Macrophage Stimulating Protein May Promote Tubular Regeneration after Acute Injury

MSP Plasma Levels Increased in Patients with AKI

We studied 25 critically ill patients with acute renal failure (ARF) included in the Failure group of ADQI-RIFLE (Acute Dialysis Quality Initiative-Risk, Injury, Failure, Loss of Kidney Function, End Stage Kidney Disease) criteria,²⁵ and we collected plasma for MSP determination in an early phase (days 1 through 3; n = 10) and in a late phase (days 7 through 14; n = 25) of ARF. Patients showed an average SOFA (Sequential Organ Failure Assessment) score and an estimation of mortality risk not significantly different from those observed in the whole ICU population with ARF treated during 1 yr (n = 118; Table 1). In comparison with healthy volunteers (n = 10), ICU patients with ARF presented significantly higher MSP plasma levels (Figure 1A). The levels of MSP were significantly higher in the late than in the early phase of ARF. In addition, in patients with ARF, the MSP levels were significantly higher than in the ICU population with preserved renal function (n = 25; Figure 1A). No significant differences in the APACHE (Acute Physiology and Chronic Health Evaluation) II score was observed in the groups ICU early ARF (22.2 ± 1.1), ICU late ARF (21.8 ± 1.9), and ICU without ARF (21.0 ± 0.7). MSP levels were normal in patients with chronic renal failure (CRF; n = 10); however, when the same patients with CRF underwent renal transplantation, a significant increase of MSP was observed in the first week after the procedure (kidney Tx week 1) to decrease when a stable graft function was reached (kidney Tx week 8; Figure 1A).

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Figure 1.

Evaluation of MSP plasma levels in patients with ARF and of MSP release from TEC cultured in detrimental conditions. (A) ELISA analysis of MSP plasma levels (ng/ml) in healthy volunteers (Healthy; n = 10), in critically ill patients with normal renal function (ICU; n = 10), in the early phase (ICU early ARF; n = 10) or in the late phase (ICU late ARF; n = 25) of ARF, and in patients with CRF (CRF Pre Tx; n = 10) before or in the first or the eighth week after kidney transplantation. Results are expressed as average ± 1 SD. ANOVA with Newmann-Keuls multicomparison test was performed: *P < 0.05 ICU, ICU early ARF, ICU late ARF, or Kidney Tx week 1 versus Healthy; ^§P < 0.05 ICU early ARF or ICU late ARF versus ICU; ^#P < 0.05 ICU late ARF versus ICU early ARF, ^†P < 0.05 Kidney Tx week 1 versus CRF Pre Tx or Kidney Tx week 8. (B) ELISA determination of MSP (pg/ml) in supernatants of TEC exposed to subcytotoxic dosages of cisplatin (CIS; range 0.01 to 1 μg/ml) for 24 h. Results are expressed as average ± 1 SD of three different experiments. ANOVA with Dunnet multicomparison test was performed: *P < 0.05 cisplatin 0.05, 0.1, 0.5, or 1.0 μg/ml versus vehicle alone. (C) ELISA determination of MSP (pg/ml) in supernatants of TEC cultured in condition of serum deprivation or with a cytotoxic dosage of cisplatin (5 μg/ml) at different time points. Results are expressed as mean ± 1 SD of three different experiments. ANOVA with Newmann-Keuls multicomparison test was performed: *P < 0.05 6 h without FCS or with 5 μg/ml cisplatin versus vehicle alone; ^§P < 0.05 12 or 24 h without FCS versus 6 h without FCS; ^#P < 0.05 12 or 24 h with 5 μg/ml cisplatin versus 6 h with 5 μg/ml cisplatin. (D through F) Representative immunofluorescence micrographs of MSP staining in TEC cultured for 6 h in basal conditions (D), in serum deprivation (E), or with 5 μg/ml cisplatin (F). Nuclei were counterstained by 1 μg/ml propidium iodide. Five experiments were performed with similar results. Magnification, ×100.

Enhanced Production of MSP by Human Tubular Epithelial Cells Exposed to Detrimental Conditions

Exposure of immortalized tubular epithelial cells (TEC) to subcytotoxic dosages of cisplatin (0.01 to 1.00 μg/ml) for 24 h resulted in a significant increase of MSP in the supernatants with respect to vehicle alone (Figure 1B). No apoptosis of TEC was observed with these dosages of cisplatin (data not shown). In contrast, a cytotoxic dosage of cisplatin (5 μg/ml) induced an early MSP production at 6 h followed by a decrease at 12 and 24 h (Figure 1C) in concomitance with the appearance of apoptosis. Similarly, serum deprivation increased MSP production by TEC at 6 h followed by a significant decrease at 12 and 24 h (Figure 1C). These data were confirmed by immunofluorescence analysis (Figure 1, D through F) showing an enhanced intracytoplasmic expression of MSP 6 h after serum deprivation (Figure 1E) or treatment with 5 μg/ml cisplatin (Figure 1F). Similar results were obtained with primary TEC (data not shown).

TEC Expressed RON Receptor

By reverse transcription–PCR (RT-PCR), we found the expression of RON mRNA in both primary and immortalized TEC (Figure 2A). Western blot (Figure 2B), FACS (Figure 2, C and D), and immunofluorescence (Figure 2, E and F) confirmed the expression of RON protein by TEC. After MSP stimulation of immortalized TEC, RON was phosphorylated (Figure 2G). The Western blot analysis showed the knockdown of MET (HGF receptor) and RON proteins by specific small interference RNA (siRNA) in immortalized TEC (Figure 2H). These results were confirmed by RT-PCR analysis (data not shown).

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Figure 2.

Detection of RON expression by TEC. (A) Representative RT-PCR analysis of RON on human primary (pTEC) and immortalized (iTEC) TEC. (B through F) Representative micrographs of Western blot (B), FACS (C and D), and immunofluorescence (E and F) analysis showing the presence of RON protein on both pTEC and iTEC. (G) Representative Western blot analysis of RON expression and its phosphorylation (P-RON) in presence of vehicle alone or of 10 ng/ml MSP. Three experiments were performed with similar results. (H) Representative Western blot analysis of MET and RON knockdown in iTEC transiently transfected with specific siRNA after 48 h, when the cells were used for the in vitro experiments (lane 1, wild-type iTEC; lane 2, siRNA MET iTEC; lane 3, siRNA RON iTEC; lane 4, control siRNA iTEC). β-Actin was used as control for protein loading. Three experiments were performed with similar results.

MSP and RON Were Upregulated in Tubules after Glycerol-Induced AKI in C57Bl/6 Mice

Control C57Bl/6 mice that were administered an injection of vehicle alone showed normal histology (Figure 3A) and levels of serum creatinine (sCr) and urea (sU; sCr 0.22 ± 0.09 mg/dl; sU 31.66 ± 4.25 mg/dl). In contrast, in mice that were administered an injection of glycerol, we observed after 3 d marked signs of tubular injury (Figure 3A) characterized by hyaline cast formation, widespread necrosis, cytoplasmic vacuolization, mitochondrial alterations, and loss of the brush border.²⁶ At day 3, the maximal rise of sCr and sU was found (sCr 1.53 ± 1.01 mg/dl; sU 367.68 ± 56.32 mg/dl). After 10 d, mouse kidneys showed features of spontaneous tubular regeneration (Figure 3A), confirmed by the decline of biochemical data (sCr 0.33 ± 0.11 mg/dl; sU 78 ± 6 mg/dl) to complete recovery of normal morphology 21 d after injury (Figure 3A). By immunohistochemistry, control C57Bl/6 mice showed a weak tubular staining for both RON and MSP (Figure 3A). RON and MSP staining was almost completely abrogated 3 d after glycerol injection (Figure 3A). By contrast, at day 10, during the phase of regeneration, tubules showed a marked upregulation of RON and MSP staining to decrease to normal levels after 21 d (Figure 3A). These data were confirmed by semiquantitative analysis of average fluorescence intensity (Figure 3B) and Western blot analysis of kidney lysates (Figure 3C) for each time point considered. In mice, the plasma levels of MSP before injury were 1.82 ± 0.22 ng/ml. MSP plasma levels decreased in concomitance with the peak of tubular injury (day 3 0.54 ± 0.16 ng/ml) and increased in the regenerative phase (day 10 7.5 ± 1.23 ng/ml) to decline thereafter (day 21 3.75 ± 0.94 ng/ml). By confocal microscopy, MSP co-localized with megalin, suggesting its expression by proximal TEC (Figure 4). No co-localization of MSP was observed with Tamm-Horsfall protein and aquaporin-2, suggesting its absence or its minimal expression in the thick ascending limb of Henle's loop and in collecting ducts (Figure 4).

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Figure 3.

MSP and RON expression in C57Bl/6 mouse kidneys after glycerol-induced AKI. (A) Representative micrographs of hematoxylin-eosin and immunohistochemistry staining for RON or MSP in kidney sections of C57Bl/6 control mice or of mice after 3, 10, or 21 d after glycerol-induced injury. Nuclei were counterstained by 0.5 mg/ml Hoechst (G, location of glomeruli). Results are representative of six control mice that were administered an injection of vehicle alone and of six mice with glycerol-induced AKI at all time points considered. (B) Semiquantitative analysis of RON and MSP average fluorescence intensity in 10 nonsequential kidney sections at all time points considered. ANOVA with Newmann-Keuls multicomparison test was performed: *P < 0.05 days 3 and 10 RON/MSP versus control (CTRL) RON/MSP; ^§P < 0.05 day 10 RON/MSP versus day 3 RON/MSP. (C) Representative Western blot analysis of RON and MSP expression on tubular lysates of kidneys f mice that were killed at different time points and obtained after separation from glomeruli (lane 1, CTRL; lane 2, day 3; lane 3, day 10; lane 4, day 21). β-Actin was used as control for protein loading. Magnifications: ×100 in A; ×600 in insets.

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Figure 4.

Confocal microscopy analysis of MSP co-localization with markers of different tubular segments in the regenerative phase after glycerol-induced injury. Representative confocal microscopy images of kidney sections of mice after 10 d from glycerol-induced injury showing the co-localization of MSP with the proximal tubular cell marker megalin. No co-localization of MSP with the thick ascending limb of Henle's loop marker Tamm-Horsfall protein (THP) and with the collecting duct marker aquaporin-2 (AQP-2) was observed. In merge micrographs, nuclei were counterstained by 0.5 μg/ml Hoechst. Five experiments were performed with similar results. Magnifications: ×650 for megalin; ×400 for THP and AQP-2.

MSP Exerted a Proliferative and Antiapoptotic Effect on TEC

TEC were incubated with increasing dosages of MSP (0.1 to 100.0 ng/ml) or with HGF (10 ng/ml) as positive control. In preliminary experiments based on XTT assay, we found that MSP increased the cell number in primary and immortalized TEC culture (Figure 5A). The following experiments were performed with immortalized TEC. The 5-bromo-2′-deoxy-uridine (BrdU) incorporation into DNA after MSP stimulation indicated a significant increase of proliferation detectable at the dosage of 1 ng/ml, with a peak at 50 ng/ml (Figure 5B). The co-incubation with the phosphatidylinositol-3-kinase inhibitor wortmannin (0.1 μM) or with PD98059 (20 μM), a specific inhibitor of Mek that activates extracellular signal–regulated kinase (ERK),²⁷ resulted in a significant decrease of MSP-induced proliferation (Figure 5B). MSP-induced proliferation was not inhibited by the co-incubation with a blocking anti-MET antibody or in TEC transfected with MET siRNA (Figure 5B). In contrast, MSP-induced proliferation was blocked in TEC transfected with RON siRNA (Figure 5B). MSP (10 ng/ml) increased TEC viability also in the presence of 5 μg/ml cisplatin (data not shown). This protective effect was due to the inhibition of cisplatin-induced apoptosis. Indeed, as shown by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay (Figure 5C), MSP significantly reduced apoptosis of TEC cultured in condition of serum deprivation or with 5 μg/ml cisplatin. The antiapoptotic effect of MSP was partially inhibited by wortmannin or PD98059 (Figure 5C). MSP-induced resistance to apoptosis was not affected by co-incubation with a blocking anti-MET antibody or in TEC transfected with MET siRNA (Figure 5C). In contrast, MSP protection from cisplatin-induced apoptosis was blocked in TEC transfected with RON siRNA (Figure 5C). The antiapoptotic activity of MSP was further confirmed by the disappearance of chromatin fragmentation (Figure 5C, inset) and by propidium iodide staining (Figure 5, D and E). The effect of MSP was not restricted to cisplatin-induced cell death, because a significant decrease of apoptosis was also observed in TEC incubated with cyclosporine (1 μg/ml), FK506 (1 μg/ml), LPS (50 ng/ml), or TNF-α (30 ng/ml; data not shown).

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Figure 5.

MSP induced TEC proliferation, inhibited cisplatin-induced TEC injury, and enhanced Fas-mediated apoptosis by modulating caspase activity. (A) Data from XTT-based assay of time-course increase in cell number of primary (open symbols) and immortalized (black symbols) TEC. MSP (10 ng/ml) induced a significant increase of cell number of both pTEC and iTEC cultures at all time points considered (12, 24, and 48 h). HGF (10 ng/ml) was used as positive control. (B) BrdU-based assay of proliferation of iTEC. MSP induced a significant increase of proliferation at dosages >1 ng/ml (*P < 0.05 MSP 1, 10, 20, 50, or 100 ng/ml versus vehicle). Wortmannin (0.1 μM) or PD98069 (20 μM) significantly reduced MSP-induced TEC proliferation (^§P < 0.05 MSP 10 plus wortmannin or MSP 10 + PD98059 versus MSP 10). Incubation with a blocking anti-MET antibody or TEC transfection with MET siRNA did not influence MSP-induce proliferation (*P < 0.05 MSP 10 + anti-MET or MSP 10 siRNA MET versus vehicle, P > 0.05 MSP 10 + anti-MET or MSP 10 siRNA MET versus MSP 10). In contrast, TEC transfection with RON siRNA completely abolished MSP-induced proliferation (^#P < 0.05 MSP 10 siRNA RON versus MSP 10). Data are expressed as average ± 1 SD of three different experiments. ANOVA with Newman-Keuls multicomparison test was performed. (C) TUNEL assay showing the antiapoptotic effect of 10 ng/ml MSP or HGF (positive control) on TEC exposed to serum deprivation or cisplatin treatment. With respect to normal culture conditions (vehicle/FCS+), serum deprivation (vehicle/FCS−) or cisplatin (CIS 5 μg/ml) significantly increased TEC apoptosis (*P < 0.05 vehicle/FCS− or CIS versus vehicle/FCS+). MSP and HGF significantly inhibited TEC apoptosis induced by serum deprivation (^§P < 0.05 HGF or MSP versus vehicle/FCS−) or by cisplatin (^#P < 0.05 CIS + HGF or CIS + MSP versus CIS). Wortmannin and PD98059 significantly inhibited the MSP-associated antiapoptotic effect (^†P < 0.05 CIS + MSP + wortmannin or CIS + MSP + PD98059 versus CIS + MSP). Incubation with a blocking anti-MET antibody or TEC transfection with MET siRNA did not influence MSP-induced resistance to apoptosis (^#P < 0.05 CIS + MSP + anti-MET or CIS + MSP siRNA MET versus vehicle, P > 0.05 CIS + MSP + anti-MET or CIS + MSP siRNA MET versus CIS + MSP). In contrast, TEC transfection with RON siRNA completely abolished MSP-induced protection from apoptosis (^†P < 0.05 CIS + MSP siRNA RON versus CIS + MSP). (C inset) Representative micrograph of DNA electrophoresis showing the typical chromatin fragmentation of apoptotic TEC incubated with 5 μg/ml (lane 1) or 10 μg/ml (lane 2) cisplatin. Fragmentation was absent in DNA extracted from TEC exposed to 5 μg/ml cisplatin in the presence of 10 ng/ml HGF (positive control, lane 3) or MSP (lane 4). (D and E) Representative micrographs of propidium iodide-stained TEC exposed to 5 μg/ml cisplatin in absence (D) or presence (E) of 10 ng/ml MSP. (F) ELISA data showing the significant reduction of cspase-3, -8, and -9 activities induced by 10 ng/ml MSP on cisplatin-treated TEC. HGF (10 ng/ml) was used as positive control. Caspase data are given as average ± 1 SD of spectrophotometer analysis of three different experiments and expressed as percentage variation in comparison with unstimulated TEC. ANOVA with Newmann-Keuls multicomparison test was performed (P < 0.05 *caspase-3, ^§caspase-8, and ^#caspase-9 CIS + MSP or CIS + HGF versus CIS). (G) Data from TUNEL assay showing that MSP inhibited sensitization to Fas-induced apoptosis in cisplatin-treated TEC. Addition of agonistic anti-Fas IgM (100 ng/ml) or supernatants collected by COS cells transiently transfected with Fas-L cDNA (COS Fas-L) but not supernatants of mock-transfected COS cells (COS mock) enhanced cisplatin-induced TEC apoptosis (*P < 0.05 CIS versus vehicle and ^#P < 0.05 CIS + anti-Fas IgM or CIS + COS Fas-L versus CIS). Apoptosis was significantly inhibited by addition of 10 ng/ml MSP (^§P < 0.05 CIS + MSP versus CIS; ^†P < 0.05 CIS + MSP + anti-Fas IgM versus CIS + anti-Fas IgM; ^‡P < 0.05 CIS + MSP + COS Fas-L versus CIS + COS Fas-L). Supernatants derived from mock-transfected COS cells did not worsen cisplatin-induced TEC apoptosis. The addition of MSP to COS mock supernatants significantly reduced TEC apoptosis (⁺P < 0.05 CIS + MSP + COS mock versus CIS + COS mock). In addition, anti-Fas IgM and supernatants of COS Fas-L transfectants did not influence the rate of TEC apoptosis in the absence of cisplatin. TUNEL data are expressed as average number of apoptotic cells/field (×100 magnification) ± 1 SD of three different experiments. ANOVA with Newman-Keuls multicomparison test was performed. Magnification, ×200 in D and E.

MSP Reduced Caspase Activity, Fas Upregulation, and Fas-Enhanced Apoptosis and Modulated Akt/ERK Phosphorylation and Bcl-xL Expression in Cisplatin-Treated TEC

MSP (10 ng/ml) induced a significant reduction of caspase-3, -8, and -9 activity in cisplatin-treated TEC (Figure 5F). Agonistic anti-Fas IgM antibody (100 ng/ml) or supernatants from COS cells transfected with human Fas ligand (Fas-L) cDNA²⁸ induced a significant increase of apoptosis in cisplatin-treated TEC but not in cells maintained in basal culture conditions (Figure 5G). The apoptotic rate of cisplatin-treated TEC was not affected by supernatants derived from mock-transfected COS (Figure 5G). MSP significantly inhibited the enhancement of apoptosis induced by anti-Fas IgM or COS Fas-L supernatants. These data suggest that MSP inhibited the sensitization of cisplatin-treated TEC to Fas-mediated apoptosis. Moreover, FACS (Figure 6, A through C) and immunofluorescence (Figure 6, D through F) showed that MSP significantly reduced the expression of Fas on cisplatin-treated TEC. As shown by Western blot and related densitometric analysis (Figure 6G), MSP prevented the decrease of Bcl-xL expression as well as of Akt and ERK phosphorylation induced by serum deprivation or cisplatin.

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Figure 6.

MSP decreased Fas and upregulated Bcl-xL expression and Akt/ERK phosphorylation in cisplatin-treated TEC. (A through F) Representative FACS (A through C) and immunofluorescence (D through F) analysis of Fas expression in TEC in different experimental conditions. Fas was slightly expressed on unstimulated TEC (A and D). Cisplatin (5 μg/ml) significantly induced the overexpression of Fas (B and E) that was almost completely abrogated by treatment with 10 ng/ml MSP (C and F). For FACS analysis, Kolmogorov Smirnov statistical test was performed. In D through F, nuclei were counterstained by 1 μg/ml propidium iodide. Five experiments were performed with similar results. (G) Representative Western blot analysis of P-Akt, Akt, P-ERK, ERK, and Bcl-xL expression in TEC exposed to various stimuli (lane 1, vehicle/FCS+; lane 2, vehicle/FCS−; lane 3, FCS−plus 10 ng/ml HGF; lane 4, FCS− plus 10 ng/ml MSP; lane 5, 5 μg/ml cisplatin; lane 6, 10 μg/ml cisplatin; lane 7, 5 μg/ml cisplatin plus 10 ng/ml HGF; lane 8, 5 μg/ml cisplatin plus 10 ng/ml MSP). MSP-induced Akt and ERK phosphorylation was confirmed by densitometric analysis expressed as P-Akt/Akt ratio (▪) or P-ERK/ERK ratio (□). β-Actin was used as control for protein loading. Three experiments were performed with similar results. Magnification, ×100 in D through F.

MSP Induced Vimentin Upregulation and Pax-2 Expression in Surviving TEC

We investigated the effect of MSP on the expression of the mesenchymal marker vimentin and of the embryonic protein Pax-2. TEC basally expressed vimentin that was almost completely lost after 24 h of serum withdrawal (Figure 7A) or in the presence of 5 μg/ml cisplatin (Figure 7C). By contrast, vimentin was re-expressed in serum-deprived (Figure 7B) and cisplatin-treated (Figure 7D) TEC after stimulation with 10 ng/ml MSP. TEC did not express in basal conditions, as well as after serum deprivation or cisplatin treatment, the Pax-2 embryonic antigen. When treated with MSP, TEC expressed Pax-2 as detected by nuclear immunostaining (Figure 7, E through H) and Western blot analysis (Figure 7I).

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Figure 7.

MSP induced upregulation of vimentin, and Pax-2 expression in TEC survived to serum deprivation or cisplatin treatment. (A through D) Representative micrographs of vimentin expression in TEC cultured with various stimuli. The mesenchymal marker vimentin was slightly detectable in TEC cultured for 24 h in the absence of FCS (A) or in presence of 5 μg/ml cisplatin (C). Addition of 10 ng/ml MSP induced the upregulation of vimentin in both serum-deprived (B) and cisplatin-treated (D) TEC. Nuclei were counterstained by 1 μg/ml propidium iodide. (E through H) representative micrographs of Pax-2 expression in TEC exposed to various experimental conditions. The embryonic nuclear marker Pax-2 was absent in TEC after 24 h of serum withdrawal (E) or incubation with 5 μg/ml cisplatin (G). MSP (10 ng/ml) induced a marked increase of intranuclear staining for Pax-2 in both serum-deprived (F) and cisplatin-treated (H) TEC. (I) Representative Western blot analysis of lysates from TEC incubated with various stimuli confirming the MSP-induced Pax-2 upregulation (lane 1, vehicle/FCS+; lane 2, vehicle/FCS−; lane 3, FCS− plus 10 ng/ml HGF; lane 4, FCS-plus 10 ng/ml MSP; lane 5, 5 μg/ml cisplatin; lane 6, 10 μg/ml cisplatin; lane 7, 5 μg/ml cisplatin plus 10 ng/ml HGF; lane 8, 5 μg/ml cisplatin plus 10 ng/ml MSP). β-Actin was used as control for protein loading. Three experiments were performed with similar results. Magnification, ×100.

MSP Maintained the Functional Integrity of Epithelial Monolayers in Cisplatin-Treated TEC

Cisplatin induced a significant decrease of TEC transepithelial resistance, suggesting altered cell polarity. MSP re-established normal transepithelial resistance in cisplatin-treated TEC (Figure 8A). In addition, MSP induced a significant increased adhesion of TEC to the extracellular matrixes fibronectin, type IV collagen, and Matrigel (Figure 8B). Cisplatin induced the downregulation of the tight junction protein zonula occludens-1 (ZO-1) that was prevented by MSP (Figure 8, C through E). Moreover, in the presence of MSP, cisplatin-treated TEC preserved the expression of molecules typical of fully differentiated proximal TEC such as alkaline phosphatase and aminopeptidase A (data not shown) and re-established the presence of the endocytic receptor megalin (Figure 8, F through H), a phenomenon probably responsible for the maintained ability of these cells to internalize FITC-labeled albumin (Figure 8, I through K).

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Figure 8.

MSP preserved TEC transepithelial resistance (TER), ability to adhere to extracellular matrices, tight junction protein/megalin expression, and internalization of FITC-labeled albumin. (A) Analysis of TER as assessment of cell polarity. Cisplatin (CIS; 5 μg/ml) significantly reduced TER values (*P < 0.05 CIS versus vehicle), whereas 10 ng/ml MSP restored normal TER values in cisplatin-treated TEC. HGF (10 ng/ml) was used as positive control (^§P < 0.05 CIS + HGF or CIS + MSP versus CIS). Data are given as average TER values of three different experiments ± 1 SD. TER values were normalized for the area of the membrane used in the experimental procedure. ANOVA with Newman-Keuls multicomparison test was performed. (B) Adhesion of TEC to extracellular matrices. Incubation for 6 h with 10 ng/ml MSP or with the positive control HGF (10 ng/ml) induced a significant increase of TEC adhesion to plates previously coated with fibronectin, type IV collagen, or Matrigel (P < 0.05 *fibronectin, ^§type IV collagen, and ^#Matrigel HGF or MSP versus vehicle alone). Data are expressed as average ± 1 SD of three different experiments. ANOVA with Newman-Keuls multicomparison test was performed. (C through E) Representative immunofluorescence micrographs showing the tight junction protein ZO-1 expression in presence of vehicle alone (C), 5 μg/ml cisplatin (D), or 5 μg/ml cisplatin + 10 ng/ml MSP (E). (F through K) Representative FACS analysis of megalin expression (F through H) and FITC-albumin internalization (I through K) by TEC exposed to various stimuli. In normal culture conditions, TEC showed the presence of megalin on their surface (F) and the ability to internalize FITC-albumin (I). Incubation with 5 μg/ml cisplatin significantly reduced megalin expression (G) and albumin uptake (J). In contrast, the addition of 10 ng/ml MSP to cisplatin-treated TEC restored both megalin expression (H) and albumin internalization (K). Kolomogorov Smirnov statistical analysis was performed in three different experiments. Similar results were obtained with HGF (data not shown), suggesting that the effects on TER, adhesion to extracellular matrices, tight junction protein/megalin expression, and internalization of FITC-labeled albumin were not specific for MSP. Magnification, ×100.

MSP Induced Re-distribution of Cytoskeleton Fibers and Dosage-Dependent TEC Migration

Compared with vehicle alone (Figure 9), 10 ng/ml MSP induced the re-distribution of actin cytoskeleton fibers and increased TEC migration that peaked at 2 h and remained significantly higher throughout all of the observation period (Figure 10A). MSP-induced TEC migration was dosage dependent with a significant increase found at dosages ≥1 ng/ml and peaking at 50 ng/ml (Figure 10B). The addition of wortmannin (0.1 μM) or PD98059 (20 μM) significantly reduced MSP-induced migration (Figure 10C). Blocking anti-MET antibody or MET siRNA transfection did not prevent MSP-induced migration (Figure 10C). In contrast, migration was abolished in TEC transfected with RON siRNA (Figure 10C). Moreover, MSP and HGF but not EGF induced a significant increase of scattered migration on extracellular matrices (Figure 10D).

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Figure 9.

MSP induced TEC migration and redistribution of cytoskeleton fibers. TEC stimulated for 12 h with vehicle alone or 10 ng/ml EGF (negative control) did not show the ability to migrate into a wound generated in a monolayer to create a cellular mechanical stress. In contrast, in the presence of 10 ng/ml MSP or HGF (positive control), TEC showed a marked enhancement of migration. Micrographs show the start (0) and the end (12 h) points of a representative migratory experiment recorded by time-lapse microscopy. Five experiments were performed with similar results. In accordance with migration data, incubation with 10 ng/ml MSP and HGF but not EGF or vehicle alone induced the redistribution of actin cytoskeleton that lost the typical stress fiber staining. Magnifications: ×100 for 0 and 12 h; ×200 for actin.

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Figure 10.

MSP-induced TEC migration was time and dosage dependent, partially inhibited by wortmannin or PD98059, and not influenced by extracellular matrices. (A) Time-course migration induced by various growth factors on TEC. In comparison with vehicle alone, MSP (10 ng/ml) and the positive control HGF (10 ng/ml) induced a significant increase of TEC migration at all time points considered (*P < 0.05 HGF or MSP versus vehicle alone at 2, 6, and 12 h). The negative control EGF (10 ng/ml) did not induce a significant increase in TEC motility. (B) Dosage-response migration induced by MSP on TEC. A significant increase of TEC migration was detected at dosages ≥1 ng/ml (*P < 0.05 MSP 1, 10, 20, 50, or 100 ng/ml versus vehicle alone). (C) Wortmannin (0.1 μM) or PD98069 (20 μM) significantly reduced MSP-induced TEC migration (*P < 0.05 MSP versus vehicle, ^§P < 0.05 MSP plus wortmannin or MSP + PD98059 versus MSP alone). HGF (10 ng/ml) was used as positive control (*P < 0.05 HGF versus vehicle). Incubation with a blocking anti-MET antibody or TEC transfection with MET siRNA did non influence MSP-induced migration (*P < 0.05 MSP + anti-MET or MSP siRNA MET versus vehicle, P > 0.05 MSP + anti-MET or MSP siRNA MET versus MSP). In contrast, TEC transfection with RON siRNA completely abolished MSP-induced migration (^#P < 0.05 MSP siRNA RON versus MSP). (D) Effect of MSP on migration of TEC cultured on extracellular matrices. Incubation with 10 ng/ml MSP significantly increased the migration of TEC cultured on plates previously coated with 20 μg/ml human fibronectin, type IV collagen, or Matrigel. HGF (10 ng/ml) was used as positive control. (P < 0.05 *fibronectin, ^§type IV collagen, or ^#Matrigel HGF or MSP versus vehicle alone). In all migration experiments, data are given as speed average (μm/h) ± 1 SD of three different experiments.

MSP Induced Scattering and Branching Morphogenesis of TEC Cultured on Matrigel-Coated Plates

TEC cultured for 24 h without FCS on Matrigel-coated plates formed round cell aggregates (Figure 11A). Addition of 10 ng/ml MSP resulted in scattering and branching morphogenesis of TEC (Figure 11B).

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Figure 11.

MSP induced scattering, branching morphogenesis, and tubulogenesis in vitro and in vivo after subcutaneous xenograft of lentiviral-transduced TEC carrying GFP (TEC GFP) in SCID mice. (A and B) Representative micrographs of morphogenesis of TEC cultured on Matrigel-coated plates in different experimental conditions. After 24 h, unstimulated TEC formed round cell aggregates (A), whereas MSP induced scattering and branching morphogenesis (B). Micrographs are representative images of six different experiments. (C through F) In vivo tubulogenesis of lentiviral-transduced TEC-GFP subcutaneously injected into Matrigel plugs in SCID mice. Unstimulated cells formed some tubular-like structures (C) that were significantly increased in the presence of 10 ng/ml MSP (D). Cisplatin (5 μg/ml) completely altered normal tubular-like structure with disappearance of GFP-positive cells (E). In contrast, MSP restored the ability of cisplatin-treated cells to form tubular-like structures (F), showing the presence of tubules with a pseudolumen and preserved polarity (F, inset). Micrographs are representative images of six different experiments. (G) Count of tubular-like structures in 10 nonsequential Matrigel sections containing TEC-GFP in various experimental conditions. With respect to vehicle alone, MSP induced a significant increase of tubular-like formations (*P < 0.05 MSP versus vehicle alone). By contrast, cisplatin challenge resulted in a significant decrease of tubular-like structures (*P < 0.05 CIS versus vehicle alone) that was partially inhibited by MSP addition (^§P < 0.05 CIS + MSP versus CIS). ANOVA with Newman-Keuls multicomparison test was performed. (H) TUNEL assay of Matrigel sections containing TEC-GFP in the presence of different stimuli. In the presence of cisplatin (5 μg/ml), a significant increase of apoptotic cells was detected (*P < 0.05 CIS versus vehicle). Addition of 10 ng/ml MSP resulted in a significant decrease of apoptosis either in serum-deprived (*P < 0.05 MSP versus vehicle alone) or in cisplatin-treated (^§P < 0.05 CIS + MSP versus CIS) cells. Data are expressed as average number of apoptotic cells/field. ANOVA with Newman-Keuls multicomparison test was performed. Magnifications: ×100 in A through H; ×600 in F inset.

MSP Induced Tubulogenesis In Vivo

We injected subcutaneously into Matrigel plugs in SCID mice TEC transduced by a lentiviral vector expressing GFP. These cells (TEC-GFP) spontaneously formed tubular-like structures (Figure 11C) that were significantly increased in the presence of 10 ng/ml MSP (Figure 11, D and G). By contrast, cisplatin completely altered TEC-GFP morphogenesis, inducing apoptosis and loss of GFP expression (Figure 11, E, G, and H). Addition of MSP on cisplatin-treated TEC-GFP partially restored normal morphogenesis (Figure 11F), showing tubular-like structures with a pseudolumen and a preserved cell polarity (Figure 11F, inset). In addition, MSP significantly decreased the number of apoptotic cells per field (Figure 11H) and preserved the immunostaining for RON, megalin, and ZO-1 (Figure 12), suggesting a role for this growth factor in maintaining cellular functional integrity and a correct tubular remodeling required for in vivo tubulogenesis.

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Figure 12.

MSP maintained markers of tubular cell integrity in cisplatin-treated TEC-GFP after subcutaneous xenograft in SCID mice. Representative confocal microscopy images of TEC-GFP injected subcutaneously into Matrigel plugs in SCID mice in the presence of 5 μg/ml cisplatin and 10 ng/ml MSP. TEC-GFP formed tubular-like structures characterized by staining for human class I HLA and by maintained expression of RON, of the endocytic receptor megalin, and of the tight junction protein ZO-1. In merge micrographs, nuclei were counterstained by 0.5 μg/ml Hoechst. Six experiments were performed with similar results. Magnification, ×600.

Patients

We enrolled in the study 25 critically ill patients who had been admitted to ICU. The inclusion criterion was the presence of ARF with the need for renal replacement therapy. Selected patients were included in the “failure” group in relation to their sCr levels or urinary output in accordance to the ADQI-RIFLE criteria.²⁵ The severity of illness was assessed by APACHE (Acute Physiology and Chronic Health Evaluation) II and by SOFA (Sequential Organ Failure Assessment) score at the moment of ARF onset. The estimation of mortality risk in the group was performed by the Acute Tubular Necrosis-Individual Severity Score (ATN-ISS) system developed by Liano et al.⁵² Patients with CRF, chronic liver failure, hepatorenal syndrome, congestive heart failure, neoplastic diseases, collagenopathies, glomerulopathies, and diabetes were excluded from the study. Plasma samples were drawn in the early phase (days 1 through 3; n = 10) and in the late phase (days 7 through 14; n = 25) of ARF. MSP plasma levels were also studied in 25 critically ill patients with normal renal function (exclusion from ADQI-RIFLE criteria), 10 patients with chronic uremia before and 1 or 8 wk after kidney transplantation, and 10 healthy volunteers.

Kidney transplant recipients were treated with the standard induction immunodepressive protocol adopted in our center: Basiliximab (20 mg) at days 0 and 4 after transplantation, tacrolimus (0.2 mg/kg body wt per d), and steroids. In selected cases at high risk for rejection, mycophenolate mofetil (1 g/d) was added to standard therapy. Tacrolimus plasma levels were maintained in the range of 17 to 20 ng/ml (weeks 1 through 4) or 12 to 15 ng/ml (weeks 5 through 12).

MSP Determination

MSP plasma levels were determined by ELISA that detected both pro-MSP and the active dimeric form (R&D Systems, Minneapolis, MN; and Ray Biotech Inc., Norcross, GA). MSP was determined in supernatants of TEC cultured in various experimental conditions. Results were calculated after the generation of a standard curve with appropriate controls and given as averages ± 1 SD.

Murine AKI Model

Animal care and treatment were conducted in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Acute toxic tubular injury in C57Bl/6 mice was induced by intramuscular injection of 7.5 ml/kg body wt 50% glycerol solution (Sigma, St. Louis, MO), as described previously.²⁶ Renal function was assessed by determination of sCr and sU levels using an automated chemistry analyzer (Beckman Instruments, Fullerton, CA). Mice were killed after 3, 6, 10, and 21 d from AKI induction. Six mice for each experimental group were examined. As controls, six mice were administered an injection of vehicle alone. Kidneys were formalin fixed and paraffin embedded for histology and immunohistochemistry. Immunostaining for MSP and RON was examined semiquantitatively in 10 nonsequential sections by measurement of fluorescence intensity by digital image analysis (Casti Imaging, Venezia, Italy) using a low-light video camera (Leica DC100, Wetzlar, Germany). Results are given as relative fluorescence intensity with respect to fluorescence spontaneously originated by tissue background. Co-localization of MSP with markers of proximal tubules, thick ascending limb of Henle's loop, and collecting ducts (respectively anti- megalin, Tamm-Horsfall protein, and aquaporin-2 antibodies from Santa Cruz Biotechnology, Santa Cruz, CA) was performed by confocal microscopy (LSM5 PASCAL; Zeiss, Jena, Germany). The modulation of MSP and RON expression was confirmed by Western blot analysis of tubular cell lysates after separation from glomeruli.

Human Tubular Cell Isolation and Culture

Primary cultures of human TEC (pTEC) were obtained from kidneys removed by surgical procedures from patients affected by renal carcinomas by a technique previously described.²⁸ An immortalized cell line (iTEC) generated by infection with a hybrid Adeno5/SV40 virus and previously characterized²⁸ was used to confirm and to extend the experiments carried out with pTEC. Both cellular types were grown with RPMI 1640 (Life Technologies, Grand Island, NY) containing 10% FCS (Hyclone, Logan, UT) and 2 mM glutamine (Life Technologies). The purity of TEC cultures was assessed on the basis of cell characterization, according to published criteria.²⁸ TEC showed negative staining for von Willebrand factor, minimal staining for desmin and vimentin, and marked staining with antibodies directed to cytokeratins and actin. TEC were also positive for markers of fully differentiated proximal TEC such as alkaline phosphatase, aminopeptidase A, and megalin. In addition, TEC showed an enhanced cAMP production after stimulation with 100 nM parathyroid hormone.²⁸

Detection of RON Receptor on TEC

Viability and Proliferation Assays

TEC were cultured on 24-well plates (Falcon Labware, Oxnard, CA) at a concentration of 5 × 10⁴ cells/well and incubated with appropriate agonists and 10 μM BrdU (Roche Diagnostics, Mannheim, Germany) or 250 μg/ml XTT (Sigma, St. Louis, MO) in a medium lacking phenol red. The absorption values, respectively, at 405 nm (BrdU) or at 450 nm (XTT) were measured in an automated spectrophotometer at different time points. In selected experiments, the PI3K inhibitor wortmannin (0.1 μM), PD98059 (20 μM), a specific inhibitor of Mek that activates ERK, or a blocking anti-MET antibody (R&D Systems) was used. All experiments were performed in triplicate.

Detection of Apoptosis

Caspase-3, -8, and -9 Activities

The activities of caspase-3, -8, and-9 were assessed by ELISA (Chemicon, Temecula, CA) on the basis of the spectrophotometric detection of the cromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA, which is recognized by caspases. TEC lysates were diluted with an appropriate reaction buffer, and DEVD-pNA was added at a final concentration of 50 M. Samples were then analyzed in an automated ELISA reader at a wavelength of 405 nm. Each experiment was performed in triplicate.

Immunofluorescence Studies

After appropriate stimuli, cultured TEC were fixed in ethanol/acetic acid 2:1 and stained with antibodies directed to human Fas (Upstate Biotechnology), vimentin (Sigma), pan-cytokeratins (Sigma), Pax-2 (Covance, Princeton, NJ), E-cadherin (Dako, Glostrup, Denmark), albumin (R&D Systems), ZO-1, MSP, megalin, alkaline phosphatase, or aminopeptidase A (all from Santa Cruz Biotechnology). After incubation with primary antibodies, samples were washed with 1× PBS and incubated with appropriated Alexa Fluor–conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) for 30 min, RT when needed. In selected experiments, cytoskeleton actin fiber distribution was evaluated by binding of 2 μg/ml FITC-conjugated phalloidin (Sigma). All samples were counterstained by 1 μg/ml propidium iodide or 0.5 μg/ml Hoechst for 30 s, mounted with anti-fade mounting medium (Vector Laboratories, Burlingame, CA), and examined by UV light microscopy.

FACS Analysis

For FACS analysis, cells were detached with EDTA and stained for 1 h at 4°C with primary antibodies directed to human Fas or megalin or with an irrelevant control antibody. After extensive washing, cells were incubated with Alexa Fluor–conjugated secondary antibodies for 45 min at 4°C where appropriate. All incubation periods were performed using a medium containing 0.25% BSA and 0.0016% sodium azide. At the end of staining, cells were newly washed, fixed in 1% paraformaldehyde, and subjected to FACS analysis (Becton Dickinson, Mountain View, CA).

Western Blot Analysis

For Western blot analysis, cells were detached with EDTA and lysed with a 50-mM Tris-HCl lysis buffer containing 1% Triton X-100, 10 μM/ml leupeptin, 10 μM PMSF, and 100 U/ml aprotinin. After centrifugation of TEC lysates at 15000 × g, protein contents of the supernatants were measured by the Bradford method: 30 μg of protein per lane was subjected to SDS-PAGE (10%) and electroblotted onto nitrocellulose membrane filters. Blots were subsequently blocked with 5% nonfat milk in 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 0.1% Tween. Membranes were incubated overnight at 4°C with antibodies directed to Akt, P-Akt, ERK, P-ERK, Bcl-xL (Santa Cruz Biotechnology), or Pax-2 at a concentration of 500 ng/ml. After extensive washing with 0.1% Tween, blots were stained for 1 h, RT with horseradish peroxidase–conjugated protein A (200 ng/ml; Amersham, Buckingamshire, UK), washed again with 0.1% Tween, developed with ECL detection reagents (Amersham), and exposed to X-Omat film (Eastman Kodak, Rochester, NY).

Evaluation of Transepithelial Electrical Resistance

Transepithelial electrical resistance was used as an indicator of cell polarity. Cells were plated in transwells on collagen-coated polycarbonate membranes (Corning Costar Corp., Cambridge, MA) and allowed to reach confluence before the addition of different stimuli. An epithelial volt-ohm meter (EVOM; World Precision Instruments, Sarasota, FL) was used to determine transepithelial electrical resistance values as described previously.⁵³ All measures were performed in triplicate and normalized for the area of the membrane.

TEC Adhesion to Extracellular Matrixes

Adhesion to extracellular matrices was evaluated on 24-well culture plates previously coated for 6 h with 20 μg/ml human fibronectin, type IV collagen, or Matrigel (Becton Dickinson, Oxford, UK). Nonspecific adhesion was blocked by incubation for 2 h with 2% BSA diluted in 1× PBS. TEC were exposed to different stimuli for 6 h at 37°C in conditions of slight agitation.³³ Thereafter, aliquots of stimulated cells were added to the wells and allowed to adhere for 2 h at 37°C. Supernatants were then removed, and attached cells were subjected to the XTT-based assay as reported previously.

Uptake of FITC-Conjugated Albumin

Protein uptake was studied after incubation of TEC with 50 mg/ml FITC-conjugated human albumin at 37°C for 2 h. After FITC-albumin challenge, TEC were extensively washed with ice-cold 1× PBS and analyzed by FACS.⁵⁴

Migration and Morphogenesis Assays

Migration was evaluated as the speed at which TEC migrated into a wound generated in a confluent monolayer to simulate a condition of mechanical stress. Cell motility was observed during a 12-h period under a Nikon Diaphot inverted microscope with a ×10 phase-contrast objective in an attached, hermetically sealed Plexiglas Nikon Np-2 incubator at 37°C. Migration was analyzed after recording on a JVC-ICCD video camera with digital saving of different images at 30-min intervals using the Micro-Image analysis system (Casti Imaging). Cellular migration was evaluated by marking the position of the nucleus of >30 cells per microscopic field for each experimental condition.⁵⁵ Speed average expressed in μm/h was calculated as the ratio of straight-line distance between the starting and ending points of observation divided by the time of observation. In selected experiments, the PI3K inhibitor wortmannin (0.1 μM), PD98059 (20 μM), a specific inhibitor of Mek that activates ERK, or a blocking anti-MET antibody (R&D Systems) was used. Moreover, we evaluated scattered cell migration after seeding TEC on plates previously coated with 20 μg/ml fibronectin, type IV collagen, or Matrigel.⁵⁶ Morphogenesis assay was performed on TEC cultured onto Matrigel for 72 h in the presence of various stimuli.

RNA Interference

In selected experiments, TEC were seeded on six-well plates and RON siRNA, MET siRNA, or relative control siRNA (80 pM) was introduced according to the manufacturer's instructions (Santa Cruz Biotechnology). After 48 h, the effective suppression of specific mRNA and proteins was verified by RT-PCR and by immunofluorescence and Western blot analysis. Subsequently, the cells were used to evaluate proliferation, resistance to apoptosis, and migration.

Generation of Lentiviral Vectors and TEC Infection

Viral production was performed as described previously.^57,58 Viral preparation titers were determined by p24 ELISA (Alliance, PerkinElmer, Wellesley, MA) and by GFP titer determination on 293T cells. Titers were typically 1E10 transduction units per ml in concentrated vector preparations. For gene marking, TEC were plated on 24-well plates in normal culture conditions and then transduced overnight with a lentivector carrying a CMV-GFP expression cassette at multiplicity of infection of approximately 20. Culture medium was replaced with fresh medium the next day. Transduction was confirmed by UV light microscopy and FACS analysis. The cell line generated was named TEC-GFP.

Subcutaneous Xenografts of TEC-GFP into Matrigel Plugs in SCID Mice

Subcutaneous xenotransplantation of TEC-GFP in Matrigel plugs was performed to evaluate the tubulogenic effect of MSP in vivo. Briefly, Matrigel deprived from growth factors (Becton Dickinson) was maintained at −20°C until use and thawed at 4°C overnight immediately before implantation. TEC-GFP (5 × 10³ cells) were re-suspended in 250 μl of fresh medium without FCS and mixed to 500 μl of Matrigel on ice using cooled pipette tips in the presence of various stimuli. All samples were subcutaneously implanted into the posterior limbs of SCID mice.⁵⁹ After 1 wk, mice were killed and Matrigel plugs were retrieved for examination. Confocal microscopy analysis was performed to detect tubulogenesis and the expression of human class I-HLA, RON, megalin, and ZO-1 on TEC-GFP. In addition, TUNEL assay by using a peroxidase-conjugated antibody was performed to detect apoptotic cells into Matrigel plugs.

Statistical Analysis

All data of various experimental procedures are expressed as averages ± 1 SD. Statistical analysis was performed by ANOVA with Newman-Keuls or Dunnet multicomparison test where appropriate.