Nitric Oxide Increases Albumin Permeability of Isolated Rat Glomeruli via a Phosphorylation-Dependent Mechanism
Bing Li,
Jian Yao,
Tetsuo Morioka and
Takashi Oite
Department of Cellular Physiology, Institute of Nephrology, Niigata University School of Medicine, Niigata, Japan.
Correspondence to Dr. Takashi Oite, Department of Cellular Physiology, Institute of Nephrology, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. Phone: 0081-25-227-2156; Fax: 0081-25-227-0769; E-mail:oite{at}med.niigata-u.ac.jp
ABSTRACT. Nitric oxide (NO) has been implicated in the inductionof proteinuria in acute inflammatory glomerulonephritis andin the increased vascular permeability seen in various otherdisease conditions. The complicated interactions of NO withother factors in vivo hinder analysis of the mechanisms involved.By use of a recently introduced method for measuring albuminpermeability (Pa) in isolated glomeruli, the question of whetherNO has a direct effect on the permeability barrier of glomerulartufts was examined and the potential mechanisms were explored.Exposure of isolated glomeruli to three NO donors, s-nitroso-N-acetyl-penicillamine(SNAP), (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate(DETA-NONOate), and sodium nitroprusside, all increased thePa. This action of NO was time- and concentration-dependentand could be mimicked by 8-bromoguanosine 3', 5'-cyclic monophosphate.Western blot analysis of the proteins from NO donor-treatedglomeruli revealed an increase of phosphotyrosine levels ofproteins of molecular mass about 120 and 70 kD. The demonstrationthat pretreatment of glomeruli with the tyrosine kinase inhibitor,genistein, could largely prevent the effect of SNAP and DETA-NONOateconfirmed the crucial role of tyrosine phosphorylation in theNO-induced increase of Pa. Furthermore, the tyrosine phosphataseinhibitor, phenylarsine oxide (PAO), could mimic the actionof NO on Pa. NO-enhanced tyrosine phosphorylation was furtherconfirmed by immunofluorescence staining, where positive cellsin SNAP- and PAO-treated glomeruli were much more frequent thanthat in controls. By use of dual-label staining in combinationwith podocyte specific marker, nephrin, it was observed thatmost of the phosphorylated positive cells corresponded to podocytes.These results suggest that NO impairs the glomerular permeabilitybarrier through a tyrosine phosphorylation-dependent mechanism.
Nitric oxide (NO) is an indispensable molecule with a varietyof biologic functions. It has been shown to regulate multiplecellular functions, including smooth muscle cell relaxation,neurotransmission, and macrophage-induced cytotoxicity, as wellas cell proliferation and apoptosis (1–3). In the kidney,the presence of NO under physiologic conditions was confirmedby microdialysis studies (4). Low concentrations of NO, releasedby glomerular endothelial cells, play a pivotal role in regulatingmicrovascular hemodynamics, in concert with antagonistic vasoactivesubstances such as angiotensin II and endothelin (5). Underpathologic conditions, NO generation in the glomeruli is markedlyenhanced because of the induction of NO synthase expressionin intrinsic glomerular cells and infiltrating, activated macrophages(5). In acute inflammation in glomeruli, enhanced productionof NO was closely related to the induction of proteinuria. BlockingNO synthase expression with the specific inhibitor L-NMMA profoundlyattenuated proteinuria in the anti-Thy 1 antibody induced modelof glomerulonephritis (6), which indicates that NO was involvedin the disturbance of the glomerular permeability barrier, leadingto proteinuria.
Direct induction of vascular and epithelial hyperpermeabilityby NO has been reported elsewhere (7–9). However, informationconcerning the effect of vasoactive substances on glomerularpermeability is still incomplete. The complicated interactionsof NO with other relevant factors make it extremely difficultto analyze its actions and mechanisms in detail in vivo. Inthe present work, we examine whether NO has a direct effecton the glomerular capillary permeability barrier, using themethod for measuring albumin permeability (Pa) in isolated glomerulideveloped by Savin et al. (10). Furthermore, we investigatedthe potential mechanisms of NO-mediated glomerular hyperpermeabilty.
Reagents
S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside(SNP), (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate(DETA-NONOate), 8-bromoguanosine 3', 5'-cyclic monophosphate(8-Br-cGMP), phenylarsine oxide (PAO), genistein, protaminesulfate, bovine serum albumin (BSA), aprotinin, pepstatin, sodiumorthovanadate, phenylmethylsulfonyl fluoride, and poly-L-lysinewere obtained from Sigma (St. Louis, MO). Dulbecco’ modifiedEagle medium (DMEM) was ordered from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan).
Experimental Animals
Normal male Sprague-Dawley rats (170 to 250 g body weight) withfree access to Purina chow and water were used in all experiments.
Isolation of Glomeruli
Sprague-Dawley rats were anesthetized with ether and kidneyswere immediately removed. The renal capsules were removed, andthe outer 1 to 2 mm of the renal cortex was excised and cutinto fine fragments. The glomeruli were then isolated in DMEMthat contained 6% BSA by standard sieving techniques as describedelsewhere (10–13). At each step, the tissue was abundantlyrinsed in DMEM that contained 6% BSA. The PH of the medium wasadjusted to 7.4 before use. The whole procedure for isolationof glomeruli was carried out within 30 min at room temperature.
Incubation Medium and Washing Medium
The incubation medium was identical to the isolation medium(see above) except for the addition of the agents to be tested.The washing medium was DMEM that contained 1% BSA. The pH ofeach medium was adjusted to 7.4 before use.
Measurement of Glomerular Volume Change
Glomeruli, incubated in 6% BSA medium with or without varioustest agents, were allowed to adhere to an observation chambercoated with poly-L-lysine (1 mg/ml) for 5 to 10 s. Unattachedglomeruli were removed by gentle washing with fresh isolationmedium. Adherent glomeruli were viewed in a microscope, andthen the image was captured for their initial images by computer.All selected glomeruli were free of Bowman’ capsules.After an initial period of observation, the medium was replacedwith washing medium that contained 1% BSA. The change of mediumfrom 6% to 1% BSA produced an oncotic gradient across the glomerularcapillary wall and resulted in an influx of fluid and an increasein glomerular capillary volume. Volume changes in the glomerulisubsequent to the applied oncotic gradient occurred within 5s and were maintained for at least several minutes under bothcontrol and experimental conditions. Repeat images were obtained2 to 3 min after a change of medium. The area of each glomeruluswas automatically measured by use of National Institutes ofHealth software. The initial and final volumes of each glomeruluswere calculated from the area (S) by use of the formula V =3/4(S/)3/2. Volume change (V) was calculated as V = (Vfinal- Vinitial)/Vinitial x 100. At least ten glomeruli from threeor more rats were studied in each experiment.
Calculation of Albumin Reflection Coefficient (a)and Pa
The calculation of a and Pa was done on the basis of the detailsreported by Savin et al. (10–13). Because the increasein V is proportional to the oncotic gradient across the capillarywall, a, the ratio of V of experimental to control glomeruliin response to an identical oncotic gradient can be calculatedas follows: a = Vexperiment/Vcontrol. Pa was calculated froma and defined as Pa = 1 - a. When the reflection coefficienta is 0, albumin moves at the same rate as water, and Pa is 1.0.When a is 1.0, albumin cannot cross the membrane and Pa is 0.
Western Blot Analysis
Samples of isolated rat glomeruli were lysed in RIPA buffer(50 µM Tris-HCL [pH 7.5], 150 µM NaCL, 1% TritonX-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate) thatcontained 25 µg/ml aprotinin, 2 mM sodium orthovanadate,25 µg/ml leupeptin, 2 mM phenylmethysulfonyl fluoride,and 50 mM sodium fluoride for 30 min on ice. Lysates were clarifiedby centrifugation at 15,500 x g for 15 min at 4°C. The proteinconcentration of the supernatant was measured with a Bio-RadProtein Assay Kit. The supernatant was mixed with 2x samplebuffer (114 mM Tris [pH 6.8], 9% glycerol, 2.7% sodium dodecylsulfate, 0.02% bromophenol blue, and 4.5% mercaptoehtanol),boiled for 5 min, subjected to 7.5% sodium dodecyl sulfate-polyacrylamidegel electrophoresis, and transferred onto nitrocellulose membranes.Nonspecific binding sites were blocked by phosphate-bufferedsaline (PBS)-0.1% Tween 20 supplemented with 3% BSA for 1 hat room temperature. The membrane was probed with a horseradishperoxidase-labeled anti-phosphotyrosine antibody (Santa CruzBiotechnology, Santa Cruz, CA) diluted 1:5000 for 1 h at roomtemperature. After extensive washing with three changes of PBS-0.1%Tween 20, immunoreactive proteins were visualized with an enhancedchemiluminescense detection system, followed by exposure toKodak x-ray film (Eastman Kodak, Rochester, NY).
Immunocytochemistry
Isolated rat glomeruli were preincubated with either 400 µMSNAP for 1 h or 1 µM PAO for 15 min or left as untreatedcontrols. The medium was removed, and then the isolated ratglomeruli were rinsed in PBS and fixed in 3% paraformaldehydefor 20 min at 4°C. Free aldehyde groups were blocked withammonium chloride (50 mM in PBS, 20 min, 4°C). Isolatedglomeruli were then permeabilized with 1% Triton X-100. Afterblocking the nonspecific binding with fetal calf serum (FCS)(20 min, 20°C), glomerular suspension was incubated withmouse monoclonal anti-phosphotyrosine (PY20) antibody (SantaCruz Biotechnology) diluted 1:100 in 1% FCS-PBS for 1 h at 37°C.Then glomerular suspension was incubated for 1 h at 37°Cwith a 1:100 dilution of FITC-conjugated rabbit anti-mouse-IgG(Santa Cruz Biotechnology) in PBS that contained 1% FCS. Fordouble immunostaining with anti-PY20 antibody and a podocytespecific marker, glomerular suspension was at first incubatedwith mouse monoclonal anti-PY20 antibody, followed by the incubationwith FITC-anti-mouse IgG, as described above. Then glomerularsuspension was incubated with rabbit anti-rat nephrin antibody(14), diluted 1:200 in 1% FCS-PBS for 1 h at 37°C, washedwith PBS, and then incubated with tetramethyl rhodamine B isothiocyanate-conjugatedgoat anti-rabbit IgG (Dako, Glostrup, Demark) before final washing.Glomerular specimens treated were mounted on the slide glassand examined by use of confocal laser scanning microscope (MRC1024,Bio-Rad). Controls with irrelevant secondary or primary antibodiesdid not exhibit significant immunostaining.
Cell Viability
Excessive NO is well known to be cytotoxic. Therefore, it iscritical to rule out the possibility of impairment of cell viabilityby NO donors as well as other experimental agents, which wouldcompromise the glomerular integrity and result in a false increasein Pa. For this purpose, the MTT assay was performed. Incubationof isolated rat glomeruli with SNAP (500 µM), SNP (1 mM),CGMP (500 µM), and PAO (10 µM) for 4 h producedno significant difference in the activity of mitochondria enzymecompared with the untreated control.
Statistical Analyses
Average Pa was calculated from 10 glomeruli randomly selectedfrom each group under each experimental condition. Each experimentwas repeated at least three times. Values were compared by useof the unpaired t test. All values were expressed as mean ±SEM, and P < 0.05 was considered to be significant.
NO Increased the Pa in Isolated Rat Glomeruli
Glomeruli were incubated for 3 h at 37°C with three structurallyunrelated NO donors, SNAP (400 µM), DETA-NONOate (400µM), and SNP (1 mM), and the Pa was then measured. Asshown in Figure 1, these three different NO donors all causeda significant increase in Pa (0.55 ± 0.06, 0.51 ±0.09, and 0.43 ± 0.05, respectively) compared with theresults in control experiments (0 ± 0.06). As a representative,SNAP was chosen for the following experiments.
Figure 1. Effect of three different NO donors on albumin permeability (Pa) of isolated rat glomeruli. Isolated rat glomeruli were treated with different nitric oxide (NO) donors (400 µM S-nitroso-N-acetyl-penicillamine [SNAP], 400 µM[Z]-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate [DETA-NONOate], and 1 mM sodium nitroprusside [SNP]) or left untreated as controls for 3 h. All results were expressed as mean ± SEM (n = 3). **P < 0.01 versus control.
Incubation of glomeruli with 400 µM SNAP caused a time-dependentincrease of Pa. As shown in Figure 2A, there was a slight increasein Pa after 30 min (0.12 ± 0.08), compared with controls(0 ± 0.05). A significant increase in Pa was evidentafter only 1 h of incubation (0.26 ± 0.13), and the peak(0.45 ± 0.03) was reached at 3 h.
Figure 2. Time- and concentration-dependent effect of SNAP on Pa of isolated rat glomeruli. (A) Isolated rat glomeruli were either treated with 400-µM concentrations of SNAP or left untreated as controls for different time periods. (B) Isolated rat glomeruli were either treated with different concentrations of SNAP or left untreated as controls for 3 h. All results were expressed as mean ± SEM (n = 3). *P < 0.05 versus control, **P < 0.01 versus control.
Glomeruli were incubated for 3 h at 37°C with differentconcentrations of SNAP, and Pa was estimated. As shown in Figure 2B,SNAP increased Pa in a concentration-dependent manner. Atthe concentration of 150 µM it already significantly enhancedPa, and the maximal increase was seen at a concentration 450µM.
To demonstrate that the action of SNAP on Pa was really dueto its released NO, rather than the other metabolite products,we have compared the ability of freshly prepared and the oldSNAP medium (preincubated with assay medium at 4°C over24 h) in inducing glomerular permeability. Because SNAP spontaneouslygenerates NO in aqueous media and has a half-life of only 5h, little NO would be left in old SNAP medium. As expected,treatment of glomeruli with freshly prepared 400 µM SNAPmedium for 3 h enhanced Pa (0.49 ± 0.06), whereas old400 µM SNAP medium had little effect (0.03 ± 0.04),compared with the untreated control (0 ± 0.07).
cGMP Mimicked the Effect of SNAP on Pa
Most of the actions of NO are mediated via a cGMP signalingpathway (1–3). It was reasonable to speculate that cGMPmight also mediate the enhancing effect of NO on glomerularpermeability. To test this, glomeruli were incubated for 3 hwith 8-Br-cGMP (500 µM) or SNAP (500 µM). As shownin Figure 3, both SNAP and 8-Br-cGMP significantly increasedthe Pa compared with controls.
Figure 3. Effect of 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) on Pa of isolated rat glomeruli. Isolated rat glomeruli were treated with either SNAP (500 µM) or cGMP (500 µM) or left untreated as controls for 3 h. All results were expressed as mean ± SEM (n = 3). **P < 0.01 versus control.
NO Impairs the Glomerular Permeability Barrier through a Tyrosine Phosphorylation-Dependent Mechanism
Many studies have reported that the endothelial and epithelialbarrier functions correlates with tyrosine phosphorylation levels(15–21). Therefore, we asked whether phosphorylation wasalso involved in the NO-induced disruption of the glomerularpermeability barrier. Treatment of isolated rat glomeruli withSNP (1 mM), DETA-NONOate (400 µM), and SNAP (400 µM)for 30 min all caused an increase of the phosphotyrosine levelsof the proteins in the molecular mass at 120 and 70 kD (Figure 4).The increase in tyrosine phosphorylation after SNAP treatmentwas time-dependent, reached a maximum at 30 min, and persistedfor at least 2 h (Figure 5). In addition, a dephosphorylationof protein in the molecular mass at 40 kD (Figure 5) was observed.
Figure 4. Effect of three different NO donors on tyrosine phosphorylation in isolated rat glomeruli. Isolated rat glomeruli were treated with different NO donors (1 mM SNP, 400 µM DETA-NONOate, and 400 µM SNAP) for 30 min and lysed with RIPA. Lysate was blotted with anti-phosphotyrosine antibody (PY20). Blot shown is a representative of three independent experiments. Right arrows indicate two major bands of increased tyrosine phosphorylation.
Figure 5. Time-dependent effect of SNAP on tyrosine phosphorylation in isolated rat glomeruli. Isolated rat glomeruli were incubated with 400 µM SNAP for the lengths of time indicated, lysed with RIPA, and immunoblotted with recombinant anti-phosphotyrosine antibody (PY20). Blot shown is from one of three experiments producing similar results. Right arrows indicate two major bands of increased tyrosine phosphorylation.
To further document that NO perturbs the glomerular permeabilitybarrier via mechanisms that involve tyrosine phosphorylation,we examined whether tyrosine kinase inhibitor genistein couldreverse the hyperpermeability induced by NO donors. As shownin Figure 6A, genistein (100 µM) itself did not alterthe Pa (-0.02 ± 0.07). However, preincubation with genisteinfor 1 h significantly prevented the SNAP-elicited incrementof Pa. This effect of genistein was concentration-dependent;10 µM genistein has already caused a 60% inhibition ofthe SNAP-induced increase of Pa, and 100 µM genisteinalmost completely reversed the effect of SNAP. The hyperpermeabilityelicited by another NO donor, DETA-NONOate, could also be significantlyblocked by genistein (Figure 6B). These observations suggesta close correlation between NO-induced glomerular hyperpermeabilityand protein tyrosine phosphorylation.
Figure 6. Effect of genistein, a tyrosine kinase inhibitor, on the hyperpermeablity induced by NO donors in isolated rat glomeruli. (A) Glomeruli were preincubated with the indicated concentrations of genistein for 1 h and then exposed to 400 µM SNAP for additional 3 h. All results were expressed as mean ± SEM (n = 3). (B) After preincubation with 100 uM genistein for 1 h, glomeruli were treated with different NO donors (400 µM SNAP and 400 µM DETA-NONOate) for an additional 3 h. **P < 0.01 versus control, #P < 0.01 versus SNAP or DETA-NONOate.
The crucial role of tyrosine phosphorylation in the NO-inducedincrease of Pa was further confirmed by the observation thatpretreatment of glomeruli with the tyrosine phosphatase inhibitor,PAO, could mimic the action of SNAP on the albumin permeability.The phosphatase inhibitor caused a significant increase of permeabilityin a time- and concentration-dependent fashion (Figure 7). Thiseffect of PAO occurred rapidly and was most pronounced at 2h (Figure 7A). A significant increase of Pa could be observedat a concentration as low as 0.1 µM (Figure 7B). Enhancementof tyrosine phosphorylation in isolated rat glomeruli by PAOwas confirmed by Western blot (Figure 8). Treatment of isolatedrat glomeruli with PAO caused time- and dose-dependent increasesin the tyrosine phosphorylation. With 1 µM PAO, the maximalincrease was detected at 30 min after incubation and was maintainedfor at least 2 h (Figure 8A). The major bands that were phosphorylatedwere of mass at 120 and 70 kD, similar to that induced by NOdonors. A dephosphorylation of protein in the molecular massat 40 kD (Figure 8) was also observed, as shown in Figure 5.
Figure 7. Phenylarsine oxide (PAO) mimicked the action of SNAP on Pa in isolated rat glomeruli. (A) Isolated rat glomeruli were treated either with 1 µM PAO or left as untreated controls for the time periods indicated. (B) Isolated rat glomeruli were either treated with indicated concentrations of PAO or left as untreated controls for 2 h. All results were expressed as mean ± SEM (n = 3). *P < 0.05 versus control, **P < 0.01 versus control.
Figure 8. Enhancement of tyrosine phosphorylation in isolated rat glomeruli by PAO. (A) Isolated rat glomeruli were incubated with 1 µM PAO for the indicated time periods and lysed with RIPA. Lysate was blotted with anti-phosphotyrosine antibody (PY20). The blot shown is a representative of three independent experiments. Right arrows indicate two major bands of increased tyrosine phosphorylation. (B) Isolated rat glomeruli were stimulated with the indicated concentrations of PAO for 15 min and lysed with RIPA. Lysate was blotted with anti-phosphotyrosine antibody (PY20). The blot shown is a representative of three independent experiments. Left arrows indicate two major bands of increased tyrosine phosphorylation.
To further demonstrate the effect of NO on tyrosine phosphorylationin glomeruli, we carried out immunofluorescence staining withanti-PY20 IgG on isolated rat glomeruli using a confocal laserscanning microscope. In the control group, a low level of stainingfor phosphotyrosine was detected. In contrast, after treatmentof glomeruli with SNAP (400 µM) for 1 h or PAO (1 µM)for 15 min, an obvious increase of phosphotyrosine stainingcould be observed. The phosphotyrosine positive cells in SNAP-and PAO-treated glomeruli were much more frequent than thatin controls (Figure 9). To clarify the property of the glomerularcells expressing enhanced tyrosine phosphorylation, we havedone dual-label staining (Figure 10) with anti-PY20 antibody(FITC labeled, green), in combination with a glomerular epithelialspecific marker, anti-rat nephrin antibody (tetramethyl rhodamineB isothiocyanate-labeled, red). Computer-aided superimpositionof the two fluorescence images revealed that most of the positivelyphosphorylated cells in SNAP- and PAO-treated glomeruli werecolocalized with the rat nephrin (yellow, arrows).
Figure 9. Immunocytochemistry showing the effect of SNAP and PAO on tyrosine phosphorylation in isolated rat glomeruli. Isolated rat glomeruli were preincubated with either 400 µM SNAP for 1 h or 1 µM PAO for 15 min or left as untreated controls. Isolated rat glomeruli were then stained with anti-PY20 antibody as described in the Materials and Methods section.
Figure 10. Distribution of tyrosine phosphorylation after treatment of SNAP and PAO in isolated rat glomeruli. Isolated rat glomeruli were preincubated with either 400 µM SNAP for 1 h or 1 µM PAO for 15 min or left as untreated controls. Isolated rat glomeruli were then double stained with a monoclonal anti-PY20 antibody (FITC labeled, green) and a polyclonal rabbit anti-rat nephrin antibody (tetramethyl rhodamine B isothiocyanate-labeled, red), as described in the Materials and Methods section. Colocalization of anti-PY20 and anti-rat nephrin antibodies generates a yellow fluorescence (arrows), which indicates that most of positively phosphorylated cells in SNAP- or PAO-treated glomeruli (green, in Figure 9) were podocytes.
In this study, we provide evidence that NO has the potentialto disrupt the glomerular barrier function, as reflected byincreased albumin permeability in an in vitro assay system.Furthermore, we demonstrated that this action of NO is relatedto augmented phosphotyrosine levels of some glomerular proteins,which suggests the involvement of functional protein(s) in NO-inducedglomerular hyperpermeability.
The assay for albumin permeability in isolated glomeruli usedin this paper was established by Savin et al. (10–13).A modified method, based on the same principle but using a multisizerinstead of a video recording system for the measurement of glomerularvolume, has also been reported (22). We have employed protocolssimilar to those of Savin et al. (10–13) with some modificationsregarding image treatment. For analyzing the microscope imagesof isolated glomeruli, pictures of the glomeruli were directlydigitized into a computer via a CCD camera. The area of eachglomerulus was automatically measured with the help of the NationalInstitutes of Health image analysis software program. This approachavoids the tedious work of measuring glomerular diameters manuallyand is simpler and more objective.
Exposure of isolated glomeruli to three structurally unrelatedNO donors, SNAP, DETA-NONOate, and SNP, all resulted in an enhancementof Pa. A cytotoxic effect of the NO donors and NO itself couldbe excluded as a major cause of the increased Pa, for a numberof reasons. First, treatment of glomeruli with NO donors didnot influence the enzymatic activity of mitochondria, as judgedin the MTT assay. Second, the effect of SNAP and DETA-NONOateon Pa could be largely reversed by pretreatment of glomeruliwith a tyrosine kinase inhibitor. Third, the concentrationsof SNAP employed (80 to 500 µM) were consistent with thoseused by others (up to 1 mM) when testing for anti-mitogenicand permeability-enhancing effects of NO donors on culturedcells (3,23–26). Therefore, these results can be takento indicate that NO has the potential to regulate glomerularalbumin permeability.
Addition of 8-Br-cGMP, a stable analog of cGMP, to the assaysystem mimicked the action of NO and suggests that cGMP signalingmay be associated with the enhancing effect of NO on glomerularpermeability. The role of cGMP signaling in NO-induced microvascularand epithelial hyperpermeability has been documented elsewhere(8,27). However, the participation of cGMP-independent mechanismscannot be excluded. The impairment of the permeability barrierby cAMP has been reported elsewhere (28). The products resultingfrom the interaction of NO with other oxygen radicals have alsobeen demonstrated to be able to affect the permeability of epithelialcells (23,26). The exact role of these factors in the NO-inducedenhancement of glomerular Pa remains to be addressed.
In this study, Western blot analysis of the proteins from threeNO donors treated glomeruli revealed an enhancement of phosphotyrosineproteins of molecular weights around 120 and 70 kD. The timecourse of tyrosine phosphorylation induced by SNAP was closelycorrelated with the degree of Pa enhancement seen. Furthermore,we confirmed, using two kinds of NO donor, SNAP and DETA-NONOate,that the action of NO on Pa could be largely prevented by thetyrosine kinase inhibitor, genistein. In addition, incubationof isolated glomeruli with the tyrosine phosphatase inhibitor,PAO, mimicked the action of SNAP on albumin permeability. Onthe basis of these findings, it seems likely that tyrosine phosphorylationof certain glomerular proteins is a key event in the initiationof glomerular hyperpermeability. This notion is consistent withthe results of recent study that used cultured endothelial cells,which showed that disturbance of the permeability barrier wasrelated to the increased phosphorylation of focal adhesion proteins(29). Tyrosine phosphorylation is dynamically regulated by thecompeting activities of protein tyrosine kinases and proteintyrosine phosphatases. It has been demonstrated that NO is ableto inhibit the activity of protein tyrosine phosphatases inone of the glomerular cell types, mesangial cells (30), underthe assumption that NO may cause an increased phosphorylationof certain proteins on tyrosine residues.
The question as to why increased tyrosine phosphorylation ofcertain glomerular proteins could lead to the disruption ofthe glomerular barrier remains to be addressed. The glomerularpermeability barrier is composed of the glomerular basementmembrane, as well as glomerular epithelial and endothelial cells.It is considered that any disruption of the normal architectureof the glomerular barrier components, such as degradation ofglomerular basement membrane by proteinases and/or disturbancesof the cell-to-cell and cell-to-matrix interactions, could leadto enhanced albumin permeability (6,31–34). The phosphotyrosine-containingproteins have been found to be concentrated mainly along theregions of cell-to-cell and cell-to-matrix interaction, especiallythe proteins found in focal adhesion, adherent junctions, andtight junctions (35–37). Accumulating evidence demonstratesa critical role of tyrosine phosphorylation in the regulationof cell-to-cell and cell-to-matrix interactions and in the disintegrationof the permeability barrier (18,20,21,29,32,38,39). In the culturedepithelial cell line, MDCK, PAO-induced hyperpermeability wasassociated with increased phosphorylation of the tight junctionprotein, ZO-1 (21). Enhanced protein phosphorylation at thelocation of tight junctions and at the basal membrane foot processesof isolated glomeruli was also reported in nephrotic rats (32).In this study, the exact properties of the enhanced phosphotyrosineproteins having molecular weights around 120 and 70 kD are unclear.In addition, treatment of glomeruli with PAO or NO donors couldalso result in an obvious dephosphorylation of protein at themolecular mass 40 kD (Figure 5 and 8). The property of thisprotein also remains uncertain. Because the hyperpermeabilityelicited by NO could be completely prevented by preincubationof glomeruli with kinase inhibitor genistein (Figure 6), itis less likely that this dephosphorylation contributes to theincreased glomerular albumin permeability. Immunohistochemicalstaining of phosophotyrosine proteins revealed that most ofthe positive cells in SNAP-treated glomeruli were situated inthe surface region of the glomeruli. Using dual-label stainingin combination with podocyte specific marker, nephrin, we demonstratedthat most of the phosphotyrosine-positive cells correspondedto podocytes. A recent study on cultured podocytes has revealedthat dephosphorylation of tyrosine proteins was necessary formaintaining the normal architecture of the foot processes andattachment of podocytes onto the underlying extracellular matrix(33). Taken together, it is highly likely that glomerular visceralepithelial cells are involved in NO-induced tyrosine phosphorylation,although participation of endothelial and mesangial cells cannotbe excluded. Molecular and cellular identification of enhancedphosphotyrosine protein(s) will be a focus of our future research.
In summary, we have demonstrated that NO has the ability toimpair the glomerular permeability barrier via a mechanism relatedto tyrosine phosphorylation of glomerular proteins. If tyrosinephosphorylation of specific functional proteins is a commonsignal pathway, leading to impairment of the glomerular barrierfunction and pharmacologic and selective modulations at theglomerular phosphotyrosine level might represent a new strategyin the treatment of certain glomerular diseases.
Acknowledgments
Part of the results of this paper were presented in 33rd AnnualMeeting of American Society of Nephrology. This study was supportedby research grants from the Promotion of Niigata UniversityResearch Projects, Tsukada Foundation, Japan-China Medical Association,and a Grant-in-Aid for scientific research (12671032) as wellas one for the encouragement of young scientists (13770598)from the Ministry of Education, Science, Sports and Culture,Japan. The authors wish to thank Dr. S. Batsford, Instituteof Medical Microbiology and Hygiene, Freiburg University, Germany,for critical review and suggestions for our manuscript and K.Kamata for excellent technical assistance. We are grateful toDr. Kawachi, Department of Molecular Biology, Institute of Nephrology,Niigata University, Japan, for providing rabbit anti-nephrinantibody.
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Received for publication October 18, 2000.
Accepted for publication July 8, 2001.
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Top
Abstract
Introduction
(S/
V) was calculated as 
a)and Pa
120 and 70 kD (Figure 4). The increase in tyrosine phosphorylation after SNAP treatment was time-dependent, reached a maximum at 30 min, and persisted for at least 2 h (Figure 5). In addition, a dephosphorylation of protein in the molecular mass at 
