Abstract

   Progressive respiratory failure is the primary cause of death in the
   coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome
   of the acute respiratory distress syndrome (ARDS), characterized by an
   initial exacerbated inflammatory response, metabolic derangement and
   ultimate tissue scarring. A positive balance of cellular energy may
   result crucial for the recovery of clinical COVID-19. Hence, we asked
   if two key pathways involved in cellular energy generation,
   AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC)
   signaling and fatty acid oxidation (FAO) could be beneficial. We tested
   the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in
   different experimental models mimicking COVID-19 associated
   inflammation in lung and kidney. We also studied two different cohorts
   of COVID-19 patients that had been previously treated with metformin.
   These drugs ameliorated lung damage in an ARDS animal model, while
   activation of AMPK/ACC signaling increased mitochondrial function and
   decreased TGF-β-induced fibrosis, apoptosis and inflammation markers in
   lung epithelial cells. Similar results were observed with two indole
   derivatives, IND6 and IND8 with AMPK activating capacity. Consistently,
   a reduced time of hospitalization and need of intensive care was
   observed in COVID-19 patients previously exposed to metformin. Baicalin
   also mitigated the activation of pro-inflammatory bone marrow-derived
   macrophages (BMDMs) and reduced kidney fibrosis in two animal models of
   kidney injury, another key target of COVID-19. In human epithelial lung
   and kidney cells, both drugs improved mitochondrial function and
   prevented TGF-β-induced renal epithelial cell dedifferentiation. Our
   results support that favoring cellular energy production through
   enhanced FAO may prove useful in the prevention of COVID-19-induced
   lung and renal damage.

   Keywords: Fibrosis, Metabolism, Mitochondria, Inflammation, COVID-19

Graphical abstract

   Boosting bioenergetics by the drugs metformin (AMPK activator) and
   baicalin (CPT1A activator) protects against COVID-19-associated
   inflammation and fibrosis in lung and kidney.

   Image 1
   [59]Open in a new tab

Highlights

     * •
       Metformin (AMPK activator) and baicalin (CPT1A activator) improved
       mitochondrial function.
     * •
       Both drugs reduced kidney fibrosis in different animal models of
       lung and kidney injury, key target organs of COVID-19.
     * •
       AMPK/ACC activation decreased TGF-β-induced fibrosis, apoptosis and
       inflammation markers in lung and renal epithelial cells.
     * •
       COVID-19 patients on previous treatment with Metformin showed
       reduced time of hospitalization and needed of intensive care.

Abbreviations

   ARDS
          acute respiratory distress syndrome

   BAL
          bronchoalveolar lavage

   BMDMs
          bone marrow-derived macrophages

   CKD
          chronic kidney disease

   COVID-19
          coronavirus disease 2019

   ECM
          extracellular matrix

   EMT
          epithelial-to-mesenchymal transition

   FAO
          fatty acid oxidation

   FAN
          folic acid nephropathy

   FBS
          fetal bovine serum

   HRPTEC
          human renal primary tubular epithelial cells

   ITS
          insulin-transferrin-selenium

   OCR
          oxygen consumption rate

   SPF
          specific pathogen free

   TECs
          tubular epithelial cells

   T2DM
          type 2 diabetes mellitus

   UUO
          unilateral ureteral obstruction

1. Introduction

   COVID-19 may affect almost any organ of the human body due to the wide
   distribution of peptidase angiotensin-converting enzyme 2 (ACE2), whose
   interaction with the virus spike protein S dictates its tropism
   [[60]1]. Lung disease is by far the most important condition
   responsible for COVID-19 morbidity and mortality [[61]2]. Once the
   virus infects epithelial cells from the upper respiratory tract, it may
   evolve to symptomatic pneumonitis by infecting ACE2-expressing type 2
   alveolar epithelial cells. In a later phase, severe disease may
   develop, involving disruption of the epithelial-endothelial barrier,
   complement deposition and an acute inflammatory reaction with a massive
   release of cytokines [[62]3], causing diffuse alveolar damage and
   respiratory insufficiency, often refractory to therapy [[63]4]. This
   state shares most of the molecular and pathophysiological features of
   acute respiratory distress syndrome (ARDS), which may be caused by an
   ample spectrum of infectious and non-infectious agents. ARDS remains a
   major clinical challenge as mortality in severe ARDS is close to 50%
   and many patients who survive it suffer disabling long-term
   complications. The absence of effective therapies in ARDS, in general
   and in its COVID-19-associated form in particular, has triggered the
   search for therapies beyond classical pharmacological approaches. Since
   the start of the COVID-19 pandemic, it became clear that the major
   cellular inflammatory response elicited by organs affected with the
   virus also impacts their metabolic behavior [[64]5]. Thus, achieving a
   positive cellular energy balance may result crucial for the recovery
   from COVID-19. We hypothesized that boosting two key pathways involved
   in cellular energy generation namely, AMP-activated protein kinase
   (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation
   (FAO), could have a protective role in different experimental models
   mimicking COVID-19-associated inflammation and fibrosis in lung and
   kidney. To test this hypothesis, we used the drugs metformin (AMPK
   activator) and baicalin (CPT1A activator). While the knowledge about
   metformin is extensive and the clinical experience with its use is very
   broad [[65]6], the FAO-enhancing flavonoid baicalin has not reached the
   clinical stage. Nevertheless, this is one of the few compounds with a
   selective capacity to enhance fatty acid oxidation (FAO) by virtue of
   its direct action on the FAO rate-limiting step catalyzed by carnitine
   palmitoyl transferases (CPTs), whose most abundant isoform in the lung
   and kidney is CPT1A [[66]7]. We studied an animal model of ARDS, a
   cellular model (BEAS-2B cells) and a set of clinical data from patients
   with COVID-19 under previous treatment with metformin. Single-cell
   analysis of databases from patients affected with COVID-19 resulted
   useful in identifying cell populations from lung and kidney expressing
   a signature of metabolic derangement. We also explored the effects of
   metformin and baicalin in two animal models of kidney injury (folic
   acid nephropathy, FAN and unilateral ureteral obstruction, UUO)
   together with kidney epithelial cells (PTEC and HKC-8 cells) exposed to
   profibrotic mediators. We found that these energy metabolism enhancers
   were capable of inducing protection from inflammation and fibrosis in
   both lung and kidney in pathogenic settings, sharing important features
   with COVID-19.

2. Results

2.1. Metformin and baicalin protect the lung from experimental ARDS

   To test the effects of metformin and baicalin we used the LPS-induced
   experimental model of ARDS as shown in [67]Fig. 1A. Treatment with each
   drug significantly protected from the establishment of fibrosis in the
   lung, as well as it markedly reduced the severity of ARDS-related
   pathological lesions and expression of fibrosis-associated genes
   ([68]Fig. 1B, C, D, E and H). No synergistic beneficial effect of
   combined effect of Baicalin and Metformin was observed (data not
   shown). Increased levels of fibronectin were also observed in lungs
   from LPS-treated mice, whereas both metformin and baicalin drastically
   reduced this effect ([69]Fig. 1F and G). While LPS was able to promote
   AMPK activation, this effect was not evident in the presence of
   metformin and baicalin ([70]Fig. 1F). Evaluation of apoptosis in lung
   slices from the ARDS model by using BCL2 Associated X (BAX)
   immunohistochemistry showed that baicalin, but not metformin, abrogated
   LPS-induced cell death ([71]Fig. 1I and J). In contrast, no effect of
   either drug was found on the increase of cell proliferation mediated by
   LPS ([72]Supplemental Figs. 1A and B).

Fig. 1.

   [73]Fig. 1
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   Metformin and baicalin administration ameliorate lung damage and
   fibrosis in an animal model of LPS-induced ARDS. (A) Timeline of the
   LPS-induced ARDS mouse model and metformin/baicalin administration. (B)
   Representative microphotographs from one mouse per group of hematoxylin
   and eosin (H&E) (upper panels), Masson Trichrome (medium panels) and
   Sirius Red (lower panels) staining of lungs from mice subjected to
   LPS-induced ARDS after metformin/baicalin treatment. Scale bars: 25 μm.
   (C) Heat map of the anatomopathological study of the lung from mice
   treated as described above. (D) Violin plots of histological
   semi-quantitative global damage evaluation of the lungs from mice
   treated as described above. (E) Quantification of Sirius Red staining
   from (B) represents the mean ± s.e.m. (F) Immunoblots depicting
   fibronectin (FN), phosphorylated AMP-activated protein kinase (p-AMPK)
   protein levels in lungs from control and LPS-induced ARDS mice after
   metformin/baicalin treatment. (G) Bar graphs represent the
   mean ± s.e.m. of fold changes corresponding to densitometric analyses
   from (F), n = 6 mice. (H) Violin plots of mRNA levels of
   fibrosis-associated genes determined by qRT-PCR in lungs from mice
   treated as described above. Bar graphs represent the mean ± s.e.m. of
   fold changes. (I) Representative micrographs of one mouse per group
   showing the expression of BAX in lung sections of mice treated as
   described above. Scale bar = 25 μm. (J) Bar graph represents the
   quantification of the mean ± s.e.m. of % of BAX positive stained area
   in lungs from mice treated as described above. n = 5–8, mice *P < 0.05,
   **P < 0.01, ***P < 0.001 compared to control lungs; ^#P < 0.05 compared
   to lungs from LPS-induced ARDS mice. Statistical analysis for more than
   two groups was done with Kruskal-Wallis test. (For interpretation of
   the references to colour in this figure legend, the reader is referred