Fig 1.
The expression of Klk is impaired in IL-17RA-/- kidney following C. albicans infection.
(A) WT and IL-17RA-/- mice (n = 4–6) were subjected to systemic C. albicans (CAF2-1 or SC5314) infection. After 48 h, kidneys were evaluated for fungal load. Data pooled from 2–3 independent experiments. (B) Heat map representing averaged intensity of expression of genes in WT and IL-17RA-/- kidneys (n = 2) at 48 h p.i. (C) Schematic representation of Kallikrein-kinin system (KKS). (D) Kidneys of WT and IL-17RA-/- mice (n = 6) were evaluated for expression of Klk1 and Klk1b27 at 48 h p.i. Data pooled from 2 independent experiments. At 72 h p.i., whole cell extracts from WT and IL-17RA-/- kidneys (n = 5–6) infected with C. albicans (E) CAF2-1 or (F) SC5314 were evaluated for Klk1 protein by western blotting. Sham infected mice received PBS. Images were quantified using ImageJ. Representative image of 1 of 2 independent experiments (E and F). For the bar diagram, data are combined from 2 independent experiments. Bars indicate mean ± S.D. P<0.05 (*), <0.01 (**), <0.001 (***). ns, not significant.
Fig 2.
IL-17-driven Klk1 expression in the kidney is necessary for immunity to C. albicans.
(A) WT mice (n = 5) were subjected to systemic candidiasis. Sham-infected mice received PBS. After 72 h, kidney sections were stained with anti-Klk1 or isotype control Abs. Black arrows indicate Klk1 staining. Photomicrographs are representative of 2 independent experiments. Original magnification: 100X. (B) Primary RTEC from C57Bl6/J mice were treated ± IL-17 (50 ng/ml and 200 ng/ml), TNFα (5 ng/ml) or IL-17 (200 ng/ml) + TNFα (5 ng/ml) for 24 h. Expression of Klk1 and Klk1b27 was assessed by qPCR. Bars represent mean ± S.D. Data are representative of 4 independent experiments. (C) WT mice (n = 5) were injected with Ad-IL-17 or Ad-ctrl (1x109 pfu). Six days post-injection, kidney sections were stained with anti-Klk1 or isotype control Abs. Inset: Klk1 staining in RTEC and negative staining in glomerulus (GM) and blood vessels (BV). Photomicrographs are representative of 2 independent experiments. Original magnification: 200X (upper panel) and 400X (lower panel). (D) WT and IL-17RA-/- (n = 14) mice were injected with Ad-Klk1 or Ad-ctrl 72 h prior to systemic C. albicans infection. Sham-infected WT and IL-17RA-/- mice (n = 3) received Ad-Klk1 only. Survival was assessed over 14 d. Data pooled from 3 independent experiments. (E) WT (n = 8) mice were injected with Ad-Klk1 or Ad-ctrl 72 h prior to systemic C. albicans infection. Fungal burden was assessed at 72 h p.i. Each dot represents one mouse, and horizontal bars indicate mean. Data are combined from 2 independent experiments. P < 0.05 (*), <0.01 (**), <0.0001 (****). ns, not significant.
Fig 3.
Bradykinin confers renal protection to IL-17RA-/- mice following disseminated C. albicans infection.
(A) IL-17RA-/- mice (n = 14) were treated with bradykinin (300 nmol/kg/day) or PBS starting on day -1 (relative to infection). On day 0, mice were administered C. albicans i.v. and evaluated for survival over 14 d. WT mice were infected with C. albicans and left untreated. Sham-infected IL-17RA-/- mice were given bradykinin only or left untreated (n = 3). (B) WT mice (n = 10–11) were treated with Bdkrb1 (R715; 1 mg/kg/day) and Bdkrb2 (HOE140; 1mg/kg/day) antagonists or PBS starting 1 day prior to infection and then daily for 14 d. Sham-infected WT mice were treated with the antagonists only (n = 3). Mice were evaluated for survival over 14 d. Data are pooled from 2 independent experiments for (A) and (B). P <0.05 (*), <0.01 (**).
Fig 4.
Bradykinin alleviated kidney injury and preserved renal function in C. albicans infected mice.
WT mice (n = 14–20) were either left treated ± bradykinin (300 nmol/kg/day) starting day -1 (relative to infection). Mice were infected with C. albicans strains (A) (CAF2-1) or (B) (SC5314). Sham-infected WT mice were treated ± bradykinin (n = 3–5). Survival was assessed over 14 d. Data are pooled from (A) 4 and (B) 2 independent experiments. At day 7 p.i., mice were evaluated for (C) angioedema development in the hind paw (n = 4–7) and (D) serum BUN and creatinine levels (n = 6–10). Data pooled from 2–3 independent experiments. Each dot represents one mouse and the bars indicate mean. At day 7 p.i., kidney sections were evaluated for (E) histopathology and inflammatory cell influx by PAS staining (n = 8) and (F) NGAL expression by IHC (n = 6–8). Black arrows indicate tubular damage and atropy; * indicates inflammatory cell influx. Representative photomicrographs from 2 independent experiments. Original magnification: 100X. (G) Cell lysates of kidney homogenates (n = 5–6) were evaluated for NGAL by western blotting. Images were enumerated using ImageJ. Representative image of 1 of 2 independent experiments. Bars indicate mean ± S.D and combined from 2 independent experiments. P <0.05 (*), <0.01 (**). <0.001 (***), <0.0001 (****). ns, not significant.
Fig 5.
Minimal impact of bradykinin treatment on fungal clearance and inflammatory cells influx in the C. albicans infected kidney.
WT mice were treated ± bradykinin (300 nmol/kg/day) or PBS on day -1 (relative to infection). (A) Kidneys were evaluated for fungal load on days 3 (n = 3–4) and 7 (n = 8–11) p.i. (B) At day 7 p.i. (n = 4–6), kidney infiltrating neutrophils (Gr1+) and macrophages (F4/80+) (gated on CD45+) cells were evaluated by flow cytometry. Data are combined from 2 independent experiments for (A) and (B). Each dot represents one mouse, and the bars indicate mean. (C) BM-derived neutrophils and BMDMs from WT mice were incubated in vitro with unopsonized C. albicans yeast ± bradykinin for 3 h, and fungal load was determined by plating culture supernatants. Percentage of C. albicans killed by neutrophils and macrophages is shown. (D) IL-6 in RTEC conditioned media was assessed after 24 h bradykinin treatment. (E) IL-6 and TNFα and (F) nitrite levels in the supernatants of BMDMs treated ± bradykinin or LPS (1 ng/ml) for 24 h. Data are representative of 3 independent experiments for (C-F). Bars indicate mean ± S.D. p <0.01 (**). ns, not significant.
Fig 6.
Apoptosis of kidney-resident cells is reduced in bradykinin treated mice in candidiasis.
WT mice were treated with bradykinin (300 nmol/kg/day) starting 1 day prior to infection and daily for 7 days. Sham-infected WT mice were treated with bradykinin. (A) At day 7 p.i. (n = 4–12), early and late apoptotic kidney resident cells (gated on CD45- cells) were quantified by AnnexinV and PI staining. (B) Frozen kidney sections (n = 4–8) were subjected to TUNEL staining and counterstained with DAPI. White arrows indicate TUNEL+ cells. The number of TUNEL+ cells was quantified in 15 randomly selected high powered fields (400X). Original magnification: 100X. (C) Frequency of cleaved Caspase-3+ kidney-resident cells (gated on CD45- cells) were quantified by flow cytometry (n = 4–6). Solid histogram: Negative control; Open histogram: DEVD-FMK staining. (D) Serial kidney sections (n = 4–6) were stained for cleaved Caspase-3. (E) Sorted kidney-resident cells (CD45-) (n = 6–7) were evaluated for the expression of Bcl-xL and Bax mRNA by qPCR. Data pooled from 2 and 3 independent experiments for (A-D) and (E), respectively. Each dot represents one mouse and bars indicate mean. Bar indicates mean ± S.D. P <0.05 (*), <0.01 (**), <0.0001 (****). ns, not significant.
Fig 7.
Combination of fluconazole and bradykinin increased survival of C. albicans infected mice.
(A) Experimental design for combinatorial fluconazole and bradykinin therapy strategy (B) WT mice (n = 16) were infected with C. albicans. Infected mice were treated with two doses of FLC only (5 mg/kg at 2 and 24 h p.i.), bradykinin only (300nmol/kg starting 1 day prior to infection and daily for 14 days) or a combination of FLC and bradykinin. Sham-infected mice (n = 3) were treated with FLC, bradykinin or both. Survival was assessed over 14 d. Data are combined from 3 independent experiments. p<0.05 (*), p>0.001 (***), p>0.0001 (****).