Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells

Marie Essig, Fabiola Terzi, Martine Burtin, and Gérard Friedlander

Institut National de la Santé et de la Recherche Médicale U-426 and Department of Physiology, Faculté de Médecine Xavier Bichat, Université Denis Diderot-Paris 7, F-75018 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effects of flow-induced mechanical strains on the phenotype of proximal tubular cells were addressed in vivo and in vitro by subjecting LLC-PK1 and mouse proximal tubular cells to different levels of flow. Laminar flow (1 ml/min) induced a reorganization of the actin cytoskeleton and significantly inhibited the expression of plasminogen activators [tissue-type (tPA) activity: 25% of control cells; tPA mRNA: 70% of control cells; urokinase (uPA) mRNA: 56% of control LLC-PK1 cells]. In vivo, subtotal nephrectomy (Nx) decreased renal fibrinolytic activity and uPA mRNA content detectable in proximal tubules. Nx also induced a reinforcement of the apical domain of the actin cytoskeleton analyzed by immunofluorescence. These effects of flow on tPA and uPA mRNA were prevented in vitro when reorganization of the actin cytoskeleton was blocked by cytochalasin D and were associated, in vitro and in vivo, with an increase in shear stress-responsive element binding activity detected by an electrophoretic mobility shift assay in proximal cell nuclear extracts. These results demonstrate that tubular flow affects the phenotype of renal epithelial cells and suggest that flow-induced mechanical strains could be one determinant of tubulointerstitial lesions during the progression of renal diseases.

kidney; tubulointerstitium; plasminogen activators; cytoskeleton; shear stress


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A REDUCTION OF RENAL MASS IS followed by a progressive destruction of remaining functional nephrons even after the apparent resolution of the initial injury. This deterioration of renal structures is observed in a large number of renal diseases (25) and involves glomerular and tubulointerstitial damage. Tubulointerstitial damage is a major prognosis factor because a strong correlation between the intensity of tubulointerstitial lesions and the degree of reduction of renal function has been observed in various nephropathies such as glomerulonephritis, diabetic nephropathy, and renal mass reduction (1, 18, 25, 28, 31). Tubulointerstitial damage results in part from an imbalance between extracellular matrix (ECM) production and proteolysis. One of the proteases involved in ECM proteolysis is plasmin, which is formed from plasminogen after cleavage by the two plasminogen activators, tissue-type (tPA) and urokinase (uPA), along the fibrinolytic pathway. Inhibition of the fibrinolytic pathway has been shown to be associated with glomerulosclerosis or interstitial fibrosis in various renal diseases (14) and to correlate with the severity of impairment of renal function during chronic renal failure (7).

Several recent studies in endothelial cells have focused on the potent role of mechanical stress induced by vascular flow in regulating endothelial cell functions. Ion channel properties, growth factor synthesis, and tPA expression have been demonstrated to be modified by vascular flow (9, 13, 23, 33). In kidney, epithelial cells are subjected to the urinary flow all along the tubule, and numerous studies have established that modifications of tubular flow affect ion channel characteristics (37). Recently, it has been demonstrated that urinary flow affects the synthesis of nitric oxide in collecting duct cells (5).

On the basis of these observations, we hypothesize that tubular flow could modify the phenotype of proximal tubular cells and could be involved in the modification of ECM remodeling observed after renal mass reduction, because a threefold increase in glomerular filtration and proximal tubular flow rate was evidenced soon after subtotal nephrectomy by micropuncture studies (19, 42). We report here that various flow conditions induced, in vitro and in vivo, a decrease in fibrinolytic activity and a reorganization of the cytoskeleton of proximal tubular cells. Our results suggest that this effect of flow on the fibrinolytic system occurs most likely through the reorganization of the cytoskeleton and is associated with the transactivation of shear stress-responsive elements (SSREs).


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Cell Culture

Mouse proximal tubule (MPT) cells were isolated from 3- to 4-wk-old C57Bl/6 mice as described (21). Briefly, kidneys were removed aseptically from anesthetized mice. The cortex was separated from the medulla and incubated for 30 min in a 1 mg/ml collagenase solution. Homogeneous populations of nephron segments were separated on a Percoll gradient (Percoll 42%; centrifugation 17,000 rpm for 30 min at 4°C). The F4 layer, composed almost exclusively of proximal tubules, was seeded on glass slides (76 × 26 mm) in culture medium [Ham's F-12/Dulbecco's modified Eagle's medium (1:1, vol/vol), 25 mM HEPES, 21.5 mM HCO3, 1 mM Na pyruvate, 10 ml/l of a 100× nonessential amino acid mixture, 4 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 nM Na selenite] supplemented with 5 µg/ml insulin, 35 µg/ml transferrin, 5 nM triiodothyronine, 0.5 µM retinoic acid, 25 ng/ml prostaglandin E1, and 50 nM dexamethasone. Fetal calf serum (1%) was present in the medium for the first 48 h.

LLC-PK1 cells established by Hull et al. (20) were grown on glass slides (76 × 26 mm) in hormone-free culture medium supplemented with 5% fetal calf serum.

For flow experiments, glass slides seeded with a confluent monolayer of MPT or LLC-PK1 cells were assembled into a parallel plate chamber, forming a flow channel (0.5 × 20 × 70 mm) connected to recirculating flow loops. A laminar flow was generated by the hydrostatic pressure difference between a high and a low reservoir. The flow system was kept at 37°C and ventilated with 95% humidified air-5% CO2.

Cells were sheared during 2, 4, or 24 h in 15 ml of serum- and hormone-free culture medium. At the end of the flow, supernatants were collected and concentrated to 1 ml using Centriflo columns (Amicon, Beverly, MA). Cells were lysed in 200 µl of 0.5% Triton X-100/PBS buffer.

Animal Experiments

Experiments were conducted on 8-wk-old C57Bl/6 mice. Mice were anesthetized with a mixture of 2% Rompun (xylazine; 6 µg/g body wt, Bayer, Leverkusen, Germany) and Clorketam 1000 (ketamine; 120 µg/g body wt, Etoquinol, Lirre, France). Control mice were subjected to a sham operation (Sham) with decapsulation of both kidneys. As previously described (38), 75% subtotal nephrectomy (Nx) was performed by excision of the right kidney and the two poles of the left kidney. At death, the kidneys were removed, cut into two halves, and either immediately frozen for immunofluorescence and zymographic analyses or used for proximal tubule isolation. Each group contained five animals and was analyzed 8 days after surgery.

Proximal Tubule Isolation

Proximal tubules were isolated from one kidney as previously described (16). Briefly, the kidney was kept at 4°C in Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES, pH 7.4, and 5 mM D-glucose (HBSS-HEPES). The cortex was separated from the medulla and then dilacerated by pressure (Dounce homogenizer no. 7). Proximal tubules were isolated by filtering on mesh (75 µm) to retain glomeruli, washed twice with HBSS-HEPES, and frozen at -80°C until extraction of nuclear proteins or RNA.

Immunofluorescence

Cells grown on glass coverslips or 4-µm-thick frozen kidney sections were fixed in 4% formaldehyde/PBS for 10 min and permeabilized in 0.1% Triton X-100/PBS for 5 min at room temperature. Cells were incubated with either tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (1:1,000/PBS) for 30 min at 37°C or a monoclonal anti-human vinculin antibody for 1 h at 37°C (1:200/PBS-1% BSA) revealed by a FITC-conjugated secondary antibody. After two washes in PBS, coverslips were mounted using the DAKO Faramount mounting medium. Stained cells were stored in the dark at 4°C until analysis with a Nikon microscope.

Zymography

For zymography of cell culture supernatants or cytosols, 1 µg of total proteins was mixed with nonreducing sample buffer. After separation of the proteins on a 10% acrylamide gel, the gel was soaked in 2.5% Triton X-100 for 1 h and layered onto a fibrin-agarose gel containing bovine plasminogen-rich fibrinogen (7 mg/ml), bovine thrombin (40 U/ml), and agarose (1%), as previously described (30). Human tPA (Biopool), human uPA (Urokinase, Hoechst), and rat urine, which contains high amounts of uPA, were used as controls. In situ kidney zymography was performed on 7-µm-thick frozen sections by layering glass slides onto a 0.75-mm fibrin-agarose gel as described by Sappino et al. (35). Zymograms were allowed to develop for 3-48 h at 37°C. Gel photographs were scanned with an imaging densitometer and quantified using the National Institutes of Health (NIH) Image 1.6 software program. Quantification of the renal lysis zone detected by in situ zymography was realized by analysis of three independent slides for each kidney. Analyzed surface area was identical in Sham and Nx animals. Data are expressed as the percentage of control values.

Immunoblotting

Immunoblotting of supernatants or lysates was carried out by standard techniques. SDS-PAGE was carried out according to the method of Laemmli (27).Twenty micrograms of total proteins were mixed with nonreducing sample buffer and separated on a 10% acrylamide gel under nonreducing conditions. Separated proteins were electrotransferred to a nitrocellulose membrane (Bio-Rad). A 10% solution of nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) was used to block nonspecific binding during 1 h at 37°C. The membrane was then incubated 1 h at 37°C with the primary antibody [anti-tPA (1:500 in 10% milk-PBST) and anti-uPA (1:200 in 10% milk-PBST)] and then washed three times with PBST. The membrane was next incubated 1 h at 37°C with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Labs, West Grove, PA) and washed six times with PBST. Immunoreactive protein bands were detected using the enhanced chemiluminescence method (ECL kit, Amersham, Buckinghamshire, UK). Photographs of the membranes were scanned by an imaging densitometer and quantified using the NIH Image 1.6 software program. Protein concentrations were determined by the method of Bradford with the use of the Coomassie protein assay reagent from Pierce (Rockford, IL).

RNA Isolation and Analysis

Proximal tubule suspensions and MPT cells. Total RNA was extracted from freshly isolated proximal tubules prepared from one kidney or from one glass slide of MPT cells, as previously described by Chomczynski and Sacchi (6) using tRNA (40 µg) as the carrier. The RNA pellet was diluted in 20 µl H2O, and 10 µl were used to synthesize cDNA by reverse transcription using 400 U Moloney murine leukemia virus RT (M-MLV; GIBCO BRL) and random hexamers as primers in a final volume of 40 µl. cDNAs were analyzed by PCR using the murine ribosomal S14 RNA as reference (forward primer: 5'-ATCAAACTCCGGGCCACAGGA-3'; reverse primer: 5'-GTGCTGTCAGAGGGGATGGGG-3'; product length: 133 bp) and murine uPA primers (forward primer: 5'-AAGAATGCATGGTGCATGACTGC-3'; reverse primer: 5'-TCACAGTCTGAACCAAACGG-3'; product length 514 bp). To rule out a possible contamination by human S14, we sequenced the PCR product and compared it with the published sequence of murine S14. This comparison showed 100% homology. Final concentrations and amounts in the PCR mixture were as follows: 500 nM S14 primers, 200 nM uPA primers, 250 nM dNTPs, 1.5 mM MgCl2, 2 or 10 µl cDNA, 1 U Taq polymerase, and reaction buffer as supplied in the kit (final volume: 50 µl). cDNA was denatured for 5 min at 95°C and then amplified using cycling parameters as follows: S14: 95°C, 30 s; 59°C, 30 s; 72°C, 30 s; uPA: 95°C, 30 s; 56°C, 30 s; and 72°C, 30 s. Either 30, 35, and 40 cycles, respectively, were performed. Amplified products were separated on 1% agarose gel electrophoresis and visualized by ethidium bromide/ultraviolet light. Gels were scanned by an imaging densitometer and quantified using the NIH Image 1.6 software program.

LLC-PK1 cells. Total RNA extracted from one glass slide of LLC-PK1 cells was analyzed by Northern blot. Twenty micrograms of total RNA were electrophoresed on 1% agarose-18% formaldehyde denaturing gels and transferred overnight to a nylon membrane (Hybond N, Amersham). After baking (2 h at 80°C), the membranes were prehybridized for 30 min at 65°C in hybridization buffer [5× standard sodium citrate (SSC), 5× Denhardt's, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA]. The murine tPA cDNA was a gift from R. Lijnen (Leuwen, Belgium). The porcine uPA cDNA was synthesized by RT-PCR using primers, starting at positions 1198 and 1613 of the mRNA porcine sequence, respectively (forward: 5'-AAGTCACCACCAAAATGCTGTG-3'; reverse: 5'-CCCAGCACCCAGACTTGTATC-3'). The 436-bp fragment was subcloned in the pGEM-T Easy vector as described by the manufacturer (Promega, Madison, WI), and the uPA cDNA fragment was excised by EcoRI digestion. Murine beta -actin cDNA probe was used to control for of mRNA loading. cDNAs were labeled by random priming using [alpha -32P]dCTP as described by the manufacturer (PrimeIt, Stratagene, La Jolla, CA). The membranes were hybridized overnight at 65°C, washed twice with 2× SSC, 0.1% SDS at 25°C, then with 0.1× SSC, 0.1% SDS at 60°C. Autoradiography was performed at -80°C using Biomax films (Kodak) and intensifying screens. Radioactivity was quantified using an Instantimager (Packard).

Electrophoretic Mobility Shift Assay

Cells collected after exposure to flow or freshly isolated proximal tubules were lysed for 10 min in 400 µl of ice-cold hypotonic buffer {[in mM] 10 HEPES, 10 KCl, 1.5 MgCl2, 1 EDTA, 2 dithiothreitol, and 1 polymethylsulfonyl fluoride (PMSF), as well as 5 µM leupeptin and 5 µM aprotinin} as described (39). Nuclei were collected by centrifugation (3,500 g, 15 min), and nuclear proteins were eluted for 30 min by using 20 µl of hypertonic elution buffer [(in mM) 20 HEPES, 1 KCl, 1.5 MgCl2, 0.2 EDTA, 2 dithiothreitol, and 1 PMSF, as well as 25% glycerol, 5 µM leupeptin, and 5 µM aprotinin]. After centrifugation (15,000 g, 20 min), supernatants containing the eluted nuclear proteins were frozen at -80°C until analysis. Oligonucleotides used in the electrophoretic mobility shift assay experiments were as follows: shear stress-responsive element (SSRE)1: 5'-CTCGGCTCTCAGAGACCCCCTAAGCGCC-3' and 5'-GCGCTTAGGGGGTCTCTGAGAGCCGGAA-3'; SSRE2: 5'-TCAGCTCGGCTCTCAGAGACCCCCTAAGCGCC-3' and 5'-TCAGGCGCTTAGGGGGTCTCTGAGAGCCGAG-3'; mutated (m)SSRE: 5'-CTCGGCTCTACACTGTAGCATAAGCGCC-3'; and activator protein-1 (AP-1): 5'-CGCTTGATGACTCAGCCGGAA-3' (24). Annealed SSRE1 and mSSRE oligonucleotides were end-labeled by [gamma -32P]dATP using the T4 DNA kinase (GIBCO BRL). Because nuclear extracts prepared from proximal tubules demonstrated high phosphatase activity, we used a second SSRE probe (SSRE2), which was end-labeled by [alpha - 32P]dCTP using the Klenow DNA polymerase fragment (GIBCO BRL). Preliminary experiments demonstrated a similar pattern of migration between SSRE1 and SSRE2 probes. Five micrograms of nuclear proteins were hybridized with 32P-labeled oligonucleotides (20,000 counts/min) in a binding buffer (10 mM HEPES-KOH, 250 mM KCl, 0.2 mM EDTA, 10% glycerol, 2.5 mM dithiothreitol, 0.05%, Nonidet P-40, 1 µg/µl dIdC) for 30 min at 4°C. In supershift studies, rabbit polyclonal anti-p50 and anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) were added 10 min before the addition of the probe. DNA-protein complexes were separated on a 5% acrylamide gel in 0.5× Tris-borate-EDTA buffer at 150 V for 2 h. Gels were dried before radioactivity was quantified with the use of an Instantimager, and autoradiography was performed using standard procedures.

Materials

Insulin, transferrin, dexamethasone, prostaglandin E1, retinoic acid, triiodo-L-thyronine, sodium selenite, type 1 collagenase, cytochalasin D, TRITC-phalloidin, bovine fibrinogen, and bovine thrombin were purchased from Sigma (St. Louis, MO). Percoll was from Pharmacia (Uppsala, Sweden). Agarose was from Eurobio (Les Ulis, France). Polyclonal antibodies anti-porcine tPA, anti-human tPA, and anti-mouse uPA were from Biopool (Umea, Sweden). SDS, 30% acrylamide/Bis (37.5:1) solution, and nitrocellulose membranes were from Bio-Rad. Culture media and reagents were from GIBCO BRL (Grand Island, NY). Plastic ware was from Costar (Cambridge, MA). Animals were from Iffa Credo.

Statistical Analyses

Statistical analyses of the data were performed using one-way analysis of variance followed by a protected least-significant difference test. Statistical significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Effects of Flow on the Cytoskeleton and Fibrinolytic Activity in Cultured Proximal Cells

To evaluate the effect of flow on the phenotype of proximal tubular cells, we stimulated MPT cells in a laminar flow chamber and analyzed their cytoskeletal organization and fibrinolytic system. MPT cells subjected to flow showed an increased pinocytosis (not shown) without loss of cell viability, an aspect that was also described in endothelial cells (10). As previously described for epithelial cells, the actin cytoskeleton of unsheared MPT cells demonstrated numerous pronounced cytosolic actin stress fibers (Fig. 1A). Laminar flow (1 ml/min) for 24 h induced a disappearance of the cytosolic actin stress fibers and a reinforcement of the lateral actin network. More potent flow (5 ml/min) induced a further reinforcement of the actin lateral network.


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Fig. 1.   Organization of the actin cytoskeleton of mouse proximal tubule (MPT) cells subjected to flow analyzed by fluorescence microscopy [×400 (A), ×600 (B)]. Flow induced a disappearance of cytosolic actin stress fibers and the reinforcement of the lateral actin network. C and D: representative zymography of 3 independent experiments (C) and representative semiquantitative RT-PCR of 2 independent experiments (D) for urokinase plasminogen activator (uPA) activity and mRNA in static or sheared MPT cells. Flow (1 ml/min) induced a decrease in uPA activity and mRNA content.

With regard to the fibrinolytic system, the major PA activity detectable in MPT cells supernatants or cytosols involved uPA. Laminar low (1 ml/min) for 24 h induced a large decrease in uPA activity detected by zymography (Fig. 1C) that was associated with a rapid decrease in mRNA abundance evidenced after 4 h of flow (Fig. 1D).

To further analyze the characteristics of the modulation of the tubular fibrinolytic system by flow, we used LLC-PK1 cells, which are known to exhibit phenotypic features of proximal tubular cells (20). With regard to fibrinolytic activity, tPA activity was the major PA activity detectable in LLC-PK1 supernatants and cytosols (Fig. 2A). However, as previously described (12), uPA activity could be rapidly induced by 100 nM antidiuretic hormone [arginine vasopressin (AVP)] (Fig. 2A). Thus further analysis of uPA expression was performed in the presence of 100 nM AVP. Laminar flow (1 ml/min) for 24 h induced a major inhibition of tPA activity detected in cytosols and supernatants by zymography (Fig. 2A). tPA activity of sheared cells reached 45 and 25% of control values in cytosol and supernatant, respectively (Fig. 2B). Laminar flow also induced a decrease in uPA activity detected in supernatants in the presence of AVP. The decrease in tPA and uPA activity induced by flow was associated with a decrease in PA antigen and mRNA visualized by immunoblot and Northern blot. The single band of 64 kDa, corresponding to the porcine tPA detected by immunoblot analysis in control cells, decreased under flow condition (Fig. 2C) and reached only 53% of control values in sheared cells (Fig. 2D). On the other hand, uPA antigen could not be detected by immunoblot using either human or mouse anti-uPA antibodies. With regard to mRNA content, laminar flow for 4 h induced a decrease in both tPA and uPA mRNA (Fig. 2E). tPA-to-beta -actin and uPA-to-beta -actin ratios in sheared cells reached 70 and 56% of control values for tPA and uPA, respectively (Fig. 2, F and G).


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Fig. 2.   Fibrinolytic system expression in static or sheared LLC-PK1 cells. A: representative zymography evaluating fibrinolytic activity. B: quantification of tissue-type plasminogen activator (tPA) lysis zone (n = 3). C and D: representative immunoblot and quantification of tPA antigen expression (n = 3), respectively. Representative Northern blot (E) and quantification of tPA-to-beta -actin mRNA (n = 9; F) and uPA-to-beta -actin mRNA (n = 9; G) ratios are also shown. Values are means ± SE expressed as the percentage of static values. Flow (1 ml/min) induced an inhibition of tPA and uPA expression in LLC-PK1 cells.*P < 0.05.

Experiments using various levels of flow (300 µl/min, 600 µl/min, and 1 ml/min) and static conditions revealed that only the highest value of flow (1 ml/min) could induce a significant decrease in tPA or uPA mRNA content (300 µl/min, 108 and 102% of control values, and 600 µl/min, 129 and 100% of control values, for tPA-to-beta -actin and uPA-to-beta -actin ratios respectively) (Fig. 3A). At last, reversibility of the phenotypic changes induced by flow was assessed by leaving LLC-PK1 cells successively in a static condition for 12 h, in flow conditions (1 ml/h) for 12 h, and then in a static condition for 12 h (Fig. 3B). As shown by zymographic analysis, inhibition of tPA activities induced by 12 h of shear stress was reversed when cells returned to a static condition.


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Fig. 3.   Dose-response (A) and reversibility (B) of flow-induced inhibition of the fibrinolytic system in LLC-PK1 cells. A: LLC-PK1 cells were subjected to different levels of flow (no flow, 300, 600, and 1,000 µl/min) for 4 h, then lysed to analyze uPA and beta -actin mRNA content by Northern blot. Values are means ± SE expressed as the percentage of static values of 4 independent experiments.*P < 0.05. B: LLC-PK1 cells were incubated for 12 h in a static condition (period 1), then subjected to static (A) or flow (1 ml/min; B) conditions for 12 h (period 2), then returned to a static condition for 12 h (period 3). At the end of each period, supernatants were collected and concentrated for analysis by zymography. Inhibition of the fibrinolytic activity induced by flow was reversed by return to a static condition.

In Vivo Effects of Flow

In Sham mice, high fibrinolytic activity was detected in kidney slices by in situ zymography (Fig. 4A). As previously described (35), this activity was inhibited by 1 mM amiloride, suggesting that uPA was the predominant renal PA. Subtotal nephrectomy (i.e., Nx rats) was associated with an inhibition of the renal fibrinolytic activity, which occurred as early as 1 day after surgery (not shown) and became significant 8 days after surgery (Fig. 4A). Quantification of renal lysis zones showed that renal fibrinolytic activity in Nx mice reached only 35% of Sham values (Fig. 4B).


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Fig. 4.   Renal fibrinolytic activity of kidney slices from sham-operated (Sham) animals or from animals that underwent subtotal nephrectomy (Nx). In situ zymography was performed 8 days after surgery. Shown are representative zymography (A) and quantification (B) of renal lysis zone observed in zymography in absence of amiloride. Quantification was done on 3 slices/kidney from 3 and 2 kidneys for Sham and Nx groups, respectively. Values are means ± SE expressed as the percentage of Sham values. *P < 0.05. C-E: uPA mRNA content in proximal tubules isolated from Nx and Sham animals analyzed by RT-PCR using S14 mRNA as reference. cDNA of static MPT cells was used as a positive control [control (+)] whereas PCR mixture without cDNA was used as a negative control. Shown are representative RT-PCR (C) and quantification (D) of uPA (open circle , Nx; triangle , Sham) and S14 (filled bars, Nx; open bars, Sham) signals in Nx and Sham samples, using 2 µl of RT reaction product. E: representative RT-PCR using various volumes of RT reaction product in Nx and Sham animals to equalize S14 signals. Values are means ± SE expressed as the percentage of positive control [control (+)] values (n = 3). Nx induced a decrease in uPA activity and mRNA content in proximal tubules.

To evaluate whether inhibition of uPA activity observed in Nx animals could result from a decrease in uPA mRNA, we analyzed uPA mRNA content in proximal tubules of Nx animals compared with Sham animals. Amplification (30, 35, and 40 cycles) of 2 µl of RT reaction product showed that uPA mRNA content was decreased in Nx animals compared with Sham animals, whereas S14 signals were not significantly different (Fig. 4, C and D). This decrease was also evidenced when volumes of RT reaction product were chosen to equalize S14 signals in Nx and Sham animals (Fig. 4E). These results suggested that inhibition of uPA activity in Nx animals could result from a decrease in uPA mRNA abundance.

With regard to the organization of the cytoskeleton, the actin network in proximal tubular cells of Sham animals consisted of long, thick fibers oriented along the axis of the brush border, in thin fibers in the terminal web, and in basolateral stress fibers anchoring in focal adhesion contacts (Fig. 5A). Nx induced a reinforcement of actin fluorescence in the brush border and in the terminal web, which became punctated and striated (Fig. 5B).


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Fig. 5.   Organization of the actin cytoskeleton in proximal tubules of Sham or Nx animals analyzed by fluorescence microscopy (×400). Brush border (&cjs3616;), terminal web (not-right-arrow ), and basolateral stress fibers (triangle ) are shown. Nx showed a reinforcement of apical (brush border and terminal web) actin fluorescence.

Intracellular Pathway Involved in Modulation of the Fibrinolytic System by Flow

We and others have previously demonstrated that the expression of the fibrinolytic system could be modified by the organization of the cytoskeleton. In rat proximal tubular cells and in LLC-PK1 cells, disruption of the actin cytoskelton induced by cytochalasin D produced an increase in tPA and uPA expression (3, 16). Because flow-induced inhibition of uPA was associated with a reorganization of the cytoskeleton in MPT cells, we evaluated the link between flow-induced modification of PA and cytoskeletal organization in LLC-PK1 cells by submitting cells to flow in the absence or presence of cytochalasin D, which blocks cytoskeletal reorganization. In the absence of cytochalasin D, flow (1 ml/min) for 24 h induced a reorganization of the cytoskeleton in LLC-PK1 cells with reinforcement of the lateral domain of the actin filament network (Fig. 6B) compared with unsheared cells (Fig. 6A). Cytochalasin D (1 µM) induced a disruption of actin filaments evidenced in both static and sheared cells (Fig. 6, C and D). With regard to tPA and uPA mRNA content, cytochalasin D completely prevented the decrease in tPA and uPA mRNA induced by flow (Fig. 6E).


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Fig. 6.   Organization of the actin cytoskeleton in static (A and C) or sheared (B and D) LLC-PK1 cells in the absence (A and B) or presence (C and D) of cytochalasin D (1 µM) analyzed by fluorescence microscopy (×400). Flow (1 ml/min) for 4 h induced the reinforcement of the lateral actin network (black-triangle), which is inhibited in the presence of cytochalasin D. E: uPA and tPA mRNA expression under static or flow conditions (1 ml/min) for 4 h in the absence or presence of cytochalasin D (1 µM). Left: representative Northern blot. Right: quantification of uPA-to-beta -actin (n = 5) and tPA-to-beta -actin mRNA ratios (n = 5). Values are means ± SE expressed as the percentage of static values. Cytochalasin D prevented the inhibition of uPA and tPA mRNA induced by flow. *P < 0.05.

In endothelial cells, the flow-induced increase in platelet-derived growth factor (PDGF) synthesis was related to an increase in the activity of transcriptional factors binding a specific DNA sequence called the SSRE (34). We analyzed whether such an increase in SSRE binding activity could be detected in sheared LLC-PK1 cells. In nuclear extracts of LLC-PK1 cells, flow (1 ml/min for 2 h) induced an increase in binding to probes specific for the SSRE (Fig. 7A). This increase was inhibited in the presence of excess nonlabeled SSRE probe and not by the nonspecific AP-1 probe. Furthermore, no binding to a mSSRE probe could be detected (Fig. 7A). Altogether, these results showed that the binding was specific for the SSRE. We next evaluated the SSRE binding activity present in vivo in proximal tubules of Sham and Nx animals. As observed in LLC-PK1 cells, nuclear extracts from proximal tubules of Nx animals showed a pronounced increased binding to SSRE-specific probes compared with nuclear extracts from Sham animals (Fig. 7B). Because some experiments in endothelial cells suggest that one of the transcriptional factors binding to the SSRE is the nuclear factor(NF)-kappa B complex, composed mostly of two subunits, the p50 and p65 proteins (24), we preincubated nuclear extracts of Nx animals with anti-p50 or anti-p65 antibodies. In nuclear extracts of Nx animals, the major signal of SSRE binding activity was inhibited in the presence of anti-p50 or anti-p65 antibodies in the binding reaction, suggesting the presence of a p50-p65 NF-kappa B complex (Fig. 7B).


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Fig. 7.   A: binding of nuclear proteins from static and sheared LLC-PK1 to 32P-labeled shear stress-responsive element (SSRE)1- or 32P-labeled mutated (m)SSRE (20,000 counts/min)-specific probe in the presence of an excess (20, 50, 100×) of nonlabeled SSRE or activator protein (AP)-1 probe. Flow (1 ml/min) induced a specific increased binding to the SSRE probe. B: binding of nuclear proteins from proximal tubules freshly isolated from Sham and Nx animals, to 32P-labeled SSRE2 (20,000 counts/min)-specific probe in absence or presence of anti-p50 or anti-p65 antibodies. Nx animals showed an increase in SSRE binding activity, which was prevented by either anti-p50 or anti-p65 antibodies.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phenotypic modifications induced by flow, first demonstrated in vascular cells, are now a growing field of research in many other cell types layered by a fluid film, such as renal epithelial cells, chondrocytes, or osteocytes (40). In endothelial cells, vascular flow was reported to induce modifications of the nitric oxide-endothelin balance, to increase the synthesis of many growth factors, and to modify the expression of adhesion molecules (9). These effects of flow were demonstrated to result from shear stress, a mechanical strain that induced the reorganization of the cytoskeleton (17) and the stiffening of the cells (11). Modification of the vascular shear stress at the atheromatous plaque level is one of the mechanisms contributing to the progression of vascular lesions in cardiovascular diseases (9).

Renal epithelial cells are subjected to urinary flow, which could be highly modified during various physiological or pathological conditions, and knowledge of phenotypic modifications of tubular cells induced by flow could help in an understanding of the pathophysiology of renal diseases. Our study described two phenotypic modifications induced by flow in proximal tubular cells: the reorganization of the cytoskeleton and the inhibition of the fibrinolytic system.

The reorganization of the cytoskeleton shown in our study in epithelial cells is not identical to what is observed in endothelial cells. In the latter cells, the major event is the alignment of actin stress fibers, which could be evidenced in vitro and in vivo in different vessels (17). Such an alignment does not exist in proximal cells. In contrast, tubular epithelial cells subjected to flow seem to reinforce the apical and lateral domains of actin filaments, which are fundamental elements of the cytoskeleton in epithelial cells. It is noteworthy that the actin network in cell cultures in a static condition, composed of many cytosolic stress fibers, differs strikingly from that observed in vivo in proximal cells. Cytosolic stress fibers were not induced by a specific support because they were observed under various coating conditions (gelatin, collagen type I, silaned and nonsilaned glass). The presence of cytosolic stress fibers has been previously described in epithelial cells from various origins cultured under usual (static) conditions and was related to the dedifferentiation of the cells (4). Our results suggest that the absence of flow takes part in the appearance of these cytosolic stress fibers.

We analyzed the expression of the fibrinolytic system in proximal cells because, in addition to reabsorptive capacities, proximal cells take part in the remodeling of the ECM surrounding the tubular structure and because tubulointerstitial fibrosis resulting from abnormal ECM remodeling is one of the major prognosis factor in renal diseases. In proximal cells, both plasminogen activators, tPA and uPA, are modified by flow, whereas only modulation of tPA expression by flow was already documented in endothelial and vascular smooth muscle cells (13, 33). To our knowledge, this study is the first report of an effect of flow on uPA expression. In epithelial proximal tubular cells, the evolution of tubular fibrinolytic activity under flow was different from that observed in endothelial cells because Diamond et al. (13) demonstrated that vascular flow induced an increase in tPA activity. In contrast, our study was in agreement with that of Papadaki et al. (33), who showed that tPA mRNA of vascular smooth muscle cells was decreased by flow. These discrepancies could result from the differences in cell types but also from the level of the mechanical strains exerted on cells.

The mechanical strains resulting from flow depend on the viscosity of the fluid, the value of the flow, and the internal ray of the structure. In the vascular bed, they range from 5 to 100 dyn/cm2 (9). In contrast, smooth muscle cells are subjected to lower mechanical strains, with a range under 5 dyn/cm2. In proximal tubules, the accurate value of mechanical strains could not be calculated. In fact, only the flow in the initial portion of the proximal tubule, corresponding to the single-nephron glomerular filtration rate (SNGFR), could be known because urinary flow is modified all along the tubule in consequence of tubular reabsorption. Furthermore, Bonvalet et al. (2) demonstrated that the internal ray of the proximal tubule also decreases all along the tubule and that only the linear velocity of the tubular fluid is constant in the proximal tubules. This previous study allows us to evaluate the tubular fluid velocity resulting from various SNGFRs and to cells subjected to a laminar flow of similar fluid velocity. In our laminar flow chambers, flow values corresponding to SNGFRs of 30 and 90 nl/min were 300 µl/min and 1 ml/min, respectively, the latter leading to a mechanical stress of 0.17 dyn/cm2, which is a far lower value than those used in endothelial or vascular smooth muscle cells studies.

In vivo, inhibition of the fibrinolytic system and reorganization of the cytoskeleton were also observed in Nx animals compared with Sham animals. Subtotal nephrectomy is known to induce a threefold increase in glomerular and tubular flow in the remaining nephrons (19, 42), and modifications of mechanical strains have been involved in phenotypic changes of mesangial cells and the appearance of glomerulosclerosis (8, 41). Our study focused on proximal tubule alterations by using freshly isolated proximal tubule suspensions that allowed us to differentiate glomerular and tubular modifications. The inhibition of the fibrinolytic system and the reorganization of the actin cytoskeleton of proximal tubular cells that we observed in vivo in Nx animals were closely related to what was evidenced in vitro in MPT or LLC-PK1 cells subjected to flow. Furthermore, these modifications were associated with an increased SSRE binding activity in nuclear extracts of proximal cells, demonstrating modifications of mechanical strains exerted on proximal cells in Nx animals. Altogether, these results suggest that the inhibition of the fibrinolytic system and the reorganization of the cytoskeleton observed in Nx animals could result from effects of increased tubular flow and modifications of mechanical strains exerted on proximal cells.

Mechanisms underlying the modulation of the fibrinolytic system by tubular flow are not elucidated, but it is likely that the reorganization of the cytoskeleton, which was observed under flow conditions, is instrumental. Organization of the cytoskeleton was previously proved to be one of the modulators of the fibrinolytic components in different cell lines. In LLC-PK1 cells, Nagamine et al. (3, 22, 29) demonstrated that disruption of the cytoskeleton by cytochalasin D induced an increase in uPA expression mediated by activation of the extracellular signal-regulated kinase pathway and the AP-1 transcription factor (3, 22, 29). In previous work, we demonstrated that disruption of the actin cytoskeleton induced by inhibition of geranylgeranylated Rho proteins resulted in an increase in the fibrinolytic system of endothelial and epithelial proximal tubular cells (15, 16). Flow conditions, which have been demonstrated in endothelial cells to induce a reinforcement of the cytoskeleton (11), could be the mirror image of these previous observations. Indeed, flow-induced inhibition of the fibrinolytic system was associated in vivo and in vitro with a reorganization of the cytoskeleton evidenced by immunofluorescence. Furthermore, blocking this reorganization by the use of cytochalasin D prevented the decrease in uPA and tPA mRNA in LLC-PK1 cells subjected to flow. These results confirm the close relationship between cytoskeleton reinforcement and the inhibition of the fibrinolytic system, but the underlying intracellular pathway involved in these phenomena remains to be identified.

As observed in endothelial cells, flow induced the activation of transcription factors binding to the specific DNA binding element SSRE in proximal epithelial cells. Activation of this element has been shown to be instrumental in the activation of PDGF and intercellular adhesion molecule-1 genes by vascular flow (34) and to result from NF-kappa B, p50-p65 heterodimer, binding (24). Our results evidenced the presence, in proximal cells stimulated by flow in vitro and in vivo, of SSRE binding activity composed, in part, of p50 and p65 proteins. Although analysis of tPA and uPA promoter could reveal some SSRE sequences, a direct role of SSRE activation in flow-induced fibrinolysis inhibition is unlikely to occur. Indeed, SSRE sequences have been generally involved in positive regulation of genes. Furthermore, PA mRNA abundance seems to depend mostly on posttranscriptional modifications affecting the stability of the mRNA. In fact, many 3'-regulatory sequences affecting uPA mRNA stability have been demonstrated in LLC-PK1 cells (26, 32). In addition, a protein destabilizing uPA mRNA and acting through binding to the 3'-end has recently been identified in pulmonary epithelial cells (36). Thus the target of the SSRE binding proteins in nuclear extracts of stimulated proximal cells needs still to be elucidated.

In conclusion, this study demonstrates for the first time that tubular flow induces a decrease in the fibrinolytic activity of proximal tubular cells. This effect was associated, in vitro and in vivo, with a reorganization of the cytoskeleton and the activation of SSRE binding activity. These results suggest that increased tubular flow could lead to alterations of tubular cell functions and could be one of the events underlying tubular damage occurring after renal mass reduction.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale IFR 02, Centre National de la Recherche Scientifique, Université Paris 7, Faculté X-Bichat, Association pour l'Utilisation du Rein Artificiel, and the Laboratoire de Recherches Physiologiques, and Société Cegetel.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Essig, Institut National de la Santé et de la Recherche Médicale U 426, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, F-75018 Paris, France (E-mail: essig{at}bichat.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 23 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 281(4):F751-F762
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