The MET-FLAM Faculty

Kathrin ELLER

Scientific Interests:

The lab of Kathrin Eller has long standing experience in GN research and is evaluating immune-mechanisms and new treatment options for the past 20 years (beyond others [3–6]). They were the first group to discover that regulatory T cells have the potential to be therapeutically used in experimental GN [5, 7]. In the past years, they further evaluated the potential of GLP-1R agonists, which are routinely used as anti-diabetic medication. GLP-1R agonists (GLP-1R), such as liraglutide, are already in clinical use for patients with type-2 diabetes and obesity. and have been shown to improve surrogate renal end points in large randomized controlled trials in type-2 diabetes (T2DM) patients [8–10]. Importantly, the renal endpoints were mainly driven by a decreased proportion of patient developing macro-albuminuria [8–10].

In line with human reno-protective effects of GLP-1RA, various murine kidney disease models showed improved outcome when treated with a GLP-1RA. On the one hand, we showed that GLP-1RA treatment with liraglutide improved the phenotype of a T-cell-mediated renal disease model, namely nephrotoxic serum nephritis [11]. A research group from Aachen provided evidence of an improved renal phenotype of db/db mice, which were upregulated for GLP-1 and its metabolites by adenovirus-transfer adenoviral overexpression of GLP-1. The improved renal phenotype by GLP-1 was independent from the glycaemic control and possibly mediated by decreased T-cell and macrophage infiltration [12].

GLP-1R is expressed in the kidney exclusively by pre-glomerular vascular smooth muscle cells and juxtaglomerular cells [13–15]. There, GLP-1 mediates natriuresis and diuresis, alters renal haemodynamics and decreases systemic blood pressure [13]. It has been speculated that anti-inflammatory capacity of GLP-1RA might further add to the improved renal outcomes since C-reactive protein levels significantly decrease by 25–60 % in patients with T2DM treated with GLP-1RA independent of changes in fasting glucose, body weight and body fat [16]. GLP1R is expressed on various immune cells and exerts anti-inflammatory effects on innate immune cells as well as T cells mice and humans [17–19].

Proposed Dissertation Topic:

Background: Glucagon-like peptide 1 (GLP-1) is a widely studied gastrointestinal incretin hormone, which stimulates insulin production and contributes to glucose metabolism in many ways [20]. GLP-1 receptor agonists (GLP-1RAs), such as liraglutide, are currently used to treat type-2 diabetes mellitus and obesity. Large randomized controlled trials demonstrate that GLP-1RAs significantly improve renal function in patients with diabetic nephropathy and reduce high-sensitive C-reactive protein significantly [9]. Data suggest that kidney protection is mediated by mechanisms beyond glycemic control. In addition to hemodynamic effects, GLP-1RAs are also speculated to have anti-inflammatory properties [13]. The GLP-1RA liraglutide was shown to inhibit the proliferation of T cells, especially of Th1 and Th17 cells, in an inflammatory-mediated experimental glomerulonephritis model, possibly by interfering with T-cell glucose metabolism [11]. Others have shown that GLP-1RAs alter monocyte activation as well as macrophage polarization [21].

Hypothesis and objectives: We hypothesize that improvement of the inflammatory-mediated kidney disease model nephrotoxic serum nephritis (NTS) by GLP-1RAs is mediated by their anti-inflammatory mechanisms. We plan to distinguish whether T cells or rather monocyte / macrophages are responsible for the GLP-1RA protective effect. Finally, glucose metabolism will be studied in both T cells as well as monocytes treated with GLP-1RAs. In detail, the PhD candidate will investigate (i) the effects of GLP-1RA on T-cell function in nephrotoxic serum nephritis (NTS) by using Rag1^−/− mice after CD4⁺ T-cells transfer of either Glp-1r^−/− or wild-type mice; (ii) the NTS phenotype in CD4⁺ T cell as well as monocyte / macrophage-specific Glp-1r^−/− mice, both of which will help to distinguish whether GLP-1RA signaling in T cells and / or monocyte / macrophages is critical for T-cell proliferation in NTS; (iii) how the GLP-1RA liraglutide interacts with glucose metabolism in T cells and monocytes using multiple methods including glycolysis assays, Seahorse analyzer, and mass spectrometry in vitro.

Methods and approaches: The recruited PhD candidate will be trained to isolate T cells from mice, to transfer them into Rag^−/− mice, and to induce NTS. The characterization of the NTS model includes detection of albuminuria, kidney function parameters such as blood urea nitrogen and immune complexes by ELISA, immune cell composition in secondary lymphoid organs and the kidney by flow cytometry, assessment of tissue damage by conventional histology, and immune cell infiltration in the kidney by immunohistochemistry. Furthermore, the PhD candidate will determine cell composition in secondary lymphoid organs and the kidney by bulk RNAseq and single-RNAseq methodology and perform T-cell proliferation assays (1st year). In parallel, the student will generate CD4⁺ T-cell- and monocyte / macrophage-specific Glp-1r^−/− mice using cre/flox mice, which will be subjected to NTS (1st and 2nd year). In addition, the PhD candidate will be trained in in vitro techniques to stimulate and polarize T cells and monocyte / macrophages and to analyze glucose metabolism using glycolysis assays, Seahorse analyzer, and mass spectrometry (3rd and 4th year). Since GLP-1RAs might have profound effects also via the blood–brain axis [7], we will also perform behaviour analysis of our NTS mice in collaboration with Aitak Farzi.

Pitfalls and alternative approaches: It is still in discussion whether GLP-1RAs do inhibit immune cells directly since myeloid cells express only low levels of the canonical GLP-1R. Thus, it might be the case that neither macrophage specific GLP-1R knock-out nor T-cell-specific GLP1-R knock-out mice show aggravated phenotypes after NTS. Two alternative pathways could still be responsible for the protective GLP-1RA effect in our NTS model. First, GLP-1RA have been shown to attenuate T-cell-mediated gut inflammation through the gut intraepithelial lymphocyte GLP-1R [6]. Thus, via influencing the gut-kidney axis, NTS might be improved by GLP-1RAs. This could be tested by using beta7^−/− mice as published by one of our collaborators Prof. Swirski [6]. Another mechanism how GLP-1RAs might improve NTS is by influencing the brain–immune axis as recently described [7]. Again, we have an ongoing collaboration with Prof. Drucker, who will help us to perform experiments to unravel this axis in our NTS model. Furthermore, we already included analysis of the behaviour of our mice with the expert help of Aitak Farzi, who is part of our consortium.

Involved Faculty members: Kathrin Eller (PI), Aitak Farzi (behaviour analysis), Dagmar Kratky (metabolic phenotyping), Stefano Angiari (T-cell metabolism), Martin Stradner (T-cell characterization) and Susanne Sattler (immune-cell phenotyping).

International Collaborations: Antal Rot (Queen Mary University of London, UK), Katalin Susztak (University of Pennsylvania, USA), Ulf Panzer (Uni Hamburg, Germany), Hans-Joachim Anders (LMU München, Germany).

Facilities: Our team currently consists of two PhD candidates, two postdocs and one technician. Our laboratory is located within the university hospital. Our division provides the required laboratory and office space, secretarial assistance, and basic laboratory facilities, such as cell culture, flow cytometry, immuno­fluorescence microscopy and more. Specific equipment for mouse kidney function evaluation are also available in our lab. Seahorse technology, metabolomics and mass spectrometry as well as bulk and single-nuclear RNAseq are also available through collaboration partners.

Preparatory Findings:

Figure 1. Liraglutide protects mice from nephrotoxic serum nephritis (NTS).
WT mice were subjected to NTS for 14 days and treated with vehicle (n = 14) or liraglutide (n = 13) from the day of immunization. (A) Histology revealed a significantly improved phenotype of NTS in mice treated with liraglutide. Representative PAS-stained kidney sections of mice treated with vehicle or liraglutide are shown. Magnification ×400. (B) Urinary albumin:creatinine ratios (µg/mg) were evaluated on day 14 after NTS induction. Liraglutide treated mice showed significantly decreased albuminuria as compared to vehicle-treated controls. *** p < 0.001; adapted from [11].

Figure 2. GLP-1R signalling decreases proliferation of T cells.
(A) T cells were isolated from C57BL/6J mice, stimulated with aCD3/CD28 and treated with liraglutide at a concentration of 60 µg/ml (n = 5) or vehicle (n = 5). Proliferation of cells was evaluated after 72 hours. The GLP-1RA liraglutide significantly inhibited proliferation of T cells in vitro. (B) Splenocytes were isolated from WT (n = 9) and Glp-1r^−/− mice (n = 9) after 14 days of NTS. Cells were stimulated in vitro by plate-coated rabbit IgG and LPS and proliferation was evaluated. Splenocytes from Glp-1r^−/− mice with NTS showed increased proliferation to both stimuli. The stimulation index is given as the ratio between the OD values of stimulated to unstimulated cells. * p < 0.05** p < 0.01*** p < 0.001; adapted from [11].

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