β2-Adrenergic receptor agonists as a treatment for diabetic kidney disease

Abstract

We have previously shown that the long-acting β₂-adrenergic receptor (β₂-AR) agonist formoterol induced recovery from acute kidney injury in mice. To determine whether formoterol protected against diabetic nephropathy, the most common cause of end-stage kidney disease (ESKD), we used a high-fat diet (HFD), a murine type 2 diabetes model, and streptozotocin, a murine type 1 diabetes model. Following formoterol treatment, there was a marked recovery from and reversal of diabetic nephropathy in HFD mice compared with those treated with vehicle alone at the ultrastructural, histological, and functional levels. Similar results were seen after formoterol treatment in mice receiving streptozotocin. To investigate effects in humans, we performed a competing risk regression analysis with death as a competing risk to examine the association between Veterans with chronic kidney disease (CKD) and chronic obstructive pulmonary disease (COPD), who use β₂-AR agonists, and Veterans with CKD but no COPD, and progression to ESKD in a large national cohort of Veterans with stage 4 CKD between 2011 and 2013. Veterans were followed until 2016 or death. ESKD was defined as the initiation of dialysis and/or receipt of kidney transplant. We found that COPD was associated with a 25.6% reduction in progression from stage 4 CKD to ESKD compared with no COPD after adjusting for age, diabetes, sex, race-ethnicity, comorbidities, and medication use. Sensitivity analysis showed a 33.2% reduction in ESKD in Veterans with COPD taking long-acting formoterol and a 20.8% reduction in ESKD in Veterans taking other β₂-AR agonists compared with those with no COPD. These data indicate that β₂-AR agonists, especially formoterol, could be a treatment for diabetic nephropathy and perhaps other forms of CKD.NEW & NOTEWORTHY Diabetic nephropathy is the most common cause of ESKD. Formoterol, a long-acting β₂-adrenergic receptor (β₂-AR) agonist, reversed diabetic nephropathy in murine models of type 1 and 2 diabetes. In humans, there was an association with protection from progression of CKD in patients with COPD, by means of β₂-AR agonist intake, compared with those without COPD. These data indicate that β₂-AR agonists, especially formoterol, could be a new treatment for diabetic nephropathy and other forms of CKD.

Keywords: diabetes; nephropathy; β-agonist.

Graphical abstract

Figure 1.

High-fat diet (HFD) mice develop significant weight gain and albuminuria. A: the schematic of the experimental plan to investigate the role of formoterol in recovery from diabetic renal disease is shown. B: HFD compared with low-fat diet (LFD) mice after 12 wk showed a significant weight gain. Representative mice from both groups are shown. C: HFD mice had albuminuria by SDS-PAGE of urine samples, whereas LFD mice had no albuminuria. The red arrow indicates albumin on the gel. Bovine serum albumin (BSA) was run on lane 2; as a positive control. n = 4 for both groups in this experiment with each lane representing a mouse. The experiments were repeated twice with similar results.

Figure 2.

Formoterol treatment results in recovery from high-fat diet (HFD)-induced diabetic renal injury. A: HFD induced a significant increase in body weight compared with a low-fat diet (LFD). B: overnight fasting glucose levels were found to be significantly higher in mice that received the HFD compared with the LFD. C: SDS-PAGE of urine samples after 7 and 14 days of formoterol treatment showed a decrease in albumin excretion in HFD mice treated with formoterol compared with HFD mice treated with vehicle alone. Red arrow points to albumin. BSA, bovine serum albumin. D: measurement of urine albumin to creatinine ratio (UACR) by ELISA showed a significant reduction in albuminuria at 7 and 14 days in HFD mice treated with formoterol compared with HFD mice treated with vehicle alone. E: HFD mice had a significantly increased kidney weight compared with LFD mice, and this was reversed with formoterol treatment. F: histology, hematoxylin and eosin (H&E) staining, of kidney sections in LFD, HFD, and HFD + formoterol mice. Bar in the full kidney sections = 800 μm. Bar in middle row = 50 μm. G and H: quantitative analysis of kidney size and glomerular tuft area showed that both were increased in HFD mice compared with LFD mice and that formoterol treatment decreased these parameters back to what was seen in LFD mice. I: histology and Masson’s trichrome staining of kidney sections in LFD, HFD, and HFD + formoterol mice examining fibrosis. Yellow arrow points to fibrosis in blue. Bar in top row = 50 μm. J: quantitative analysis showing increased fibrosis in kidneys of HFD vs. LFD mice, which was largely reversed with formoterol. K: serum creatinine increased in HFD mice compared with LFD mice and decreased with formoterol treatment. L: representative EM sections show podocyte foot process effacement (yellow *) in HFD mice but not in LFD or HFD + formoterol mice. Mitochondria were rounded in HFD mice as opposed to oblong in LFD and HFD + formoterol mice. Bar = 800 nm. M: markers of mitochondrial biogenesis in kidney lysates were evaluated by Western blotting using OXPHOS (cocktail antibodies of mitochondrial electron transport chain complex I to V) and GAPDH antibodies. GAPDH is a loading control. N: quantitative analysis showing that complex II was significantly decreased in HFD mice compared with LFD mice, and levels were restored in HFD + formoterol mice. ns, nonsignificant. All data are presented as means ± SE. These experiments were repeated twice with similar results. For F, I, and L, representative sections are shown. For C, G, M, and N, representative experiments are shown with each dot representing a mouse. For A, B, D, E, J, and K, data from all the experiments were aggregated with each dot representing a mouse. For glomerular tuft area in H, three measurements from each mouse were used. EM, electron microscopy.

Figure 3.

Formoterol accelerates the recovery from streptozotocin (STZ)-induced diabetic renal injury. A: schematic of the experimental plan to evaluate the efficacy of formoterol treatment in a type I diabetes mouse model. B: STZ-treated animals had significant increases in weight after 8 wk, which did not change after treatment for an additional 2 wk with formoterol or vehicle. C: kidney weights were not statistically different among the three groups. D: kidney/body weight was significantly higher in STZ-treated mice compared with control mice. E: 4-h fasting glucose levels were found to be significantly higher in STZ-treated mice compared with controls. F and G: measurement of urine albumin/creatinine ratios by ELISA showed a reduction in albuminuria at 14 days in STZ mice treated with formoterol compared with mice treated with vehicle alone. H: serum creatinine was increased in STZ-treated mice compared with control mice. I: blood urea nitrogen (BUN) levels were increased in STZ-treated mice compared with control mice, and formoterol decreased BUN in these mice compared with mice treated with vehicle alone. These experiments were repeated twice with similar results. For A–I, n = 5 for each group with each dot representing a mouse. ns, nonsignificant.

Figure 4.

Formoterol treatment resulted in recovery from streptozotocin (STZ)-induced diabetic renal injury. A: representative images from H&E, PAS, and Masson’s trichrome staining of sections from paraffin-embedded renal tissue. Histological analysis showed that formoterol treatment reduced fibrosis (yellow arrows) in STZ-treated mice compared with mice treated with vehicle alone. STZ mice had nodular lesions typical of diabetic nephropathy (red arrows). Formoterol treatment reversed these changes. Scale bar = 50 μm. B: quantitative analysis of the glomerular tuft area demonstrated a significant reduction in formoterol-treated mice compared with mice treated with vehicle alone. C: formoterol treatment in STZ-treated mice mice showed a significant reduction in fibrosis compared with mice treated with vehicle alone. All data are presented as means ± SE. D: representative TEM images of podocyte foot processes in control, STZ + vehicle, and STZ + formoterol-treated mice. Foot processes appeared effaced and GBM thickened in STZ-treated mice (yellow arrow). Scale bar = 2 μm. E: quantitative analysis of electron micrographs showed significant reduction in the number of slit diaphragms per micrometers in STZ-treated mice, which was reversed with formoterol. F: representative EM images of mitochondria whose shape and size were distorted following STZ and restored with formoterol. Scale bar = 200 nm. G: markers of mitochondrial biogenesis in kidney lysates were evaluated by Western blotting using OXPHOS (cocktail antibodies of mitochondrial electron transport chain complex I to V) and GAPDH antibodies. H: densitometric analysis of immunoblots showed significant increases in complexes V-ATP5A, IV-MTCO1, and I-NDUFA9 proteins in STZ + formoterol-treated kidneys compared with control kidneys and in IV-MTCO1 and I-NDUFAS proteins in STZ + formoterol treated kidneys compared with STZ + vehicle- treated kidneys (P < 0.05). All data are presented as means ± SE. I and J: a trend toward increased PGC-1α expression was found in STZ animals treated with formoterol. These experiments were repeated twice with similar results. For A, D, and F, representative sections are shown. For E and G–J, representative experiments are shown with each dot representing a mouse. For glomerular tuft area in B, three sections from each mouse were used. For fibrosis measurements in C, three sections from each mouse were used. EM, electron microscopy; H&E, hematoxylin and eosin; ns, nonsignificant; PAS, periodic acid-Schiff.

Figure 5.

Unadjusted cumulative incidence plot of ESKD and death among a retrospective cohort of older Veterans (aged 65 yr or over) by COPD status from time of incident stage 4 CKD diagnosis. CKD, chronic kidney disease; COPD, chronic obstructive pulmonary disease; ESKD, end-stage kidney disease.