Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: Shock. 2022 Oct 4;58(5):426–433. doi: 10.1097/SHK.0000000000001995

Hydrocortisone, Ascorbic Acid and Thiamine (HAT) Therapy Decreases Renal Oxidative Stress and Acute Kidney Injury (AKI) In Murine Sepsis

John Kim 1, Allan Stolarski 2, Qiuyang Zhang 1, Katherine Wee 1, Daniel Remick 1
PMCID: PMC9713586  NIHMSID: NIHMS1834815  PMID: 36445231

Abstract

Background:

Acute Kidney Injury (AKI) occurs frequently in septic patients and correlates with increased mortality. Since clinical studies investigating hydrocortisone, ascorbic acid and thiamine (HAT) have demonstrated discordant results, studies were performed using mortality stratification for therapy to identify candidates for therapy and determine mechanisms of organ injury.

Methods:

Sepsis was induced using the cecal ligation and puncture (CLP) model of sepsis with fluid and antibiotic support. Heart rate measurements obtained 6 h after CLP stratified mice into Live-Predicted (Live-P) or Die-P. Stratified mice were then randomized for treatment with HAT or Vehicle (V) given 7 h after CLP. Physiologic measurements were taken again at 24 h and mice sacrificed to collect blood and organs.

Results:

Five groups were created 1) Live-P Vehicle, 2) Live-P HAT, 3) Die-P Vehicle, 4) Die-P HAT and 5) Naïve mice. Comparisons were made to test the hypotheses that i) Die-P Vehicle mice will have significant deterioration compared to Live-P mice targeting the kidney and ii) HAT will correct these deleterious changes in Die-P mice. Compared to Live-P, Die-P mice had a significant decline in all measured physiologic parameters (heart rate, cardiac output, breath rate and temperature) which were corrected with HAT therapy (p<0.05 for all parameters). Die-P mice had declines in the ascorbic acid within the blood, peritoneal lavage and kidney homogenate compared to Live-P mice indicating consumption, and the decline was corrected with HAT. Elevated IL-6, KC, MIP-2 and IL-1RA were found in Die-P mice and decreased with HAT. Markers of endothelial cell injury (glypican 1 and 4) were elevated in the Die-P mice and these values were decreased with HAT therapy. Low oxygen levels with subsequent oxidative stress (OS) in the kidney were visualized in histologic sections using hypoxyprobe and also with carbonyl proteins and 8-isoprostaglandin F2α in kidney homogenates. Die-P mice had significant elevations of renal OSs which was ameliorated with HAT. Kidney injury was evident in the Die-P mice compared to Live-P mice with elevations in BUN and cystatin C which were significantly reduced with HAT. There was no evidence of global hypoxia or organ injury since hepatic parameters remained normal.

Conclusions:

Our data show that in CLP-induced sepsis Die-P mice have increased inflammation, oxidative stress and kidney injury. HAT therapy decreased renal oxidative stress and injury in the Die-P group when given after the onset of sepsis-induced physiologic changes.

Keywords: hypoxyprobe, IL-6, risk stratification, oxidative stress, acute kidney injury, ascorbic acid

Introduction

An estimated 49 million cases of sepsis afflict patients in the world each year1 and a third of these septic patients will develop acute kidney injury (AKI)2. Recurrent AKI may also be found in patients admitted to intensive care units (ICU)3. A study of over 4000 patients showed that nearly 65% of patients with septic shock developed AKI4. The issue of AKI in critically ill patients has become more important during the COVID pandemic where a study showed that half of COVID patients developed AKI5. Hypotension has been implicated in shock patients who develop AKI6.

While hypotension is a risk factor for AKI, the precise mechanisms of septic AKI (S-AKI) are not well established7. Early changes in renal blood flow have been demonstrated even when hypotension is not present8. Oxidative stress (OS) within the renal tubules has been implicated as a potential mechanism3. Elegant studies by Wang demonstrated that following cecal ligation and puncture (CLP) induced sepsis there is an early reduction in renal blood flow and increased OS within the renal tubules9. These studies, and several others, suggest that antioxidants could prove beneficial in the treatment of sepsis and sepsis-induced AKI10.

Ascorbic acid therapy for sepsis is controversial11 and there was initial excitement when a small clinical study showed that a combination of Hydrocortisone, Ascorbic Acid and Thiamine (HAT) substantially improved outcome in septic patients12. A meta-analysis of seven large-scale clinical trials with over 800 patients concluded that no detrimental effects were observed with HAT therapy but conclusive results showing efficacy would require additional studies13. These controversial results prompted studies in the well characterized murine model of CLP-induced sepsis to test the hypothesis that sepsis will induce renal OS and damage only in mice with a high probability of death and that HAT therapy would reduce OS and kidney damage.

Materials and Methods

Mice

Female (21~23g, N=65) and male (18~20g, N=63) outbred ICR mice (CD-1) were purchased from Envigo (Indianapolis, IN) and maintained under standard laboratory condition. All mice were housed in a temperature - and humidity-controlled room with a 12-hour dark-light cycle. Food and water were allowed ad libitum. All experiments were performed in accordance with the NIH guidelines, and approved by the Boston University Institutional Animal Care and Use Committee (IACUC). The ARRIVE checklist is included as supplemental data.

Sepsis model

We used the previously reported CLP sepsis model14 modified from the standard CLP model15. Briefly, 20 minutes prior to surgery, mice were given buprenorphine (0.05 mg/kg) subcutaneously. CLP was performed under isoflurane anesthesia using a protocol that results in 40% mortality14. Immediately after CLP, mice resuscitated with 1.0 ml of warmed normal saline and analgesia provided (buprenorphine, 0.05 mg/kg) every 12 hours for a total of 5 doses. Antibiotic therapy (imipenem, 25 mg/kg, Merck, West Point PA) in Lactated Ringers with 5% dextrose was administered starting 2 hours after CLP and continued every 12 hours for a total of 3 doses. Previous studies show approximately 40% mortality over 28 days using this model14,15.

Mortality prediction

The mice were divided into two groups, P-Die and P-Live, based on physiologic data collected at 6 hours post CLP as reported previously14,16. Mice with HR ≥620 are grouped as Predicted Live (P-Live) and mice with HR <620 are as Predicted Die (P-Die). Mice in each group were randomized for HAT or Vehicle treatment.

HAT treatment

Mice were treated with HAT as described in our previous report14. Hydrocortisone, ascorbic acid and thiamine were purchased from Sigma-Aldrich (St. Louis, MO) and doses were based on the original treatment protocol for patients12. Specifically the following doses were given: hydrocortisone: 1.5 mg/kg, vitamin C: 45 mg/kg and thiamine: 3 mg/kg. All three drugs were dissolved in 1.0 ml warm Lactated Ringer’s with 5% dextrose and given immediately after stratification followed by two additional doses before being sacrificed, 24 hours post CLP. Therapy was initiated about 7 hours after the onset of sepsis. The Vehicle group received the same volume of lactated Ringers with 5% dextrose.

Sample and Data collection

Prior to CLP, baseline information was collected including body weight, temperature, and physiologic parameters as described in our previous publication14. For all studies, physiologic parameters measured at 6, and 24 hours post CLP and blood was collected after the physiologic parameters are measured. Pimonidazole HCl (60mg/kg body weight, Hypoxyprobe™ Kit, HPI Inc, Burlington, MA) in 100uL warm normal saline was intraperitoneally injected 2 hours before sacrifice. The corresponding author was blinded to group allocation until the data were collected. Physiologic data from sham operated mice are included in figures 1 and 3. Since there were no differences between the sham operated and naïve mice, the sham group was not included in subsequent studies to reduce the number of mice.

Figure 1: CLP induced physiology changes.

Figure 1:

Open in a new tab

Non-invasive physiology parameters were measured in the mice six hours after CLP. The mice were stratified based on their Heart Rate (HR) with HR ≥620 are grouped as Predicted Live (P-Live) and mice with HR <620 are as Predicted Die (P-Die). A) shows the distribution of the HR. B) Pulse distension, C) Breath rate and D) Body temperature. HR was not statistically analyzed since that parameter defined the groups. P values calculated using Mann-Whitney test since the data were not normally distributed.

Figure 3: HAT therapy improves physiologic parameters.

Figure 3:

Open in a new tab

HAT therapy was initiated after mice were stratified. In the mice predicted to die, HAT significantly improved A) Heart rate, B) Pulse distension, C) Breath rate and D) Body temperature. The data were collected 24 hours after CLP. P values calculated using Mann-Whitney test since the data were not normally distributed with Bonferroni correction.

Twenty-four hrs post CLP, mice were euthanized by an overdose of isoflurane and blood collected into tubes containing 50μl of 3.8% sodium citrate. The peritoneum was opened and washed with 5 ml of warm Hanks Balanced Salt Solution (HBSS, Mediatech, Manassa, VA) supplemented with 10 U/ml Heparin (McKesson Packaging, Irving TX)14,17. The collected peritoneal lavage fluid was centrifuged at 500 × g for 5 minutes. The peritoneal fluid was stored at −20°C for analysis for inflammatory mediators and other injury markers. After sacrifice, lung, liver, spleen and kidney were collected. Once weighed, all organs were place in 1.0 ml ice-cold PBS containing protease inhibitor cocktail (Complete®, Roche Diag, Manheim, Germany) and 0.05% Triton X-100 (Sigma). These organs were processed for protein extraction through three 10-s rounds of homogenization and sonication followed by centrifugation at 15,000 × g for 15 min at 4°C. The supernatants were transferred to a new tube and stored at −20°C for analysis. Total protein concentration in the supernatant was determined by Bradford assay using Coomassie Plus protein assay reagent (Thermo Sci, Rockford, IL) as suggested by manufacturer.

Ascorbic Acid

Ascorbic acid concentrations were measured by a colorimetric assay as described in the manufacturer’s guide (Vitamin C Assay Kit, KT-75000, Kamiya Biomed, Seattle, WA). Vitamin C concentrations in organs were normalized to the total protein in each organ.

Inflammatory mediators, glypicans and biochemistry

The concentration of cytokines, the CXC chemokines KC and Macrophage Inflammatory Protein 2) and cystatin C were determined by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs (R&D Systems, Inc.) as previously described18. Glypican 1 and 4 were measured using ELISA kits (Cloud Clone, Wuhan China). BUN (Blood Urea Nitrogen), AST (Aspartate Aminotransferase), and Bilirubin were measured by standard clinical methods using kits from Pointe Scientific, Inc (Canton, MI) as previously described15.

Oxidative stress markers

Oxidative stress was measured in the organs using ELISA kits. Lipid peroxidation was quantified by measuring 8-isoprostaglandin F2α (Cell Biolabs, STA-337, San Diego CA) and measuring protein oxidation by protein carbonyl derivatives by ELISA (Cell Biolabs, STA-310).

Immunohistochemistry

Immediately after peritoneal lavage fluid is collected, portions of organs were removed, fixed in 10% buffered formalin and processed for immunohistochemistry using the Ventana Benchmark Discovery System (Roche). Slides were pretreated with Benchmark Ultra CC1 (Roche) at 95° C for 64 minutes and incubated with anti-pimonidazole mouse IgG1 monoclonal antibody (Hypoxyprobe™ Kit, HPI Inc, Burlington, MA) diluted 1/50 in Ventana antibody diluent with casein (Roche, Indianapolis, IN) for 60 min at 37° C. Rabbit anti-mouse linker (Abcam, Cambridge, MA) was diluted at 1:1000 using Ventana antibody diluent and Prediluted ImmPRESS® HRP goat anti-rabbit IgG Detection kit (Vector Lab, Burlingame, CA) was added. IHC was visualized by adding 3, 3′ diaminobenzidine tetrahydrochloride substrate (Discovery Chromomap DAB, Roche) and slides were counterstained with Hematoxylin II (Roche) and Bluing Reagent (Roche).

Statistical Analysis

All statistical analysis was performed using Prism Version 9 (GraphPad, San Diego, CA). Nonparametric analyses were performed since the majority of the data were not normally distributed. Sample size was determined by interim analysis of the data. The studies tested two hypotheses 1) mice predicted to die (P-Die) will have deterioration compared to the P-Live mice and 2) HAT therapy would improve the values in the P-Die mice. To test these hypotheses the P-Die and P-Live groups were compared with the Mann-Whitney test. Next the values in the P-Die HAT treated group were compared to the P-Die vehicle group. Since more than 1 statistical test was applied to the data, a Bonferroni correction was applied.

Results

Previous work from our lab demonstrated that early physiologic measurements would predict mortality in the CLP sepsis model14,16. To increase the reproducibility of these data19,20 the studies were repeated by measuring physiologic variables six hours after the onset of sepsis. Heart rate (HR) was used to classify mice as predicted to die (P-Die) or live (P-Live). Since the HR was used to categorize the mice, the differences were not statistically analyzed. Mice classified as P-Live (HR > 620 beats/minute) had significantly lower pulse distension (PD), breath rate (BR) and body temperature compared to the P-Die mice, confirming our original data14 (Figure 1).

One hypothesis for sepsis induced cell and organ injury is a reduction in ascorbic acid (vitamin C) levels, which could be corrected with HAT. To test this hypothesis, mice were stratified into P-Die or P-Live based on six hour HR levels and randomized to receive HAT or vehicle. Mice were sacrificed 24 hours after CLP and plasma, peritoneal fluid and kidneys collected. P-Die mice treated with vehicle had significantly lower levels of ascorbic acid compared to P-Live mice. HAT therapy significantly increased these levels, but only in the P-Die mice (Figure 2A). A similar pattern was found in all three compartments: blood, peritoneal fluid and kidney homogenates. These data show the strength of stratifying septic individuals since the correction of the ascorbic acid levels would not have been observed without this stratification.

Figure 2: Vitamin C concentrations were decreased in P-Die mice and reversed by HAT treatment.

Figure 2:

Open in a new tab

Six hours after CLP, mice were stratified and each group (P-die or P-Live) was randomized to receive either HAT or Vehicle (VEH)> Mice were sacrificed 24 hours after CLP (18 hours after HAT Rx). Vitamin C concentrations in kidney were normalized by per total protein concentration (mg mediator/ml/mg protein.) P values calculated with Mann-Whitney test with Bonferroni correction for multiple comparisons.

We next examined if HAT treatment would correct the physiologic derangements observed in sepsis following the protocol of six hour stratification, randomization and treatment. Measurements taken 24 hours after sepsis demonstrated that HAT significantly reversed the declines in HR, PD, BR and body temperature, but only in the P-Die mice (Figure 3). Similar to the pattern observed with the ascorbic acid levels in figure 2, no changes in the physiology measurements were observed in the P-Live mice. Subgroup analysis by sex did not show an additional benefit to a sex with HAT therapy, in this analysis or any other data in figures 3–9.

Figure 9: Liver Injury in CLP mice.

Figure 9:

Open in a new tab

Acute liver injury was evaluated by analyzing plasma AST (Panel A), plasma Bilirubin (Panel B) and hepatic hypoxic injury (Panel C) 24-hour post CLP. There were no significant differences between the groups.

Plasma cytokines were measured 24 hours after CLP to determine the impact of HAT therapy (Figure 4) since previous studies showed that plasma cytokines were elevated in mice predicted to die21. The cytokines IL-6, IL-1RA, KC and MIP-2 were significantly increased in the P-Die mice. HAT therapy reduced these elevated cytokine levels, but only in the P-die group. HAT reduced the classic pro-inflammatory cytokine IL-6, as well as the chemokines KC and MIP-2. These effects (increased levels in P-Die mice which were depressed with HAT) were also found in peritoneal lavage fluid and kidney homogenates (data not shown). HAT also reduced the anti-inflammatory cytokine IL-1RA suggesting that HAT provided a return to homeostasis rather than acting as an anti-inflammatory agent.

Figure 4: Reduced systemic expression of inflammatory mediators by HAT treatment 24 hours post CLP.

Figure 4:

Open in a new tab

Plasma cytokines levels were significantly decreased by HAT therapy initiated after stratification into predicted to die (P-die) or P-live. P values were calculated using Mann-Whitney test since the data were not normally distributed, with Bonferroni correction.

Endothelial cell injury has been hypothesized as a major contributor to organ dysfunction observed in patients with sepsis22 and elevated plasma levels of glypicans 1 (GPC1) and 4 (GPC4) have been documented23. The increases in GPC1 and GPC4 are believed to be secondary to glycocalyx shedding from the injured endothelial cells. We collected blood 24 hours after the onset of CLP induced sepsis and measured GPC1 and GPC4. Figure 5 shows that both of these markers are increased in the P-Die mice compared to the P-Live. The elevated levels were decreased in the P-Die mice with HAT therapy.

Figure 5: HAT treatment significantly reduces markers of endothelial damage, glypican 1 (Panel A) and glypican 4 (Panel B).

Figure 5:

Open in a new tab

Mice were stratified, randomized for treatment and sacrificed 24 hours after the onset of sepsis. Glypican 1 and 4 were measured in the plasma by ELISA. P values calculated with Mann-Whitney test with Bonferroni correction for multiple comparisons.

Renal injury frequently occurs in sepsis2,15 the kidney may represent a source of the glypicans. The renal tubules have high oxygen consumption due to metabolic demands9. Studies were performed to evaluate renal hypoxia during sepsis using immunohistochemistry (IHC). When tissues are exposed to hypoxia 2-nitroimidazoles (hypoxyprobe) will form adducts in the tissue which may be detected with antibodies and IHC. For our studies CLP was performed, mice stratified and then randomized for HAT therapy. Two hours before sacrifice hypoxyprobe was injected intraperitoneally, organs collected and processed. The hypoxyprobe technique has been used previously to detect hypoxia in the CLP model9,24. Figure 6 shows representative IHC where there is intense staining in the predicted to die group treated with vehicle (VEH, Panel B). Closer examination of the histologic sections shows more intense staining in the renal medulla. The staining intensity was quantified with Image J which confirmed increased hypoxia in the P-die group that was prevented with HAT in the renal medulla (Panel F). A similar pattern was found in the renal cortex. These data show that significant renal hypoxia was present in the P-Die mice.

Figure 6: Hypoxic renal injury in P-die mice rescued with HAT.

Figure 6:

Figure 6:

Open in a new tab

Kidney hypoxia was evaluated by oxidative stress-specific IHC staining. Representative immunohistochemistry images are shown. Predicted to die mice treated with HAT (Panel A) had relatively weak staining while vehicle treated P-die mice had strong staining especially prominent in the medulla. Predicted to live mice HAT (Panel C), VEH (Panel D) and Naïve mice had weak staining. Stain intensity quantified with Image J showed a significant increase in medullary staining in the Vehicle treated P-die mice compared to P-live mice (Panel F) and HAT Rx decreased the staining. Similar results were observed in the renal cortex. P values calculated with Mann-Whitney test with Bonferroni correction.

The renal hypoxia documented in figure 6 could lead to oxidative stress which has been implicated as the cause of endothelial dysfunction in sepsis, including injury to the glycocalyx22. We previously reported that HAT reduced oxidative stress in the peritoneal cells, i.e. from the site of infection14. Prior reports have indicated that the kidney shows significant oxidative damage during sepsis25 and renal injury is frequently found in septic patients2. We examined the markers of oxidative stress in kidney homogenates. HAT therapy significantly reduced both carbonyl proteins and 8-iso-prostaglandin F2α (Figure 7).

Figure 7: HAT treatment significantly reduces renal oxidative stress markers, Carbonyl protein (Panel A) and 8-Iso Prostaglandin F2α (Panel B).

Figure 7:

Open in a new tab

Mice were stratified, randomized for treatment and sacrificed 24 hours after the onset of sepsis. Kidneys were harvested and homogenized. The concentrations of markers were determined by ELISA and normalized by per total protein concentration (mg or nmol/ml/mg protein) in kidney. P values calculated with Mann-Whitney test.

We previously reported that CLP induced sepsis primarily causes renal injury and that HAT therapy improves survival. The next experiments tested whether the HAT induced decreases in hypoxia and oxidative stress could be mechanisms resulting in better renal function. The power of the stratification protocol is shown in figure 8, where the P-die mice had significantly greater plasma BUN compared to the P-Live mice. These data replicate prior work where stratification based on plasma levels of IL-6 showed that P-Die mice had greater renal damage15. HAT therapy significantly reduced the plasma BUN, but only in the P-Die mice. Peritoneal BUN levels showed a similar pattern (Figure 8, panel B). Cystatin C is a marker of renal injury, primarily injury to the glomerulus. P-Die mice had increased cystatin C which was reduced with HAT (Figure 8 Panel C). While cystatin C was reduced, the levels were not reduced as dramatically as the BUN. This correlates with the hypoxic injury in figure 6 where there was a greater hypoxyprobe staining intensity in the medulla than the cortex.

Figure 8: Acute kidney Injury in CLP mice.

Figure 8:

Open in a new tab

HAT treatment reduces the BUN concentrations in plasma (Panel A) and peritoneal lavage (Panel B), and plasma cystatin C (Panel C) in P-Die mice 24 hours after CLP. P-die mice also had significantly higher BUN and cystatin C levels than P-Live mice. P values were calculated with Mann-Whitney test with Bonferroni correction.

The low pulse distention (a surrogate for cardiac output) corrected with HAT therapy (Figure 3B) suggests that hypotension and diminished global tissue perfusion may have caused the renal injury. This hypothesis was examined by evaluating hepatic function after CLP (Figure 9). Plasma collected 24 hours after the onset of sepsis did not show significant elevations in AST (marker of hepatocyte injury, Panel A) or bilirubin as a marker of liver function (Panel B). Sections of liver examined for increased hypoxyprobe staining to determine if liver hypoxia was present did not show any significant elevations in tissue staining (Panel C).

Discussion

Our stratification protocol showed that mice predicated to die had substantial renal hypoxia, oxidative stress and renal injury. Our results are similar to Wang et al who demonstrated that in CLP sepsis there is renal oxidative stress and AKI9. These investigators showed increased hypoxia and oxidative stress in the renal tubules after CLP. Interestingly, there was little evidence of hypoxia in the renal glomeruli even though there was a reduction in the glomerular filtration rate. The hypoxia in the renal medulla may be a therapeutic target when treating sepsis patients26. It has been suggested that sepsis induced AKI has a different pathophysiology that AKI caused by just hypoperfusion7, a concept supported by our data showing specific injury to the kidney and not the liver.

The current publication shows that in mice predicted to die from sepsis, renal hypoxia leads to oxidative stress within the kidney and subsequent renal injury. This injury could be corrected with HAT therapy that was initiated after stratification. It is critical to point out that this mechanism of renal hypoxia would not have been observed if we considered all septic mice in one group (i.e. P-Die combined with P-Live). If just the HAT vs VEH groups were compared there was no difference between these groups. Sample size analysis showed that 204 mice would need to be analyzed to show that HAT protects the kidneys. Machine learning tools may define sepsis phenotypes that will help with stratification27.The importance of stratification has been highlighted during the COVID-19 pandemic where defining clinical phenotypes showed a doubling of mortality in one subgroup28. Another study examining community acquired pneumonia also found two subgroups, and only the group at higher risk of dying responded to steroid therapy29. Using the CLP peritonitis model of sepsis we have previously reported similar results, that only mice with the highest risk of dying responded to glucocorticoid therapy30. The current manuscript highlights the importance of stratification in experimental studies19.

Substantial controversy exists about whether HAT reduces mortality in septic patients. Since the initial report12 there have been retrospective cohort studies and randomized clinical trials which have been subjected to meta-analysis13,31. Both of these meta-analyses of the completed trials showed that HAT therapy was not effective, although there was no evidence of harm. The largest of these trials (VICTAS) was terminated early for administrative reasons and specifically stated that it may have been underpowered32. The study did evaluate kidney injury and HAT did not improve renal function. The limitations of the VICTAS study were addressed in an accompanying editorial that noted the underpowered study and the lack of harm in the HAT group33. For all of the clinical HAT studies, patients were not stratified for therapy and our results show that stratification will reduce the number of subjects that need to be studied.

There are two important conclusions from the current study. First, stratification based on probability of death allows therapy to be targeted to those who will benefit. Stratification allows enrollment of fewer subjects, and prior clinical studies with HAT have been underpowered13,31. Second, our results show that HAT therapy specifically protects the sepsis-induced AKI through the mechanisms of reduced oxidative stress. Our results highlight how animal models, which allow access to tissue, can provide mechanistic insights into the pathogenesis of disease.

Limitations to the current study must acknowledged. Glypican 1 and 4 were measured as markers of endothelial cell injury although several other markers such as syndecans have been used in the past. The glypicans were selected since ELISA kits were available during COVID. We did not perform immunohistochemistry to show that the elevated glypicans in the blood were derived from the kidney rather than other organs. These were not performed due to the complexity of establishing a sensitive assay, and the results would only be supporting evidence that the kidneys were injured. Electron microscopy was not performed which could have shown evidence of mitochondrial damage34 not appreciated with light microscopy. There are multiple biomarkers of renal injury35 and we only measured two.

Supplementary Material

Video File for highlighting
Download video file (5.6MB, mp4)

Acknowledgments

Supported by NIH grants UL1TR001430, S10 OD026983, R21AI147168, and T32GM098308. Michelle Wei contributed to these studies.

References

  • 1.Rudd KE, Johnson SC, Agesa KM, et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the Global Burden of Disease Study. Lancet. Jan 18 2020;395(10219):200–211. doi: 10.1016/S0140-6736(19)32989-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Peerapornratana S, Manrique-Caballero CL, Gómez H, Kellum JA. Acute kidney injury from sepsis: current concepts, epidemiology, pathophysiology, prevention and treatment. Kidney Int. November 2019;96(5):1083–1099. doi: 10.1016/j.kint.2019.05.026 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. Acute kidney injury. Nat Rev Dis Primers. July 15 2021;7(1):52. doi: 10.1038/s41572-021-00284-z [DOI] [PubMed] [Google Scholar]
  • 4.Bagshaw SM, Lapinsky S, Dial S, et al. Acute kidney injury in septic shock: clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy. Intensive Care Med. May 2009;35(5):871–81. doi: 10.1007/s00134-008-1367-2 [DOI] [PubMed] [Google Scholar]
  • 5.Trifi A, Abdellatif S, Masseoudi Y, et al. COVID-19-induced acute kidney injury in critically ill patients: epidemiology, risk factors, and outcome. Acute Crit Care. Nov 2021;36(4):308–316. doi: 10.4266/acc.2021.00934 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Panwar R, Tarvade S, Lanyon N, et al. Relative Hypotension and Adverse Kidney-related Outcomes among Critically Ill Patients with Shock. A Multicenter, Prospective Cohort Study. Am J Respir Crit Care Med. November 15 2020;202(10):1407–1418. doi: 10.1164/rccm.201912-2316OC [DOI] [PubMed] [Google Scholar]
  • 7.Gomez H, Ince C, De Backer D, et al. A unified theory of sepsis-induced acute kidney injury: inflammation, microcirculatory dysfunction, bioenergetics, and the tubular cell adaptation to injury. Shock. Jan 2014;41(1):3–11. doi: 10.1097/SHK.0000000000000052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Dyson A, Bezemer R, Legrand M, Balestra G, Singer M, Ince C. Microvascular and interstitial oxygen tension in the renal cortex and medulla studied in a 4-h rat model of LPS-induced endotoxemia. Shock. Jul 2011;36(1):83–9. doi: 10.1097/SHK.0b013e3182169d5a [DOI] [PubMed] [Google Scholar]
  • 9.Wang Z, Holthoff JH, Seely KA, et al. Development of oxidative stress in the peritubular capillary microenvironment mediates sepsis-induced renal microcirculatory failure and acute kidney injury. Am J Pathol. Feb 2012;180(2):505–16. doi: 10.1016/j.ajpath.2011.10.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.van der Slikke EC, Boekhoud L, Bourgonje AR, et al. Plasma Free Thiol Levels during Early Sepsis Predict Future Renal Function Decline. Antioxidants (Basel). Apr 19 2022;11(5)doi: 10.3390/antiox11050800 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Moss SR, Prescott HC. Current Controversies in Sepsis Management. Semin Respir Crit Care Med. October 2019;40(5):594–603. doi: 10.1055/s-0039-1696981 [DOI] [PubMed] [Google Scholar]
  • 12.Marik PE, Khangoora V, Rivera R, Hooper MH, Catravas J. Hydrocortisone, Vitamin C, and Thiamine for the Treatment of Severe Sepsis and Septic Shock: A Retrospective Before-After Study. Chest. Jun 2017;151(6):1229–1238. doi: 10.1016/j.chest.2016.11.036 [DOI] [PubMed] [Google Scholar]
  • 13.Zayed Y, Alzghoul BN, Banifadel M, et al. Vitamin C, Thiamine, and Hydrocortisone in the Treatment of Sepsis: A Meta-Analysis and Trial Sequential Analysis of Randomized Controlled Trials. J Intensive Care Med. Mar 2022;37(3):327–336. doi: 10.1177/0885066620987809 [DOI] [PubMed] [Google Scholar]
  • 14.Kim J, Arnaout L, Remick D. Hydrocortisone, Ascorbic Acid, and Thiamine (HAT) Therapy Decreases Oxidative Stress, Improves Cardiovascular Function, and Improves Survival in Murine Sepsis. Article. Shock. Apr 2020;53(4):460–467. doi: 10.1097/shk.0000000000001385 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Craciun FL, Iskander KN, Chiswick EL, Stepien DM, Henderson JM, Remick DG. Early murine polymicrobial sepsis predominantly causes renal injury. Shock. Feb 2014;41(2):97–103. doi: 10.1097/SHK.0000000000000073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Remick DG, Bolgos GR, Siddiqui J, Shin J, Nemzek JA. Six at six: interleukin-6 measured 6 h after the initiation of sepsis predicts mortality over 3 days. Shock. Jun 2002;17(6):463–7. [DOI] [PubMed] [Google Scholar]
  • 17.Mella JR, Chiswick E, Stepien D, et al. Antagonism of the Neurokinin-1 Receptor Improves Survival in a Mouse Model of Sepsis by Decreasing Inflammation and Increasing Early Cardiovascular Function. Crit Care Med. Feb 2017;45(2):e213–e221. doi: 10.1097/CCM.0000000000002075 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kim J, Merry AC, Nemzek JA, Bolgos GL, Siddiqui J, Remick DG. Eotaxin Represents the Principal Eosinophil Chemoattractant in a Novel Murine Asthma Model Induced by House Dust Containing Cockroach Allergens. J Immunol. September 1, 2001. 2001;167(5):2808–2815. [DOI] [PubMed] [Google Scholar]
  • 19.Osuchowski MF, Ayala A, Bahrami S, et al. Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS): An International Expert Consensus Initiative for Improvement of Animal Modeling in Sepsis. Shock. Oct 2018;50(4):377–380. doi: 10.1097/SHK.0000000000001212 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Hsieh T, Vaickus MH, Remick DG. Enhancing Scientific Foundations to Ensure Reproducibility: A New Paradigm. Am J Pathol. Jan 2018;188(1):6–10. doi: 10.1016/j.ajpath.2017.08.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Osuchowski MF, Welch K, Siddiqui J, Remick DG. Circulating cytokine/inhibitor profiles reshape the understanding of the SIRS/CARS continuum in sepsis and predict mortality. J Immunol. Aug 1 2006;177(3):1967–74. [DOI] [PubMed] [Google Scholar]
  • 22.Joffre J, Hellman J. Oxidative Stress and Endothelial Dysfunction in Sepsis and Acute Inflammation. Antioxid Redox Signal. November 20 2021;35(15):1291–1307. doi: 10.1089/ars.2021.0027 [DOI] [PubMed] [Google Scholar]
  • 23.Fisher J, Linder A, Bentzer P. Elevated plasma glypicans are associated with organ failure in patients with infection. Intensive Care Med Exp. Jan 07 2019;7(1):2. doi: 10.1186/s40635-018-0216-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Rocheteau P, Chatre L, Briand D, et al. Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy. Nat Commun. Dec 15 2015;6:10145. doi: 10.1038/ncomms10145 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lv D, Zhang Y, Wang C, Gu Y, Li X. Platelets Derived Transthyretin Participate in The Development of Sepsis Associated Acute Kidney Injury by Inducing Oxidative Stress and Apoptosis of Renal Tubular Epithelial Cells. Shock. May 01 2022;57(5):722–731. doi: 10.1097/SHK.0000000000001908 [DOI] [PubMed] [Google Scholar]
  • 26.Lankadeva YR, Okazaki N, Evans RG, Bellomo R, May CN. Renal Medullary Hypoxia: A New Therapeutic Target for Septic Acute Kidney Injury? Semin Nephrol. November 2019;39(6):543–553. doi: 10.1016/j.semnephrol.2019.10.004 [DOI] [PubMed] [Google Scholar]
  • 27.Stolarski AE, Kim J, Nudel J, Gunn S, Remick DG. Defining Sepsis Phenotypes-Two Murine Models of Sepsis and Machine Learning. Shock. Jun 01 2022;57(6):268–273. doi: 10.1097/SHK.0000000000001935 [DOI] [PubMed] [Google Scholar]
  • 28.Ranjeva S, Pinciroli R, Hodell E, et al. Identifying clinical and biochemical phenotypes in acute respiratory distress syndrome secondary to coronavirus disease-2019. EClinicalMedicine. Apr 2021;34:100829. doi: 10.1016/j.eclinm.2021.100829 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wittermans E, van der Zee PA, Qi H, et al. Community-acquired pneumonia subgroups and differential response to corticosteroids: a secondary analysis of controlled studies. ERJ Open Res. Jan 2022;8(1)doi: 10.1183/23120541.00489-2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Osuchowski MF, Connett J, Welch K, Granger J, Remick DG. Stratification is the key: Inflammatory biomarkers accurately direct immunomodulatory therapy in experimental sepsis*. Crit Care Med. Mar 25 2009; [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Somagutta MKR, Pormento MKL, Khan MA, et al. The Efficacy of vitamin C, thiamine, and corticosteroid therapy in adult sepsis patients: a systematic review and meta-analysis. Acute Crit Care. Aug 2021;36(3):185–200. doi: 10.4266/acc.2021.00108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sevransky JE, Rothman RE, Hager DN, et al. Effect of Vitamin C, Thiamine, and Hydrocortisone on Ventilator- and Vasopressor-Free Days in Patients With Sepsis: The VICTAS Randomized Clinical Trial. JAMA. February 23 2021;325(8):742–750. doi: 10.1001/jama.2020.24505 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Walter KL, Seymour CW. Hydrocortisone, Vitamin C, and Thiamine for Treatment of Sepsis: Making Evidence Matter. JAMA. February 23 2021;325(8):730–731. doi: 10.1001/jama.2020.26029 [DOI] [PubMed] [Google Scholar]
  • 34.Guo J, Wang R, Liu D. Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate Sepsis-Induced Acute Kidney Injury by Promoting Mitophagy of Renal Tubular Epithelial Cells. Front Endocrinol (Lausanne). 2021;12:639165. doi: 10.3389/fendo.2021.639165 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Molinari L, Del Rio-Pertuz G, Smith A, et al. Utility of Biomarkers for Sepsis-Associated Acute Kidney Injury Staging. JAMA Netw Open. May 02 2022;5(5):e2212709. doi: 10.1001/jamanetworkopen.2022.12709 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video File for highlighting
Download video file (5.6MB, mp4)

RESOURCES