other downstream mediators of the renin recruitment effect may be factors belonging to the RAAS system.
From preclinical and clinical studies RAAS inhibitors are known to decrease fibrosis, inflammation and albuminuria, [33][34][35] but they also lead to renin recruitment. 36,37 In (1) Activation leads to increased angiotensin II cleavage, which in turn leads to renin recruitment by lack of negative feedback mechanism. The product of the cleavage of angiotensin II, angiotensin 1-7, activates the Mas1 receptor resulting in antiinflammatory and antifibrotic effects. (2) Activation leads to an increase in cAMP levels resulting in increased expression of Ren1 in the VSMC of the arteries. This would lead to an increase in local RAAS components including angiotensin II, which when cleaved as proposed above would further contribute to increased angiotensin 1-7 levels suggests that renin gene expression can stimulate biological events beyond RAAS, potentially including angiogenesis and glomerulus repair, 42 which are beneficial for maintaining a healthy kidney.
The present study was limited to healthy mice and mice with nephrotoxic serum nephritis independent of the pathophysiological consequences of diabetic nephropathy, that includes hyperglycemia and hypoinsulinemia, conditions that are known to affect renal vascular injury. 43

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.