Lasmiditan Induces Mitochondrial Biogenesis in Primary Mouse Renal Peritubular Endothelial Cells and Augments Wound Healing and Tubular Network Formation

Abstract

Kidney disease (KD) is a progressive and life-threatening illness that has manifested into a global health crisis, impacting >10% of the general population. Hallmarks of KD include tubular interstitial fibrosis, renal tubular cell atrophy/necrosis, glomerulosclerosis, persistent inflammation, microvascular endothelial cell (MV-EC) dysfunction/rarefaction, and mitochondrial dysfunction. Following acute kidney injury (AKI), and/or during KD onset/progression, MV-ECs of the renal peritubular endothelial capillaries (RPECs) are highly susceptible to injury, dysfunction, and rarefaction. Pharmacological induction of mitochondrial biogenesis (MB) via 5-Hydroxytryptamine Receptor 1F (HTR1F) agonism has been shown to enhance mitochondrial function and renal vascular recovery post-AKI in mice; however, little is known about MB in relation to renal MV-ECs and RPECs repair mechanisms. To address this gap in knowledge, the in vitro effects of the potent and selective FDA-approved HTR1F agonist lasmiditan were tested on primary mouse renal peritubular endothelial cells (MRPECs). Lasmiditan increased mitochondrial maximal respiration rates, mRNA and protein expression of MB-related genes, and mitochondrial number in MRPECs. MRPECs were then exposed to pro-inflammatory agents associated with renal MV-EC dysfunction, AKI, and KD (i.e., lipopolysaccharides, transforming growth factor-β1, and tumor necrosis factor-α), in the presence/absence of lasmiditan. Lasmiditan treatment augmented MRPECs wound healing, endothelial tubular network formation (ETNF), enhanced barrier integrity, and blunted inflammatory-induced MV-EC dysfunctions. Together, these data suggest that lasmiditan induces MB and improves wound healing and ETNF of primary MRPECs in the presence/absence of pro-inflammatory agents, highlighting a potential therapeutic role for lasmiditan treatment in renal MV-EC dysfunction, AKI, and/or KD.

New & Noteworthy:

Lasmiditan, an FDA-approved HTR1F agonist, induces mitochondrial biogenesis (MB) and enhances recovery following acute kidney injury in mice. Renal microvascular endothelial cells (MV-EC) are highly susceptible to dysfunction/rarefaction post-injury. The effect of MB on MV-EC repair/recovery is unknown. We show that lasmiditan induces MB in primary mouse renal peritubular endothelial cells and improves wound healing, endothelial tubular network formation and barrier integrity after inflammatory-induced dysfunction, indicative of its potential for the treatment of kidney diseases.

Graphical Abstract

Introduction

Kidney disease (KD) has become a global health crisis, characterized by both its progressive nature and life-threatening consequences.(1) In recent years, it has become one of the leading causes of both morbidity and mortality, impacting over 10% of the general population.(2, 3) Alarmingly, the all-age mortality rate associated with KD has increased >42% since 1990.(4)

KD encompasses a broad spectrum of distinct pathophysiological disorders that compromise the functional and/or structural integrity of the kidneys.(5) Despite the diversity of these disorders, hallmark characteristics of KD include tubular interstitial fibrosis, renal tubular cell atrophy and/or necrosis, glomerulosclerosis, persistent inflammation, microvascular endothelial cell (MV-EC) dysfunction and/or rarefaction, and mitochondrial dysfunctions.(6–8) Due to vast cellular heterogeneity, substantial signaling complexity, and functional/structural intricacies of the kidney, a paucity of FDA-approved drugs for KD has resulted.(9, 10) Thus, identification of drugs to effectively treat and/or hinder the onset/progression of KD remains of vital importance.

Renal MV-ECs are integrated throughout the capillary networks of the kidney and play a vital role in regulating various physiological processes, including angiogenesis, coagulation, filtration/reabsorption, inflammation, organ perfusion, transport of solutes and lipids, etc.(8, 11, 12) Furthermore, these cells exhibit a distinct metabolic profile compared to other resident kidney cells, predominantly relying on aerobic glycolysis for the generation of adenosine 5′-triphosphate (ATP).(13) Following an episode of acute kidney injury (AKI) and/or during the onset/progression of KD, renal MV-ECs are highly prone to injury, dysfunction, and rarefaction.(8, 12, 13) The excessive production of reactive oxygen/nitrogen species (ROS/RNS) and activation of various inflammatory signaling pathways by pro-inflammatory agents (e.g., lipopolysaccharides (LPS), transforming growth factor-beta 1 (TGFβ1), tumor necrosis factor alpha (TNFα), etc.) following AKI and/or KD, enhance renal MV-ECs permeability and disrupt vascular integrity.(8, 12–14) Additionally, ROS/RNS not only induce direct damage to MV-ECs but also trigger inflammatory responses that enhance the migration of leukocytes across the endothelium, further exacerbating renal MV-ECs permeability, injury, dysfunction, and rarefaction.(14–16)

In human and animal models of AKI and KD, renal MV-EC dysfunction further promotes kidney injury, inflammation, and rarefication, all of which increase the risk of AKI recurrence and/or KD progression.(15–19) Prior studies have highlighted that the renal peritubular endothelial capillaries (RPECs) experience significant microvascular rarefaction (∼25–45%) and undergo endothelial-to-mesenchymal transition (∼10–15%) following AKI, further contributing to renal damage and KD onset/progression.(15, 16, 20) AKI and KD are often associated with a persistent reduction in mitochondrial number, function, and cellular energetics, resulting in impaired renal recovery and injury/disease progression.(16, 21–24) Conversely, pharmacological strategies aimed at stimulating mitochondrial biogenesis (MB), the production of new and functional mitochondria, have been demonstrated to expedite renal recovery and improve the function of renal proximal tubule cells and glomerular capillary MV-ECs following AKI.(25–38) However, the cellular pathways that govern RPEC repair processes following renal injury/disease and how pharmacological stimulation of MB may influence these repair mechanisms, remain unknown.

RPEC repair processes and identification of druggable targets that promote these repair processes have remained limited due to various challenges in isolating and maintaining pure populations of RPECs in primary culture, as they are prone to undergo phenotypic alterations and exhibit poor angiogenic outgrowth potential.(39) Additionally, the lack of commercially available primary RPECs, costs and time associated with isolation/monoculture of primary RPECs, and large variability in the purity/yield of isolated primary RPECs have further inhibited research progress. Recently, refined protocols for the isolation and monoculture of mouse renal peritubular endothelial cells (MRPEC) have been generated to overcome several of these obstacles and obtain MRPECs of suitable quality for downstream pharmacological and physiological experiments.(39) Here, we examined the in vitro effects of lasmiditan, a potent and selective FDA-approved HTR1F agonist known to induce MB in the renal cortices of mice, on primary MRPECs in the presence/absence of known pro-inflammatory molecules (i.e., LPS, TGFβ1, and TNFα) associated with renal MV-EC dysfunction, AKI, and KD onset/progression.(6, 22, 28, 31, 32, 40–43)

Materials and Methods

Isolation and Culture of Primary MRPECs:

Primary MRPECs were isolated and monocultured as previously described by Thompson, et al.(39) Briefly, 10-week-old C57BL/6J male mice (RRID:IMSR_JAX:000664; ~20–25 grams; The Jackson Laboratory, Bar Harbor, ME, USA) were anesthetized via isoflurane and subjected to transcardial perfusion with ~30 ml of heparinized saline [Normal saline + 100 U/ml of heparin] to remove circulating blood cells from the kidney. Following perfusion, kidneys were harvested and the renal cortices removed for enzymatic digestion and MRPEC isolation/purification. Renal cortices were digested with a collagenase-type-1 solution [DMEM/F12 media + 350 A.U./ml of collagenase-type-1 + 100 A.U./ml of DNAse-1] prior to Percoll density gradient centrifugation. Cells were then digested further with an Accutase solution [Accutase solution (1x) + 100 A.U./ml of DNAse-1]. Following digestion, cells were processed through sequential cell strainers (70-micron and 40-micron strainers, respectively) to remove large cellular aggregates and glomeruli. Cells were then subjected to one round of CD326+ (EPCAM) magnetic microbead negative selection to remove contaminating epithelial cells, followed by two positive selection cycles with CD146+ magnetic microbeads. Purified MRPECs were then resuspended, seeded, and monocultured in microvascular specific media (EGM-MV2; Lonza; Basel, Switzerland; Cat#: CC-3202) supplemented with recombinant mouse VEGF-164 protein [50 ng/ml] and Heparin [0.75 U/ml]. One day prior to functional assay experiments, MRPEC outgrowth media was replaced with serum free basal EGM-MV2 microvascular specific media.

Quantitative Real-Time Polymerase Chain Reaction Analysis of mRNA Expression:

Total RNA from MRPECs was isolated using TRIzol reagent per manufacturer’s protocol (Life Technologies; Grand Island, NY; Cat.#: 15596018). The iScript Advanced cDNA synthesis kit (Bio-Rad Laboratories; Hercules, CA; Cat.#: 1725037) was then employed to convert MRPEC RNA transcripts into cDNA, per manufacturer’s protocol. To confirm HTR1F expression in primary MRPECs, cDNA transcripts were amplified utilizing the Promega PCR Master Mix reagent (Promega; Madison, WI; Cat.#: PRM7502), per manufacturer’s protocol. PCR products were confirmed via electrophoresis on a 1% agarose gel, as previously described.(28) Additionally, RT-qPCR analyses were subsequently performed on the newly synthesized cDNA libraries via the SsoAdvanced Universal SYBR Green Supermix reagent kit (Bio-Rad Laboratories; Hercules, CA; Cat.#: 1725272) and DNA primer pairs specific to our tested genes of interest, per manufacturer’s protocol. The relative expression of mRNA was quantified via the 2^-ΔΔCT method, as previously described.(44) For all RT-qPCR analyses presented within this manuscript, β-actin was employed as the housekeeping control gene for normalization. All target genes evaluated and their respective DNA oligo primer pair sequences are listed below: ATP Synthase-β (Forward primer sequence (fwd): 5’-CTA-TGT-GCC-TGC-TGA-TGA-CC-3’; Reverse primer sequence (rev): 5’-GGA-TAG-ATG-CCC-AAC-TCA-GC-3’); B2M (fwd: 5’-CTG-GTC-TTT-CTG-GTG-CTT-GTC-3’; rev: 5’-TAT-GTT-CGG-CTT-CCC-ATT-CTC-C-3’); β-actin (fwd: 5’-GGG-ATG-TTT-GCT-CCA-ACC-AA-3’; rev: 5’-GCG-CTT-TTG-ACT-CAA-GGA-TTT-AA-3’); HTR1F (fwd: 5’-GCC-GTG-ATG-ATG-AGT-GTG-TC-3’; rev: 5’-ATC-ATC-CGA-CTC-GCT-TGT-CT-3’); PGC-1α (fwd: 5’-AGG-AAA-TCC-GAG-CGG-AGC-TGA-3’; rev: 5’-GCA-AGAAGG-CGA-CAC-ATC-AA-3’); TFAM (fwd: 5’-GCT-GAT- GGG-TAT-GGA-GAA-G-3’; rev: 5’-GAG-CCG-AAT-CAT-CCT-TTG-C-3’).

Measurement of MRPECs Oxygen Consumption Rates (OCR):

Primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. Culture media was then removed and MRPECs were washed with phosphate buffered saline (1x PBS; Gibco; Waltham, MA; Cat.#: 10010049). MRPECs were subsequently dissociated and collected via 1x Accutase dissociation (Millipore Sigma; Burlington, MA; Cat.#: A6964–100ml). Accutase digestion solution was neutralized utilizing an equivalent volume of MRPEC culture media and cells were pelleted by centrifugation at 300 g for 10 min at 4°c. Cells were resuspended in culture media and counted utilizing a TC20 automated cell counter (Bio-Rad Laboratories; Hercules, CA; Cat.#: 1450102). MRPECs were then seeded into 96-well XF-Pro M Cell Culture Microplates (Agilent Technologies; Santa Clara, CA; Cat.#: 103775–100) at a concentration of 2.0×10⁴ cells/well, as previously described.(45) The following day, each MRPEC plate was treated with either vehicle control [Normal saline + 0.1% dimethyl sulfoxide ((DMSO)]; Millipore Sigma; Burlington, MA; Cat.#: D8418–250 ml), or lasmiditan at various concentrations [1.0 nM - 100 nM] for 24 hrs. MRPECs basal and maximal OCR was measured and calculated using the Seahorse Bioscience XF-96 Extracellular Flux Analyzer, as previously described.(46) Briefly, MRPECs basal OCR was measured for three cycles prior to the injection of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; [5 μM]) (Millipore Sigma; Burlington, MA; Cat.#: C2920–10 mg) and three times following FCCP injection to calculate their maximal uncoupled OCR rate (FCCP-OCR).

MitoTracker Staining and Quantification in MRPECs:

Mitochondrial staining with MitoTracker Red CMXRos (Thermofisher; Waltham, MA; Cat.#: M7512) was conducted in accord with the manufacturer’s protocol. Briefly, primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then treated with either vehicle control [Normal saline + 0.1% DMSO] or lasmiditan [100 nM] for 24 hrs. Following the treatment period, growth media was removed and replaced with fresh pre-warmed (37°c) serum free basal EGM-MV2 microvascular specific media containing MitoTracker Red CMXRos [250 nM], per manufacturer’s protocol. MRPECs were then incubated at 37°c in a cell culture incubator supplied with 5% CO₂ for 30 min. Staining solution was then removed and replaced with EGM-MV2 microvascular specific complete growth media. MRPECs were subsequently imaged on an EVOS M5000 microscope with an Olympus 40x UPlanSApo infinity-corrected objective ((0.95NA/0.18WD); (Olympus, Tokyo, Japan; Cat.#: 1-U2B828)). MRPECs MitoTracker Red CMXRos TIFF images (8-bit) were then processed and quantified via the Mitochondrial Analyzer plugin in ImageJ (RRID:SCR_003070) for various mitochondrial attributes.

Transmission Electron Microscopy (TEM) Analyses:

Primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then treated with either vehicle control [Normal saline + 0.1% DMSO] or lasmiditan [100 nM] for 24 hrs, submerged in 2.5% glutaraldehyde Electron Microscopy Solution (Electron Microscopy Sciences; Hatfield, PA; Cat.#: 16537–15), and allowed to fixate overnight at 4°c. The following day, 2.5% glutaraldehyde fixative solution was aspirated from the samples, cells were washed once with ice-cold 1x PBS (Gibco; Waltham, MA; Cat.#:10010049) and submitted immediately to the TEM core facility at the University of Arizona in ice-cold 1x PBS for TEM sample preparation and imaging. Additionally, MRPECs were assessed ex-vivo from 10-week-old C57BL/6J male mice (~20–25 grams) that received daily dosing (via intraperitoneal injection (IP)) with either [Normal saline + 0.1% DMSO] or lasmiditan [0.3 mg/kg] over a 48 hr period. Following the treatment interval, MRPECs were isolated, prepped for TEM as described above. Images were acquired with a FEI Tecnai Spirit Transmission Electron Microscope (Hillsboro, OR) at 100 kV. TIFF images (8-bit) of each sample were acquired via a XR41-CCD digital camera (Woburn, MA) with direct magnification of 8200x and a print magnification of 15000x at 7”. For all cases, five randomized images from various regions in each sample were analyzed, and mitochondrial number per field as well as individual mitochondria area/field were calculated in a blinded manner, as previously described.(47)

Protein Isolation and Immunoblot Analyses:

Primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then treated with either vehicle control [Normal saline + 0.1% DMSO] or lasmiditan [100 nM] for 24 hrs. MRPEC protein was subsequently isolated via RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) containing Halt Protease and Phosphatase Inhibitor Cocktail (1:100; Thermofisher, Waltham, MA; Cat.#: 78447). Gel electrophoresis and immunoblot analyses were performed as previously described.(48, 49) Membranes were subsequently imaged using an Azure 600 imaging system (Azure; Dublin, CA; Cat.#: AZI600–01). Optical density was quantified using ImageJ software, as previously described.(32) Primary antibodies used for immunoblotting were purchased from: Abcam (Cambridge, MA) – eNOS [M221] (1:1,000; Abcam, Cat# ab76198, RRID:AB_1310183), p-eNOS [EPR20991] (Serine 1177; 1:1,000; Abcam, Cat# ab215717, RRID:AB_2893314), PGC-1α ((Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha), (1:1,000; Abcam, Cat# ab191838, RRID:AB_2721267)), TFAM-[mtTFA] ((Mitochondrial Transcription Factor-A); (1:1,000; Abcam, Cat# ab131607, RRID:AB_11154693)), Total OXPHOS (Oxidative Phosphorylation) rodent WB antibody cocktail (1:250; Abcam, Cat# ab110413, RRID:AB_2629281), VE-Cadherin [EPR18229] (1:1,000; Abcam, Cat# ab205336, RRID:AB_2891001). Cell Signaling (Danvers, MA) – GAPDH ((Glyceraldehyde 3-Phosphate Dehydrogenase); (1:1,000; Cell Signaling Technology, Cat# 5174, RRID:AB_10622025)). Secondary antibodies used for immunoblotting were purchased from: Abcam (Cambridge, MA) – Goat Anti-Rabbit IgG H&L (HRP) (1:10,000; Abcam, Cat# ab6721, RRID:AB_955447), Rabbit Anti-Mouse IgG H&L (HRP) (1:10,000; Abcam, Cat# ab6728, RRID:AB_955440).

MRPEC Wound Healing/Migration Capacity Assays:

Primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then harvested via Accutase dissociation and centrifuged at 300 g for 10 min at 4°c. MRPECs were seeded at a concentration of 7.0×10⁴ cells/well in Ibidi wound healing culture inserts and allowed to settle overnight, per manufacturer’s protocol. The next day, cells were treated with either vehicle control [Normal saline + 0.1% DMSO], lasmiditan [100 nM], lipopolysaccharide (LPS) [1 μg/ml], LPS + lasmiditan [1 μg/ml + 100 nM, respectively], tumor necrotic factor alpha (TNFα) [100 ng/ml], TNFα + lasmiditan [100 ng/ml + 100 nM, respectively], transforming growth factor-beta1 (TGFβ1) [10 ng/ml], or TGFβ1 + lasmiditan [10 ng/ml + 100 nM, respectively] for 24 hrs prior to the removal of the 500-micron well inserts. MRPECs were subsequently tracked and quantified over 24 hrs on an EVOS M5000 microscope under phase contrast with an EVOS 10x fluorite LWD phase-contrast objective (0.30NA/7.13WD). MRPEC wound healing/migration capacity TIFF images (8-bit), taken at 0 hrs and 24 hrs post-well insert removal, were then processed and quantified via Ibidi FastTrack AI software (MetaVi Labs), to determine total wound closure percentages. ImageJ was then used to generate representative images with delineated margins (red outlines).

MRPEC Barrier Integrity and Permeability Transendothelial Electrical Resistance (TEER) Assays:

MRPEC barrier integrity and permeability TEER assays were conducted as previously described.(49) Briefly, primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then harvested via Accutase dissociation and centrifuged at 300 g for 10 min at 4°c. Cells were subsequently resuspended in basal EGM-MV2 microvascular specific media containing either vehicle control [Normal saline + 0.1% DMSO], lasmiditan [100 nM], LPS [1 μg/ml], LPS + lasmiditan [1 μg/ml + 100 nM, respectively], TNFα [100 ng/ml], TNFα + lasmiditan [100 ng/ml + 100 nM, respectively], TGFβ1 [10 ng/ml], or TGFβ1 + lasmiditan [10 ng/ml + 100 nM, respectively], counted via a TC20 automated cell counter (Bio-Rad; Cat.#: 1450102), and then diluted to 2.0×10⁵ cells/ml. MRPECs were then seeded at a density of 2.0×10⁵ cells/well on polyester-coated trans-well inserts (Corning; Cat.#: CLS3460) with a pore size of 0.4 μm in a 12-well format and allowed to grow for 48 hrs. TEER was assessed daily over the 48 hr interval utilizing an EVOM² Epithelial/Endothelial Voltohmmeter with chopsticks electrodes (World Precision Instruments). Net resistance/treatment well (Net - Ω) was calculated by subtracting the average resistance/blank well (Ω) obtained from 12-blank well controls containing media-only, and final TEER values were calculated and reported as Ω·cm² based on the area of the well inserts, as previously described.(49)

MRPEC Endothelial Tubular Network Formation (ETNF) Matrigel Assays:

MRPEC ETNF Matrigel assays were conducted as previously described.(49, 50) Briefly, primary MRPECs were isolated and monocultured for 5–7 days, until ~80% confluency was observed. MRPECs were then treated with either vehicle control [Normal saline + 0.1% DMSO], lasmiditan [100 nM], LPS [1 μg/ml], LPS + lasmiditan [1 μg/ml + 100 nM, respectively], TNFα [100 ng/ml], TNFα + lasmiditan [100 ng/ml + 100 nM, respectively], TGFβ1 [10 ng/ml], or TGFβ1 + lasmiditan [10 ng/ml + 100 nM, respectively] for 24 hrs. MRPECs were then harvested via Accutase dissociation and centrifuged at 300 g for 10 min at 4°c. Cells were subsequently resuspended in basal EGM-MV2 microvascular specific media containing either vehicle control, lasmiditan, LPS, LPS + lasmiditan, TNFα, TNFα + lasmiditan, TGFβ1, or TGFβ1 + lasmiditan, counted on a TC20 automated cell counter (Bio-Rad; Cat.#: 1450102), and then diluted to 1.8×10⁵ cells/ml. Following dilution, 50 μl of MRPEC suspension was added to an Ibidi μ-Slide 15-Well 3D (Fitchburg, WI) cell culture slide precoated with 10 μl of Corning reduced growth factor, phenol red free, Matrigel solution (Corning, NY), per manufacturer’s protocol. MRPECs were then allowed to settle in Matrigel-coated wells over a 24 hr period prior to imaging with an EVOS 4x fluorite LWD phase-contrast objective (0.13NA/10.58WD) on an EVOS M5000 microscope under phase contrast with a light diffuser insert to aid in reducing uneven illumination. MRPEC ETNF Matrigel TIFF images (8-bit) were then processed and quantified via the Angiogenesis Analyzer plugin in ImageJ for various ETNF endothelial cell features.

Animal Guidelines:

All experimentations performed on animals in these studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona prior to the performance of said experimentation, and all precautions were made to minimize animal distress. Additionally, all animal experiments were conducted in stringent accordance with the recommendations outlined within “The Guide for the Care and Use of Laboratory Animals” curated and distributed by the National Institutes of Health.(51)

Data and Statistical Analysis:

For all experiments conducted within this manuscript N=6, where N represents one animal or primary cells isolated from one animal; N=6 was selected for this work based on a power analysis conducted for ANOVA with an effect size = 0.25, an α = 0.05, and a β=0.8. A Shapiro-Wilk test was performed on all data to confirm the normality of distribution. All data that had only two experimental groups underwent Welch’s two sample t-tests. Data that followed normal distributions and had more than two experimental groups underwent a one-way ANOVA (followed by a Tukey’s post-hoc test if F in ANOVA achieved P < 0.5 and there was no significant variance inhomogeneity). Conversely, data that did not follow normal distributions and had more than two experimental groups underwent a nonparametric ANOVA (Kruskal-Wallis H-test followed by a Dunnett’s post-hoc test if F in ANOVA achieved p< 0.5 and there was significant variance inhomogeneity). Data were analyzed using GraphPad Prism software V10.2.3 (La Jolla, CA) and a p<0.05 was considered statistically significant in all data sets. Statistical comparisons on graphs between only two experimental groups are presented in a traditional asterisks (*) format, and comparisons on graphs between more than two experimental groups are presented in the ANOVA Compact Letter Display (CLD) format. In CLD formatting, if any of the experimental groups share the same letter(s), it indicates that the P-value from the statistical analyses conducted between them was not significant (p>0.05); conversely, if the experimental groups do not share the same letter(s), it indicates that the P-value from the statistical analyses conducted between them was considered significant (p<0.05). Unless otherwise stated, all data are representative of group means ± SEM.

Results

Lasmiditan Enhances Mitochondrial Maximal Respiration Rate & Number in Primary MRPECs.

PCR was used to confirm the retention of HTR1F expression in primary MRPEC monocultures (Fig. 1A).(28) After this confirmation, lasmiditan, a potent and selective FDA-approved HTR1F agonist, (28, 31) was screened at various concentrations [1 nM - 100 nM] to determine MB inducing effects in primary cultures of MRPECs via a Seahorse XF96 analyzer, as described previously.(45) MRPECs treated with 100 nM lasmiditan exhibited a ~23% increase in FCCP-OCR when compared to vehicle-treated [normal saline + 0.1% DMSO] control cells, indicating a rise in MRPECs mitochondrial maximal respiration rate (Fig. 1B).(45) Therefore, 100 nM lasmiditan was the dose selected for all subsequent in vitro MRPEC experiments.

Figure 1|. Lasmiditan Enhances Mitochondrial Maximal Respiration Rate & Number in Primary MRPECs.

(A) PCR assessment of primary MRPEC monocultures for the expression of HTR1F. (B) Seahorse XF96 analyses showing the percent increase in MRPECs maximal mitochondrial respiration rate after FCCP [5 uM] injection, after 24 hrs of lasmiditan treatment [100 nM]. Data are representative of the average percent increase (± SEM) from basal OCR readings following FCCP [5 uM] injection across N=6 MRPEC replicates/mice and N=6 mice/treatment condition. RT-qPCR analyses of MRPECs after 24 hrs of lasmiditan treatment [100 nM] were then employed to determine relative mRNA expression changes across various mitochondrial biogenesis related genes: (C) PGC-1α, (D) TFAM, and (E) ATP Synthase-β. (F) Representative images of MRPEC monocultures after 24 hrs of lasmiditan [100nM] or vehicle treatment. Mitochondria are shown stained with MitoTracker Red CMXRos, binary processed, and then skeletonized to produce a skeleton tree for downstream Mitochondrial Analyzer Plug-in Analyses in ImageJ: (G) mitochondrial number/field, (H) mitochondrial area/field, (I) mitochondrial perimeter/field, and (J) mitochondrial mean form factor/mitochondria/field. One-Way ANOVA followed by a Tukey post-hoc correction for multiple comparisons was employed to identify significant FCCP-OCR alterations across various lasmiditan concentrations. All other data within this figure underwent Welch’s two-sample t-tests to identify significant differences in mRNA expression and mitochondrial features between treatment groups. A p-value of p≤0.05 was considered significant between treatment groups and is denoted by asterisks (*). Data are representative of the average fold change from vehicle-treated control groups (± SEM) across N=6 MRPEC isolations/treatment group where N=6 mice/treatment condition.

RT-qPCR was then used to examine alterations in the mRNA expression of various MB-related genes after 24 hrs of lasmiditan treatment. Lasmiditan-treated primary MRPEC monocultures depicted a 1.5-fold increase in PGC-1α expression, which is considered the master regulator of MB, compared to vehicle-treated controls (Fig. 1C). Additionally, the expression of TFAM, considered the master regulator of mitochondrial DNA transcription, and ATP Synthase-β, a subunit within complex-five of the mitochondrial electron transport chain (ETC), were increased in lasmiditan-treated MRPECs compared to vehicle-treated controls by 1.9-fold and 1.4-fold, respectively (Fig. 1D and 1E). Mitochondrial staining with MitoTracker Red CMXRos was then conducted to capture images of primary MRPECs mitochondrial content (Fig. 1F). Following 24 hrs of lasmiditan treatment, primary MRPEC monocultures displayed an increase in mitochondrial count/field, mitochondrial area/field, and mitochondrial perimeter/field compared to vehicle-treated controls by 1.8-fold, 1.3-fold, and 1.5-fold, respectively (Fig. 1G – 1I). Furthermore, no difference was observed in the mean mitochondrial form factor/field between groups, indicating MB induction rather than alterations in mitochondrial dynamics processes such as fusion or fission (Fig. 1J).(52)

Lasmiditan Induces Mitochondrial Biogenesis in Primary MRPECs, In Vitro & Ex Vivo.

TEM was used to determine if the increase in MRPECs mitochondrial maximal respiration rate and quantity following lasmiditan treatment was due to an induction of MB (Fig. 2A). Primary MRPECs that were treated with lasmiditan for 24 hrs exhibited an increase of 1.5-fold in mitochondrial count/field compared to vehicle-treated controls (Fig. 2B). Additionally, no difference was observed in MRPECs individual mitochondrial area/field between treatment groups, indicating a lack of mitochondrial swelling or fragmentation (Fig. 2C).

Figure 2|. Lasmiditan Induces Mitochondrial Biogenesis in Primary MRPECs, In Vitro & Ex Vivo.

Representative TEM images of primary MRPECs that received either vehicle control [Normal Saline + 0.1% DMSO] or lasmiditan [100 nM] treatment in vitro for 24 hrs. (B) Quantification of mitochondrial number/field and (C) individual mitochondria area/field from TEM images of MRPECs treated in vitro with either vehicle control or lasmiditan for 24 hrs. (D) Representative TEM images of ex vivo primary MRPECs isolated from mice that received either vehicle control or lasmiditan [0.3 mg/kg] treatment daily via I.P. injection over a 48 hr interval. (E) Quantification of mitochondrial number/field and (F) individual mitochondria area/field from TEM images of ex vivo primary MRPECs isolated from mice that received either vehicle control or lasmiditan treatment daily via I.P. injection over a 48 hr interval. Welch’s two-sample t-tests were utilized to identify significant differences in mitochondrial features between treatment groups. A p-value of p≤0.05 was considered significant between treatment groups and are denoted above by asterisks (*). Data are representative of the average raw mitochondrial counts/field or raw individual mitochondria area/field (± SEM) across five randomized and blinded images obtained per N. N=6 MRPEC isolations/treatment group where N=6 mice/treatment condition with 5 images per N=1. (A & D) - Black scale bars, 2 μm.

To assess whether the MB induction observed in primary MRPEC monocultures following in vitro lasmiditan treatment could be recapitulated in vivo, MRPECs were isolated from mice that were administered either lasmiditan [0.3 mg/kg] or vehicle control [normal saline + 0.1% DMSO] via IP injection daily over a 48 hr interval, as previously described (Fig. 2D).(32) Primary MRPECs, isolated ex vivo from mice, that received lasmiditan exhibited an increase of 1.4-fold in mitochondrial count/field compared to MRPECs obtained from vehicle-treated control animals (Fig. 2E). Similar to MRPECs treated under in vitro conditions, no difference was observed in ex vivo isolated MRPECs individual mitochondrial area/field between treatment groups (Fig. 2F). Together, these data indicate that lasmiditan induces MB in primary MRPECs both in vitro and ex vivo.(32, 47)

Lasmiditan Increases MB-Related Proteins, Mitochondrial ETC Complexes, & Endothelial Cell Integrity Markers in Primary MRPECs.

To discern whether lasmiditan alters the protein expression of various MB-related and/or endothelial-related proteins, MRPECs were harvested and processed for protein analyses after 24 hrs of either lasmiditan or vehicle treatment. Immunoblot analyses of lasmiditan-treated MRPECs exhibited an increase in expression of the MB regulator proteins PGC-1α and TFAM, by 1.3-fold and 1.4-fold, respectively, compared to vehicle-treated controls (Fig. 3A – 3C). Proceeding lasmiditan treatment, MRPECs also displayed an increase in subunits of the mitochondrial ETC complexes I, II, III, and V (i.e., NDUFB8, SDHB, UQCRC2, and ATP5A) compared to vehicle-treated controls by 1.3-fold, 1.4-fold, 1.3-fold, and 1.7-fold, respectively. Lasmiditan had no effect on the mitochondrial ETC complex IV subunit MTCO1 (Fig. 3D – 3I). Immunoblot analyses were then conducted on MRPEC functional endothelial marker eNOS (endothelial nitric oxide synthase 3) and junctional stability marker VE-Cadherin (vascular endothelial-cadherin 5) (Fig. 3J).(13) MRPECs treated with lasmiditan displayed an increase in both total eNOS and activated eNOS, indicated by phosphorylation of eNOS at activating position serine-1177 (p(s1177)-eNOS), by 1.6-fold compared to vehicle-treated controls (Fig. 3K and 3L).(31) Additionally, lasmiditan-treated MRPECs showed an increase in VE-Cadherin by 1.6-fold compared to vehicle-treated controls (Fig. 3M). Taken together, these data indicate that lasmiditan increases MB regulator proteins PGC-1α and TFAM, various subunits of the mitochondrial ETC complexes, and enhances endothelial-functional markers eNOS and VE-Cadherin in primary MRPECs, further indicative of MB induction.

Figure 3|. Lasmiditan Increases MB-Related Proteins, Mitochondrial ETC Complexes, & Endothelial Cell Integrity Markers in Primary MRPECs.

Representative MRPEC immunoblots of MB-related proteins, PGC-1α and TFAM, after 24 hrs of lasmiditan treatment. (B) Quantification of MRPECs relative PGC-1α and (C) TFAM protein abundance, after 24 hrs of lasmiditan treatment. (D) Representative MRPEC immunoblot of mitochondrial ETC complexes, after 24 hrs of lasmiditan treatment. (E) Quantification of MRPECs relative ATP5A (Subunit of Complex-5), (F) UQCRC2 (Subunit of Complex-3), (G) MTCO1 (Subunit of Complex-4), (H) SDHB (Subunit of Complex-2), and (I) NDUFB8 (Subunit of Complex-1) protein abundance, after 24 hrs of lasmiditan treatment. (J) Representative MRPEC immunoblots of endothelial cell markers, p(s1177)-eNOS, eNOS, and VE-Cadherin, after 24 hrs of lasmiditan treatment. (K) Quantification of MRPECs relative p(s1177)-eNOS (activation phosphorylation site: eNOS), (L) eNOS protein abundance, and (M) VE-Cadherin protein abundance, after 24 hrs of lasmiditan treatment. Welch’s two-sample t-tests were utilized to identify significant differences in protein abundance between treatment groups. A p-value of p≤0.05 was considered significant between treatment groups and are denoted above by asterisks (*). Data are representative of the average fold change from vehicle-treated control groups (± SEM) across N=6 MRPEC isolations/treatment group where N=6 mice/treatment condition.

Assessment of MRPEC Wound Healing, TEER, and ETNF Following LPS-exposure in the Presence/Absence of Lasmiditan.

We next sought to determine the effects of lasmiditan on primary MRPEC function in the presence/absence of various pro-inflammatory stimuli known to be associated with endothelial cell dysfunction, AKI, and KD onset/progression. Thus, various wound healing/migration, TEER barrier integrity/permeability, and ETNF assays were conducted on primary MRPECs following either lasmiditan or vehicle treatment in the presence/absence of the pro-inflammatory agent LPS [1 μg/ml].

Wound closure was assessed in MRPECs following treatment with either lasmiditan or vehicle control in the presence/absence of LPS (Fig. 4A). MRPECs treated with lasmiditan exhibited an increase of 1.2-fold in wound healing/migration capacity compared to vehicle-treated controls (Fig. 4B). Conversely, MRPECs exposed to LPS displayed a reduction in wound healing/migration capacity compared to vehicle-treated controls and lasmiditan-treated MRPECs by 23% and 38%, respectively (Fig. 4B). The observed reduction in MRPECs wound healing/migration capacity following exposure to LPS was prevented in MRPECs treated with both LPS and lasmiditan (Fig. 4B). TEER assays were then conducted to evaluate the effects of lasmiditan on MRPECs barrier integrity/permeability (Fig. 4C). After 24 hrs, MRPECs that received lasmiditan exhibited an increase in TEER by 1.3-fold compared to vehicle-treated controls, indicative of increased barrier integrity and reduced permeability (Fig. 4C). Conversely, MRPECs exposed to LPS displayed a reduction in TEER compared to vehicle-treated controls and lasmiditan-treated MRPECs by 27% and 43%, respectively, indicative of reduced barrier integrity and increased permeability (Fig. 4C). Similarly, after 48 hrs, MRPECs that received lasmiditan maintained an increase in TEER by 1.2-fold compared to vehicle-treated controls, and MRPECs exposed to LPS maintained a reduction in TEER compared to vehicle-treated controls and lasmiditan-treated MRPECs by 20% and 35%, respectively (Fig. 4C). The observed reduction in MRPECs barrier integrity and increased permeability following exposure to LPS was prevented in MRPECs treated with both LPS and lasmiditan (Fig. 4C).

Figure 4|. Assessment of MRPEC Wound Healing, TEER, and ETNF Following LPS-exposure in the Presence/Absence of Lasmiditan.

(A) 24h time course wound healing assays of MRPECs (7×10⁴ cells/well) treated with either: 1) Vehicle Control [Saline + 0.1% DMSO], 2) Lasmiditan [100 nM], 3) LPS [1 μg/ml], or 4) LPS + lasmiditan [1 μg/ml + 100 nM, respectively]. (B) Quantification of MRPECs total percent wound closure after 24 hrs of treatment. (C) Quantification of MRPECs (2×10⁵ cells/well) TEER over a 48h period following treatment. (D) Representative 4x magnification phase contrast photomicrographs of MRPECs (9×10³ cells/well) in Matrigel-based ETNF assays after 24 hrs of treatment. MRPEC Matrigel ETNF TIFF images (8-bit) were then processed and quantified via the Angiogenesis Analyzer plugin in FIJI (ImageJ) for various ETNF-related endothelial cell features: (E) total number of branches/well, (F) total number of master junctions/well, (G) total number of master segments/well, and (H) total branches length (pixels)/well. Data represents N=6/group and are expressed as means ± SEM; p<0.05 compared by one-way ANOVA, followed by a Tukey post-hoc correction for multiple comparisons, or by Kruskal-Wallis H-Test nonparametric ANOVA, followed by a Dunnett’s post-hoc correction for multiple comparisons, are considered significant. Significance between groups is displayed in Compact Letter Display (CLD) formatting, where different letters on top of bars signifies a statistical difference between groups. (A) - White scale bars, 100 μm; (D) - White scale bars, 500 μm.

Following 24 hrs of treatment with either lasmiditan or vehicle control in the presence/absence of LPS, MRPECs ETNF was assessed (Fig. 4D). MRPECs that received either lasmiditan or lasmiditan + LPS displayed an increase in the number of branches, master junctions, master segments, and total branching length by more than 2-fold compared to vehicle- and LPS-treated controls (Fig. 4E – 4H). Interestingly, no differences were observed between LPS-treated MRPECs and vehicle-treated controls (Fig. 4E – 4H). Taken together, these data indicate that following LPS exposure, MRPECs exhibit a reduction in wound healing/migration capacity and become more permeable. Conversely, lasmiditan treatment augments MRPECs wound healing and ETNF, while also enhancing barrier integrity. Importantly, lasmiditan treatment was able to blunt the LPS-induced wound healing/migration and barrier integrity dysfunctions observed in primary MRPECs.

Assessment of MRPEC Wound Healing, TEER, and ETNF Following TGFβ1-exposure in the Presence/Absence of Lasmiditan.

After utilizing various functional assays to determine the effects of lasmiditan on primary MRPECs in the presence/absence of LPS, we then conducted these assays in the presence/absence of TGFβ1 [10 ng/ml], another pro-inflammatory agent known to be associated with endothelial cell dysfunction, AKI, and KD onset/progression. Expectedly, MRPECs treated with lasmiditan exhibited an increase in wound healing/migration capacity compared to vehicle-treated controls (Fig. 5A – 5B). Conversely, MRPECs exposed to TGFβ1 displayed a reduction in wound healing/migration capacity compared to vehicle-treated controls and lasmiditan-treated MRPECs by 19% and 27%, respectively (Fig. 5B). Similar to that observed with LPS, this reduction was prevented with lasmiditan treatment (Fig. 5B). MRPECs exposed to TGFβ1 also displayed a reduction in TEER compared to vehicle-treated controls and lasmiditan-treated MRPECs at both 24 and 48 hrs, indicative of reduced barrier integrity and increased permeability (Fig. 5C). The observed reduction in MRPECs barrier integrity and increased permeability following exposure to TGFβ1 was prevented in MRPECs treated with both TGFβ1 and lasmiditan over the entire 48 hr period (Fig. 5C). Similar to that observed in the LPS experiments, MRPECs that received lasmiditan treatment displayed an increase in the number of branches, master junctions, master segments, and total branching length by more than 2-fold compared to vehicle-treated controls (Fig. 5D – 5H). Conversely, MRPECs that were exposed to TGFβ1 for 24 hrs exhibited decreased ETNF, as indicated by a reduction in the number of branches, master junctions, master segments, and total branching length compared to vehicle-treated controls by, 72%, 87%, 89%, and 90%, respectively (Fig. 5D – 5H). The observed reduction in MRPECs ETNF following exposure to TGFβ1 was negated in MRPECs treated with both TGFβ1and lasmiditan (Fig. 5D – 5H).

Figure 5|. Assessment of MRPEC Wound Healing, TEER, and ETNF Following TGFβ1-exposure in the Presence/Absence of Lasmiditan.

(A) 24h time course wound healing assays of MRPECs (7×10⁴ cells/well) treated with either: 1) Vehicle Control [Saline + 0.1% DMSO], 2) Lasmiditan [100 nM], 3) TGFβ1 [10 ng/ml], or 4) TGFβ1 + lasmiditan [10 ng/ml + 100 nM, respectively]. (B) Quantification of MRPECs total percent wound closure after 24 hrs of treatment. (C) Quantification of MRPECs (2×10⁵ cells/well) TEER over a 48h period following treatment. (D) Representative 4x magnification phase contrast photomicrographs of MRPECs (9×10³ cells/well) in Matrigel-based ETNF assays after 24 hrs of treatment. MRPEC Matrigel ETNF TIFF images (8-bit) were then processed and quantified via the Angiogenesis Analyzer plugin in FIJI (ImageJ) for various ETNF-related endothelial cell features: (E) total number of branches/well, (F) total number of master junctions/well, (G) total number of master segments/well, and (H) total branches length (pixels)/well. Data represents N=6/group and are expressed as means ± SEM; p<0.05 compared by one-way ANOVA, followed by a Tukey post-hoc correction for multiple comparisons, or by Kruskal-Wallis H-Test nonparametric ANOVA, followed by a Dunnett’s post-hoc correction for multiple comparisons, are considered significant. Significance between groups is displayed in Compact Letter Display (CLD) formatting, where different letters on top of bars signifies a statistical difference between groups. (A) - White scale bars, 100 μm; (D) - White scale bars, 500 μm.

Taken together, these data indicate that following TGFβ1 exposure, MRPECs display a modest reduction in wound healing/migration capacity, become more permeable, and exhibit a severe impairment in angiogenesis and tube formation capacity. Conversely, lasmiditan treatment improved primary MRPECs wound healing, and ETNF, while also enhancing barrier integrity following TGFβ1-induced MV-EC dysfunction.

Assessment of MRPEC Wound Healing, TEER, and ETNF Following TNF𝛂-exposure in the Presence/Absence of Lasmiditan.

Upon determining the functional effects of lasmiditan on primary MRPECs in the presence/absence of LPS and TGFβ1, we then conducted these functional assays in the presence/absence of TNFα [100 ng/ml], a third pro-inflammatory agent commonly associated with endothelial cell dysfunction, AKI, and KD onset/progression. While no differences were observed between TNFα-treated MRPECs and vehicle-treated controls, MRPECs that received either lasmiditan or lasmiditan + TNFα displayed an increase in wound healing/migration capacity compared to vehicle- and TNFα-treated controls (Fig. 6A – 6B). Similar to that observed with LPS and TGFβ1, MRPECs exposed to TNFα also displayed a reduction in TEER compared to vehicle-treated controls and lasmiditan-treated MRPECs at both 24 and 48 hrs, indicative of reduced barrier integrity and increased permeability (Fig. 6C). The observed reduction in MRPECs barrier integrity and increased permeability following exposure to TNFα was prevented in MRPECs treated with both TNFα and lasmiditan over the entire 48 hr period (Fig. 6C). Similar to that observed in both LPS and TGFβ1experiments, MRPECs that received lasmiditan again displayed an increase in the number of branches, master junctions, master segments, and total branching length by more than 2-fold compared to vehicle-treated controls (Fig. 6D – 6H). MRPECs that were exposed to TNFα for 24 hrs also exhibited decreased a ETNF, as evident by a reduction in the number of branches, master junctions, master segments, and total branching length compared to vehicle-treated controls by, 67%, 87%, 91%, and 89%, respectively (Fig. 6D – 6H). The observed reduction in MRPECs ETNF following exposure to TNFα was negated in MRPECs treated with both TNFα and lasmiditan (Fig. 6D – 6H).

Figure 6|. Assessment of MRPEC Wound Healing, TEER, and ETNF Following TNF𝛂-exposure in the Presence/Absence of Lasmiditan.

(A) 24h time course wound healing assays of MRPECs (7×10⁴ cells/well) treated with either: 1) Vehicle Control [Saline + 0.1% DMSO], 2) Lasmiditan [100 nM], 3) TNFα [100 ng/ml], or 4) TNFα + lasmiditan [100 ng/ml + 100 nM, respectively]. (B) Quantification of MRPECs total percent wound closure after 24 hrs of treatment. (C) Quantification of MRPECs (2×10⁵ cells/well) TEER over a 48h period following treatment. (D) Representative 4x magnification phase contrast photomicrographs of MRPECs (9×10³ cells/well) in Matrigel-based ETNF assays after 24 hrs of treatment. MRPEC Matrigel ETNF TIFF images (8-bit) were then processed and quantified via the Angiogenesis Analyzer plugin in FIJI (ImageJ) for various ETNF-related endothelial cell features: (E) total number of branches/well, (F) total number of master junctions/well, (G) total number of master segments/well, and (H) total branches length (pixels)/well. Data represents N=6/group and are expressed as means ± SEM; p<0.05 compared by one-way ANOVA, followed by a Tukey post-hoc correction for multiple comparisons, or by Kruskal-Wallis H-Test nonparametric ANOVA, followed by a Dunnett’s post-hoc correction for multiple comparisons, are considered significant. Significance between groups is displayed in Compact Letter Display (CLD) formatting, where different letters on top of bars signifies a statistical difference between groups. (A) - White scale bars, 100 μm; (D) - White scale bars, 500 μm.

Taken together, these data indicate that following TNFα exposure, MRPECs display enhanced permeability and exhibit a severe impairment in ETNF. Importantly, lasmiditan treatment improved primary MRPECs ETNF, while also enhancing barrier integrity following TNFα-induced MV-EC dysfunction.

Discussion

Despite the increasing incidence rates, mortality, and global concern of KD, many of these debilitating diseases, including AKI, remain progressive and/or terminal due to a lack or absence of effective FDA-approved therapeutics.(1–4, 10, 53, 54) Therefore, identification of novel and/or repurposed drugs that can effectively treat or deter the onset/progression of KD is of dire importance. Pharmacological induction of MB has been proposed as a potential therapeutic strategy to treat a multitude of acute and chronic diseases in which mitochondrial dysfunction is a hallmark of disease onset/progression, including AKI and KD.(27–37, 47, 55–63) Prior studies in animal models have demonstrated that pharmacological induction of MB via HTR1F agonism enhances wound healing, migration, and ETNF of primary renal glomerular MV-ECs, and accelerates markers of renal vascular recovery following ischemia-reperfusion induced AKI.(28, 32) However, the RPEC repair processes following injury, and the effects of pharmacological MB induction on these mechanisms remain unknown.(15, 28, 39) We hypothesized that lasmiditan, a potent and selective FDA-approved HTR1F agonist, may induce MB in primary MRPECs, and alleviate MV-EC dysfunctions under various pro-inflammatory conditions associated with renal MV-EC dysfunction, AKI, and KD onset/progression.(64–67)

Similar to previous reports in primary glomerular MV-ECs, primary MRPECs were observed to express HTR1F, and lasmiditan treatment increased mitochondrial maximal respiration rate and MB regulatory protein expression, indicative of MB induction.(28) Importantly, lasmiditan-induced MB in primary MRPECs was confirmed via MitoTracker Red CMXRos staining, in vitro TEM imaging, and ex vivo TEM imaging. To determine translatability of lasmiditan-induced MB, we assessed both in vitro and ex vivo lasmiditan-treated MRPECs. As anticipated, given known mitochondrial discrepancies between in vitro and in vivo cells regarding function, quantity, and dynamics, in vitro monocultured MRPECs exhibited reduced mitochondrial content compared to ex vivo MRPECs.(52, 68–74) Irrespective of the treatment/imaging modality utilized, however, lasmiditan treatment increased mitochondrial number in primary MRPECs compared to vehicle-treated controls, with no aberrant alterations in individual mitochondrial area/shape, further indicative of MB induction versus mitochondrial swelling, fragmentation, and/or alterations in mitochondrial fission/fusion dynamics.(28, 32, 52) In accord with prior reports of pharmacological MB induction in MV-ECs, lasmiditan-treated MRPECs were found to have increased eNOS and VE-Cadherin proteins, indicative of enhanced MV-ECs function and junctional stability.(28, 49, 67, 75)

LPS, TGFβ1, and TNFα elicit profound effects on renal MV-ECs and contribute to the MV-EC injury, dysfunction, and rarefaction observed in AKI and KD.(6, 15, 64–66, 76–79) RPECs are particularly vulnerable to injury from these pro-inflammatory mediators, resulting in a multitude of MV-EC dysfunctions, including loss of barrier integrity, increased permeability, impaired renal perfusion, reduced oxygen and nutrient delivery to tubular epithelial cells, and rarefaction. (6, 15, 20, 28, 39, 64–66, 76–79) Despite these in vivo findings, it remains unknown how each of these pro-inflammatory mediators individually contribute to the RPEC dysfunction/rarefaction observed in AKI and KD.(64, 65, 67, 75–77, 80) Primary MRPECs exposed to either LPS, TGFβ1, or TNFα displayed characteristics of MV-EC dysfunction in various functional assays (i.e., wound healing/migration, TEER barrier integrity/permeability, and ETNF assays) compared to vehicle-treated controls. Interestingly, the pro-inflammatory agents were found to induce both conserved and differential consequences regarding MV-EC dysfunctions in primary MRPECs (Table 1). LPS, TGFβ1, and TNFα markedly reduced MV-EC barrier integrity and enhanced permeability in primary MRPECs (64–67, 81–83), while, similar to in vivo reports, lasmiditan treatment enhanced barrier integrity and reduced permeability in these cells (Table 1).(28, 32, 49) In contrast, pro-inflammatory agent-dependent responses were detected across MRPEC wound-healing/migration and ETNF assays. Primary MRPECs treated with LPS or TGFβ1 displayed a reduction in wound healing/migration capacity, while no effect was observed in MRPECs treated with TNFα. Primary MRPECs treated with TNFα or TGFβ1, on the other hand, revealed a reduction in ETNF, which was not observed following LPS exposure. Importantly, not only did lasmiditan treatment alone increase wound-healing/migration and ETNF, but the pro-inflammatory agent-induced MV-EC dysfunctions were also augmented or completely abrogated in the presence of lasmiditan (Table 1). While promising, future studies will need to be conducted to determine if lasmiditan is similarly capable of ameliorating combinatorial pro-inflammatory agent-induced MV-EC dysfunctions.

Table 1|.

Summary Overview: MRPEC Functional Assays Following Vehicle Control or Lasmiditan Treatment in the Presence/Absence of Various Pro-inflammatory Agents.

MRPEC Treatment	Wound Healing / Migration Assay	TEER Assay	ETNF Assay
Vehicle Control	—	—	—
LPS	▼	▼	—
TGFβ1	▼	▼	▼
TNFα	—	▼	▼
Lasmiditan	▲	▲	▲
LPS+Lasmiditan	— ▲	—	▲
TGFβ1+Lasmiditan	— ▲	—	—
TNFα+Lasmiditan	▲	—	—

This table contains a summary overview compilation of the observed outcomes acquired from the MRPEC functional assays conducted, following either vehicle control or lasmiditan treatment in the presence/absence of various pro-inflammatory agents (i.e., LPS, TGFβ1, and TNFα). In this table, MRPECs treated with vehicle control were considered the experimental baseline for each assay conducted and are denoted by a [ — ] symbol. Groups that displayed no statistical difference from the experimental baseline following treatment are therefore also denoted by a [ — ] symbol. Groups that were observed to have a statistical reduction from the experimental baseline following treatment are represented by a [ ▼ ] symbol and are shaded blue. Groups that were observed to have a statistical increase from the experimental baseline following treatment are represented by a [ ▲ ] symbol and are shaded orange. Groups that were observed to have no statistical difference from the experimental baseline or from a group that was statistically increased from the experimental baseline following treatment are represented by both [ — ▲ ] symbols and are shaded light orange.

As the renal microvasculature is crucial for renal function and repair processes, a greater understanding of how MV-ECs function under healthy and disease conditions may provide novel insight into druggable targets that blunt/prevent microvascular dysfunction/rarefaction following kidney injury and/or during the onset/progression of KD.(15, 28, 39, 64, 65, 67, 81, 84–86) While glomerular MV-EC mechanisms and injury responses have been extensively evaluated, RPECs have remained elusive and understudied.(28, 39, 64) While follow-up studies will be required to determine the precise cellular and molecular processes that govern the conserved/differential renal MV-EC dysfunctions observed in primary MRPECs following exposure to different pro-inflammatory agents, to our knowledge, this is the first study to comprehensively examine various functional consequences of pro-inflammatory agents on primary MRPECs, and to assess how pharmacological induction of MB affects these outcomes. These presented data underscore a potential role for repurposing lasmiditan to promote mitochondrial and renal MV-EC recovery following an AKI-episode and/or during the onset/progression of KD.

Data Availability Statement.

The original contributions presented in this manuscript are included within this article, further inquiries can be directed to the corresponding author.