Solution of PHZ of 25 mg/mL (Sigma-Aldrich) was prepared in PBS immediately before use. The Fe³⁺ form of heme [hemin (ferriprotoporphyrin IX), designated as heme (Frontier Scientific Inc. or Sigma-Aldrich)] was dissolved to 20 mM in 50-mM NaOH and 145-mM NaCl, and further diluted in PBS just before use. Plasma-purified Hx was provided by CSL Behring.

All experiments were conducted in accordance with the recommendations for the care and use of laboratory animals following the ARRIVE regulations and with the approval APAFIS#3764-201601121739330v3 of the French Ministry of Agriculture. C57Bl/6 mice were from Charles River Laboratories (L’Arbresle, France). Female C57Bl/6 mice were injected i.p. with 200 µL of PBS (Gibco), or freshly prepared heme [40 µmol/kg, corresponding to 28-µg/g body weight (20)] repeated 24 h later (days 0 and 1). PHZ (900 µmol/kg, corresponding to 0.125-mg/g body weight) was injected i.p. in at day 0 and C57Bl/6 mice sacrificed by cervical decerebration at days 1, 2, or 4 at the age of 8 weeks. Only single-sex mice were used for consistency and female were selected since they are less aggressive. To test the efficacy of Hx, mice were pretreated with i.p. injection of 40 µmol/kg of human Hx 1 h before i.p. injection of 900 µmol/kg of PHZ. Concentrations and route of administration were chosen as described (19–21). Mice kidneys were recovered and snap-frozen in liquid nitrogen or fixed and included in paraffin.

The follow-up of renal function was performed by placing 6-week-old C57Bl/6 female mice (Charles Rivers) in individual metabolic cages for acclimation for 5 days, with daily weight measurement and fixed, daily amounts of food. After stabilization of body weight (day −3) metabolic parameters were measured (diuresis, amount of excrements, water and food uptake, and body weight). Urine was collected at fixed times for 3 days at baseline, and 2 days after injections. PHZ, heme, or PBS were injected at day 0. Blood was taken from the retro-orbital sinus before and at day 2 after treatment. Blood gas was measured at day 2 after treatment (Alere system, self-calibrated epoc^® BGEM Test Card). Organs were recovered at day 2. In alternative experiments in regular cages organs were recovered at day 4. Organs were snap-frozen in liquid nitrogen in Cryomatrix (Thermoscientific). Urinary protein and creatinin levels, as well as plasma urea concentrations were measured using Konelab equipment. For comparative purposes, similar experiments were performed in regular cages, with five animals per group.

Moreover, 3-μm-thick sections of fixed (PFA 4%) frozen kidneys were cut with Cryostat Leica AS-LMD. Heme oxygenase 1 (HO-1) expression was studied using rabbit anti-mouse HO-1 (Abcam, Ab13243) followed by a polymer anti-rabbit IgG-HRP (DAKO, K4003). Staining was revealed with DAB solution. Slides were scanned by Nanozoomer (Hamamatsu). Hematoxylin–Eosin, Perl’s Prussian blue, and PAS coloration were performed by routine procedures using sections of paraffin-embedded kidneys at days 1, 2, and 4. Coloration of slides was scanned by Slide Scanner Axio Scan (Zeiss).

Snap-frozen kidney sections were recovered in RLT buffer (Qiagen) + 1% β-mercaptoethanol (Gibco) and used for mRNA extraction with Qiagen RNeasy miniKit. The quality and quantity of mRNA were evaluated with bioanalyser Agilent 2100 using Agilent TNA 6000 NanoKit and if the RNA integrity number was >7 the mRNA was retrotranscribed to cDNA. Gene markers of early tubular and endothelium activation/injury relevant for hemolysis and SCD were analyzed by low-density array (LDA, ThermoFisher) including NGAL, Kim-1, HO-1, Il-6, Ki67, ICAM-1, E-selectin and P-selectin, Caspase-3, and CD31 (16, 22–25), and validated by RTqPCR for NGAL, Kim-1, HO-1, and Ki67 (ThermoFisher). Il-1β and TNF-α have been tested by RTqPCR (ThermoFisher).

Urine of the mice recovered from the metabolic cages was centrifuged and diluted 1/2 with a sample buffer containing glycerol and bromophenol blue (but not SDS) and 20 µL are deposited on 10 wells 12% gels and migrated for 40 min. Staining the gel-resolved proteins was performed with Coomassie blue.

After discoloration, bands of interest were excised from the Coomassie blue-stained SDS page gel at the same level for the PBS, heme and PHZ-treated animals. Samples were then reduced with the addition of DTT (4.2 mM, final concentration) for 45 min at 37°C, then alkylated with iodoacetamide (7.6 mM, final concentration) and subjected to in-gel tryptic digestion using porcine trypsin (Promega, France) at 12.5 ng/µL. The dried peptides extracts were dissolved in 12 µL of solvent A (2% acetonitrile, 0.1% formic acid) and analyzed by online nanoLC using an Ultimate 3000 System (Dionex) coupled to a LTQ-XL Orbitrap mass spectrometer (Thermo Fisher Scientific). Each peptide extract (5 µL) was loaded on a C18 precolumn (Acclaim PepMap C18, 5-mm length × 300-µm I.D., 5-µm particle size, 100-Å porosity, Dionex) at 20 µL/min in solvent A. After 5-min desalting, the precolumn was switched online with a C18 capillary column (Acclaim PepMap C18, 15-cm length × 75-µm ID × 3-µm particle size, 100-Å porosity, Dionex) equilibrated in solvent A. Peptides were eluted using a 0–70% gradient of solvent B (80% acetonitrile, 0.1% formic acid) during 50 min at a flow rate of 300 nL/min. The LTQ-XL Orbitrap was operated in data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap in the 400–1,600 m/z range with the resolution set to a value of 60,000. The five most intense ions per survey scan were selected for collision-induced dissociation (CID) fragmentation and the resulting fragments were analyzed in the linear trap (LTQ). Dynamic exclusion was employed within 45 s to prevent repetitive selection of the same peptide.

Peak lists extraction from XCalibur raw files were automatically performed using Proteome Discover software (version 1.4, Thermo Fisher scientific). Database searches were performed using the Mascot server v2.2.07 with the following parameters: database Mouse; enzymatic specificity: tryptic with two allowed missed cleavages; fixed modification of cysteine residues [carbamidomethylated (C)]; variable oxidation of methionine residues; 5-ppm tolerance on precursor masses and 0.6-Da tolerance on fragment ions; fragment types taken into account were those specified in the configuration “ESI-trap.”

The gels with urine proteins, resolved by electrophoresis as above were transferred to nitrocellulose membranes using iBlot equipment (Invitrogen) and stained with Rabbit polyclonal anti-pancreatic alpha amylase antibody (Abcam, ab199132) using SNAP technology (Merk Millipore), followed by immunodetection with Goat anti Rabbit IgG-HRP (Santa Cruz, 1/5,000). The signal was revealed by chemiluminescence.

The levels of α-amylase were detected in mouse plasma, using Colorimetric Amylase Assay Kit (Abcam, ab102523) using the protocol provided by the manufacturer.

Primary human umbilical vein endothelial cells (HUVEC) were cultured as described previously (8, 26) and treated with decreasing doses of PHZ: 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, 0.019 mg/mL, or TNF-α 10 ng/mL or medium only for 24 h. Cells in 24-well plates were detached with trypsine and washed with cold PBS. The cells were resuspended in 300 µL of PBS BSA 2%, dispatched in three tubes and labeled with anti-E-selectine antibody 1:50 (Immunotech, 1243) or mouse anti-VCAM1 FITC antibody 1:50 (AbD serotec, 0770) or mouse anti-ICAM-1 antibody 1:50 (Beckman, IM0544) or anti-P-selectin antibody (BIORAD, MCA796) for 30 min at 4°C. For the ICAM-1 and P-selectin staining, a goat anti-mouse PE antibody 1:100 (Beckman, IM0855)was used for 30 min at 4°C. After washing, the cells were resuspended in 100 µL of annexin-V-binding buffer (BD, 556454) and labeled with annexin-V-APC (1:100) (BD, 550474) for 15 min at room temperature and DAPI (1:100). After the incubation, 400 µL of annexin buffer was added before flow-cytometry analysis (FACS LSR II).

Results were analyzed using a statistical software package (GraphPad Prism 5) as indicated in figures legends. Briefly, * p < 0.05, ** p < 0.005, and *** p < 0.001. Multiple groups in the mRNA level analyses were compared with Kruskal–Wallis with Dunn’s test for multiple pairwise comparisons. When only two groups were compared, Mann–Whitney test was applied. The metabolic parameters were compared for the mice before and after indicated treatments using two-way ANOVA with Tukey’s test for multiple pairwise comparisons.

To find out to what extend the acute intravascular hemolysis and heme induce renal injury, we evaluated the parameters of the kidney function, placing the mice in metabolic cages. Blood gas analyses revealed that heme- and PHZ-treated mice had decreased blood hemoglobin at day 2 compared with PBS controls of about 8% (not reaching significance) and significant 46%, respectively (Figure 1A). This erythrocytes lysis was associated with a significant increase in lactate as a surrogate marker of hypoxia and cell damage (Figure 1B) and a decrease in the HCO₃⁻, a marker of metabolic acidosis (Figure 1C) in the PHZ group. The heme-treated group presented with slightly decreased blood glucose at day 2, without reaching significance (Figure 1D). Plasma Na⁺ was increased in the PHZ-treated groups (Figure 1E), while K⁺ (Figure 1F) and Ca²⁺ (Figure 1G) remained unaltered. Furthermore, we measured urea and creatinine to estimate the changes in glomerular filtration rate. Plasma creatinine concentration (Figure 1H) and the plasma urea (Figure 1I) were significantly increased in PHZ-treated mice compared with the baseline. Moreover, heme treatment also increased the plasma urea in a significant manner compared with baseline (Figure 1I). PHZ- and heme-injected mice also presented with an oliguria (24-h diuresis) at day 1 (Figure 1J) which persisted in day 2 for PHZ but normalized for heme-injected mice. These were accompanied by reduced water (Figure 1K) and food (Figure 1L) intake, resulting in a decrease of excrements (Figure 1M) and 13% weight loss at day 2 for the PHZ-injected mice (Figure 1N). Proteinuria normalized by the creatininuria was not significantly different in PHZ- and heme-treated mice compared with PBS group (Figure 1O). Alteration of kidney function parameter is most likely functional, due to the anemia and reduced food and water uptake and less likely due to renal tissue damages.

Kidneys (Figure 2A), blood as well as liver and spleen of PHZ-treated mice were dark in color, compared with PBS-injected controls, while heme-treated mice showed only mild dark coloration. To gain a deeper insight whether acute hemolysis induced tissue injury in the kidney, renal sections were analyzed by histology. By light microcopy, paraffin-embedded kidney sections of the mice treated with PBS or PHZ were similar regarding the analysis of glomeruli, that were normal with no modification of glomeruli size, endocapillary proliferation, glomeruli were not congested, and did not shown neither TMA lesions nor mesangial proliferation at day 2 (not shown) and day 4 (Figures 2B,C). No major modification of renal histology was detected, except minor but significant increase of tubular dilatation at day 4, a sign of acute tubular necrosis at day 4 (p = 0.009) (Figures 2B,C). No signs of immune cells infiltration were detected by histology at days 1, 2 (data not shown), or 4 (Figures 2B,D) and CD45 staining (data not shown). Perl’s Prussian blue staining for hemosiderin, a hallmark for hemolysis, was positive in the PHZ-injected mice (Figure 2E).

Furthermore, we analyzed at gene level a number of more sensitive markers for inflammation and tissue injury in the acute hemolysis model. Sustained expression of markers of tubular injury NGAL and Kim-1, till days 2 and 4, respectively (Figures 3A,B), and sustained expression of markers of proliferation––Ki67 and inflammation––IL-6 till day 2 (Figures 3C,D) were detected. How ever, endothelial activation E-selectin, P-selectin, and ICAM-1 (Figures 3E–G) were detected only at day 1. No difference in immune cell-produced inflammation mediators IL-1b and TNF-α, apoptosis marker Caspase-3 as well as endothelial cell junction factor CD31 was detected (not shown).

To find out whether the endothelial activation was due to intravascular hemolysis or related to direct effects of PHZ, endothelial cells (HUVEC) were exposed to PHZ in vitro for 2 h (the duration of the in vivo experiment). Increasing doses up to 0.312 mg/mL did not promote apoptosis (over 80% of the cells being annexin-V⁻ DAPI⁻), while higher doses resulted in a massive cell death (Figure 3H). In addition, incubation of PHZ for 24 h did not induce cell-surface expression of adhesion markers such as E-selectin, VCAM-1, or ICAM-1, contrary to 10 µg/mL of TNF-α (Figures 3I–K). Neither PHZ nor TNF-α triggered surface expression of P-selectin (not shown).

To find out whether these changes are heme-dependent or occur due to upstream products (as hemoglobin), we investigated expression of NGAL, Kim-1, and IL-6 in heme-treated mice at day 2. We detected a significant increase of NGAL expression (Figure 4A), about 50% weaker compared with NGAL expression in PHZ-treated mice at day 2. However, no modification of mRNA levels of Kim-1 and IL-6 (Figures 4B,C) were detected. Furthermore, the i.p. injection of the heme scavenger human plasma-derived Hx did not prevent the expression of these genes after PHZ treatment (Figures 4D–I), showing a largely heme-independent process.

We analyzed the expression of the same gene panel in liver and spleen and observed similar alterations of a set of genes in the PHZ-treated mice, indicating that this model is associated with multiorgan lesions (data not shown).

Strong upregulation of the cytoprotective enzyme HO-1 till day 4 at both gene (Figures 5A,B) and protein levels (Figure 5C) was detected in kidneys of PHZ-injected mice. Preinjection of Hx did not modify expression of HO-1 in PHZ-treated mice (Figure 5B). Treatment with heme also resulted in upregulation of this enzyme (Figure 5C). HO-1 staining was localized in the tubules. Immunohistochemistry revealed no HO-1 expression in PBS-treated mice (Figure 5C).

To find out if the tubular necrosis and the oxidative and inflammatory stress in the kidney result in alteration of proteinuria, the profile of the urinary protein content was examined by electrophoresis (Figure 6A). When the 24-h urine samples were analyzed at equal volume, without normalization to creatinine, it revealed increased total protein content in the PHZ-injected mice (in agreement with the measurement of total protein). Nevertheless, when the urine was diluted to normalize to creatinine, the protein content was similar to the PBS-treated mice (not shown). Alteration of the renal endothelium could affect the glomerular microvasculature and the filtration function of the kidney. A distinction between glomerular and tubular proteinuria could be made in experimental models based on the profile of urine electrophoresis, appearance of low-molecular weight proteins (<60 kDa) suggesting tubular damage, while high-molecular weight proteins (>60 kDa) indicating glomerular one. No apparent major imbalance of the higher and lower molecular weight proteins was revealed by the electrophoresis, when normalization to creatinine was performed, but a tendency to higher level of low-molecular weight proteins was noted (not shown).

Nevertheless, when samples were deposited at equal volume (for qualitative analyses) several proteins that were not present in the PBS controls were specifically identified in the PHZ and heme samples. To identify these proteins and find out whether they reflect alteration of the tubular or glomerular function, a mass spectrometry analyses were performed by in-gel trypsin digestion of the proteins. Urine of PHZ-treated animals was positive for meprin-α, cytoskeletal keratins, α-1-antitrypsine and α-1-microglobulin (protein AMBP), while heme-treated mice urine was positive for meprin-α only (Table 1).

The gels revealed also a presence of a thick ~55-kD band, characteristic for the heme and to less extend to PHZ-injected mice, which was absent in the PBS controls (Figure 6A). Mass spectrometry analyses revealed that the protein is pancreatic α-amylase (Table 1). In-debt peptides analyses were performed to distinguish between salivary amylase and pancreatic amylase, confirming the presence of the pancreatic isoform of the enzyme (data not shown). This result was confirmed by western blot in the PHZ-injected group (Figure 6B).

Presence of pancreatic α-amylase in the urine could suggest an acute pancreatitis, caused by heme toxicity and hemolysis. To test this hypothesis, measurement of plasmatic pancreatic α-amylase was performed and these were found indeed increased in PHZ-treated animals as compared with controls (Figure 6C).