Mechanistic target of rapamycin complex 1 (mTORC1) signaling is active in inflammation, but its involvement in septic acute kidney injury (AKI) has not been shown. mTORC1 activation (p-S6) in renal fibroblasts was increased in a mouse AKI model induced by 1.5 mg/kg lipopolysaccharide (LPS). Deletion of tuberous sclerosis complex 1 (TSC1), an mTORC1 negative regulator, in fibroblasts (Fibro-TSC1−/−) inhibited the elevation of serum creatinine and blood urea nitrogen in AKI compared with that in TSC1fl/fl control mice. Endothelin-1 (EDN1) and phospho-Jun-amino-terminal kinase (p-JNK) were up-regulated in Fibro-TSC1−/− renal fibroblasts after LPS challenge. Rapamycin, an mTORC1 inhibitor, and bosentan, an EDN1 antagonist, eliminated the difference in renal function between TSC1fl/fl and Fibro-TSC1−/− mice after LPS injection. Rapamycin restored LPS-induced up-regulation of EDN1, endothelin converting enzyme-1 (ECE1), and p-JNK in TSC1-knockdown mouse embryonic fibroblasts (MEFs). SP600125, a Jun-amino-terminal kinase (JNK) inhibitor, attenuated LPS-induced enhancement of EDN1 and ECE1 in TSC1-knockdown MEFs without a change in phospho-S6 ribosomal protein (p-S6) level. The results indicate that mTORC1–JNK-dependent up-regulation of ECE1 elevated EDN1 in TSC1-knockout renal fibroblasts and contributed to improvement of renal function in Fibro-TSC1−/− mice with LPS-induced AKI. Renal fibroblast mTORC1 plays an important role in septic AKI.

Introduction

Sepsis, a dysregulated host response to severe infection [1], has been shown to occur in 32.4–47.5% of critically ill patients with acute kidney injury (AKI). It is associated with increased AKI severity and mortality [2,3]. AKI, a frequent serious complication of sepsis, is characterized by acute failure of kidney filtration, regulation of ion and water balance, and urine production [4]. A better understanding on the pathogenesis of septic AKI would facilitate the development of novel therapies.

The mechanistic target of rapamycin (mTOR) is the catalytic subunit of two proteins, mTOR Complex 1 (mTORC1) and 2 (mTORC2) [5]. mTORC1 phosphorylates downstream targets of S6 kinase 1, and results in phosphorylation of S6 ribosomal protein [6,7]. The rapamycin-FKBP12 complex directly inhibits mTORC1, but mTORC2 is not sensitive to acute rapamycin treatment [5]. Tuberous sclerosis complex (TSC), a heterotrimeric complex comprising tuberous sclerosis complex 1 (TSC1), TSC2, and TBC1D7, is a negative regulator of mTORC1 signaling [5].

mTORC1 is active in pathophysiological processes including inflammation [8], but its involvement in septic AKI has not been described. Previous animal studies reported that p-mTOR was up-regulated in the renal cortex 48 h after lipopolysaccharide (LPS) treatment. The mTOR inhibitor temsirolimus impaired renal function at 18 h but improved it at 48 h by promoting autophagy in mice at 45 weeks of age [9]. In the current study, cell-specific ablation of TSC1 and activation of mTORC1 in fibroblasts improved renal function in an LPS-induced mouse AKI model, indicating a role of renal fibroblast mTORC1 in septic AKI.

Methods

Animal models

All animal experiments were carried out with the approval of the Southern Medical University Animal Care and Use Committee, in accordance with guidelines for the ethical treatment of animals. Equal numbers of male and female mice were used. Col1α2-creERT and TSC1flox/flox mice were both from Jackson Laboratory (Bar Harbor, ME, U.S.A.; Stock nos. 016237 and 005680, respectively). Yellow fluorescent protein (YFP) mice were a gift from Professor Tianming Gao, Southern Medical University. Conditional knockout models and their litter mate controls were injected intraperitoneally with tamoxifen (Sigma-Aldrich, St. Louis, U.S.A.) 4 mg/25 g in corn oil [10]. The same tamoxifen regimen was used with Col1α2-creERT;YFP+ mice. Rapamycin 1 mg/kg was administered intraperitoneally daily.

Creatinine, BUN, serum ions

Blood was obtained for detection of serum creatinine, BUN, sodium, and chloride from anesthetized mice by cardiac puncture.

Histological assessment

The left kidney was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin (H&E) or periodic acid Schiff (PAS). For immunostaining, sections were incubated with p-S6 (Cell Signaling Technology, Beverly, U.S.A.), TSC1 (Proteintech Group, Chicago, U.S.A.), fibroblast-specific protein-1 (FSP1; Proteintech Group), EDN1 (Abcam, Cambridge, U.K.), or phospho-Jun-amino-terminal kinase (p-JNK; Cell Signaling Technology) primary antibodies. Alexa Fluor 488- and 594-labeled secondary antibodies (Invitrogen, Carlsbad, U.S.A.) were used. Apoptosis was evaluated with a TUNEL assay kit (Promega, Madison, U.S.A.). For YFP visualization, mouse kidneys were fixed for 10 h in 4% paraformaldehyde at 4 °C and equilibrated in 30% sucrose overnight at 4 °C. The kidneys were then embedded in OCT compound, and 5 µm frozen sections were stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunostained or fluorescent images were photographed and scored as previously described by counting the positive cells in four randomly selected fields of the renal cortex [11]. The positive cells were quantified by researchers without their knowing of the groups.

Cell culture and treatment

TSC1-knockdown mouse embryonic fibroblasts (MEFs) were prepared by transfection with TSC1 siRNA (GenePharma, Shanghai, China) using Lipofectamine RNAiMAX reagent (Invitrogen) following the manufacturer’s instructions. MEFs were treated with rapamycin or SP600125 along with their controls 24 h after transfection; 12 h later, cells were cultured with or without LPS for 12 h and then harvested.

Quantitative real-time PCR

Total RNA was isolated from MEFs with Trizol (Invitrogen) and cDNA was generated with a reverse transcriptase. Real-time PCR was performed using SYBR Premix Ex Taq II kits (TaKaRa Bio, Dalian, China) following the manufacturer’s instructions. To determine the relative expression level of mRNA, each gene was normalized to the expression level of the GAPDH reference gene. The primers used in the present study are listed below: EDN1-fw 5′-GCA CCG GAG CTG AGA ATG G-3′; EDN1-rev 5′-GTG GCA GAA GTA GAC ACA CTC-3′; iNOS-fw 5′-GTT CTC AGC CCA ACA ATA CAA GA-3′; iNOS-rev 5′-GTG GAC GGG TCG ATG TCA C-3′; PTGES2-fw 5′-CCT CGA CTT CCA CTC CCT G-3′; PTGES2-rev 5′-TGA GGG CAC TAA TGA TGA CAG AG-3′; GAPDH-fw 5′-AGG TCG GTG TGA ACG GAT TTG-3′; GAPDH-rev 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′.

Western blotting analysis

The protein of cultured MEFs was extracted, and separated by gel electrophoresis, transferred to nitrocellulose membranes, and subsequently incubated with antibodies following the standard protocol [12]. The bands were quantified using the Quantity One software (ver. 4.6.2, Bio-Rad Laboratories).

Statistical analysis

Statistical analysis was performed with the SPSS (ver. 20.0, IBM Corp.) software. For unpaired variables, the differences between groups were analyzed using two-sample t-test or one-way ANOVA. For paired variables, data were analyzed using paired t-test. P-values < 0.05 were considered statistically significant.

Results

Enhancement of mTORC1 activity in the renal fibroblasts of LPS-induced AKI mice

AKI was induced in C57BL/6J mice at 8 weeks of age by intraperitoneal injection of 5 mg/kg LPS [13,14]. Elevated serum creatinine and blood urea nitrogen (BUN) 14 h later confirmed successful establishment of the AKI sepsis model (Figure 1A). The number of p-S6+ interstitial renal cells increased, which indicated mTORC1 activity was up-regulated by LPS treatment (Figure 1B). Immunofluorescent co-staining with FSP1 and p-S6 antibodies confirmed that the interstitial cells were most likely to be fibroblasts [15]. Significant increase in the number of FSP1+ and p-S6+ double-positive cells suggested that mTORC1 was activated in renal fibroblasts in septic AKI (Figure 1C).

p-S6 was elevated in renal fibroblasts of mice with AKI induced by 5 mg/kg LPS

Figure 1
p-S6 was elevated in renal fibroblasts of mice with AKI induced by 5 mg/kg LPS

(A) Serum creatinine and BUN in C57BL/6J AKI and control mice (n = 5). (B) p-S6 (S235/S236) staining in C57BL/6J mouse kidneys in the AKI and control groups (n = 5). Arrows indicate p-S6+ interstitial cells. Arrowheads indicate p-S6 interstitial cells. Scale bar, 50 µm. Quantitative scoring of the staining results is shown in the histogram. (C) FSP1 and p-S6 (S235/S236) immunolabeling in C57BL/6J mouse kidneys from the AKI and control groups (n = 5). Arrows indicate FSP1+ p-S6+ fibroblasts. Scale bars, 20 µm. Quantitative scoring of the staining results is shown in the histogram. Data are means ± SEM. *P < 0.05.

Figure 1
p-S6 was elevated in renal fibroblasts of mice with AKI induced by 5 mg/kg LPS

(A) Serum creatinine and BUN in C57BL/6J AKI and control mice (n = 5). (B) p-S6 (S235/S236) staining in C57BL/6J mouse kidneys in the AKI and control groups (n = 5). Arrows indicate p-S6+ interstitial cells. Arrowheads indicate p-S6 interstitial cells. Scale bar, 50 µm. Quantitative scoring of the staining results is shown in the histogram. (C) FSP1 and p-S6 (S235/S236) immunolabeling in C57BL/6J mouse kidneys from the AKI and control groups (n = 5). Arrows indicate FSP1+ p-S6+ fibroblasts. Scale bars, 20 µm. Quantitative scoring of the staining results is shown in the histogram. Data are means ± SEM. *P < 0.05.

Generation of fibroblast-TSC1−/− mice

The involvement of mTORC1 in the renal fibroblasts of septic AKI model mice was investigated in tamoxifen-inducible conditional knockout mice. The TSC1 gene was specifically deleted in fibroblasts (Fibro-TSC1−/−) by the Cre-loxP system using Col1α2-creERT and TSC1flox/flox mice (Figure 2A). The schedule of tamoxifen injections is shown in Figure 2B and mouse-tail DNA was genotyped with PCR analysis (Figure 2C). The results of the PCR assay of genomic DNA from kidney indicated efficient recombination of the floxTSC1 allele in the presence of the Col1α2-creERT transgene after tamoxifen injection (Figure 2D) [16].

Conditional deletion of TSC1 in fibroblasts

Figure 2
Conditional deletion of TSC1 in fibroblasts

(A) Protocol for generating mice with fibroblast-specific deletion of TSC1. (B) The figure shows the experimental procedures involved in TSC1fl/fl and Col1a2-creERT;TSC1fl/fl mice (except for the procedure specifically indicated). (C) Representative results of PCR genotyping using mouse tail DNA. (D) PCR assay of genomic DNA isolated from the kidneys of TSC1fl/fl and Col1a2-creERT;TSC1fl/fl mice with or without tamoxifen treatment. (E) YFP expression in the kidneys of Fibro-YFP+ mice in the 1.5 mg/kg LPS and control groups. Arrows indicate vascular poles of the glomeruli. Asterisks indicate blood vessel lumens. (F) FSP1 and TSC1 immunolabeling in TSC1fl/fl and Fibro-TSC1−/− kidneys (n = 5). The arrow indicates a FSP1+ TSC1+ fibroblast. Arrowheads indicate FSP1+ TSC1 fibroblasts. The histogram shows the staining scores. (G) p-S6 (S235/S236) staining in TSC1fl/fl and Fibro-TSC1−/− kidneys (n = 5). Arrows indicate p-S6+ interstitial cells. The histogram shows the staining scores. (H) Right kidney weight and kidney-to-body weight ratio of TSC1fl/fl and Fibro-TSC1−/− mice (n = 5). (I) Serum creatinine and BUN in TSC1fl/fl and Fibro-TSC1−/− mice 8 weeks after the first tamoxifen injection (n = 8). The figure shows the experimental procedure. Scale bars (EG), 20 µm. Data are means ± SEM. *P < 0.05.

Figure 2
Conditional deletion of TSC1 in fibroblasts

(A) Protocol for generating mice with fibroblast-specific deletion of TSC1. (B) The figure shows the experimental procedures involved in TSC1fl/fl and Col1a2-creERT;TSC1fl/fl mice (except for the procedure specifically indicated). (C) Representative results of PCR genotyping using mouse tail DNA. (D) PCR assay of genomic DNA isolated from the kidneys of TSC1fl/fl and Col1a2-creERT;TSC1fl/fl mice with or without tamoxifen treatment. (E) YFP expression in the kidneys of Fibro-YFP+ mice in the 1.5 mg/kg LPS and control groups. Arrows indicate vascular poles of the glomeruli. Asterisks indicate blood vessel lumens. (F) FSP1 and TSC1 immunolabeling in TSC1fl/fl and Fibro-TSC1−/− kidneys (n = 5). The arrow indicates a FSP1+ TSC1+ fibroblast. Arrowheads indicate FSP1+ TSC1 fibroblasts. The histogram shows the staining scores. (G) p-S6 (S235/S236) staining in TSC1fl/fl and Fibro-TSC1−/− kidneys (n = 5). Arrows indicate p-S6+ interstitial cells. The histogram shows the staining scores. (H) Right kidney weight and kidney-to-body weight ratio of TSC1fl/fl and Fibro-TSC1−/− mice (n = 5). (I) Serum creatinine and BUN in TSC1fl/fl and Fibro-TSC1−/− mice 8 weeks after the first tamoxifen injection (n = 8). The figure shows the experimental procedure. Scale bars (EG), 20 µm. Data are means ± SEM. *P < 0.05.

The location of Cre activity was determined by visualization of YFP in Col1α2-creERT;YFP+ mice after tamoxifen administration, and was found to reside in the renal interstitium, but not in renal tubules (Figure 2E). YFP-positive cells were seen at the glomerular vascular poles near the afferent and efferent arterioles of the glomeruli and around blood vessels, but not in the vascular tissues (Figure 2E).

TSC1 is an upstream negative regulator of mTORC1. Loss of TSC1 activates mTORC1 and then up-regulates p-S6. Double immunolabeling of FSP1 and TSC1 found TSC1 expression in FSP1+ cells in TSC1fl/fl mice, but not in FSP1+ cells in Fibro-TSC1−/− mice, indicating specific deletion of TSC1 in fibroblasts (Figure 2F). The expression of p-S6 was enhanced in the renal interstitium of Fibro-TSC1−/− mice (Figure 2G).

The weight of the right kidney or the ratio of right kidney weight to body weight in TSC1fl/fl and Fibro-TSC1−/− mice 15 days after the first tamoxifen injection was not significantly different (Figure 2H). Eight weeks after the first tamoxifen injection, the serum creatinine and BUN in TSC1fl/fl and Fibro-TSC1−/− mice were similar (Figure 2I). The results show that deletion of TSC1 in fibroblasts did not result in kidney injury.

Improvement of renal function in Fibro-TSC1−/− LPS-induced AKI model mice

An LPS-induced AKI model was developed to assess whether specific deletion of TSC1 in fibroblasts affected renal function during septic AKI (Figure 2B). Fibro-TSC1−/− and TSC1fl/fl mice had similar responses to a 5 mg/kg LPS challenge (Figure 3A). However, 14 h after administration of a reduced dose of 1.5 mg/kg LPS [9]. Serum creatinine and BUN were significantly lower in Fibro-TSC1−/− mice than those in TSC1fl/fl mice, indicating improvement of renal function in the Fibro-TSC1−/− mice (Figure 3B). Serum chloride increased after injection of 1.5 mg/kg LPS, but the difference in TSC1fl/fl and Fibro-TSC1−/− mice was not significant (Figure 3B). There were no between-group differences in serum sodium or body weight (Figure 3B).

Reduction in serum creatinine and BUN after 1.5 mg/kg LPS challenge in Fibro-TSC1−/− mice compared with TSC1fl/fl mice

Figure 3
Reduction in serum creatinine and BUN after 1.5 mg/kg LPS challenge in Fibro-TSC1−/− mice compared with TSC1fl/fl mice

(A) Serum creatinine and BUN in TSC1fl/fl and Fibro-TSC1−/− mice with AKI induced by 5 mg/kg LPS (n = 4). (B) Serum creatinine, BUN, sodium (Na+), chloride (Cl), and body weights of mice in the experimental groups (n = 5–11). (C) FSP1 and p-S6 (S235/S236) immunolabeling in kidneys from the experimental groups (n = 5). Arrows indicate FSP1+ p-S6+ fibroblasts. The histogram shows the staining scores. Scale bars, 20 µm. (D) Serum creatinine, BUN, and body weights of mice in the experimental groups (n = 8–9). Rapa, rapamycin (mTORC1 inhibitor). Data are means ± SEM. *P < 0.05.

Figure 3
Reduction in serum creatinine and BUN after 1.5 mg/kg LPS challenge in Fibro-TSC1−/− mice compared with TSC1fl/fl mice

(A) Serum creatinine and BUN in TSC1fl/fl and Fibro-TSC1−/− mice with AKI induced by 5 mg/kg LPS (n = 4). (B) Serum creatinine, BUN, sodium (Na+), chloride (Cl), and body weights of mice in the experimental groups (n = 5–11). (C) FSP1 and p-S6 (S235/S236) immunolabeling in kidneys from the experimental groups (n = 5). Arrows indicate FSP1+ p-S6+ fibroblasts. The histogram shows the staining scores. Scale bars, 20 µm. (D) Serum creatinine, BUN, and body weights of mice in the experimental groups (n = 8–9). Rapa, rapamycin (mTORC1 inhibitor). Data are means ± SEM. *P < 0.05.

Compared with those in TSC1fl/fl mice in the control group, p-S6+ fibroblasts in Fibro-TSC1−/− mice were increased, which further confirmed the specific knockout of TSC1 in fibroblasts (Figure 3C). Even at the lower dosage of 1.5 mg/kg LPS, the number of p-S6+ fibroblasts increased in TSC1fl/fl mice compared with that in controls (Figure 3C). As expected, Fibro-TSC1−/− mice treated with 1.5 mg/kg LPS had the most FSP1+ and p-S6+ double-positive cells (Figure 3C).

Fourteen days after the first injection of tamoxifen, AKI was induced in Fibro-YFP+ mice by 1.5 mg/kg LPS. The mice were killed 14 h later for visualization of YFP, which was still observed in the renal interstitium, but not in the renal tubules (Figure 2E). YFP-positive cells were also seen at the glomerular vascular poles and around blood vessels, but not in the vessel tissues (Figure 2E). The results showed that LPS did not change the location of Cre activity.

Rapamycin abolishes the improvement in 1.5 mg/kg LPS-induced Fibro-TSC1−/− mice

To determine whether the protective effect of TSC1 deletion was caused by mTORC1 activation, mice were treated with an mTORC1 inhibitor, rapamycin, before 1.5 mg/kg LPS injection (Figure 2B). The decreased expression of p-S6 after rapamycin treatment confirmed the inhibition of mTORC1 (Figure 3C), and with rapamycin treatment there were no differences in serum creatinine or BUN in TSC1fl/fl and Fibro-TSC1−/− (Figure 3D). The treatment groups did not differ in body weight (Figure 3D).

Cell injury in LPS-induced AKI

The morphological changes in both TSC1fl/fl and Fibro-TSC1−/− mice given 5 mg/kg LPS included tubular degeneration and occasional tubular dilatation without tubular cast formation (Figure 4A). Very few morphological abnormalities were seen in mice given 1.5 mg/kg LPS (Figure 4A). Apoptotic nuclei accumulated in mice given 5 mg/kg LPS or rapamycin plus 1.5 mg/kg LPS, and TSC1fl/fl mice given 1.5 mg/kg LPS (Figure 4B). Fewer apoptotic cells were observed in Fibro-TSC1−/− than those in TSC1fl/fl mice given 1.5 mg/kg LPS (Figure 4B). Neither fibroblast-specific TSC1 deletion nor 1.5 mg/kg LPS treatment changed the size, shape, or color of the kidneys (Figure 4C).

Cell injury associated with LPS-induced AKI

Figure 4
Cell injury associated with LPS-induced AKI

(A) Representative images of H&E and PAS staining in kidneys from the experimental groups. Arrows indicate degenerated tubules. Asterisks indicate dilated tubules. (B) TUNEL staining in kidneys from the experimental groups (n = 4–5). The histogram shows the staining scores. (C) Gross specimens of the left kidneys from the experimental groups. Scale bars (A, B), 50 µm. Data are means ± SEM. *P < 0.05.

Figure 4
Cell injury associated with LPS-induced AKI

(A) Representative images of H&E and PAS staining in kidneys from the experimental groups. Arrows indicate degenerated tubules. Asterisks indicate dilated tubules. (B) TUNEL staining in kidneys from the experimental groups (n = 4–5). The histogram shows the staining scores. (C) Gross specimens of the left kidneys from the experimental groups. Scale bars (A, B), 50 µm. Data are means ± SEM. *P < 0.05.

Endothelin-1 up-regulation in renal fibroblasts of LPS-treated Fibro-TSC1−/− mice

Sepsis is characterized by circulatory disorders, including decreased systemic vascular resistance and microcirculation dysfunction [4], and three vasoactive factors or their synthases were evaluated in MEFs. Prostaglandin E synthase-2 (PTGES2) expression was unchanged, but real-time PCR revealed a two-fold up-regulation of endothelin-1 (EDN1) in MEFs after 6 h of LPS stimulation (Figure 5A). LPS also increased the expression of inducible nitric oxide synthase (iNOS) at 24 h in a dose-dependent manner (Figure 5A). The expression of EDN1 protein in MEFs was increased at 12 and 24 h following 1 or 10 μg/ml LPS treatment (Figure 5B). Small interfering RNA (siRNA) decreased TSC1 expression in MEFs and compared with that in negative control MEFs treated with 1 μg/ml LPS, knockdown of TSC1 up-regulated EDN1 at 12 h (Figure 5C). Rapamycin treatment significantly inhibited mTORC1 activation in MEFS as shown by down-regulation of p-S6, and it decreased EDN1 expression in TSC1-knockdown MEFs (Figure 5C).

Up-regulation of EDN1 in TSC1-knockdown MEFs treated with 1 μg/ml LPS

Figure 5
Up-regulation of EDN1 in TSC1-knockdown MEFs treated with 1 μg/ml LPS

(A) Relative gene expression of PTGES2, iNOS, and EDN1 in MEFs determined with real-time PCR (n = 3). (B) Western blot assays of iNOS and EDN1 expression in MEFs in the experimental groups (n = 3). Elevation of EDN1 in MEFs at 12 and 24 h after LPS treatment. The histograms show the densiometric analysis. (C) Western blot assays of TSC1, p-S6 (S235/S236), S6, and EDN1 in MEFs from the experimental groups (n = 3). Rapamycin rescued LPS-induced EDN1 enhancement in TSC1-knockdown MEFs. The histograms show the densiometric analysis. Data are means ± SEM. *P < 0.05.

Figure 5
Up-regulation of EDN1 in TSC1-knockdown MEFs treated with 1 μg/ml LPS

(A) Relative gene expression of PTGES2, iNOS, and EDN1 in MEFs determined with real-time PCR (n = 3). (B) Western blot assays of iNOS and EDN1 expression in MEFs in the experimental groups (n = 3). Elevation of EDN1 in MEFs at 12 and 24 h after LPS treatment. The histograms show the densiometric analysis. (C) Western blot assays of TSC1, p-S6 (S235/S236), S6, and EDN1 in MEFs from the experimental groups (n = 3). Rapamycin rescued LPS-induced EDN1 enhancement in TSC1-knockdown MEFs. The histograms show the densiometric analysis. Data are means ± SEM. *P < 0.05.

In vivo, EDN1 was up-regulated in the renal fibroblasts of TSC1fl/fl mice by 1.5 mg/kg LPS, and the up-regulation was significantly increased in Fibro-TSC1−/− mice treated with 1.5 or 5 mg/kg LPS (Figure 6A). Rapamycin pretreatment attenuated EDN1 enhancement in Fibro-TSC1−/− mice (Figure 6A). Bosentan, an EDN1 antagonist at the endothelin-A and endothelin-B receptor level, confirmed that the improvement resulted from enhancement of EDN1. Bosentan, 100 mg/kg in 5% gum arabic, administered intragastrically 1 h before 1.5 mg/kg LPS [17] prevented the improvement of renal function in Fibro-TSC1−/− mice (Figure 6B).

Up-regulation of EDN1 in renal fibroblasts in Fibro-TSC1−/− mice treated with LPS

Figure 6
Up-regulation of EDN1 in renal fibroblasts in Fibro-TSC1−/− mice treated with LPS

(A) FSP1 and EDN1 immunolabeling in kidneys (n = 4-5). Arrows indicate FSP1+ EDN1+ fibroblasts. The histogram shows the staining scores. (B) Serum creatinine and BUN of TSC1fl/fl and Fibro-TSC1−/− mice treated with 100 mg/kg bosentan 1 h before injection of 1.5 mg/kg LPS (n = 5). Scale bars, 20 µm. Data are means ± SEM. *P < 0.05.

Figure 6
Up-regulation of EDN1 in renal fibroblasts in Fibro-TSC1−/− mice treated with LPS

(A) FSP1 and EDN1 immunolabeling in kidneys (n = 4-5). Arrows indicate FSP1+ EDN1+ fibroblasts. The histogram shows the staining scores. (B) Serum creatinine and BUN of TSC1fl/fl and Fibro-TSC1−/− mice treated with 100 mg/kg bosentan 1 h before injection of 1.5 mg/kg LPS (n = 5). Scale bars, 20 µm. Data are means ± SEM. *P < 0.05.

Enhancement of p-JNK in the renal fibroblasts of Fibro-TSC1−/− mice treated with 1.5 mg/kg LPS

In the synthesis of EDN1, the final step is selective hydrolysis of Trp21–Val22 by endothelin converting enzyme-1 (ECE1) [18]. ECE1 was up-regulated in TSC1-knockdown MEFs by LPS challenge (Figure 7). Pretreatment with rapamycin prevented LPS-induced ECE1 accumulation (Figure 7). Previous studies have reported that nuclear factor (NF)-κB, Jun-amino-terminal kinase (JNK), and extracellular signal-regulated protein kinases (Erk)1/2 were involved in ECE1 regulation [19,20]. The expression of p-NF-κB p65 subunit, p-JNK, and p-Erk1/2 was assayed, p-JNK was enhanced in LPS-treated TSC1-knockdown MEFs, but p-NF-κB p65, and p-Erk1/2 expression in TSC1-knockdown and negative control MEFs remained unchanged following LPS treatment (Figure 7). Compared with TSC1-knockdown MEFs treated with LPS alone, rapamycin reduced p-JNK expression (Figure 7).

Enhancement of p-JNK expression in TSC1-knockdown MEFs treated with 1 μg/ml LPS

Figure 7
Enhancement of p-JNK expression in TSC1-knockdown MEFs treated with 1 μg/ml LPS

Western blot assays of TSC1, ECE1, p-NF-κB p65 (Ser536), NF-κB p65, p-JNK (Thr183/Tyr185), JNK, p-Erk1/2 (Thr202/Tyr204), and Erk1/2 in MEFs from the experimental groups (n = 3). Rapamycin rescued LPS-induced p-JNK enhancement in TSC1-knockdown MEFs. The histograms show the densiometric analysis. Data are means ± SEM. *P < 0.05.

Figure 7
Enhancement of p-JNK expression in TSC1-knockdown MEFs treated with 1 μg/ml LPS

Western blot assays of TSC1, ECE1, p-NF-κB p65 (Ser536), NF-κB p65, p-JNK (Thr183/Tyr185), JNK, p-Erk1/2 (Thr202/Tyr204), and Erk1/2 in MEFs from the experimental groups (n = 3). Rapamycin rescued LPS-induced p-JNK enhancement in TSC1-knockdown MEFs. The histograms show the densiometric analysis. Data are means ± SEM. *P < 0.05.

p-JNK expression was greater in Fibro-TSC1−/− than that in TSC1fl/fl mice following 1.5 mg/kg LPS treatment; rapamycin attenuated the enhanced expression (Figure 8A). To determine the role of JNK, the effect of SP600125, a JNK inhibitor, on LPS-induced ECE1 and EDN1 increase was investigated. Pretreatment with 10 μM SP600125 attenuated the increase in ECE1 and EDN1 expression in LPS-stimulated TSC1-knockdown MEFs; p-S6 expression was not changed (Figure 8B). This suggests that mTORC1 acts on the upstream of JNK in enhancement of LPS-induced EDN1 expression.

Enhancement of p-JNK expression in renal fibroblasts of Fibro-TSC1−/− mice treated with 1.5 mg/kg LPS

Figure 8
Enhancement of p-JNK expression in renal fibroblasts of Fibro-TSC1−/− mice treated with 1.5 mg/kg LPS

(A) FSP1 and p-JNK (Thr183/Tyr185) immunolabeling in kidneys (n = 5). Arrows indicate FSP1+ p-JNK+ fibroblasts. The histogram shows the staining scores. (B) Western blot assays of TSC1, ECE1, P-S6 (S235/S236), S6, and EDN1 expression in MEFs from the experimental groups (n = 3). SP600125 (a JNK inhibitor) decreased LPS-induced ECE1 and EDN1 enhancement in TSC1-knockdown MEFs; p-S6 was not changed. The histograms show the densiometric analysis. Scale bars, 20 µm. Data are means ± SEM. *P < 0.05.

Figure 8
Enhancement of p-JNK expression in renal fibroblasts of Fibro-TSC1−/− mice treated with 1.5 mg/kg LPS

(A) FSP1 and p-JNK (Thr183/Tyr185) immunolabeling in kidneys (n = 5). Arrows indicate FSP1+ p-JNK+ fibroblasts. The histogram shows the staining scores. (B) Western blot assays of TSC1, ECE1, P-S6 (S235/S236), S6, and EDN1 expression in MEFs from the experimental groups (n = 3). SP600125 (a JNK inhibitor) decreased LPS-induced ECE1 and EDN1 enhancement in TSC1-knockdown MEFs; p-S6 was not changed. The histograms show the densiometric analysis. Scale bars, 20 µm. Data are means ± SEM. *P < 0.05.

Discussion

Injection of an exogenous toxin like LPS and intestinal leakage produced by cecal ligation and puncture are frequently used animal sepsis models. LPS can induce systemic hypotension and decrease the glomerular filtration rate. Low doses of LPS, such as 5 mg/kg, do not cause systemic hypotension, but do decrease glomerular filtration rate and filtration fraction [13,14,21]. It can thus be used to study renal responses during the initial phase of sepsis without hypotension. AKI has been confirmed by changes in BUN or creatinine in some [22–24] but not all studies [25,26]. As cecal ligation and puncture-induced AKI may not be reproducible, a low-dose LPS model was used in the present study.

The pathological description of septic AKI is inconsistent. In some patients, the histology of normal glomeruli and renal tubules was preserved [27], and no evidence of increased tubular injury or apoptosis was seen in a sheep model of septic AKI [28]. However, some studies reported tubules with small vacuoles or flattened epithelia in LPS-induced AKI [29,30]. In the present study, degenerated and dilated tubules and an increase in apoptotic cells were seen with a 5 mg/kg LPS challenge. Tubular injury was absent from mice given 1.5 mg/kg LPS, but the number of apoptotic cells was increased in TSC1fl/fl mice. The results indicated the presence of dose-dependent cell injury in LPS-induced AKI.

The hemodynamic characteristics of sepsis include general arterial dilation and consequent decline in systemic vascular resistance [31]. Previous studies emphasized systemic hypotension, renal vasoconstriction, and ischemia–reperfusion injury as the primary mechanisms of septic AKI. Recently, septic AKI was shown to occur in the setting of renal vasodilatation and increased renal blood flow [32]. A better understanding of the pathological mechanisms of septic AKI is needed to account for differences from the traditional ischemia paradigm. Sepsis causes profound alterations in the microvascular circulation throughout the body, characterized by increasing heterogeneity of flow. There is a decrease in capillaries with continuous blood flow along with a concomitant increment of capillaries with intermittent or no flow. The renal microcirculation of septic AKI is altered in a similar fashion [33]. The changes in microcirculation create regions of hypoperfusion and hypoxia that cause cell and tissue injury [34,35]. The glomerular ultrafiltration rate is proportional to the pressure gradient from the glomerular capillary to the tubular space [36]. Causes of loss of glomerular filtration rate in septic AKI may include dominant efferent, compared with afferent, arteriole dilation, with subsequent decrease in glomerular filtration pressure (intraglomerular hypotension), intrarenal hemodynamic alterations (periglomerular shunting), excessive inflammatory activation, or any combination of the aforementioned. Septic infection up-regulates iNOS activity and NO production, causing dilation of efferent arterioles and inducing periglomerular shunting. The decreased flow through the glomerular capillaries reduces the glomerular filtration rate [37].

EDN1 is a 21 amino acid peptide derived from Big EDN1, a 39 amino acid peptide precursor, by ECE1 [18]. It is a potent vasoconstrictor [38] secreted by the basolateral surface of vascular endothelial cells [39]. In many species, the plasma concentration of EDN1 is ∼1 pM, which is two orders of magnitude below the pharmacological threshold. Its half-life under healthy conditions is ∼1 min [40,41]. These characteristics indicate that EDN1 is not a circulating hormone but acts as an autocrine or paracrine factor at discrete sites [40]. Systemic endothelin receptor blockade decreases peripheral vascular resistance and blood pressure in humans, which suggests that endogenous EDN1 helps maintain vascular tone [42]. Renal vessels are sensitive to EDN1 [43], which modulates vasoconstriction of afferent and efferent glomerular arterioles. The sensitivity and response are greater in efferent than those in afferent arterioles [44,45].

In the present study, 1.5 mg/kg LPS up-regulated EDN1 in mouse renal fibroblasts. Specific knockout of TSC1 enhanced EDN1 up-regulation and improved renal function, and bosentan prevented improvement of AKI in Fibro-TSC1−/− mice. In addition, Cre activity was present in the renal interstitium at the glomerular vascular poles and adjacent to blood vessels of Fibro-YFP+ mice. Overall, the results are consistent with the protective activity of renal fibroblasts in AKI induced by 1.5 mg/kg LPS. The paracrine secretion of EDN1 may oppose LPS-induced vasodilatation to maintain normal intrarenal blood flow and microcirculation, reduce cell injury, and support renal function. EDN1 may close shunting pathways by constricting the vessels. Its differential effect on afferent and efferent glomerular arterioles could increase intraglomerular pressure to maintain the filtration rate. Preliminary data (Supplementary Figure S1) on the blood flow velocities of renal interlobar arteries in 1.5 mg LPS-induced AKI mice confirmed that the peak-systolic blood flow velocities (Vmax) of interlobar arteries in Fibro-TSC1−/− mice were significantly higher than that in TSC1fl/fl mice. This result indicated that EDN1 constricted the afferent and efferent glomerular arterioles. Further study will be carried out on renal hemodynamics.

The findings add to the understanding of the mechanism of septic AKI by accounting for maldistribution of blood caused by vasodilation. We also found that specific TSC1 knockout in 5 mg/kg LPS-treated mice enhanced the up-regulation of EDN1 but did not improve renal function. This may be due to renal morphological abnormalities caused by 5 mg/kg LPS, resulting in more severe renal impairment than that caused by 1.5 mg/kg LPS, exceeding the protective threshold of EDN1. The study findings are consistent with a recent report that sirolimus, a rapamycin analog, significantly decreased p-JNK expression in the brain tissue of mice with MPT-induced neurotoxicity [46].

In summary, mTORC1–JNK-dependent up-regulation of ECE1 elevated EDN1 in TSC1-deleted renal fibroblasts, which contributed to the improvement of renal function in Fibro-TSC1−/− mice with AKI induced by 1.5 mg/kg LPS (Figure 9). Fibroblast mTORC1 was active in this mouse model of septic AKI.

Diagram of the proposed mechanism of action in TSC1-deleted renal fibroblasts in this mouse model of LPS-induced AKI.

Clinical perspectives

  • The involvement of mTORC1 signaling in septic AKI has not been shown.

  • Our results indicate that mTORC1–JNK-dependent up-regulation of ECE1 elevated EDN1 in TSC1-knockout renal fibroblasts and contributed to improvement of renal function in Fibro-TSC1−/− mice with LPS-induced AKI.

  • The data allow us to further understand the roles of renal fibroblasts and EDN1 in septic AKI, and facilitate the development of novel therapies.

We thank Prof. Tianming Gao for the gift of YFP mice, and Kangyan Liang, Kang Tan, Zhi Xiong, Caixia Wang, and Minyu Xie for their excellent technical assistance.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Author contribution

Junhui Shen, Yue Zhang, Zhenguo Chen, Zhong-Kai Cui, and Xiaochun Bai designed the study. Junhui Shen conducted study and data acquisition. Junhui Shen, Fang Yao, Kai Li, Yue Zhang, Zhenguo Chen, Yuxia Zhou, Song Xu, Yuwei Zhang, Wenqing Jiang, Hanbin Zhang, Kaifen Tan, and Anling Liu were involved in analysis and interpretation of data. Junhui Shen, Zhong-Kai Cui, and Xiaochun Bai involved in drafting the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 81625015, 81530070, 31529002, 81772406] and the State Key Development Program for Basic Research of China [2015CB553602].

Abbreviations

     
  • AKI

    acute kidney injury

  •  
  • BUN

    blood urea nitrogen

  •  
  • ECE1

    endothelin converting enzyme-1

  •  
  • EDN1

    endothelin-1

  •  
  • Erk1/2

    extracellular signal-regulated kinases 1 and 2

  •  
  • p-Erk1/2

    phospho-extracellular signal-regulated kinases 1 and 2

  •  
  • FSP1

    fibroblast-specific protein-1

  •  
  • H&E

    hematoxylin and eosin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • JNK

    Jun-amino-terminal kinase

  •  
  • p-JNK

    phospho-Jun-amino-terminal kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • MEFs

    mouse embryonic fibroblasts

  •  
  • mTOR

    mechanistic target of rapamycin

  •  
  • mTORC1

    mechanistic target of rapamycin complex 1

  •  
  • NF-κB

    nuclear factor κB

  •  
  • p-NF-κB

    phospho-nuclear factor κB

  •  
  • PAS

    periodic acid Schiff

  •  
  • PTGES2

    prostaglandin E synthase-2

  •  
  • siRNA

    small interfering RNA

  •  
  • S6

    S6 ribosomal protein

  •  
  • p-S6

    phospho-S6 ribosomal protein

  •  
  • TSC

    tuberous sclerosis complex

  •  
  • TSC1

    tuberous sclerosis complex 1

  •  
  • YFP

    yellow fluorescent protein

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Supplementary data