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. Author manuscript; available in PMC: 2026 Oct 14.
Published in final edited form as: Am J Physiol Renal Physiol. 2026 Sep 18;331(5):F673–F684. doi: 10.1152/ajprenal.00160.2026

Glucagon receptor agonist aggravates a murine model of Balkan nephropathy

Sadhana Kanoo 1, Ony Arujo Galdino 1, Natalia Lopez 1, Young Chul Kim 1,2, Haiyan Zhang 2, Souradipta Ganguly 2,3, Debanjan Dhar 2,3, Helen A Goodluck 1, Scott Culver Thomson 1,2, Volker Vallon 1,2
PMCID: PMC13660735  NIHMSID: NIHMS2212003  PMID: 42789963

Abstract

Combining agonists of glucagon receptors (GCGRA) and GLP1 receptors (GLP1RA) is explored for treatment of obesity, but little is known on kidney protection by GCGRA. To gain further insights, mice with Western diet-induced obesity were first treated for 18 days with a GLP1RA or a GCGRA, and then exposed to tubule-toxic aristolochic acid (AA), a murine model of Balkan nephropathy. Prior to AA application, GLP1RA reduced food intake by ~30% and body weight by ~16% vs. vehicle with no significant changes in non-fasted blood glucose levels or plasma creatinine or urea. GCGRA reduced body weight by ~6% without changing food intake or plasma creatinine associated with hyperglycemia (230 vs 143 mg/dl) and 14% lower plasma urea concentrations versus vehicle. Among AA treated groups, GLP1RA improved some parameters but overall did not robustly attenuate kidney injury. GCGRA enhanced kidney mRNA expression of Rev-Erba, an inhibitory transcription factor previously implicated in glucagon-induced suppression of glucose transporter SGLT2, suppression of inflammation/fibrosis, but aggravation of AA-induced kidney injury. GCGRA indeed reduced kidney expression of Sglt2, inflammatory markers (incl. Il6, Ccl2, Tnfa and histological inflammation score), and also tubular injury marker KIM-1 and profibrotic Col1a1 and Sirius red fibrosis staining. GCGRA, however, sensitized towards AA-induced kidney injury based on reduction in body weight and hematocrit, and higher levels of plasma creatinine, urea and ammonia, kidney NGAL and p21 mRNA expression, histological acute tubular injury score, and mortality. In conclusion, GCGR agonism can aggravate a murine model of Balkan nephropathy, possibly involving Rev-Erba-induced gene suppression.

Keywords: Glucagon receptor agonist, GLP1 receptor agonist, Western diet, aristolochic acid, Balkan nephropathy

New and Noteworthy

This study suggests that a glucagon receptor agonist (GCGRA) can aggravate a murine model of Balkan nephropathy induced by the nephrotoxin aristolochic acid. Mechanistically, this is associated with GCGRA-induced upregulation of the inhibitory transcription factor Rev-Erba, which has previously been linked to promoting kidney injury in this model.

Graphical Abstract

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Introduction

Agonists of the glucagon receptor (GCGRA) are an emerging class of drugs with therapeutic potential in obesity, type 2 diabetes (T2D), and metabolic dysfunction-associated steatohepatitis (MASH; formerly nonalcoholic steatohepatitis [NASH]), particularly developed for the combination with a glucagon like peptide 1 receptor agonist (GLP1RA). Naïve glucagon has a half-life of minutes and primarily targets the GCGR although the structural similarity with glucagon-like peptide GLP1 also allows glucagon to act as a weak agonist on the GLP1R (1). In comparison, the emerging class of GCGRA are monospecific for GCGR and long-acting. GLP1RA have well-established kidney and cardiac protective effects in patients with T2D and chronic kidney disease (CKD)(2). The potential of GCGRA for kidney protection, however, remains less clear.

The physiology of glucagon counters the blood glucose lowering effects of insulin (3). Glucagon also facilitates liver formation and kidney excretion of urea to maintain nitrogen homeostasis. This includes an increase in GFR and thereby filtered urea and inhibition of tubular urea reabsorption (3). In accordance, glucagon infusion in metabolically well controlled, short-term insulin-dependent male diabetic individuals enhanced GFR by 6–11% (4), and tubular knockdown of GCGRA in mice enhanced plasma urea levels (5). Excessive urea levels have been linked to cardiovascular and overall morbidity and mortality (6). The impact of a GCGRA on urea levels in CKD is not known.

Experimental studies suggest an inhibitory impact of glucagon on proximal tubule transport. Glucagon infusion in rats attenuated proximal tubule NaCl and fluid reabsorption (7), which is thought to facilitate urea excretion by lowering the rise in luminal concentrations of urea and thereby the driving force for reabsorption (3). A GCGRA reduced the expression of proximal tubule glucose transporter SGLT2 in mice with opposite effects noted for an inhibitory glucagon receptor antibody (8). Indeed, the physiological adaptation to less filtered glucose during fasting includes glucagon-induced down-regulation of proximal tubule SGLT2 expression and activity; the underlying mechanism involves glucagon induction of the inhibitory transcription factor Rev-Erba (9). Likewise suggesting inhibition of a proximal tubular transport process, glucagon infusion in short-term insulin-dependent diabetic individuals enhanced urinary beta 2-microglobulin excretion (urinary albumin excretion remained unchanged)(4), with the same pattern observed in healthy individuals (10).

With regard to kidney protection, studies in mice with tubular knockdown of the GCGR provided conflicting results: one study reported oxidative stress, enhanced inflammasome activity, and excess lipid accumulation in the kidney and doubling of UACR associated with kidney deposition of collagen and fibronectin and a modest increase in plasma creatinine in aged mice (5). In contrast, another study provided evidence that tubular absence of GCGR is kidney protective in response to streptozotocin-induced hyperglycemia in heminephrectomized mice on a high fat diet with a similar protective effect induced by a human monoclonal antibody that functions as a competitive antagonist of GCGR, while kidney outcome was worsened by twice daily i.p. application of glucagon (11).

Therapeutic kidney data for GCGRA in humans are sparse. After a promising phase 2a study with the dual GLP1RA and GCGRA cotadutide (12), a subsequent phase 2b dose-finding study in patients with T2D and CKD (eGFR of 20 or more and under 90 mL/min per 1.73 m2 and UACR over 50 mg/g) showed that both cotadutide and the GLP1RA semaglutide improved body weight and blood glucose control and left eGFR unaffected versus placebo (13). Patients on the highest dose of cotadutide were 14 times more likely to experience resolution of macroalbuminuria to microalbuminuria, despite comparable reductions in glucose, body weight, and blood pressure compared with semaglutide (13). The study is suggestive of kidney protective benefits of cotadutide, possibly in part due to its glucagon agonism.

Acute kidney injury (AKI) is itself a risk factor for CKD and worsens the CKD prognosis and quality of life (14, 15). Aristolochic acid (AA)-induced nephropathy is a form of nephrotoxic AKI that is caused by ingestion of AA-contaminated herbal medicines or wheat and is the cause of Balkan endemic nephropathy (16, 17). Repeated exposure to AA leads to fibrosis of the tubular interstitium and development of a CKD-like phenotype (18). The current study compared the effect of a GLP1RA and a long-acting GCGRA on kidney outcome in a murine model of Balkan nephropathy.

Methods

Animals:

Information provided was guided by the “Animal Research: Reporting of In Vivo Experiments Guidelines Checklist” (19). All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD), and were reviewed and approved by the local Institutional Animal Care and Use Committee (Veterans Affairs San Diego Healthcare System). 5-week-old male C57BL/6J mice (RRID:IMSR_JAX: 000664) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed in the same temperature-controlled room with a 12:12 hr light/dark cycle and free access to chow and tap water.

Exposure of mice to Western diet, drugs, and AA:

The study protocol is shown in Fig. 1. At 8–10 weeks of age, all mice were switched for the rest of the study to a Western diet [“WD”, TD88137, Teklad Diets, Envigo: 42% kcal from fat, high in saturated fatty acids (>60% of total fatty acids), high sucrose (34% by weight)]. After 5.5 weeks on WD, body weight and blood glucose were not different between groups prior to starting agonist treatment (Suppl. Fig. 1A). Mice were started on the following treatments applied by daily s.c. application between 9–11 am and continued for the rest of study: GLP1R agonist semaglutide (7.5 nmol/kg), GCGR agonist BI2130086 (30 nmol/kg)(20), or Vehicle (50 mM phosphate buffer pH 7.0 and 5% mannitol at 5 uL/g)(21) with continuous dose adjustment to body weight. The last injections were given the day before harvest.

Fig. 1: Experimental plan.

Fig. 1:

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AA distributes primarily in vascular space and extracellular fluid space (volume of distribution of ~0.1L/kg), and has a high plasma protein binding, which facilitates delivery to the kidney. GLP1RA and GCGRA lower body weight primarily by reducing body fat mass, and thus should have minimal influence on the distribution volume of AA. Therefore, the dose of AA was adjusted to the body weight before initiation of drug/vehicle treatment: after 2.5 weeks on drug/vehicle treatments, a fixed dose (2.5 mg/kg body weight) and similar amount of aristolochic acid (AA, A9451, Sigma-Aldrich, St. Louis, MO) was given per injection every 3 days for a total of 7 injections to each group (Suppl. Fig. 1B).

Blood and urine collection and organ harvest:

The first two blood collections (80 μL each) were performed under brief isoflurane anesthesia: before the first AA injection (“Basal”) and two days after the last AA injection (“After inj”). A third blood collection (160 μL) was performed before organ harvest 6–11 days after the last AA injection (“Harvest”) under deep terminal anesthesia (i.p. cocktail of ketamine [100 mg/kg, Henry Schein Animal Health, Dublin, OH] and xylazine [10 mg/kg, Bimeda-MTC Animal Health, Cambridge, ON, Canada]). All blood samples were collected between 9–11 am by posterior orbital plexus puncture into heparinized capillaries, which were sealed and centrifuged to measure hematocrit, and plasma was stored at −80°C for further analysis. After the third blood collection, kidneys, heart, and liver were removed under deep anesthesia, weighed, and flash frozen in liquid nitrogen. Urine was obtained by gently grabbing the mice and inducing spontaneous urination, collected in a clean petri dish (Corning Incorporated, Corning, NY) or by bladder puncture under terminal anesthesia.

Measurement of plasma creatinine, urine markers of glomerular and tubular injury, and plasma markers of liver function:

Blood glucose levels were determined by tail snip and glucometer (Contour glucometer, Bayer, Mishawaka, IN) in non-fasted and conscious mice between 9–10 am. Plasma creatinine concentration was determined by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the O’Brien Center for Acute Kidney Injury Research at the University of Alabama at Birmingham (Birmingham, AL). Plasma urea (DIUR-100, Bioassay Systems, Hayward, CA) and ammonia (EnzyChrom ammonia/ammonium assay kit, BioAssay Systems, Hayward, CA) were measured according to manufacturer’s instructions. Plasma concentrations of alanine aminotransaminase (ALT), alkaline phosphatase (ALP), and cholesterol were measured using the Abaxis/Zoetis Mammalian Liver Profile system (catalog# 10023223). Urine albumin (Bethyl Laboratories, Montgomery, TX) and urine kidney injury molecule KIM-1 and NGAL (both assays by R&D Systems, Minneapolis, MN) were determined using ELISA assays, following the manufacturer’s instructions and concentrations normalized to creatinine, which was measured by a kinetic modification of Jaffe’s reaction (ThermoFisher Scientific, Waltham, MA). Urine osmolality was measured using a Vapro vapor pressure osmometer 5520 (Wescor, Logan, UT).

Kidney real-time PCR:

As described (22, 23), total RNA from whole kidney was isolated using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD). Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). For real-time PCR, TaqMan Universal PCR Master Mix (Applied Biosystems, Warrington, UK) and TaqMan Gene Expression Assay primers (Thermo Scientific) were used in a QuantStudio 6 Real-Time PCR System. The reaction protocol was as follows: 2 min at 50° and 10 min at 95° with 40 cycles of 15 s at 95° and 1min at 60°. Each experiment was performed in duplicate using the primers listed in Table 1. The expression of target genes was normalized to that of house-keeping genes hypoxanthine-guanine phosphoribosyltransferase (Hrpt) and ribosomal protein L19 (Rpl19).

Table 1.

Real-time PCR primers used

Target Gene Assay ID
AQP2 (aquaporin 2) Mm00437575_m1
Ccl2 (MCP1) Mm00441242_m1
Col1a1 Mm00801666_m1
Havcr1 (KIM-1) Mm00506686_m1
Hprt Mm00446968_m1
Il6 Mm00446190_m1
Lcn2 (NGAL) Mm01324470_m1
Lrp2 (megalin) Mm01328171_m1
Nphs1 (nephrin) Mm01176615_g1
p16 (Cdkn2a) Mm00494449_m1
p21 (Cdkn1a) Mm01303209_m1
Rpl19 Mm02601633_g1
Slc5a2 (SGLT2) Mm00453831_m1
Slc12a1 (NKCC2) Mm01275821_m1
Slc12a3 (NCC) Mm00490213_m1
Tnfa Mm00443258_m1
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Ccl2, C-C motif chemokine ligand 2; MCP-1, monocyte chemoattractant protein-1; Col1a1, collagen type I-α1; Havcr1, hepatitis a virus cellular receptor 1; KIM-1, kidney injury molecule-1; Hprt, hypoxanthine phosphoribosyltransferase 1; Il6, interleukin-6; Lcn2, Lipocalin-2; NCC, sodium chloride cotransporter; NGAL, neutrophil gelatinase-associated lipocalin; NKCC2, sodium-potassium-2 chloride cotransporter; Lrp2, low density lipoprotein receptor-related protein 2; p16 Cdkn2a, cyclin-dependent kinase inhibitor 2A; p21 Cdkn1a, cyclin-dependent kinase inhibitor 1A; Rpl19, ribosomal protein L19; SGLT2, sodium glucose cotransporter 2; Tnfa, tumor necrosis factor alfa.

Histological Analysis:

Formalin-fixed paraffin-embedded kidney and liver samples (5 μm) were prepared for histological analyses by the University of California-San Diego Tissue Technology Shared Resource (supported by National Cancer Institute Cancer Center Support Grant P30-CA-23100). Sirius Red (Cat# NC9039835, Fisher Scientific) staining was performed to evaluate kidney fibrosis area in scanned images as described (24). To this end, the ratio of red color area to total renal cortex area was measured using ImageJ software (ImageJ, RRID:SCR_003070) with the investigator blinded to study groups.

Tubular injury analysis and semi quantification was performed by a renal pathologist (H.Z.) blinded to experimental groups as described (23, 25, 26). Periodic acid-Schiff (PAS; Sigma-Aldrich, St. Louis, MO) staining was used for this purpose. Glomerular injury score: Severity of Scale of 0–4 (hypertrophy, mesangial matrix expansion, fibrosis/sclerosis and basement membrane thickening). 0: normal; 1: mild increase in mesangial cellularity and matrix; 2: moderate increase in mesangial cellularity and matrix; 3: severe increase in mesangial cellularity and matrix/with occasional segmental sclerosis; 4: diffuse segmental sclerosis, global glomerulosclerosis /hyalinized end-stage glomeruli. Tubular analysis consisted of estimating the total amount of tubular and tubulointerstitial damages in the cortical regions of the kidney. Evaluated acute and chronic injuries included tubular dilatation, loss of brush border, intratubular/medullary casts, thickening of tubular basement membrane and tubular atrophy, and interstitial fibrosis. The scoring scale was defined as follows: 0: normal tubular architecture, 1: >0–25% of tubules and the tubulointerstitium are injured, 2: >25–50% of tubules and the tubulointerstitium are injured, 3: >50–75% of tubules and the tubulointerstitium are injured, and 4: >75% of tubules and the tubulointerstitium are injured (23, 25, 26). The histological scoring for interstitial inflammation primarily detected lymphocytes, and is shown as percent area of inflammation: 0: <10%, 1: 10–25%, 2: 26–50%, 3: 51–75%, 4: >75%.

To analyze liver lipid content, immunofluorescence was used to determine the liver perilipin-2 positive area. Perilipin-2, also called adipophilin or ADRP, is a coat protein found on the surface of cellular lipid droplets. Perilipin-2 immunohistochemistry and immunofluorescence staining are widely used as sensitive tissue markers to visualize lipid accumulation (27). Immunofluorescence was performed as described (28) using anti-perilipin-2 antibody-conjugated with CoraLite Plus 488 (1:200, CL488–15294, Proteintech) ), and the % perilipin-2 positive lipid droplet area was determined from 5 regions of interest per sample using QuPath (29). Liver inflammatory foci were quantified in H&E-stained liver sections using QuPath v0.7.0 by an investigator blinded to experimental group. A supervised pixel classifier was trained to identify inflammatory aggregates, with hepatic parenchyma used as non-inflammatory or exclusion classes. Classifier-positive regions were converted into individual detection objects using a predefined minimum-area threshold and visually reviewed to remove false-positive detections. Inflammation was quantified as inflammatory foci per um2 of analyzed liver tissue and as inflammatory focus area (%), calculated as the summed inflammatory-focus area divided by the total analyzed tissue area × 100.

Statistical analysis:

Data are presented as means ± SE and dots show individual values. Values that were more than two SDs above or below the average were considered outliers and excluded from analysis. Data were log-transformed when doing so enhanced data normality. To establish the effect of AA and the drugs, groups were compared to the vehicle AA group by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. An overall significance level of P < 0.05 was considered as statistically significant.

Results

Response to GLP1RA and GCGRA prior to first aristolochic acid injection:

Application of GLP1RA reduced food intake (by ~30%) and body weight (by ~16%) vs. veh (Fig. 2A&B). GLP1RA did not alter blood glucose, plasma urea and creatinine, or hematocrit (Fig. 2C-F), and modestly reduced urine albumin (UACR) and NGAL to creatinine ratio with no significant effect on urine KIM-1 (Fig. 2G-I).

Fig. 2: Response to the 2 agonists prior to first aristolochic acid injection.

Fig. 2:

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A: Food intake profile and mean food intake during drug/vehicle; B: Body weight profile and change in body weight in response to drug/vehicle; C: Blood glucose levels day 18, non-fasted; D: plasma urea; E: plasma creatinine; F: hematocrit; G: urine albumin/creatinine ratio; H: urine NGAL to creatinine ratio; I: urine KIM-1 to creatinine ratio. Individual values and/or means±SE are shown. To establish the effect of the drugs, the 2 drug groups were compared with vehicle (veh) by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=7–18 mice/group. *P < 0.05. NS, not significant.

GCGRA reduced body weight by ~6% vs veh without altering food intake (Fig. 2A&B). GCGRA increased blood glucose levels and lowered plasma urea vs veh (Fig. 2C & D). GCGRA did not change plasma creatinine, hematocrit, or UACR, and, like the GLP1RA, modestly lowered urine NGAL without changing KIM-1 to creatinine ratios (Fig. 2E-I).

Characterization of murine Balkan nephropathy model:

Aristolochic acid injection in the drug-vehicle group (AA veh) lowered food intake and body weight vs control vehicle (control veh)(Fig. 3A&B), associated with higher kidney weight (Fig. 3G), unchanged heart weight, and lower absolute liver weight vs control veh (Suppl. Fig. 1C&D). As signs of impaired kidney function, AA reduced urine osmolality after the last AA injection (Fig. 3C), reduced hematocrit (Fig. 3D&H) and increased plasma creatinine levels (Fig. 3E&I) after the last AA injection and at organ harvest vs control veh, and raised plasma urea and ammonia at harvest while blood glucose levels were not changed (Fig. 3J-L). AA enhanced UACR (Fig. 3M) while glomerular injury score remained normal (Fig. 4A, D) associated with higher kidney mRNA expression of nephrin (Fig. 5A), which in this short term experiment may reflect an attempt for podocyte repair (30). Suggesting tubular injury, AA veh presented higher urinary KIM-1 to creatinine ratios after the last AA injection (Fig. 3F) and higher acute tubular injury scores in cortex and outer and inner stripe of outer medulla (OSOM, ISOM) vs control vehicle at harvest (Fig. 4B, D & E). This was associated with downregulation of mRNA expression of proximal tubule transporters Lrp2 (megalin) and Slc5a2 (SGLT2), unchanged expression of thick ascending limb sodium-potassium-2 chloride cotransporter Slc12a1 (NKCC2) and collecting duct water channel AQP2 (aquaporin2), and an increase in distal convoluted tubule sodium chloride cotransporter Slc12a3 (NCC) (Fig. 5A). mRNA expression of tubular injury markers Havcr1 (KIM-1), Lcn2 (NGAL) and p21 were increased (Fig. 5B). The inflammatory and fibrotic responses were modest. AA enhanced kidney mRNA markers of inflammation (Il6, Ccl2, Tnfa) and senescence (p16)(Fig. 5C) without a detectable rise in kidney interstitial inflammation score by pathologist (Fig. 4C). AA veh showed enhanced kidney cortex Sirius red fibrosis staining and kidney Col1a1 mRNA expression vs control vehicle (Fig. 6A&B).

Fig. 3: Effects of aristolochic acid (AA) and modulation by the 2 agonists – food intake, body weight and markers of kidney function.

Fig. 3:

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A: Food intake profile and mean food intake with/after AA; B: Body weight profile and change in body weight in response to AA at harvest; C-F: all parameters determined at 2 days after last AA injection; C: urine osmolality; D: change in hematocrit; E: plasma creatinine; F: urine KIM-1 to creatinine ration; G-M: all parameters determined at harvest; G: kidney wet weight; H: change in hematocrit; I/J: plasma urea and ammonia levels; L: blood glucose; M: urine albumin to creatinine ratio. To establish the effect of AA and of the drugs, AA veh was compared to the other 3 groups by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=5–12 mice/group, except N=2 and 3/group for urine albumin and urine KIM-1 to creatinine ratio in GCGRA due to limited volume. *P < 0.05. NS, not significant.

Fig. 4: Effects of AA and modulation by the 2 agonists – histological glomerular and tubular injury and inflammation scores.

Fig. 4:

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All scores by pathologist blinded to study groups. A: glomerular injury score; B: tubular injury score in cortex and outer and inner stripe medulla (OSOM, ISOM); C: interstitial inflammation score; D: representative pictures in kidney cortex, dilated tubules with loss of epithelial cells/containing casts (*) or necrotic debris (**); black arrow - interstitial fibrosis; yellow arrow - interstitial inflammation; E: representative pictures in outer medulla, (*) dilated tubules containing casts; black arrow-interstitial fibrosis; yellow arrow-interstitial inflammation. See methods for further scoring details. To establish the effect of AA and of the drugs, AA veh was compared to the other 3 groups by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=6–10 mice/group. *P < 0.05. NS, not significant.

Fig. 5: Effects of AA and modulation by the 2 agonists – kidney expression of glomerular, tubular transport and injury, and pro-inflammatory markers and Rev-Erba.

Fig. 5:

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A-D: Kidney mRNA expression, normalized to house-keeping genes Hprt and Rpl19 and the control vehicle group. To establish the effect of AA and of the drugs, AA veh was compared to the other 3 groups by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=5–10 mice/group. *P < 0.05. NS, not significant.

Fig. 6: Effects of AA and modulation by the 2 agonists – markers of kidney fibrosis.

Fig. 6:

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A: representative pictures of Sirius red fibrosis staining and analysis; B: kidney mRNA expression of Col1a1, normalized to house keeping genes Hprt and Rpl19 and the control vehicle group. To establish the effect of AA and of the drugs, AA veh was compared to the other 3 groups by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=6–10 mice/group. *P < 0.05. NS, not significant.

GLP1RA had little impact on AA-induced kidney injury:

Following AA application, GLP1RA group showed similar food intake and AA-induced body weight reduction compared with AA veh (Fig. 3A&B), associated with lower blood glucose levels (Fig. 3L). The described deleterious kidney effects of AA were largely unaffected by GLP1RA (Fig. 3–6). Exceptions include that GLP1RA lowered urine KIM-1 to creatinine ratios after the last AA injection vs AA veh (Fig. 3F), slightly lowered kidney mRNA expression of proinflammatory Ccl2 (Fig. 5C), and lowered Sirius red kidney cortex fibrosis staining without a significant effect on kidney Col1a1 mRNA expression (Fig. 6A&B). Compared with AA veh, the AA GLP1RA group had lower kidney, heart and liver weight (Fig. 3G, Supp. Fig. 1C&D) roughly in proportion to the absolutely lower body weight at harvest, reflecting body weight effects of GLP1RA before AA application (Fig. 3B).

GCGRA sensitized mice to AA:

In GCGRA-treated mice, AA induced a stronger and sustained reduction in body weight compared with AA veh associated with lower food intake, particularly after the first week of AA treatment (Fig. 3A&B). In veh and GLP1RA groups, 10 of 12 (83%) and 10 of 11 (91%) mice survived until harvest in response to AA, whereas in GCGRA, only 6 of 11 (55%) mice survived. To prevent losing more mice in the latter group, the total number of injections was reduced for all groups from the original protocol of 8 injections (22, 23, 31) to 7 injections and harvest performed 6 days (GCGRA) and 11 days (other groups) after the last AA injection instead of planned 21 days. The AA GCGRA group had lower kidney, heart and liver weight at harvest vs AA veh, roughly in proportion to the lower body weight (Fig. 3G, Supp. Fig. 1C&D, Fig. 3B). AA eliminated the hyperglycemia observed in GCGRA vs veh group prior to AA (Fig. 2C & Fig. 3L), possibly in part due to the lower food intake and/or AA interfering with GCGRA-induced hepatic gluconeogenesis. AA GCGRA had similar UACR (Fig. 3M; urine only obtained from 2 AA GCGRA mice) and glomerular injury score compared with AA veh (Fig. 4A, D) associated with a further increase in kidney mRNA expression of nephrin (Fig. 5A), a potential podocyte stress signal.

Suggesting enhanced kidney injury, GCGRA caused greater AA-induced hematocrit reduction (Fig. 3D&H) and higher levels in plasma creatinine after the last AA injection and a trend at harvest (Fig. 3E&I). Prior to AA application plasma urea levels were lower in GCGRA vs veh group (Fig. 2D). In contrast, plasma urea levels were higher and were associated with elevated plasma ammonia levels in GCGRA vs veh in response to AA (Fig. 3J&K). AA GCGRA had higher acute tubular injury scores in cortex and outer stripe and inner stripe of outer medulla (OSOM, ISOM) vs AA veh (Fig. 4B, D&E), associated with higher kidney mRNA expression of p21, a cell cycle inhibitor induced by DNA damage, as well as the tubular injury marker NGAL (Lcn2) (Fig. 5B).

Previous work implicated the repressive transcription factor Rev-ErbA (Nr1d1) in promoting AA-induced kidney injury in a mouse model (32) and in glucagon-induced repression of kidney Sglt2 (Slc5a2) mRNA expression (9). In the current study, the described signs of enhanced tubular injury in the AA GCGRA group were associated with enhanced kidney mRNA expression of Rev-Erba while kidney Sglt2 (Slc5a2) mRNA expression was repressed compared with AA veh (Fig. 5A&D). Consistent with a proposed glucagon-induced shift in sodium and water transport to the more distal tubule (3), the AA GCGRA showed increased kidney mRNA expression of Slc12a1, Slc12a3, and Aqp2 compared with AA veh (Fig. 5A). Urine osmolality was similar in the AA GCGRA vs the AA veh group at 2 days after the last AA injection (Fig. 3C) suggesting similar renal fluid conservation.

Unexpectedly, the AA GCGRA group showed lower urinary to creatinine ratios of the tubular injury marker KIM-1 after the last AA injection (Fig. 3F) and lower kidney mRNA expression of Kim-1 (Havcr1) at harvest vs AA veh (Fig. 5B). Moreover, the GCGRA group showed lower kidney mRNA expression of proinflammatory genes (Il6, Ccl2, Tnfa) and the senescence gene p16 (Fig. 5C) associated with a lower interstitial inflammatory score compared with AA veh (Fig. 4C, D&E), as well as lower kidney mRNA expression of Col1a1 associated with less Sirius red fibrosis staining (Fig. 6A&B). Thus, pretreatment with GCGRA induced mixed signals regarding the kidney response to AA. With regard to liver effects, AA modestly increased plasma levels of alkaline phosphatase (ALP) without significantly changing plasma levels of alanine aminotransferase (ALT) or cholesterol; all determined 2 days after last AA injection (Fig. 7A). This was associated at harvest with lower liver weight (Supp. Fig. 1D) and a trend for lower liver lipid content (assessed by perilipin-2 immunostaining) (Fig. 7B) as well as a modest increase in liver inflammatory foci (Fig. 7C). Compared with AA veh, the AA GLP1RA and the AA GCGRA group had similar levels of plasma ALP and ALT, associated with reduced liver weight and lipid content. The AA GCGRA group, but not the GLP1RA group, also had lower plasma cholesterol levels and liver inflammatory foci compared with AA veh (Supp. Fig. 1D and Fig. 7A-C).

Fig. 7: Effects of AA and modulation by the 2 agonists – liver effects.

Fig. 7:

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A: plasma levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and cholesterol; B: liver perilipin-2 analysis by immunostaining and representative pictures; C: analysis and representative pictures of liver inflammatory foci (per μm2 of analyzed liver tissue and as inflammatory focus area (%), calculated as the summed inflammatory-focus area divided by the total analyzed tissue area × 100). To establish the effect of AA and of the drugs, AA veh was compared to the other 3 groups by 1-way ANOVA; if significant, this was followed by pairwise comparisons and correction for multiple comparisons (Holm-Sidak). If Normality failed, Kruskal-Wallis One Way Analysis of Variance on Ranks was used. N=5–10 mice/group. *P < 0.05. NS, not significant.

Discussion

The current study in Western diet-fed mice observed expected phenotypes of the GLP1RA and the GCGRA prior to AA application, consistent with target engagement. This included reduced food intake and body weight in response to GLP1RA (33) and a robust hyperglycemia and lower plasma urea concentrations in response to GCGRA, which did not lower food intake and showed numerically lesser body weight reduction than GLP1RA. Glucagon levels and GCGR activation are known to increase during hypoglycemia and in response to an increase in protein intake to enhance hepatic gluconeogenesis and facilitate nitrogen excretion by enhancing urinary urea excretion (5, 34). The body weight loss in response to GCGR activation can involve a reduction in food intake but in rodents an increase in energy expenditure and promotion of fat oxidation can dominate (35, 36). Prior to AA application, neither of the drugs significantly affected hematocrit, plasma creatinine, and urine KIM-1 to creatinine ratios. The GLP1RA but not the GCGRA reduced UACR while both modestly lowered urinary NGAL to creatinine ratios, a marker of tubule stress/injury in Western-diet fed obese mice, consistent with both distinct and similar effects on the kidney.

The murine model of AA-induced Balkan nephropathy showed the expected kidney phenotypes consistent with primary tubulotoxicity (16–18, 22, 23, 31). AA is taken up by proximal tubular cells and then causes DNA damage, which triggers a DNA damage response and acute tubular injury. DNA damage response by p21 is a hallmark of AA-induced tubular injury (37). Only symptomatic treatments are available for Balkan nephropathy, and few studies explored conditions that modify kidney susceptibility to AA, including recent studies showing partial protection by inhibition of SGLT2 (22), which induces a fasting-like metabolism, whereas a Western diet aggravated the model (31).

GLP1RA treatment did not robustly affect AA-induced kidney injury, although some parameters were improved compared with vehicle-treatment (see Results). In comparison, GCGRA treatment sensitized the animals to AA. This included higher kidney levels of p21 mRNA expression and signs of enhanced tubular injury, associated with greater body weight and hematocrit reduction, but also higher plasma levels of creatinine, urea and ammonia, and higher mortality. GCGRA-induced stimulation of hepatic amino acid metabolism and ureagenesis may become detrimental when kidney function is impaired resulting in urea and ammonia accumulation, which, as uremic toxins, may contribute to food intake suppression and body weight loss (38, 39). Vice versa, facilitating urinary nitrogen excretion by inhibiting the proximal tubule amino acid transporter Slc6a19 (B0AT1) improved AA-induced kidney phenotypes (23).

The potential of a GCGRA to aggravate kidney injury through effects on the tubular receptor has been indicated by studies using a Ksp-cadherin (expressed exclusively in tubular epithelial cells in the kidney) promoter-driven Gcgr-specific short hairpin RNA construct carried by adeno-associated virus-9 and injected in situ directly into the kidneys of mice (11). The maneuver reduced Gcgr mRNA levels by ~80% in proximal tubule epithelial cells and by ~50% in the kidney, with no effect on hepatic Gcgr. Such a tubular GCGR knockdown in mice had no obvious basal kidney phenotype but protected from kidney injury and lipid accumulation induced by the combination of a high fat diet, heminephrectomy, and streptozotocin-induced hyperglycemia and nephrotoxicity. The study further reported a similar protective effect by a human monoclonal antibody that functions as a competitive antagonist of GCGR, while kidney outcome was worsened by twice daily i.p. application of glucagon (11).

GCGRA enhanced kidney NGAL response, a primary marker of distal tubule injury, but reduced KIM-1 response to AA, a primary marker of proximal tubule injury. This may reflect a glucagon-induced shift in transport burden from proximal tubule to thick ascending limb and distal convoluted tubule, which is part of the glucagon physiology to facilitate urinary urea excretion (3): less proximal tubular sodium and water transport reduces the urea gradient needed for passive urea reabsorption, while enhanced distal tubule sodium transport compensates, and increased thick ascending limb sodium transport via NKCC2 and collecting duct AQP2-mediated water permeability ensure medullary hypertonicity and water retention to excrete the urea with minimal water loss. Considering the higher cortical tubular injury score in the AA GCGRA group, however, a lower KIM-1 response could also be due to greater loss of KIM-1-forming proximal tubule mass.

GCGRA upregulated kidney mRNA expression of the inhibitory transcription factor Rev-Erba and downregulated kidney Sglt2. Suggesting the possibility that the first event contributed to the second, previous work mechanistically linked glucagon-induced Rev-Erba expression to a suppression of kidney SGLT2 expression and function (9). Rev-Erba has been implicated in promoting AA-induced kidney injury in a mouse model, incl. the rise in plasma creatinine and kidney Ngal (Lcn2) mRNA expression (32), phenotypes promoted by the GCGRA in the current study. Likewise, the inhibitory transcription factor Rev-Erba has been shown to repress inflammatory genes Il6 and Ccl2 as well as p16 (40, 41) and shown to induce anti-fibrotic effects (40), phenotypes induced by GCGRA in response to AA. Future studies will need to test the exact role of Rev-Erba in response to GCGR agonists. This will include the hypothesis that upregulation of Rev-Erba suppresses protective aspects of the acute kidney injury response, including potential protective roles of some of the proinflammatory players but also of KIM-1 to limit the injury. While KIM-1 is a sensitive measure of tubular injury (42) it also confers a phagocytic phenotype on epithelial cells important for kidney recovery (43). A higher KIM-1/serum creatinine ratio in patients with biopsy-confirmed acute tubular injury was associated with better kidney recovery over 6 months (44). Vice versa, increased sensitivity to ischemia-reperfusion kidney injury in a mouse strain with accelerated aging was associated with a blunted kidney KIM-1 response (45). The observation that GCGRA increased p21 expression may reflect greater DNA damage, considering the fact that Rev-Erba itself can also suppress p21 (46).

Limitations and perspectives include the restriction of experiments to male mice, and future studies will need to explore effects also in female sex. In this regard, application of a long-acting glucagon analog for 8 weeks to female C57Bl/6JRj mice increased blood glucose, mesangial cell area, and mean UACR, while a long-acting glucagon receptor antibody (antagonist) reduced blood glucose and mesangial cell area (8). The current AA model modestly increased liver inflammatory foci associated with minor effects on plasma liver enzymes, and a lower liver weight associated with a trend for reduced liver lipid content. The latter may reflect lower food intake in the AA model. The GCGRA reduced plasma cholesterol levels, consistent with the literature (47, 48), and based on effects on liver enzymes and liver lipid content and inflammatory foci, did not worsen the liver phenotype. Nevertheless, since glucagon has robust effects on liver metabolism, future studies will need to determine whether a GCGRA can enhance or reduce liver metabolism of AA to kidney toxic aristolactam I (49). Also, what is the role of tubular vs hepatic GCGR for the observed phenotype? Rev-erbα is a circadian clock-regulated gene and important for circadian rhythm (50). Does a GCGRA through Rev-Erba interfere with the circadian rhythm with effects on metabolism and the cardiovascular system? The current study focused on the murine Balkan nephropathy model. What is the relevance for other forms of kidney injury, including acute kidney injury induced by other nephrotoxins, ischemia and/or sepsis, which all have clinical relevance. Also, the current study employed a rather robust GCGR agonist dose/tone. A very important question will be whether the lower glucagon tone induced by the novel combination drugs tested in obesity, T2D, MASH, and CKD will interfere with the described pathways and how this is affected by combining a GCGRA with a GLP1RA.

In conclusion, GCGR agonism can worsen outcome in a murine model of Balkan nephropathy. Further studies are needed to proof a proposed deleterious role of Rev-Erba-induced gene suppression in this setting.

Supplementary Material

Supplemental material can be found at https://doi.org/10.6084/m9.figshare.31975344

Supplemental Figure 1: Additional measurements

Grant support

The authors were supported by National Institutes of Health (NIH) Grants R01DK112042, R01DK132690, University of Alabama at Birmingham/University of California-San Diego O’Brien Center of Acute Kidney Injury NIH Grant U54 DK137307, the Department of Veterans Affairs (all to S.C.T and V. V.), and an investigator-initiated research project funded by Boehringer Ingelheim, Biberach (to V. V.).

Footnotes

Conflict of Interest Statement

Over the past 12 months, V.V. received grant support for investigator-initiated research from Boehringer-Ingelheim, Jnana Therapeutics, Novo-Nordisk, and Maze Therapeutics and served as a consultant and received honoraria from Casdin Capital, Checkpointlp, BridgeBio, GondolaBio, Google Venture, and PrimaryInsight. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

Data Availability Statement

All data generated or analyzed during this study are included in this article or can be requested by enquiry directed to the corresponding author.

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Data Availability Statement

All data generated or analyzed during this study are included in this article or can be requested by enquiry directed to the corresponding author.

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