Effects of the treatment with the Mas receptor agonist, AVE 0991, and the AT1 receptor blocker, Losartan, on adriamycin-induced renal injury.

<p>Adriamycin (ADR, 10 mg/kg) was injected in the tail vein of Balb/c mice. Animals were treated daily with vehicle (VE, filtered water), AVE0991 (AVE, 3 mg/kg) or Losartan (LOS, 10 mg/kg) by gavage from days 7 to day 14 day after ADR injection. The sham group received a single injection of NaCl 0.9% in the tail vein and was treated with filtered water. Microalbuminuria (A) and serum albumin levels (B) were evaluated on day 14 in n = 6–10 mice per group. The panel also shows urinary levels of TGF-β (C) in n = 4–5 mice per group. (*) for P<0.05 when compared to VE group and (#) for P<0.05 when compared to sham group.</p

Time course of adriamycin-induced renal dysfunction.

<p>Adriamycin (10 mg/kg) was injected in the tail vein of Balb/c mice. The following parameters were evaluated at days 7, 14 and 21 after ADR injection: mean body weight (g), systolic blood pressure (mmHg), albumin/creatinine ratio, serum albumin (g/dL), urinary and serum creatinine (mg/dl). Results are mean ± SEM of 6–10 mice per group. (*) for P<0.01 when compared to day 0.</p

mRNA expression of <i>Mas</i>, <i>AT1</i> and ACE2 in the kidneys after injection of adriamycin.

<p>In A, <i>Mas</i> and <i>AT1</i> mRNA levels were evaluated in the kidney before (Sham) and 7, 14 and 21 days after injection of adriamycin (ADR, 10 mg/kg). In B, <i>Mas</i> and <i>AT1</i> mRNA levels at day 14 in kidneys of mice injected with adriamycin that were treated with vehicle (VE) or losartan (LOS, 10 mg/kg). In C, ACE2 mRNA levels at day 14 in kidneys of mice injected with adriamycin that were treated with vehicle (VE) or losartan (LOS, 10 mg/kg). The dotted line across the graphs represents levels in control animals. Renal mRNA levels of receptors and ACE2 were estimated by real time PCR. Results are mean ± SEM of n = 5 mice per group. (*) for P<0.05 when compared to sham group.</p

Adriamycin-induced morphological changes in glomerular and tubular regions of the kidney.

<p>Representative photographs of PAS-stained glomerular and tubular regions noticed in control mice (Sham, A, B), and 7 (C, D), 14 (E, F) and 21 (G, H) days after injection of adriamycin (10 mg/kg). Glomerular damage (large arrows), tubule-interstitial changes, atrophy of tubular epithelial cells (arrowheads) and tubular enlargement (thin arrows, insert) increased in the renal cortex from day 7 (D) to day 14 (F, insert, arrow). Global sclerosis was observed in many glomeruli (E, asterisks). Resorption droplets were present in tubular cells at day 14 (panel F, thin arrows). Histological changes stabilized on day 21 (G, H). Original magnification 40× objective.</p

Histological changes and index of tubulointertitial and glomerular injury of ADR-induced nephrosis at day 14.

<p>Adriamycin (10 mg/kg) was injected in tail vein of Balb/c mice, on day 0 in vehicle-treated, VE (filter water), AVE0991-treated, AVE, (AVE 0991, 3 mg/kg) and Losartan-treated, LOS groups (losartan, 10 mg/kg). Representative photographs of PAS stained of glomerular and tubular regions were obtained at 14^th day. All mice were treated by gavage, daily, 7^th to 14^th day after ADR-induction. Glomerular (large arrow) and tubular injuries (thin arrows) observed in vehicle-treated mice (A–B) were attenuated by treatment with AVE 0991 (C–D) or Losartan (E–F). Original magnification 4× objective (A, C) and 40× (B, D). The indexes of tubulointerstitial (G) and glomerular (H) injuries were graded in a blind manner, as described in methods section. Symbols represent results in single animals and the trace is median value for 5–8 animals. (*) for P<0.05 when compared to 14^th day after ADR-induction group and (#) for P<0.05 when compared to sham group.</p

Treatment with losartan protects from adriamycin-induced renal damage in wild type (Mas^+/+) but not Mas deficient (Mas^−/−) mice.

<p>Adriamycin (10 mg/kg) was injected in the tail vein of Mas^+/+ (A–D) and Mas^−/− (E–H) FVBN mice. Animals were treated with water (VE, A–B and E–F) or Losartan (LOS, 10 mg/kg, C–D and G–H) and morpohological changes evaluated at day 14 after adriamycin injection. Glomerular (large arrow) and tubular injuries (thin arrows) are showed. Indices of tubulointerstitial (I) and glomerular (J) injuries were graded in a blind manner, as described in the Methods section. PAS-stained sections and magnification 10× (A, C, E, G) and 40× (B, D, F, H). Symbols represent results in single animals and the trace is median value. (*) for P<0.05 when compared to VE-treated group and (#) for P<0.05 when compared to sham group.</p

Disease parameters in C57BL/6 mice infected with an adapted strain of DENV-3.

<p>(A) WT mice (<i>n</i> = 6 mice per group) were inoculated with different inoculums of adapted-DENV-3 (i.p) and lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. In Figs (B–L) WT mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection mice were culled and blood and tissues were collected for the following analysis: (B) Change in body weight was expressed as percentage of initial weight loss. (C) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (D), hematocrit was expressed as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (E) Changes in vascular permeability in the liver and lungs are shown as µg Evans blue per 100 mg of tissue (left and right panels, respectively). (F) Shows changes in Systolic blood pressure from baseline until day 7 after infection expressed as Δ of blood pressure in mmHg. (G) AST (left panel) and ALT (right panel) activity determination in plasma of control and DENV-3-infected mice was shown as U/dL of plasma. (H–L) Concentrations of IL-6, TNF-α, IFN-γ IL-12/23p40 and IL-18, quantified by ELISA. Results are shown as pg per mL (serum) or pg per 100 mg (tissue). All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND – not detectable. NA – not assessed. NI- Not-infected. dpi – days post-infection.</p

IFN-γ controls NOS2-mediated NO production during adapted-DENV-3 infection.

<p>(A–C) WT mice (<i>n</i> = 6 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p) and 3, 5 or 7 days after infection, mice were culled and tissue were collected for the following analysis: (A) Determination of NOS2 RNA expression by qPCR in spleen of control and DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (B) Determination of NOS2 staining by immunohistochemistry in liver sections of control and DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. (C) Esplenocytes were incubated with DAF-2DA and fluorescence determined. Results are expressed as fold increase in fluorescence over stained cells of naive mice. (D–E), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and tissues were collected for the following analysis: (D) Determination of NOS2 RNA expression by qPCR in spleen of WT and IFN-γ^−/− DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (E) Determination of NOS2 staining by immunohistochemistry in liver sections of WT and IFN-γ^−/− DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. Results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control naive mice. # for P<0.05 when compared to WT infected mice. 10LD₅₀ corresponds to 1000 PFU of DENV-3. 1LD₅₀ corresponds to 100 PFU of DENV-3. dpi – days post-infection. NI – Not-infected.</p

Characterization of virologic and histopathological parameters in C57BL/6j mice upon adapted-DENV-3 infection.

<p>(A–D) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection, mice were culled and blood and tissues were collected for the following analysis: (A–C) Viral loads were recovered from the spleen, liver and blood, respectively. Results are shown as the log of PFU per g of tissue or per mL of blood. (D) Shows virus NS1 antigen serum levels by ELISA and expressed as O.D. (E–F) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the seventh day of infection mice were culled and liver collected for the following analyses: (E) Liver was collected, formalin-fixed and processed into paraffin sections. Serial sections from each tissue were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a, and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. (F) shows semi-quantitative analysis of hepatic damage and Hematoxylin & Eosin staining of liver sections of control and DENV-3-infected mice, seven days after infection (Scale Bar - 400 µm). The images presented are representative of an animal on the seventh day of infection. In (G) Viral inoculum (10LD₅₀ or 1000 PFU) was heat inactivated (Heat, 56°C, 60 min) or treated with UV light (UV, 15 min) before inoculation in C57BL/6j mice. Lethality was evaluated every 12 hours for 14 days. (H) WT mice (<i>n</i> = 6 mice per group) were pretreated i.p with 100 µL of Anti-DENV-3 antiserum or control serum (pre-immune serum) before inoculation of 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p). Lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. Results are expressed as mean ± SEM (except for A–C, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND- not detected. NI- not-infected. dpi- days post-infection. NC – Negative control. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

IFN-γ production is required for host resistance to adapted-DENV-3 primary infection.

<p>(A) WT mice (<i>n</i> = 4 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and seven days later, mice were culled, and splenic cells isolated for assaying IFN-γ production by cellular staining with labeled antibodies and FACS analysis. Results are expressed as % of IFN-γ-positive cells in each population. (B) WT and IFN-γ^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (C–J), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and blood and tissues were collected for the following analysis: (C–D) Viral loads were recovered from the blood (C), spleen and liver (D, left and right panels), respectively. Results are shown as the log of PFU per mL of blood or per g of tissue. (E) Serial sections from each liver were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma, shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and IFN-γ^−/− DENV-3-infected mice, five days after infection. Scale bars - 400 µm. The images presented are representative of an animal on the fifth day of infection. All results are expressed as mean ± SEM (except for C–D, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # fo P,0.05 when compared to WT infected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. 1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. ND – not detectable. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p