Podocytes are sensitive to fluid shear stress in vitro

【摘要】  Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm 2 resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK 1 cells) after 20 h. At 0.015-0.25 dyne/cm 2, lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear zonula occludens-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm 2, the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and -actinin. Because the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors that were all without effect on podocyte number under static conditions. At 0.25 dyne/cm 2, however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces a reorganization of the actin cytoskeleton and activates specific tyrosine kinases that are required to withstand fluid shear stress.

【关键词】  glomerular filtration force actin cytoskeleton

PODOCYTES ARE EXPOSED TO RELEVANT mechanical forces, arising from glomerular capillary pressure and glomerular filtration ( 12 ). Glomerular capillary pressure generates wall tension in glomerular capillaries that needs to be counterbalanced by endothelial cells (ECs) and podocytes. Because glomerular ECs possess only a faint actin cytoskeleton, the actin filaments in the foot processes of podocytes are likely to predominantly stabilize the capillary wall. Elevated glomerular pressure is transmitted to and sensed by podocytes as an increase in mechanical stress; i.e., podocytes are stretched to a greater extent. Glomerular filtration exerts a different type of force on podocytes. The flow of the primary filtrate through the filtration slits between the interdigitating foot processes and the flow over the apical surface of podocytes generate a defined level of fluid shear stress.

Increased glomerular capillary pressure leads to focal segmental glomerulosclerosis (FSGS) in animal models ( 11, 23, 25, 41 ) and is also thought to induce or to accelerate FSGS in humans ( 4 ). We and others have shown that podocytes are sensitive to stretch, affecting the cytoskeleton, cell cycle, hormone production, signal transduction, and gene expression in podocytes ( 10, 13, 14, 27, 29, 34, 36 ). In contrast, the effect of fluid shear stress has never been studied in podocytes, although, in addition to ECs ( 6 ), tubular epithelial cells are known to be sensitive to fluid shear stress ( 9, 15, 28 ).

Beyond the glomerular filtration barrier, the ultrafiltrate flows to the urinary orifice of Bowman's capsule, streaming over the surface of podocyte cell bodies. Podocytes possess a complex cell shape as they are attached to several capillaries with their major processes. To minimize flow resistance and forces exerted by fluid shear stress on the cell membrane, podocyte cell bodies appear to adopt a streamlined morphology. Adaptation of podocyte shape to filtrate flow would require that podocytes are able to sense shear stress. In addition to a role of fluid shear stress in normal podocyte biology, fluid shear stress may be involved in podocyte detachment. Under several pathological conditions, viable podocytes detach from glomerular capillaries and can be recovered in the urine ( 18, 19, 32, 35, 37, 46 ).

In the present study, we exposed podocytes of a recently generated murine cell line ( 38 ) to defined laminar shear stress in flow chambers. We found that podocytes respond in a highly sensitive fashion to shear stress, with reorganization of the actin cytoskeleton and with detachment at higher rates of shear stress. Furthermore, specific tyrosine kinases appear to play a crucial role in the adaptation of podocytes to shear stress.

MATERIALS AND METHODS

Cell culture. Cultivation of a recently generated conditionally immortalized mouse podocyte cell line was performed as reported ( 38 ). In brief, podocytes were maintained in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany) supplemented with 10% FBS (Boehringer Mannheim, Mannheim, Germany), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Life Technologies). To proliferate podocytes, cells were cultivated at 33°C (permissive conditions), and the culture medium was supplemented with 10 U/ml mouse recombinant -interferon (Life Technologies) to enhance expression of the temperature-sensitive large T antigen. To induce differentiation, podocytes were maintained at 38°C without -interferon (nonpermissive conditions) for at least 1 wk. Expression of WT-1 was routinely checked by immunofluorescence (Santa Cruz Biotechnology, Heidelberg, Germany). LLC-PK 1 renal epithelial cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. For shear stress experiments in the custom-built flow chamber and for immunofluorescence studies, podocytes were seeded on glass coverslips, which were coated with a solution of 0.1 mg/ml mouse collagen IV (BD Bioscience, Bedford, MA) for 30 min at room temperature. For observation of living cells, podocytes were cultivated in commercially available flow chambers (Ibidi µ-Slides; Integrated Bio Diagnostics, Munich, Germany).

Application of fluid shear stress. Two different flow chamber systems were operated to perform shear stress experiments. A custom-built flow chamber system and the commercially available Ibidi µ-Slides were used. Fluid flow was generated by an analog 4 channel tubing pump (REGLO Analog, Ismatec Switzerland). It circulated a total of 50 ml of culture medium within a closed-loop silicone tubing system (Ismatec, Wertheim-Mondfeld, Germany; VWR International, Darmstadt, Germany). For experiments in the custom-built flow chamber, differentiated mouse podocytes were seeded on a collagen IV-coated 35-mm coverslip as described above. Three to five days elapsed until the cells were well attached and had grown to a confluent monolayer. The coverslip was then installed in the flow chamber. The actual flow channel was formed by a silicone gasket that was placed on the coverslip before the coverslip was mounted in between a plexiglas top and bottom. A rectangular hole in the gasket set the dimensions of the flow channel (length: 32 mm, width w : 5 mm). Depending on which gasket was used, the height h of the channel could be altered from 0.5 to 1.5 mm. That way, shear stress rates could be modified. In addition to channel height, flow rate Q was adjusted to obtain a specified rate of shear stress calculated as = 6 · Q /( w · h 2 ) ( 5 ); denotes the viscosity of the medium and was set to 1 centipoise. The applied shear stress rates ranged from 0.015 to 0.25 dyne/cm 2. Experiments in the custom-built flow chamber were performed in the incubator at 37°C for 20 h. To observe effects of shear stress on living podocytes, the Ibidi µ-Slide was used. The flow channel of the µ-Slide (length: 50 mm, width: 5 mm, height: 0.4 mm) comes industrially coated with collagen IV. Differentiated podocytes were cultivated in the flow chamber for 3-5 days before the experiments, until the podocytes had formed a confluent monolayer.

Immunofluorescence. Cells were fixed inside the flow chamber using either methanol for 20 min at -20°C or 2% paraformaldehyde and 4% sucrose in PBS for 10 min at room temperature. The custom-built flow chamber system was disassembled after fixation before the cells were washed with PBS for 3 min and permeabilized with 0.3% Triton X-100 in PBS for 8 min. Cells in the µ-Slide were treated within the slide. Cells were rinsed again with PBS before nonspecific binding sites were saturated in blocking solution (2% FBS, 2% bovine serum albumin, and 0.2% fish gelatin in PBS) for 30 min. Incubation with primary antibodies was performed for 1 h at room temperature. Antibodies directed against following proteins were applied: WT-1 (Santa Cruz Biotechnology), -actinin, vinculin (both from Sigma, Deisenhofen, Germany), cortactin (kindly provided by Dr. X. Zhan, American Red Cross, Rockville, MD), and zonula occludens-1 (ZO-1; BD Transduction Laboratories, Palo Alto, CA). Antigen-antibody complexes were visualized with Cy3-conjugated secondary antibodies (Dianova, Hamburg, Germany). F-actin was visualized using fluorochrome (Alexa 488)-conjugated phalloidin (Molecular Probes, Eugene, OR). After cells had been washed with PBS, they were incubated for 30 min with secondary antibodies. After the incubation, the cells were washed with PBS and water. Coverslips were then mounted on glass slides using 15% Mowiol (Calbiochem, San Diego, CA). After staining in the µ-Slide, cells were conserved with 30% glycerol in PBS. For detection of apoptosis, cells were stained with 0.1 mg/ml Hoechst 33342 (Sigma) for 10 min after fixation to visualize nuclear condensation or fragmentation. Cell numbers were determined counting intact nuclei.

Microscopy. Living podocytes were studied, and phase-contrast time-lapse movies were obtained with an inverted microscope (DM IRBE; Leica Microsystems, Bensheim, Germany) equipped with a cooled CCD camera (Photonic Science, Robertsbridge, UK) and OpenLab software (Improvision, Coventry, UK). An air stream incubator (ASI 400, Nevtek, Burnsville, VA) kept a stable temperature of 37°C during experiments. Time-lapse examinations were usually performed for 3 h at a time. Due to the operating speed of the pump and a smaller height of the flow channel, shear stress rates reached 1.75 dyne/cm 2 in the µ-Slide. Additional experiments with lower shear stress rates had been performed in the µ-Slide to verify its comparability to the custom-built flow chamber system. Fixed specimens were viewed under an inverted widefield and confocal fluorescence microscope (DM IRBE and TCS SP, Leica).

Scanning electron microscopy. Kidneys of Wistar rats in pentobarbital sodium anesthesia were perfusion-fixed through the abdominal aorta with 3% glutaraldehyde in PBS supplemented with 0.05% picric acid. Immediately after perfusion, kidneys were removed, cut into slices, and immersed in the same fixative for 1 day. Coronal slices of 3-mm thickness were dried by the critical-point technique, mounted on aluminum stubs with silver conductive paint, sputter-coated with gold, and examined in a Philipscan 500 at 25 kV.

Inhibitors. In some experiments, the following inhibitors (Merck Biosciences, Bad Soden, Germany, and Alexis, Grünberg, Germany) were present throughout the period of application of fluid shear stress: genistein ( 1, 2 ) and AG 82 (tyrphostin A25) ( 1, 47 ), both competitive broadband tyrosine kinase inhibitors; daidzein, an inactive genistein analog ( 1 ); PP2, a Src family tyrosine kinase inhibitor ( 42 ); and PD153035, an EGF receptor tyrosine kinase inhibitor ( 16 ). For time-lapse experiments, podocytes were incubated with the inhibitor for 1 h before application of shear stress.

Anti-Thy1.1 glomerulonephritis. Anti-Thy1.1 glomerulonephritis was chosen to serve as an in vivo model of elevated fluid shear stress on podocytes. Due to mesangiolysis, the glomerular tuft loses its mechanical stability. Glomerular capillaries come into close contact with the urinary orifice, where podocytes are exposed to higher levels of fluid shear stress (cf. APPENDIX and Fig. 1 ). Tissue from a previous study on anti-Thy1.1 glomerulonephritis in rats was utilized ( 24 ). In this previous study, anti-Thy1.1 glomerulonephritis was induced by injection of the monoclonal antibody OX-7 and kidneys were fixed by perfusion at various time points after antibody injection. Semithin sections were cut and stained with methylene blue.

Fig. 1. Magnitude of fluid shear stress on podocyte surface estimated by model calculation. Fluid shear stress on podocyte surface due to filtrate flow in Bowman's space was calculated for 3 different sets of s (i.e., the distance between the glomerular tuft and Bowman's capsule) as a function of position z (from the vascular pole z = 0 to the urinary pole z = 1 of the glomerular tuft). The model and the parameters are described in the APPENDIX.

Data analysis. Data are presented as means ± SE if not otherwise indicated. ANOVA was used for statistical analysis. Dunnett's and the Student-Newman-Keuls methods were employed for comparison against the control group and multiple comparisons, respectively. A P value <0.05 was considered to indicate statistical significance.

RESULTS

To estimate the magnitude of shear stress occurring in the mouse glomerulus due to glomerular filtration, a model calculation was used (cf. APPENDIX ). According to this model calculation, fluid shear stress rates of up to 0.3 dyne/cm 2 seemed to be reasonable to be encountered by the surface of podocytes in the mouse glomerulus ( Fig. 1 ).

Under experimental conditions of 20 h of fluid shear stress, cell morphology changed and podocyte loss occurred ( Fig. 2 ). Under static conditions, podocytes showed the typical picture of a dense, confluent epithelial monolayer ( Fig. 2 ) ( 38 ). The cells possess a rounded shape, and only a minority of the cells show lamellipodia. After 20 h of low fluid flow at 0.015 dyne/cm 2, the monolayer breaks up and intercellular gaps appear. The podocytes shift to a more motile cell shape, and the number of lamellipodia increases, yet no significant podocyte loss occurs. After 20 h of shear stress at 0.25 dyne/cm 2, the former monolayer shows great gaps between the cell bodies resulting from significant cell loss and cell migration. At shear stress rates of 0.5 dyne/cm 2 applied for 20 h, the few remaining podocytes ( 16%) look severely damaged. Intact parts of a monolayer cannot be found. Podocytes were stained with Hoechst 33342 to check for nuclear condensation or fragmentation, which are hallmarks of apoptosis. The staining showed no increase in apoptotic cells up to 0.25 dyne/cm 2, indicating viable podocytes under experimental conditions (data not shown).

Fig. 2. Morphology and cell number of podocytes exposed to shear stress. Podocytes were exposed to various rates of shear stress for 20 h. Increasing rates of shear stress lead to intercellular gap formation (arrows) at lower rates and to cell loss at higher rates. Cell number was determined by counting intact nuclei. The number of intact cells starts to significantly drop at 0.25 dyne/cm 2; only a few cells remain at 0.5 dyne/cm 2. Image width is 410 µm. Values are means ± SE; n = 3-9 experiments. * P < 0.05 vs. static conditions.

In addition to podocytes, LLC-PK 1 cells were examined in the flow chamber. LLC-PK 1 cells express the phenotype of epithelial cells of the proximal tubule and had previously been described to be sensitive to fluid shear stress ( 15 ). Similar to the observations in proximal tubule cells in that study, we witnessed a reinforcement of actin filaments at areas of cell-cell contact after applying shear stress (data not shown). Furthermore, we saw an increased appearance of microvilli on the apical surface after the experiments. At a shear stress rate of 0.25 dyne/cm 2 for 20 h, the continuity of LLC-PK 1 monolayers was not affected, and no intercellular gaps occurred (data not shown). These results lead to the conclusion that podocytes are highly sensitive to fluid shear stress. They seem to be much more vulnerable compared with LLC-PK 1 cells, which withstand higher shear stress rates.

Next, the cytoskeletal organization of F-actin, vinculin, -actinin, and cortactin after application of shear stress for 20 h was examined in podocytes and compared with unsheared controls. F-actin organization changed severely. Transversal stress fibers were found regularly in control podocytes. After treatment with shear stress, transversal stress fibers were drastically reduced and a cortical actin network showed. F-actin concentration also remained high at cell-cell contacts ( Fig. 3 ). Vinculin, an adaptor protein at focal adhesions and adherens junctions, colocalized with F-actin under static conditions. In podocytes exposed to shear stress, the strict colocalization of vinculin with F-actin was lost, and vinculin exhibited a diffuse cytoplasmic distribution ( Fig. 3 ). Vinculin changes seemed to follow a typical pattern. When podocytes were stained earlier during an experiment, after 4 h of shear stress, the first changes were noticeable in the cell center. Vinculin here already was diminished, whereas cortical vinculin still showed as distinct spots. In contrast to vinculin, the intensity of -actinin at cell junctions was enhanced by shear stress ( Fig. 3 ). Cortactin, which is involved in the regulation of actin nucleation, was predominantly localized in lamellipodia and ruffles in podocytes exposed to low shear stress, indicating that low shear stress activates podocyte motility ( Fig. 3 ). These cytoskeletal changes were reversible. As podocytes that had been sheared for 20 h were cultivated for 2 additional days before being stained for actin and vinculin, they expressed the regular vinculin pattern of static control podocytes. Furthermore, the intercellular gaps vanished and the monolayer closed up. These results point to a high mechanosensitivity in podocytes to shear stress. The podocytes react with a change in cell shape and reorganization of their cytoskeletal network.

Fig. 3. Actin cytoskeleton in podocytes exposed to shear stress. Podocytes were exposed to various rates of shear stress for 20 h. Under static conditions, podocytes possess prominent stress fibers, and vinculin is present in focal adhesions (arrow). Application of shear stress results in reduction of stress fibers, loss of vinculin from focal adhesions, enhanced recruitment of -actinin to cell-cell contacts (arrowheads), and increased motility visible in the prominent cortactin staining of cell margins at 0.015 dyne/cm 2. Image width is 250 µm (24 µm for insets ).

Because we observed marked formation of lamellipodia at low shear stress, we examined the cell contacts between podocytes by staining for ZO-1, which is localized at the slit diaphragm in situ. In static control podocytes, ZO-1 was distributed in a linear pattern along cell-cell contacts ( Fig. 4 ). Despite marked lamellipodia formation, the linear staining pattern of ZO-1 was maintained at low shear stress ( Fig. 4 ), indicating that low shear stress does not disrupt the junctional continuity of podocytes. At high shear stress, the integrity of cell-cell contacts was lost, as demonstrated by the discontinuous and punctate distribution of ZO-1 ( Fig. 4 ).

Fig. 4. Zonula occludens-1 (ZO-1) distribution in podocytes exposed to shear stress. Podocytes were exposed to various rates of shear stress for 20 h. ZO-1 is distributed in a linear pattern along cell-cell contacts under static and low shear stress conditions. At a shear stress rate of 0.25 dyne/cm 2, the continuous staining pattern is lost. Image width is 220 µm (20 µm for insets ).

Subsequently, podocytes were studied by time-lapse microscopy for immediate effects of fluid shear stress. For these experiments, the Ibidi µ-Slide flow chamber was used, because it allowed observation with inverse microscopy through its extra thin, transparent plastic bottom. Within 15 min of shear stress, the majority of podocytes showed massive membrane ruffling ( Fig. 5 and supplementary video; the online version of this article contains supplemental data), accompanied by enhanced macropinocytotic activity ( 44 ). Often, more than one ruffle at a time appeared per cell. Some podocytes also showed sequences of ruffles emerging, fading away, and emerging again. Newly formed cytoplasmic vesicles became visible during ruffling. Furthermore, increased cell motility with lamellipodia formation could be observed. The homogeneous, dense picture of the monolayer disappeared within 1 h. Intriguingly, the ruffling could be triggered again through fluid flow after a phase of no shear stress of 30 min. This scrutiny proves a direct and immediate effect of fluid shear stress on podocytes.

Fig. 5. Formation of circular ruffles in podocytes in response to shear stress. Phase-contrast time-lapse microscopy revealed that circular ruffles (arrowheads) form in response to shear stress (cf. video supplement). Significant formation of ruffles is observed at 12 min after start of fluid flow, and ruffling activity remains constant thereafter. Image width is 410 µm. Values are means ± SE; n = 5 experiments. * P < 0.05 vs. t = 0.

Because tyrosine kinases are crucially involved in membrane ruffling and cell motility, the results of the flow chamber experiments suggested an activation of tyrosine kinases in podocytes by shear stress. Therefore, we tested several tyrosine kinase inhibitors under shear stress conditions. Specifically, a possibly altered F-actin distribution and increased podocyte loss after 20 h at shear rates of 0.25 dyne/cm 2 were looked at. All inhibitors did not affect cell number and morphology under static conditions ( Fig. 6 ). In the presence of 60 µM genistein and 100 µM AG 82 (tyrphostin A25), only 26 ± 14 and 0 ± 0% of viable podocytes were recovered, respectively. Both substances are competitive broadband tyrosine kinase inhibitors. In contrast, 80-90% of podocytes were recovered in the presence of solvent (0.1% DMSO), 60 µM daidzein (inactive genistein analog), 10 µM PP2 (Src family tyrosine kinase inhibitor), or 0.5 µM PD153035 (EGF receptor tyrosine kinase inhibitor).

Fig. 6. Cell number of podocytes exposed to shear stress in the presence of tyrosine kinase inhibitors. Podocytes were cultured for 20 h under static conditions or exposed to 0.25 dyne/cm 2 shear stress in the absence or presence of various tyrosine kinase inhibitors. In the presence of genistein and AG 82, the number of intact cells was markedly reduced under conditions of shear stress. Values are means ± SE; n = 3-10 experiments. * P < 0.05 vs. untreated cells at 0.25 dyne/cm 2.

F-actin distribution was not changed by tyrosine kinase inhibitors under static conditions ( Fig. 7 ). The above-described alterations in the F-actin network under shear stress (reduction of stress fibers, reinforcement of cell-cell contacts, etc.) could also be seen in podocytes treated with those inhibitors that did not lead to cell loss ( Fig. 7 ). In the presence of genistein and AG 82, however, the actin cytoskeleton of podocytes exposed to shear stress was severely altered ( Fig. 7 ), probably as a result of increased cell damage.

Fig. 7. Actin cytoskeleton in podocytes exposed to shear stress in the presence of tyrosine kinase inhibitors. Podocytes were treated for 20 h with various tyrosine kinase inhibitors under static conditions or under conditions of shear stress. None of the tyrosine kinase inhibitors altered the F-actin distribution in podocytes under static conditions. After exposure to shear stress in the presence of genistein and AG 82, the actin cytoskeleton was severely altered and cytoskeletal ghosts were observed. Image width is 250 µm.

Finally, we provide two examples that illustrate the significance of fluid shear stress for podocyte biology in vivo. Under physiological conditions, podocyte cell bodies adopt a peculiarly streamlined shape between the glomerular capillaries ( Fig. 8, left ). As a result of the streamlined morphology of podocytes, the individual lobules of the glomerular tuft possess a markedly smooth surface. Renal diseases with urinary excretion of podocytes serve as a second example. For instance, podocyte loss occurs in patients with diabetic nephropathy, FSGS, and active glomerulonephritis ( 18, 19, 32 ). We examined kidney sections of rats, in which anti-Thy1.1 glomerulonephritis had been induced 2 days before. The support of glomerular capillaries is weakened in anti-Thy1.1 glomerulonephritis due to mesangiolysis. Thus some capillaries protrude into the orifice of the proximal tubule. Podocytes covering these protruding capillaries are obviously deformed and dragged along by the enhanced shear stress at the urinary pole, indicating the impact of shear stress on podocytes in vivo ( Fig. 8, right ). Podocytes traveling along the tubular lumen were frequently observed in histological sections, indicating relevant detachment of podocytes in this model.

Fig. 8. Fluid shear stress acting on podocytes in situ. The scanning electron micrograph of a rat glomerulus illustrates the streamlined shape of cell bodies and major processes of podocytes under physiological conditions ( left ). In anti-Thy1.1 glomerulonephritis ( right ), some of the podocytes that cover capillaries without mesangial support are dragged into the orifice of the proximal tubule by shear stress (arrow). Anti-Thy1.1 glomerulonephritis was induced 2 days before tissue fixation. Image width is 90 ( left ) and 220 µm ( right ).

DISCUSSION

A variety of cells have been reported to be responsive to shear stress, of which ECs are probably studied best. Located on the inner surface of blood vessels, ECs are obviously exposed to shear stress arising from blood flow. As a reaction to shear stress, ECs are known to elongate and flatten, their cell outline and cytoskeleton become highly oriented in the direction of force ( 20, 22, 33 ). Focal contacts and adherence junctions are restructured ( 33 ). Despite intense study, the nature of the shear stress-sensing mechanism remains unclear even in ECs ( 6, 7 ).

We now add the podocyte as a flow-sensitive cell. Due to their natural function, podocytes are also exposed to fluid flow. The flow of the ultrafiltrate between the capillaries of the glomerular tuft and between the glomerular tuft and the parietal epithelium generates shear stress on the podocyte surface. The glomerular filtration rate as well as glomerular geometry can be highly modified during various physiological or pathological conditions. Could relevant forces of shear stress arise in the glomerulus in vivo, and would these forces be able to affect the podocytes? We first addressed these questions using a model calculation to estimate the shear stress rates that could correspond to actual in vivo values in the urinary space. The major factors determining the rate of shear stress on the surface of podocytes are glomerular filtration rate and the distance between Bowman's capsule and the glomerular tuft. However, exact values for this distance are unknown. To recognize this fact, we calculated the shear stress for various parameter sets (cf. APPENDIX ). Note that the resulting shear stress rates are by far lower than those encountered by ECs in blood vessels and applied to cultured ECs in experiments, reaching up to 100 dynes/cm 2 ( 39 ).

In contrast to the cell types studied so far, shear stress-induced alterations of the cytoskeleton appeared very differently in podocytes. Unlike in ECs, F-actin stress fibers were diminished and an alignment of podocytes in the direction of flow could not be found. Along with the reduction of stress fibers, vinculin distribution was also altered significantly. Accumulation of vinculin at focal adhesion sites was lost in response to shear stress. It seems likely that due to remodeling of focal adhesions under shear stress, vinculin was withdrawn. Murphy-Ullrich ( 31 ) describes a cell condition with selective loss of vinculin and -actinin from the focal adhesion plaque while the integrin-ECM protein link was left unaffected. This state is termed "intermediate adherence," and it is necessary for cell motility. Sheared podocytes indicated an increase in cell motility by an enhanced localization of cortactin at their cell periphery and frequent appearance of lamellipodia. Cortactin is known to associate only with highly dynamic F-actin subsets ( 21 ). Interestingly, enhanced cell motility does not disrupt cell-cell contacts at low shear stress, as demonstrated by an unaltered distribution of ZO-1. High cell motility was further confirmed in time-lapse studies where podocytes appeared to switch to a motile phenotype within minutes after the onset of high shear rates and break-up of the confluent monolayer. At the same time, within 15 min after the start of the experiments, massive membrane ruffling occurred (cf. supplemental video). Macropinocytosis goes along with cell surface ruffling ( 44 ), and de novo formation of vesicles could indeed be observed frequently under shear stress.

Cell motility and membrane ruffling can be triggered through growth factor treatment ( 26, 43 ). In response to mechanical stress, phosphorylation of the platelet-derived growth factor (PDGF) receptor is induced in smooth muscle cells ( 40 ). Similar to a mechanism of mechanotransduction in bronchial epithelial cells that has recently been discovered by Tschumperlin et al. ( 45 ), growth factors in the extracellular basolateral podocyte compartment, deposited by constitutive secretion or shedding, could be released at very low shear rates. Furthermore, Greenwood and co-workers ( 17 ) saw a selective loss of vinculin and -actinin from focal adhesion sites in fibroblasts after stimulating PI3-kinase with PDGF. These findings collectively suggest that growth factor-associated signal transduction pathways might play a role in the response of podocytes to shear stress.

Podocyte loss in the flow chamber occurred already under relatively low shear stress rates and progressed with increasing force. The shear stress arising from fluid flow seemed too small to be the direct cause for cell detachment. An increase in apoptosis due to experimental treatment could not be confirmed after nuclear staining with Hoechst 33342. One could argue that apoptotic cells tend to detach anyway and therefore got dragged away with the current before being counted. However, we did not observe any podocyte in the process of apoptosis. Thus it seems more likely that in response to shear stress podocytes tend to assume an intermediate adhesive state to facilitate increased cell motility, as suggested by DiMilla et al. ( 8 ). Intermediate adhesiveness may result in an increased frequency of detachment. What seems fatal on first sight may well turn out to be a smart adaptation. To distribute well-balanced mechanical force to cell-matrix adhesions, the podocyte must rearrange the large adhesion plaques that are frequently found under static conditions.

To adopt a streamlined shape under physiological conditions ( Fig. 8 ), podocytes must be able to sense fluid shear stress. Increased shear stress at a defined area on the podocyte surface might induce reorganization of the cytoskeleton until the morphological changes will result in a decrease in shear forces. Eventually, podocytes have to weaken the adhesion to some capillaries to rearrange major processes via "migration." The high sensitivity of podocytes to shear stress, the induction of a migratory, intermediate adhesive phenotype, and the reorganization of the actin cytoskeleton in a nonpolarized fashion would fit this hypothesis.

Podocyte detachment is observed in vivo. Lost podocytes appear in the urine in various renal diseases including FSGS, diabetic nephropathy, Henoch-Schönlein purpura nephritis, and Alport's syndrome ( 18, 19, 32, 46 ). The number of podocytes counted correlates with disease activity. Interestingly, podocytes appearing in the urine are viable and can be cultivated ex vivo ( 35, 37, 46 ). However, it remains unclear why podocytes detach from the basement membrane. It seems unlikely that podocytes get dragged from the glomerular basement membrane by filtrate flow in unaffected, healthy kidneys, because control patients shed only senescent podocytes, if any at all ( 19, 46 ). However, architectural changes within the glomerulus, as those we witnessed in kidney sections of rats with anti-Thy1 glomerulonephritis, may result in increased shear stress for those podocytes that protrude into the orifice of the proximal tubule. Increased shear stress might then trigger cytoskeletal reorganization, elevating the probability of podocyte detachment.

Nephrotic symptoms go along with less stable podocyte adhesion. At the same time, glomerular phosphotyrosine is increased ( 3 ). Podocyte loss in our experiments was increased through treatment with certain tyrosine kinase inhibitors; namely, genistein and AG 82 dramatically increased cell loss under fluid flow. Cell numbers were not altered in static controls or by treatment with daidzein, an inactive genistein analog, under shear stress. This indicates an involvement of specific tyrosine kinases that are required to withstand shear stress. Although even in ECs the signal transduction for shear stress has not been fully unraveled, tyrosine kinases are certainly playing an important role in the transduction of shear stress ( 6 ). Conversely, the ability of podocytes for circular ruffling was not inhibited by any of the used inhibitors, including PD153035, genistein, and AG 82 (Friedrich C, Endlich N, and Endlich K, unpublished observations). Although Krueger et al. ( 26 ) have proposed that circular ruffles are involved in the reorganization of the actin cytoskeleton, circular ruffles appear to play a minor role in the adaptation of podocytes to shear stress. Thus the mechanism, by which genistein and AG 82 induce podocyte loss specifically in response to shear stress, remains unknown at present.

In summary, we show that podocytes respond highly sensitively to fluid shear stress. Unique alterations were found in cell shape and cytoskeletal architecture after application of shear stress compared with other cell types studied so far. A highly motile podocyte phenotype with frequent appearance of lamellipodia and ruffles emerged in response to shear stress. Increasing shear stress resulted in progressive loss of podocytes. Inhibitor experiments demonstrated that specific tyrosine kinases are required to withstand fluid forces. Furthermore, our report provides a new aspect to podocyte detachment from the glomerular basement membrane under disease conditions.

APPENDIX

To estimate the shear stress encountered by the surface of podocytes in vivo, a simplified geometry of the glomerulus was assumed ( Fig. 9 ). The glomerular tuft was represented by a sphere with radius R T, separated from a spherical Bowman's capsule by the distance s. The coordinate z * was introduced to describe the axis from the vascular pole ( z * = 0) to the urinary pole ( z * = 2 R T ). Assuming a homogenous exit of filtrate over the surface of the glomerular tuft, the total filtrate flow F that passes through Bowman's space at the level z * is given by

where SNFR denotes single-glomerulus filtration rate, A 2* is tuft surface area at level z *, A T is total tuft surface area, and f is the fraction of the SNGFR that reaches the proximal tubule via Bowman's space. The fraction 1 - f of the SNGFR flows through the intercapillary spaces within the tuft, reaching the proximal tubule directly. The mean flow velocity m of the filtrate in Bowman's space depends on the filtrate flow F and the cross-sectional area B of Bowman's space at level z * according to m = F / B. The cross-sectional area between two spheres of diameter R T and R T + s at level z * with perpendicular orientation to the tuft surface can be calculated from standard geometric formulas as

This yields for the mean flow velocity m

where z * has been replaced by the dimensionless coordinate z = z */2 R T ( z = 0 at the vascular pole and z = 1 at the urinary pole).

Fig. 9. Schematic drawing of the simplified glomerular geometry used in the model calculation. The glomerular tuft was represented by a sphere with radius R T, separated from a spherical Bowman's capsule by the distance s. The dimensionless coordinate z = z */2 R T describes the axis from the vascular pole ( z = 0) to the urinary pole ( z = 1). The red arrows indicate the flow of the ultrafiltrate, where SNFR denotes single-glomerulus filtration rate.

The shear stress, acting on the surface of the glomerular tuft, is the product of viscosity and shear rate. The shear rate is the first derivative of the velocity profile with respect to the spatial coordinate ( = d /d x ). The velocity profile of laminar flow between two parallel plates of infinite size was taken to estimate the shear rate at the tuft surface. Shear stress at the tuft surface is then given by

Thus shear stress at the tuft surface is a function of the viscosity of the filtrate, of the fraction of the single glomerulus filtration rate flowing through Bowman's space, of the tuft diameter, of the width of Bowman's space, and of the position between the vascular and urinary pole.

The following values describing the mouse glomerulus were used: = 1 centipoise, f = 0.2, SNFR = 12 nl/min ( 30 ), and R T = 50 µm. Because exact values for s are unknown, shear stress rates were evaluated for different values of s : s = 6 µm, linearly increasing s from s = 2 µm (at z = 0) to 8 µm (at z = 1), and linearly increasing s from s = 3 µm (at z = 0) to 10 µm (at z = 1).

GRANTS

This study was supported by Deutsche Forschungsgemeinschaft Grant En 329/7-3.

ACKNOWLEDGMENTS

We thank Claudia Kocksch for technical assistance, Thomas Berger for construction of the flow chamber, and Ilona Dirks and Rolf Nonnenmacher for graphical work.

【参考文献】
  Akaishi Y, Hattori Y, Yoshimoto K, Kitabatake A, Yasuda K, and Kanno M. Involvement of tyrosine phosphorylation in the positive inotropic effect produced by H 1 -receptors with histamine in guinea-pig left atrium. Br J Pharmacol 130: 907-915, 2000.

Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, and Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262: 5592-5595, 1987.

Bains R, Furness PN, and Critchley DR. A quantitative immunofluorescence study of glomerular cell adhesion proteins in proteinuric states. J Pathol 183: 272-280, 1997. <a href="/cgi/external_ref?access_num=10.1002/(SICI)1096-9896(199711)183:3

Bidani AK and Griffin KA. Long-term renal consequences of hypertension for normal and diseased kidneys. Curr Opin Nephrol Hypertens 11: 73-80, 2002.

Chun TH, Itoh H, Ogawa Y, Tamura N, Takaya K, Igaki T, Yamashita J, Doi K, Inoue M, Masatsugu K, Korenaga R, Ando J, and Nakao K. Shear stress augments expression of C-type natriuretic peptide and adrenomedullin. Hypertension 29: 1296-1302, 1997.

Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 75: 519-560, 1995.

Davies PF, Zilberberg J, and Helmke BP. Spatial microstimuli in endothelial mechanosignaling. Circ Res 92: 359-370, 2003.

DiMilla PA, Stone JA, Quinn JA, Albelda SM, and Lauffenburger DA. Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength. J Cell Biol 122: 729-737, 1993.

Du Z, Duan Y, Yan Q, Weinstein AM, Weinbaum S, and Wang T. Mechanosensory function of microvilli of the kidney proximal tubule. Proc Natl Acad Sci USA 101: 13068-13073, 2004.

Durvasula RV, Petermann AT, Hiromura K, Blonski M, Pippin J, Mundel P, Pichler R, Griffin S, Couser WG, and Shankland SJ. Activation of a local tissue angiotensin system in podocytes by mechanical strain. Kidney Int 65: 30-39, 2004.

Dworkin LD, Hostetter TH, Rennke HG, and Brenner BM. Hemodynamic basis for glomerular injury in rats with desoxycorticosterone-salt hypertension. J Clin Invest 73: 1448-1461, 1984.

Endlich N and Endlich K. Stretch, tension and adhesion-adaptive mechanisms of the actin cytoskeleton in podocytes. Eur J Cell Biol 85: 229-234, 2006.

Endlich N, Kress KR, Reiser J, Uttenweiler D, Kriz W, Mundel P, and Endlich K. Podocytes respond to mechanical stress in vitro. J Am Soc Nephrol 12: 413-422, 2001.

Endlich N, Sunohara M, Nietfeld W, Wolski EW, Schiwek D, Kranzlin B, Gretz N, Kriz W, Eickhoff H, and Endlich K. Analysis of differential gene expression in stretched podocytes: osteopontin enhances adaptation of podocytes to mechanical stress. FASEB J 16: 1850-1852, 2002.

Essig M, Terzi F, Burtin M, and Friedlander G. Mechanical strains induced by tubular flow affect the phenotype of proximal tubular cells. Am J Physiol Renal Physiol 281: F751-F762, 2001.

Fry DW, Kraker AJ, McMichael A, Ambroso LA, Nelson JM, Leopold WR, Connors RW, and Bridges AJ. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265: 1093-1095, 1994.

Greenwood JA, Theibert AB, Prestwich GD, and Murphy-Ullrich JE. Restructuring of focal adhesion plaques by PI 3-kinase regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin. J Cell Biol 150: 627-642, 2000.

Hara M, Yanagihara T, and Kihara I. Urinary podocytes in primary focal segmental glomerulosclerosis. Nephron 89: 342-347, 2001.

Hara M, Yanagihara T, Takada T, Itoh M, Matsuno M, Yamamoto T, and Kihara I. Urinary excretion of podocytes reflects disease activity in children with glomerulonephritis. Am J Nephrol 18: 35-41, 1998.

Ives CL, Eskin SG, and McIntire LV. Mechanical effects on endothelial cell morphology: in vitro assessment. In Vitro Cell Dev Biol 22: 500-507, 1986.

Kaksonen M, Peng HB, and Rauvala H. Association of cortactin with dynamic actin in lamellipodia and on endosomal vesicles. J Cell Sci 113: 4421-4426, 2000.

Kataoka N, Ujita S, and Sato M. Effect of flow direction on the morphological responses of cultured bovine aortic endothelial cells. Med Biol Eng Comput 36: 122-128, 1998.

Kretzler M, Koeppen-Hagemann I, and Kriz W. Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat. Virchows Arch 425: 181-193, 1994.

Kriz W, Hahnel B, Hosser H, Ostendorf T, Gaertner S, Kranzlin B, Gretz N, Shimizu F, and Floege J. Pathways to recovery and loss of nephrons in anti-Thy-1 nephritis. J Am Soc Nephrol 14: 1904-1926, 2003.

Kriz W, Hosser H, Hahnel B, Simons JL, and Provoost AP. Development of vascular pole-associated glomerulosclerosis in the Fawn-hooded rat. J Am Soc Nephrol 9: 381-396, 1998.

Krueger EW, Orth JD, Cao H, and McNiven MA. A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells. Mol Biol Cell 14: 1085-1096, 2003.

Lewko B, Endlich N, Kriz W, Stepinski J, and Endlich K. C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress. Kidney Int 66: 1001-1008, 2004.

Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM. Effect of flow and stretch on the [Ca 2+ ] i response of principal and intercalated cells in cortical collecting duct. Am J Physiol Renal Physiol 285: F998-F1012, 2003.

Martineau LC, McVeigh LI, Jasmin BJ, and Kennedy CR. p38 MAP kinase mediates mechanically induced COX-2 and PG EP4 receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol Renal Physiol 286: F693-F701, 2004.

Meneton P, Ichikawa I, Inagami T, and Schnermann J. Renal physiology of the mouse. Am J Physiol Renal Physiol 278: F339-F351, 2000.

Murphy-Ullrich JE. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J Clin Invest 107: 785-790, 2001.

Nakamura T, Ushiyama C, Suzuki S, Hara M, Shimada N, Ebihara I, and Koide H. Urinary excretion of podocytes in patients with diabetic nephropathy. Nephrol Dial Transplant 15: 1379-1383, 2000.

Noria S, Xu F, McCue S, Jones M, Gotlieb AI, and Langille BL. Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress. Am J Pathol 164: 1211-1223, 2004.

Petermann AT, Hiromura K, Blonski M, Pippin J, Monkawa T, Durvasula R, Couser WG, and Shankland SJ. Mechanical stress reduces podocyte proliferation in vitro. Kidney Int 61: 40-50, 2002.

Petermann AT, Krofft R, Blonski M, Hiromura K, Vaughn M, Pichler R, Griffin S, Wada T, Pippin J, Durvasula R, and Shankland SJ. Podocytes that detach in experimental membranous nephropathy are viable. Kidney Int 64: 1222-1231, 2003.

Petermann AT, Pippin J, Durvasula R, Pichler R, Hiromura K, Monkawa T, Couser WG, and Shankland SJ. Mechanical stretch induces podocyte hypertrophy in vitro. Kidney Int 67: 157-166, 2005.

Petermann AT, Pippin J, Krofft R, Blonski M, Griffin S, Durvasula R, and Shankland SJ. Viable podocytes detach in experimental diabetic nephropathy: potential mechanism underlying glomerulosclerosis. Nephron Exp Nephrol 98: e114-123, 2004.

Schiwek D, Endlich N, Holzman L, Holthofer H, Kriz W, and Endlich K. Stable expression of nephrin and localization to cell-cell contacts in novel murine podocyte cell lines. Kidney Int 66: 91-101, 2004.

Schnittler HJ, Schneider SW, Raifer H, Luo F, Dieterich P, Just I, and Aktories K. Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress. Pflügers Arch 442: 675-687, 2001.

Shaw A and Xu Q. Biomechanical stress-induced signaling in smooth muscle cells: an update. Curr Vasc Pharmacol 1: 41-58, 2003.

Simons JL, Provoost AP, Anderson S, Troy JL, Rennke HG, Sandstrom DJ, and Brenner BM. Pathogenesis of glomerular injury in the fawn-hooded rat: early glomerular capillary hypertension predicts glomerular sclerosis. J Am Soc Nephrol 3: 1775-1782, 1993.

Slack JK, Adams RB, Rovin JD, Bissonette EA, Stoker CE, and Parsons JT. Alterations in the focal adhesion kinase/Src signal transduction pathway correlate with increased migratory capacity of prostate carcinoma cells. Oncogene 20: 1152-1163, 2001.

Suetsugu S, Yamazaki D, Kurisu S, and Takenawa T. Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev Cell 5: 595-609, 2003.

Swanson JA and Watts C. Macropinocytosis. Trends Cell Biol 5: 424-428, 1995.

Tschumperlin DJ, Dai G, Maly IV, Kikuchi T, Laiho LH, McVittie AK, Haley KJ, Lilly CM, So PT, Lauffenburger DA, Kamm RD, and Drazen JM. Mechanotransduction through growth-factor shedding into the extracellular space. Nature 429: 83-86, 2004.

Vogelmann SU, Nelson WJ, Myers BD, and Lemley KV. Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol 285: F40-F48, 2003.

Yano Y, Geibel J, and Sumpio BE. Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain. Am J Physiol Cell Physiol 271: C635-C649, 1996.

作者单位：1 Department of Anatomy and Cell Biology I, University of Heidelberg, Heidelberg; and 2 Department of Anatomy and Cell Biology, Ernst Moritz Arndt University, Greifswald, Germany