MMP9 and SCF protect from apoptosis in acute kidney injury

Abstract

Apoptosis of tubular epithelial cells is a hallmark of acute kidney injury (AKI), but the cellular events preceding apoptosis in this setting are incompletely understood. Because matrix metalloproteinase 9 (MMP9) degrades matrix components involved in cell survival, we studied the role of MMP9 in AKI. In the mouse model of folic acid-induced AKI, we observed a marked increase of MMP9 activity in the S3 segment of the proximal tubule (S3PT), correlating with the apoptotic phase. MMP9 deficiency increased apoptosis and the severity of renal lesions and substantially delayed recovery of renal function. MMP9-/- mice exhibited significant apoptosis in the S3PT and the intercalated cells of the collecting duct (I-CD), whereas wild-type mice exhibited none in these segments. Stem cell factor (SCF), an MMP9 substrate, was identified in the S3PT, and its receptor, c-Kit, was expressed in both the S3PT and I-CD. MMP9 released the soluble form of SCF (sSCF) from kidney cells in vivo and in vitro. In addition, SCF inhibited apoptosis of tubular cells in vitro, rescued MMP9-/- S3PT and I-CD from apoptosis in vivo, and improved renal function. An ischemia-reperfusion model of AKI produced similar results. In patients with AKI, urinary sSCF increased with acute tubular necrosis but not with prerenal azotemia. In conclusion, these data show that MMP9 protects the S3 segment of the proximal tubule and the I-CD from apoptosis in AKI, most likely by releasing sSCF.

Figure 1.

Effect of MMP9 deficiency on renal function, tubular dilation, and apoptosis at different time points after FA injection. (A) Serum creatinine level in MMP9+/+ (□) and MMP9−/− ( ) mice injected with bicarbonate vehicle (Control) or FA. (B) Percentage of dilated tubules in MMP9+/+ and MMP9−/− kidney sections. Five microphotographs were taken from six different MMP9−/− and MMP9+/+ kidneys. (C) Percentage of apoptotic nuclei. Six microphotographs were taken from six different MMP9+/+ and MMP9−/− kidneys. Data are means ± SEM. **P < 0.001 versus MMP9+/+ injected mice; *P < 0.01 versus MMP9+/+ injected mice. Magnification, ×400.

Figure 2.

Impact of MMP9 deficiency on the distribution of apoptosis along tubule segments 18 h after FA injection. (A and B) Representative kidney sections from MMP9+/+ (A) and MMP9−/− (B) mice stained with TUNEL method (apoptotic nuclei, green) and CD10 (S3 segment, red). Note that apoptosis is observed in S3 segment of proximal tubules in MMP9−/− kidney only. (C through D′) Representative collecting duct sections from MMP9+/+ (C and C′) and MMP9−/− (D and D′) mice stained with TUNEL method and DBA lectin, a marker of principal cells (C and D) or H⁺ATPase, a marker of intercalated cells (C′ and D′). The number of apoptotic nuclei is increased in MMP9−/− compared with MMP9+/+ collecting ducts (D versus C). In MMP9+/+ mice, apoptotic nuclei are observed only in principal cells (C), whereas in MMP9−/− mice, they are also seen in intercalated cells (D, arrow, and D′). (E) Percentage of apoptotic nuclei in S3PT I-CD and P-CD. Six microphotographs of six different kidneys’ samples from MMP9+/+ and MMP9−/− mice. Bar = 100 μm in A, B, C, and D and 60 μm in C′ and D′. Data are means ± SEM. **P < 0.001 versus MMP9+/+ injected mice.

Figure 3.

Induction of MMP9 in MMP9+/+ mice 18 h after injection of FA. (A) Western blot performed with 20 μg of protein extracts from control MMP9+/+ kidney or kidney from MMP9+/+ mice 18 h after injection of FA. Quantitative analysis of five blots, using β-actin as an internal control, showed that MMP9 protein expression was increased by six-fold in mice treated with FA compared with control mice injected with vehicle. (B) Zymograms performed with protein extracts from MMP9+/+ (lanes 1 and 3) and MMP9−/− (lanes 2 and 4) control kidneys (lanes 1 and 2) and kidneys injected with FA (lanes 3 and 4). Quantitative analysis of six zymograms showed that MMP9 enzymatic activity was increased by nine-fold in mice treated with FA compared with control mice injected with vehicle. (C through F) Expression of MMP9, revealed by FITC, in representative paraffin kidney sections of control MMP9+/+ mice (C and D) and of MMP9+/+ mice injected with FA (E and F). (C and E) Proximal tubule S3 segment was stained with CD10 and revealed by TRITC. Immunoreactive MMP9 (green) was induced 18 h after FA injection (E), whereas it was barely detected in control kidney (C). (D and F) Principal cells from collecting duct were stained with DBA lectin conjugated to TRITC. Bar = 120 μm in C and E and 100 μm in D and F. Data are means ± SEM. ***P < 0.001.

Figure 4.

(A through I) Immunoreactive SCF (A through D) and c-Kit (E through I) expression (FITC, green) in MMP9+/+ paraffin kidney sections. (A and E) SCF (A) and c-Kit (E) expression in normal kidney. (B through D and F through I) Expression in kidney 18 h after FA injection. (B and F) The localization of SCF (B) and c-Kit (F) at the apex of dilated tubules is similar to A and E, respectively. (C and G) Proximal tubules were stained with megalin and revealed by TRITC. The corticomedullary junction is delineated by a dotted line with the medulla in the lower part of the figure. Note that both SCF (C) and c-Kit (G) co-localized with megalin in medulla only. (D) Co-localization of SCF and MMP9 revealed by TRITC in proximal tubules. (H and I) Expression of c-Kit in collecting duct cells. Collecting duct cells were stained in red with DBA lectin, a marker of principal cells (H) or with H⁺ATPase, a marker of intercalated cells (I). Note that c-Kit was observed in intercalated cells only, where it co-localized with H⁺ATPase. Bar = 100 μm in A through D, H, and I; and 140 μm in E through G.

Figure 5.

Sensitivity of membrane SCF to MMP9 proteolysis in vitro. (A) ELISA of sSCF in the culture medium of confluent proximal tubular cell line incubated with 4-aminophenylmercuric acetate in gelatinase substrate buffer (Control) or with activated MMP9 at increasing concentrations (+MMP9 80 ng/ml, +MMP9 160 ng/ml, and +MMP9 320 ng/ml). Data are means ± SEM. ***P < 0.001 control. (B) Western blot analysis of SCF expression in cell extracts (lanes 1 through 3) and conditioned media (lanes 4 through 6) of RC-SV1 control cells (lanes 1 and 4) and cells incubated with 160 ng/ml (lanes 2 and 5) and 320 ng/ml (lanes 3 and 6) recombinant activated MMP9. Recombinant MMP9 decreased expression of the 49-kD intact form of SCF in cell extracts (compare lanes 2 and 3 with lane 1), whereas it increased the release of the 22-kD sSCF in culture medium (compare lanes 5 and 6 with lane 4).

Figure 6.

Effect of MMP9 deficiency on plasma and urine SCF levels. (A) sSCF concentration in plasma of MMP9+/+ (□) and MMP9−/− ( ) mice injected with bicarbonate vehicle (Control) or with FA at 18 h. SCF concentration was significantly lower (P < 0.005; n = 5) in the plasma of MMP9−/− compared with MMP9+/+ mice. (B) Urinary sSCF normalized to urinary protein in MMP9+/+ (□) and MMP9−/− () mice injected with vehicle (Control) or FA. Urinary SCF was not significantly different between control MMP9+/+ and MMP9−/− mice. It significantly increased in both groups of FA-injected mice compared with their respective controls (MMP9+/+ mice P < 0.001; MMP9−/− mice P < 0.002; n = 6), but the increase was significantly less in MMP9−/− mice compared with MMP9+/+ mice. ***P < 0.001 (n = 6).

Figure 7.

Effect of sSCF on apoptosis in human collecting duct cell line in vitro, (A) Percentage of apoptotic nuclei in cells grown in media supplemented (Normal) or not (Basic) with FCS and hormones and in cells grown in basic medium supplemented with soluble recombinant SCF (Basic + SCF). (B) Concentration of active caspase-3 in cells grown in Normal, Basic and Basic media supplemented with SCF. Note that apoptosis (assessed by both methods) was induced in cells grown in Basic medium and inhibited by addition of SCF. Data are means ± SEM. ***P < 0.001.

Figure 8.

Effect of sSCF on renal function, renal histology, and apoptosis. (A) Serum creatinine level in MMP9+/+ (□), MMP9−/− ( ) and MMP9−/− mice rescued with SCF (▪) 18 h, 72 h, and 15 d after FA injection. Serum creatinine concentrations were not statistically different in MMP9+/+ mice and in MMP9−/− mice injected with recombinant SCF. Data are means ± SEM, ***P < 0.001. (B) Percentage of dilated tubules in MMP9+/+, MMP9−/−, and MMP9−/− mice rescued with SCF 18 and 72 h after FA injection. Five microphotographs taken from six different kidneys were analyzed. Percentage of dilated tubules was not statistically different in MMP9+/+ mice and in MMP9−/− mice injected with rSCF. Data are means ± SEM. ***P < 0.001. (C through E) Photomicrographs of representative paraffin kidney sections stained with periodic-acid Schiff from MMP9+/+ (C), MMP9−/− (D), and MMP9−/− mice rescued with SCF (E) 18 h after FA injection. Bar = 170 μm. (F and G) Percentage of apoptotic cells in S3PT and I-CD. Six microphotographs of six different kidneys sampled from MMP9+/+, MMP9−/−, and MMP9−/− mice rescued with SCF were analyzed 18 h after FA injection (F) and 24 h after reperfusion in the clamped kidney (G). Apoptosis observed in S3PT and I-CD was not statistically different in MMP9+/+ mice and in MMP9−/− mice injected with recombinant SCF+. Data are means ± SEM, ***P < 0.001; **P < 0.01 versus MMP9+/+ or MMP9−/− injected with SCF.

Figure 9.

(A and B) Detection of SCF in urine of patients with AKI by Western blotting (A) and ELISA (B). (A) Western blot performed with 50 μg of total protein from urine of patients with tubular necrosis (lanes 1 through 4), patients with prerenal azotemia (lanes 5 through 8), and control healthy individuals (lanes 9 through 12). Note that SCF was observed only in urine of patients with tubular necrosis. (B) Urinary sSCF concentration normalized to protein in healthy individuals (control), patients with prerenal azotemia, and patients with tubular necrosis. Data are means ± SEM. ***P < 0.001 versus urine of control subjects or patients with prerenal azotemia (n = 4).