Cellular signaling and proliferative action of AVP in mesangium of SHR: Effect of low density lipoprotein

Kidney International, Vol. 50 (1996), pp. 1506—1514

Cellular signaling and proliferative action of AVP in mesangium of SHR: Effect of low density lipoprotein S-E ISHIKAWA, JKUYO KusA1cc, MIN0RI HIGASHIYAMA, SH0IcHIR0 NAGASAKA, TAIC&1o SAITO, KAZUFUMI HONDA, and TOSHIKAZU SAIT0 Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, Tochigi, Japan

Cellular signaling and proliferative action of AVP in mesangium of augments the cellular signaling and proliferative action of AVP, SHR: Effect of low density lipoprotein. The present study was undertaken and an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A

to determine whether low density lipoprotein (LDL) modulates the cellular action of arginine vasopressin (AVP) in cultured glomerular mesangial cells of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The AVP-induced cellular signal transduction, including inositol 1,4,5-trisphosphate (1P3) production, fura-2 intracellular calcium measurements and cellular alkalinization, was significantly greater in cells of SHR than those of WKY. This is based on an increase in AVP V1 receptor number in cells of the SHR. Also, the AVP activation of mitogen-activated protein (MAP) kinase and [3H}thymidine incorporation was significantly exaggerated in cells of SHR compared with

those of WKY. LDL at a concentration of 10 /Lg/ml augmented the cellular signaling and proliferative action of AVP in cells of WKY, but not in those of SHR. Since [3H]AVP receptor binding was not affected by the

LDL pretreatment, LDL modulates the signal transduction between a location distal to the AVP receptors and proximal from the production of 1P3 and diacylglycerol. These results indicate that an increase in AVP receptor capacity has a profound effect on the AVP-induced cellular signaling and proliferation, and that LDL has a slight alteration on the action of AVP in glomerular mesangial cells of SHR.

(ITIMG Co A) reductase conversely diminishes its action in the cultured rat glomerular mesangial cells [15, 16]. In a homologous cells of vascular smooth muscle, hormonally activated Na/H exchange is profoundly increased in the cells of spontaneously hypertensive rats (SHR) as compared to those of normotensive Wistar-Kyoto rats (WKY) [17, 18]. Such an enhanced activation is found with angiotensin II and AVP, and is mediated via the enhancement of phosphatidylinositol hydrolysis

and [Ca2]i mobilization [19, 20]. The enhancement at least depends on an increase in the receptor capacity of cells derived from SHR [21]. However, such an augmentation of the cellular action of vasoconstrictor hormones has yet to be explored in glomerular mesangial cells. The present study was undertaken to determine whether gbmerular mesangial cells derived from SHR have a distinct character of responding to the vasoconstrictor hormone AVP versus those derived from WKY rats. Also, whether or not such an environmental factor as LDL could modulate the cellular action

Arginine vasopressin (AVP) initiates a series of phosphatidylinositol breakdown mediated through V1 receptors in glomerular mesangial cells [1—31. Its hydrolysis results in an accumulation of inositol 1,4,5-trisphosphate (1P3) and diacylglycerol, leading to mobilization of cellular free calcium ([Ca2]i) from endoplasmic reticulum and stimulation of protein kinase C, respectively [4, 5].

of AVP in cultured glomerular mesangial cells of SHR was

In addition, AVP promotes cellular growth of mesangium, by activating protein kinase C [6, 7]. AVP activates mitogen-activated protein (MAP) kinase and induces thymidine incorporation and c-fos and c-/nyc proteins in glomerular mesangial cells and

Methods

homologous cells of the vascular smooth muscle [6-101. There is increasing evidence to suggest that abnormalities in lipoprotein

metabolism may influence the pathogenesis of cardiovascular diseases, including the glomeruloscierotic disorder [11]. Both lipoprotein metabolism and lipoprotein itself have an ability to exert direct hormonal actions in vascular beds [12, 13]. There are

examined. This maneuver may clarify if the enhancement by LDL of the cellular action of AVP could be different between these two types of cells, because of the genetic background of vascular and glomerular responsiveness.

Cell culture

The experimental procedure was similar to that described in

our previous studies [3, 15], modified from the method of Burlingtron and Cronkite [22] and Scharschmidt and Dunn [23]. Male SHR and WKY (12 weeks old) were used. Kidneys were removed under sterile conditions, and cortical tissues were cut away from the medulla. They were minced with 1 ml of physiological saline solution (PSS; 140 m'vi NaCI, 4.6 mM KCI, 1 mM

also low density lipoprotein (LDL) receptors and scavenger MgCl,, 2 mivi CaCI2, 10 m glucose and 10 mrvi Hepes, pH 7.4) by receptors in glomeruli [14]. Recently, we demonstrated that LDL a sharp razor blade. The minced renal cortical tissues were incubated with 3 ml of collagenase (1 mg/mI; Worthington Received for publication February 12, 1996 and in revised form June 24, 1996 Accepted for publication June 24, 1996

Biochemicals, Freehold, NJ, USA) for 60 minutes at 37°C. They were passed through a series of steel sieves with decreasing pore sizes (60 and 200 mesh) with the glomeruli appearing on top of the 200 mesh sieve. The glomeruli were collected into culture

© 1996 by the International Society of Nephrology

tubes and were centrifuged at 500 g for four minutes at room 1506

1507

ishikawa et at: A VP action in SHR mesangium

temperature. After aspirating the supernatants, the pellets were resuspended with Dulbecco's Modified Eagle's Minimal Essential Medium (DMEM; Flow Laboratories, McLean, VA, USA) supplemented with 20% fetal bovine serum, 100 U/mI penicillin, and 100 sg/ml streptomycin. The dispersed glomeruli were harvested into 35 x 10-mm plastic dishes with the medium and kept in a humidified incubator at 37°C under 95% air and 5% CO2. After the cultured cells were confluent, they were subcultured

of 0.2 ml 50% trichloroacetic acid, and then the cells were scraped

using disposable plastic chips for Eppendorf. The suspensions containing the disrupted cells were centrifuged using a microcentrifuger. The supernatants were washed five times with ether and the water-soluble fractions were brought to pH 7.0 using 1 N NaOH and stored at —20°C until the analysis. The pellets were

dissolved with 1 ml 0.1 N NaOH containing 1% SDS and stored at 4°C for protein assay. The water-soluble fractions were applied to using Ca2-free and Mg2 -free Hank's solution containing columns containing 1 ml of Dowex (1-x8, formate form; Muro0.025% trypsin and 0.01% EDTA. The dispersed cells were machi Kagaku, Kogyo, Tokyo, Japan) and serially eluted 10 times collected into culture tubes and centrifuged at 500 g for four with 2-ml aliquots of H20, Borax (5 mM disodium tetraborate, 60 minutes at room temperature. The pellets were resuspended in m sodium formate), 0.2, 0.4 and 1.0 M ammonium formate in 0.1 DMEM containing 20% fetal bovine serum, penicillin and strep- M formic acid. This maneuver separated inositol, glycerophostomycin, and cultured in a humidified incubator. The cultured phatidylinositol, inositol-1-phosphate, inositol bisphosphate and cells at the third through tenth passages were subjected to the 1P3, respectively. 1P3 fraction includes 1,3,4-1P3, 1,4,5-1P3, and I ,3,4,5-inositol tetrakisphosphate. Samples were collected into following studies on days 7 to 10 of the subculture. For measurements of [Ca2Ii and cellular pH (pHi), the cells scintillation counter vials and counted using a liquid scintillation were cultured on thin glass slides (13 mm in diameter; Matsunami counter. Kogyo Co., Osaka, Japan). The cultured cells were grown in 35 X Measurement of fCa2'7, 10-mm plastic dishes to study AVP receptor study, 1P3 production and MAP kinase activity. Also, they were grown on 24-well tissue The experimental procedure was similar to that used in our

culture clusters (Costar, Cambridge, MA, USA) to measure previous reports [26, 27]. The cells were preincubated with the thymidine incorporation.

medium containing 10 jsg/ml LDL for 24 hours and then rinsed twice with 1 ml PSS. They were loaded with 5 ISM fura-2/AM A VP receptor binding (Dojin Biochemicals, Kumamoto, Japan) for 60 minutes at 37°C The experimental procedure was modified from the method of in a volume of 0.25 ml PSS containing 10 jig/mI LDL. Control Fishman et al [24]. Cells were grown on 35 X 10-mm plastic group of cells were performed with the vehicle in a same manner. dishes. Cells were exposed for 24 hours to the medium containing After aspiration of the fura-2/AM solution, the glass slides were 10 sg/ml human LDL (Biomedical Technologies, Stoughton, MA, rinsed and then placed in a 1 >< 1-cm quartz cuvette with the aid USA) or the vehicle before the start of experiments. They were of a special holder in a fluorescence spectrophotometer (CAFrinsed twice with 2 ml ice-cold binding buffer (119.2 miu NaC1, 3.0 110; Japan Spectroscopic Co., Tokyo, Japan). The dual-wavemM KC1, 1.2 m'vi MgSO4, 1.0 mivi CaCI2, 1.2mM KH2PO4, 10 mM length excitation method for measurement of fura-2 fluorescence glucose, and 25 mr'i Hepes, as well as 0.1% BSA, pH 7.4) and then incubated with 1 ml ice-cold binding buffer containing 0.2 to 20 X

was used. The fluorescence was monitored at 500 nm, with excitation wavelengths of 340 and 380 nm in the ratio mode. The

i0 M [3HIAVP (sp act 67.6 Ci/mmol; New England Nuclear, effectors of AVP and endothelin-1 (Peptide Institute, Osaka, Wilmington, DE, USA) in the presence or absence of 1 X 10 NI Japan) were added after a stable fluorescence signal (R) was AVP (Sigma, St. Louis, MO, USA) at 4°C for 60 minutes. achieved. From the ratio of fluorescence at 340 and 380 nm, the Non-specific binding was determined in the presence of 1 X i0 [Ca2]i was determined as described by Grynkiewicz, Poenie and M AVP. The buffer also contained 10 pg/ml LDL which was Tsien [28], using the following expression: similar to that during the preincubation. The control group was — carried out with the vehicle in a similar manner. After the [Ca2]i (nM) = K, x [(R — Rmin)/(Rm R)] X incubation, the cells were rinsed four times with 2 ml of ice-cold

binding buffer, followed by the addition of 1 ml 0.1 N NaOH containing 1% sodium dodecylsulfate (SDS). The radioactivity was counted with a liquid scintillation counter (Model LSC-671; Aloka, Tokyo, Japan). Protein was measured by the method of Lowry et al [25]. Measurement of inositol irisphosphate

The experimental procedure was similar to that described in

where R is the ratio of fluorescence of the sample at 340 and 380 were determined as described previously nm, and Rrnax and [26]. The term is the ratio of fluorescence of fura-2 at 380 nm in

zero and saturating Ca2' concentrations. Kd is the dissociation constant of fura-2 for Ca2', assumed to be 224 nM at 37°C [28]. Measurement of pHi

The experimental procedure was similar to that described

previously [29]. The study was carried out in HCO3-frec buffer using PSS. The cells were preincubated with the medium containwith 2 ml of inositol-free DMEM and incubated with 2 ml of ing 10 jig/mI LDL for 24 hours and then rinsed twice with I ml inositol-free DMEM containing 5 jsCi/ml [myo-3H]inositol (sp act PSS. They were loaded with 2 jiM 2',7'-bis-(2-carboxymethyl)5 19.1 Ci/mmol; Amersham Tnt., Amersham, Buckinghamshire, (and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM; MoUK) for 24 hours in a humidified incubator. The medium also lecular Probes, Inc., Eugene, OR, USA) for 60 minutes at 37°C in our previous reports [15, 161. Cells grown on 35 X 10-mm plastic dishes were used in the present study. The cells were washed twice

contained 10 or 100 ig/ml LDL or the vehicle. At the time of

a volume of 0.25 ml PSS containing 10 jig/mI LDL. Control group

experiment, the cells were rinsed twice with 2 ml of PSS and then

of cells was performed with the vehicle in a same manner. The complete intracellular hydrolysis of BCECF/AM to BCECF was judged by changes in the excitation and emission spectra. The

incubated with 0.8 ml of PSS containing I X i0 and I x 10 M AVP for 10 seconds. The reaction was stopped by the addition

1508

Ishikawa et al: A VP action in SHR mesangium

fluorescence was monitored at 530 nm, with excitation wavelengths of 450 and 500 rim in the ratio mode. After measurement of the basal pHi level, AVP was added. The fluorescence signal was calibrated at several pH values (6.6, 7.0 and 7.4) in the KCI 1.0

solution (140 mtvi KCI, 4.6 ms NaCI, 1 msi MgCl2, 2 mx CaCI2, 10

m glucose and 10 mt Hepes) containing the K/H ionophore nigericin (10 j.g/ml). MAP kinase assay

ci) ci)

The experimental procedure was similar to that used in our previous studies [15, 16]. Cells grown on 35 x 10-mm plastic

C

dishes were incubated with serum-free DMEM in the presence or

0

absence of 10 .tg/ml LDL 24 hours before the start of the experiments. The cells were rinsed twice with 2 ml of PSS. The cells were incubated for 10 minutes at 37°C with 1 ml of 1 >< 10

and 1 >< 10 M AVP. Also, the study was performed with 1 x iO M endothelin-1 and I x i0— M platelet-derived growth factor (PDGF) BB (Upstate Bioteehonolgy, USA). After aspiration of the effector solutions, the cells were exposed to 0.5 ml of the solution containing 20 ms'i Tris, 5.6 m'vi /3-glyccrophosphate, 10 mM EGTA, 10 mst MgCl2, 0.1 mi'vi NaF, 2 mivi DTT, 1 mM NaVO3, 20 rg/ml aprotinin, and 1 mivi PMSF, pH 7.5. Thereafter, Fig. 1.

the dishes were immediately put on dry ice. The extracts were collected into microcentrifuge tubes by disposable plastic chips for

Eppenclorf. After centrifugation, the supernatants were transferred to plastic tubes and kept at —20°C until the time of assay. Glass tubes containing 0.06 ml of the assay mixture (40 mrvt Tris,

0

10

Bound, x 1011M/mg protein Scatchard analysis of /3JJJ.4 VP binding to the cultured glomerular

mesangial cells den ved from STIR and WKY. Symbols are: (•) and (0) show

the groups of cells of SHR, pretreated for 24 hours with vehicle and 10 jLg/ml LDL, respectively; (U) and (0) show the groups of cells of WKY, pretreated for 24 hours with vehicle and 10 .g/ml LDL, respectively. Values are the means of triplicate determinations obtained in a single experiment.

40 mivi MgCl2, 2.5 mg/mI myelin basic protein [Sigma], 0.5 mii ATP, and 0.5 .rCi 32P-yATP [sp act >10 Ci/mmol; Amersham]) were incubated at 25°C for 15 minutes. Then 0.04 ml of samples were added and the mixtures were incubated for an additional 10 cluster were collected and protein contents were measured by the minutes at 25°C. The mixtures were transferred onto glassfiber method of Lowry et al [25]. filters (Whatman 2.4-cm GF/C; Whatman, Maidstone, UK) by Statistics using disposable plastic chips for Eppendorf. The filters were then put into ice-cold 10% trichloroacetic acid containing 50 mivi All values of 1P3 production, [Ca2]i, pHi, MAP kinase activity sodium pyrophosphate, and shaken gently for 20 minutes. This and [3H]thymidine incorporation were analyzed by an analysis of maneuver was repeated four times. The filters were immersed in multiple variance and Neuman Keuls' range test. P < 0.05 was ice-cold ethanol for 20 minutes. After the filters were exposed to considered significant. diethylether, they were put into counting vials containing 10 ml of Results scintillation solution. The radioactivity was counted by using Figure 1 shows the Scatchard analysis of [3H]AVP receptor Aloka liquid scintillation counter. Also, cellular protein of all binding to the cultured glomerular mesangial cells derived from samples were measured by the method of Lowry et al [25]. SHR and WKY. K1 and were 1.39 0.17 >< 10'° M and Measurement of thymidine incolporation 0.2 >< 10° M/mg protein in the cells derived from WKY, 3.9 The experimental procedure was similar to that described respectively. B,,a of SHR was 10.4 0.6 X 10 M/mg protein, previously [15, 16]. Cells grown on 24-well tissue culture clusters which was greater than that in the cells from WKY, whereas there were used to assess DNA replication. At confluency, the cells were was no difference in Kd values between SHR and WKY (1.33

synchronized to the quiescent state by incubation in 1 ml of 0.12 X 10°vs. 1.39 0.17 X 10°M).AsshowninFigurel,the values in serum-free DMEM. After 24 hours, cells were exposed to the 10 jig/mI LDL pretreatment did not affect K1 and varying stimuli which were dissolved in the serum-free DMEM. both cultured glomerular mesangial cells of SHR and WKY. Whether or not LDL modulates the AVP-induced increase in The medium contained [3H]thymidine (1 jiCi/well; sp act 80.8 Ci/mmol; New England Nuclear). The stimuli included I x 10—8 1P3 production in the cultured rat glomerular mesangial cells was to 1 X l0' M AVP and 10 igJml LDL. The cells were incubated examined. As shown in Figure 2, AVP per se significantly inwith 1 ml of the effector for an additional 24 hours in the creased 1P3 production in both glomerular mesangial cells of SHR humidified incubator. Thereafter, the cells were rinsed four times and WKY. Its production was greater in the cells of SHR than with PSS and immersed with 0.5 ml of 10% trichloroacetic acid. that in the cells of WKY (1 X 10 M AVP; 1.72 0.04 vs. 2.36 They were collected into counting vials by disposable plastic chips 0.09 X I0 CPM/mg protein, P < 0.01). Such an AVP-induced for Eppendorf, and with 10 ml of scintillation solution then added. increase in 1P3 production was significantly enhanced by the The radioactivity was counted by using Aloka liquid scintillation 24-hour pre-exposure of cells of WKY to 10 jig/mI LDL(1 X i0 counter. Cells grown on several wells of 24-well tissue culture M AVP; 1.72 0.04 vs. 2.19 0.06 X i04 CPM/mg protein, P <

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Ishikawa et al: A VP action in SHR mesangium

addition of AVP. The 1 X iO M AVP-induced cellular alkalinization was significantly greater in cells of SHR than that in cells of WKY (+0.15 0.02 vs. +0.10 0.01, P < 0.05), whereas the AVP-induced cellular acidification was not different in cells of

B

A 3

e

SHR and WKY. The 24 hour exposure to 10 pg/ml LDL

I

significantly augmented the 1 x iO M AVP-induced cellular

*

alkalinization in cells of WKY (+0.10 0.01 vs. +0.17 0.01, P < 0.01), but not in cells of SHR. The initial cellular acidification was not affected by LDL.

Figure 6 shows the AVP-activated MAP kinase in cultured glomerular mesangial cells derived from SHR and WKY. One X io— and I X iO M AVP significantly activated MAP kinase, an activation significantly greater in cells of SHR than that in cells of WKY (1 X 106 M AVP; 28.7 1.2 vs. 98.6 6.7 pmol/mg protein, P < 0.01). When cells were pretreated for 24 hours with 10 jig/mI LDL, the AVP-activated MAP kinase was significantly

F ______ Vehicle

7

AVP, -log M

6

enhanced in cells of WKY (1 x 10—6 M AVP; 28.7

1.2 vs. 38.7

2.2 pmol/mg protein, P < 0.01), but it was not found in cells -+——

Vehicle

7

6

AVP, -log M

Fig. 2. Effect of LDL on the A VP-induced increase in 1P3 production in the

cultured glomerular mesangial cells of WKY (A) and SHR (B). Symbols are: (S) control group of cells; (0) cells pretreated with 10 ig/ml LDL for 24

hours. *P < 0.01 versus the control group. Values are means SEM, N 6.

0.01). However, in the cells of SHR the 10 jrg/ml LDL pretreatment did not alter its production. Similar results were obtained with 100 j.g/ml LDL. The 100 1rtg/ml LDL pretreatment augmented the 1 >< 10 M AVP-induced increase in 1P3 production

derived from SHR. The basal activity of MAP kinase was greater

in cells of SHR than that in cells of WKY.

We examined whether LDL modulates the AVP-induced [3H]thymidine incorporation into cultured glomerular mesangial cells of SHR and WKY (Fig. 7). In both cells derived from SHR and WKY, the AVP-induced [3Hjthymidine incorporation was obtained in a dose-dependent manner. Mesangial cells of SHR had greater [3H]thymidine incorporation than those of WKY (1 X

iO M; 1.47 0.10 vs. 3.39 0.13 X iO CPM/mg protein, P < 0.01). However, the pretreatment with 10 j.Lg/ml LDL did not affect the AVP-induced [3H]thymidine incorporation in cells of SHR, whereas its pretreatment significantly enhanced in cells of WKY. Lastly, we examined the effect of 10 jig/mI LDL on endothelin-

in cells of WKY, but not in SHR (data not shown). The basal 1-induced increases in [Ca2Ji and MAP kinase in the cultured levels of 1P3 production remained unchanged in the cells pre- glomerular mesangial cells from WKY. One X i0 M endothetreated with 10 ig/ml LDL for 24 hours. lin-1 increased [Ca2]i from 92.6 6.8 to 384.6 21.5 nM (N = Figure 3 depicts the effect of LDL on the AVP-induced increase 6, P < 0.01; Fig. 8). When cells were exposed for 24 hours to 10 in [Ca2Ii in cultured rat glomerular mesangial cells of SHR and jig/ml LDL, the 1 X 10 ' M endothelin-1-induced increase in WKY. As shown in the left side, AVP increased [Ca2]i in a [Ca2]i was enhanced (384.6 21.5 vs. 557.2 29.1 nrvt, P < dose-dependent manner. The LDL pretreatment significantly 0.01). Also, 1 X iO- M endothelin-1 activated MAP kinase from 2.1 to 31.8 3.7 pmol/mg protein (N = 4, P < 0.01), that augmented its mobilization of [Ca2]i (1 X 10 M AVP; 240.9 13.2 3.7 vs. 50.1 4.8 20.3 vs. 549.8 66.2 nrt, P < 0.01). In glomerular mesangial cells was augmented by 10 jig/mI LDL (31.8 of SHR, the AVP-induced increase in [Ca2 ]i was greater than pmol/mg protein, P < 0.05; Fig. 9). Similar results were obtained that in the cells of WKY (1 x 10 M AVP; 240.9 20.3 vs. with PDGF-BB (Fig. 9). One X 10 M PDGF-BB increased 574.9 76.8 nM, P < 0.01). However, the enhancement by LDL MAP kinase from 11.9 1.8 to 34.1 3.9 pmol/mg protein (P < of AVP mobilization of [Ca2 I ]i was not so evident, though the 0.01), and the pretreatment with 10 jig/ml LDL enhanced the 1 X 10 M AVP-induced [Ca2]i was significantly higher in cells action of PDGF-BB (34.1 3.9 vs. 48.8 2.7 pmol/mg protein, pretreatcd with LDL than that in the cells of SHR treated with the P < 0.05). vehicle for LDL. Also, the enhancement by LDL of the AVP-mobilized [Ca21i is shown in Figure 4. When cells were preexposed to LDL for 24

Discussion

In the present study we confirmed that AVP stimulates the

hours, the greater response of [Ca2]i to 1 X i0 M AVP was breakdown of phosphatidylinositol, mediated through V1 recepfound with the higher concentration of LDL in both cells of SHR

tors, in glomerular mesangial cells derived from SHR and WKY

and WKY. In contrast, the basal levels of [Ca2]i remained

[1—3]. Such a signal transduction by AVP promoted [Ca2ji mobilization, resulting in contraction of glomerular mesangial cells [30]. The early mobilization of [Ca2 ji is derived from both intra- and extracellular Ca2, and the sustained phase depends to a great extent on extracellular Ca2 [3]. AVP produced a biphasic

unchanged in the absence and presence of LDL.

Effect of LDL on the AVP-induced alteration in pHi in the cultured glomerular mesangial cells of SHR and WKY is depicted in Figure 5. As shown in our previous report [3], AVP caused an initial cellular acidification in three minutes, followed by a sustained cellular alkalinization in glomerular mesangial cells. The

change in pHi, namely, an early cellular acidification, followed by a sustained cellular alkalinization. We have already reported that

maximal pHi was obtained about six to eight minutes after the the change in pHi is closely related to that in cellular Na

1510

Ishikawa et al: A VP action in SHR mesangium

A

B

1200

* 1000

1000

800

800 +

c'J

* *

600

600

0 400

400

200

200

—+— 0

0 Vehicle

9 AVP,

8

-log M

7

I

Vehicle

I

I

9

8

Fig. 3. Effect of LDL on the A VP-induced increase in [Ca2 Ji in cultured glomerular mesangial cells derived from WKY (A') and SHR (B). Symbols are: (•) control group of cells; (0) cells pretreated with 10 pg/mI LDL for 24 hours. < 0.01 versus the control group. Values are means SEM, N = 6.

AVP, —log M

concentration, which also depends on cellular second messenger

[Ca2]i [3, 31]. In addition, AVP promoted cellular growth of mesangium, as AVP activated MAP kinase and stimulated

1200

[3fllthymidine incorporation into the cultured glomerular mesan-

gial cells of SHR and WKY. MAP kinase is serine/threoninespecific kinase and is downstream of protein kinase C in the cellular signal transduction pathway of AVP [7, 9, 10]. The present study clearly demonstrated that the cellular action of AVP was markedly greater in glomerular mesangial cells of SHR than those of WKY. Their differences were found in all the

1000

steps of signal transduction, including 1P3 production, [Ca2]i

800

*

concentration, cellular alkalinization, MAP kinase activation and [3H]thymidine incorporation. 1P3 production can be indicating the + activity of phospholipase C. As shown in Figure 1, the Bmax of 0 SHR was 2.7 times greater than that of WKY, whereas the Kd was similar in both groups of cells. These results therefore indicate that such a distinct receptor capacity is a causitive factor for the exaggerated difference in cellular signaling of AVP in glomerular mesangial cells, derived from SHR and WKY. Similar results were obtained in a homologous cells of vascular smooth muscle cells

*

600

400

[21]. Kd values were 1.33 X 10_b M and 1.39 X 10_bo M in mesangial cells from SHR and WKY, respectively, values 3.6 to 3.7 times different from the K of 3.7 >< I 0' M in mesangial cells of SD rats [15]. We have to recognize the calculation mistake in the previous paper [15].

In the cultured glomerular mesangial cells of WKY LDL enhanced the cellular signaling of AVP. LDL augmented the

AVP-induced increase in 1P3 production, without any alteration in [3HIAVP receptor binding. Therefore, LDL modulates the signal

transduction between distal to the AVP receptors and proximal from the production of 1P3 and diacylglycerol. The LDL pretreatment did not affect the basal levels of W3 and [Ca2 1i. These results confirm our previous findings in the cultured rat glomerular mesangial cells showing LDL augments the cellular signaling of AVP [15]. In an additional study, LDL also enhanced PDGFBB-activated MAP kinase in glomerular mesangial cells from WKY. PDGF-BB has tyrosine kinase receptors, and it links to

200

0 Vehicle

0.1

1

10

100

LDL, p.g/mI Fig. 4. LDL enhances the 1 X io— MA VP-induced increase in [Ca2 Ji in the cultured glomerular mesangial cells of SHR (•, 0) and WKY ( LI). The cells were prcincubated for 24 hours with the indicated concentrations of LDL. (•, •) Basal [Ca2]i, (0, 0) 1 x 10 M AVP-induced [Ca2*Ii. *P < 0.01 versus the vehicle. Values are means SEM, N = 6.

activation of G protein-coupled phospholipase C [32]. This may

be the reason why LDL enhanced the proliferative action of PDGF-BB, as well as that of AVP and endothelin-I. A study

1511

Ishikawa Ct at: AVP action in SHR mesangium

+0.2

+0.1

I0 0 Fig. 5. Effect of LDL on thel X 107 MA VP—0.1

WKY SHR Sustained alkalinization

WKY SHR Initial acidification

E

100

100

80

80

60

0.

LDL. < 0.01 versus controls. Values are means

SCM, N = 6.

B

A

0

induced changes in cellular pH (pHi) in the cultured glomendar mesangial cells of SHR and WKY. The graph shows the minimal and maximal pHi as pHi. Symbols are: (U) the control group of cells; () the LDL groups of cells, prcincubated for 24 hours with 10 sgiml

*

*

60

ci)

'I) C

0

40

40

20

20

0

Fig. 6. Modulation by LDL of the A VP-activated MAP kinase in the cultured glomerular mesangial cells of WKY (A) and SHR (B). Symbols are:

0 Vehicle 1 x 1 07u AVP 1 x 1 06M AVP

Vehicle 1 x 1 0'u AVP 1 x 1 0u AVP

(U) the control groups of cells; () the LDL groups of cells, pretreated for 24 hours with 10 sg/ml LDL. *P < 0.01 versus the control. Values are means SCM, N = 6.

suggested that the action site of LDL is phosphatidylinositol by LDL of AVP action in cells of SHR. As mentioned earlier, the bisphosphate-specific phospholipase C [lfl. However, such an cellular signal transduction of AVP is markedly augmented in augmentation by LDL was not found in the cultured glomerular SHR as compared to that of WKY. These results suggest that an mesangial cells of SHR. The 24 hours exposure of cells to LDL increase in AVP receptor number markedly augments the AVPhad no effect on the AVP-induced increase in 1P3 production, induced cellular signaling and proliferation, and that LDL could MAP kinase activation, and [3H]thymidine incorporation, as well not alter the action of AVP in glomerular mesangial cells derived as [3H]AVP receptor binding. Scannapieco et al 1331 reported that from SHR. the receptor binding of LDL is significantly greater in a homoloThere was some discrepancy in the AVP-induced increase in gous cells of vascular smooth muscle derived from SHR than 1P3 production and [Ca2 Ii in glomerular mesangial cells of SHR. those from WKY, but this might not account for the enhancement The LDL pretreatment enhanced the AVP-induced increase in

Ishikawa et al: AVP action in SHR mesangium

1512

A

B

4

4

g:3

3

O) CE

*

5 u2 >2 x ''

2

//

I

1

0

0 Vehicle

8

7

Vehicle

6

8

7

6

Fig. 7. Modulation by LDL of the A VP-induced increase in [3HJthymidine inco/poration into the cultured glomerular mesangial cells of WKY (A) and SHR (B). Symbols are: (S) the control groups of cells; (0) the LDL groups of cells, pre-exposed for 24 hours to 10 /.Lg/ml LDL. *p < 0.01 versus the control. Values are

means

SEM, N = 4.

AVP, —log M

AVP, —log M

P<0.01

600

C

400

0

200

ization, MAP kinase activity and [3H)thymidine incorporation depend on a protein kinase C-mediated pathway, the increase in [Ca2]i may not affect these parameters. The enhanced mobilization of AVP-induced [Ca2]i by LDL may contribute to cellular contraction of SHR mesangium. However, we cannot interpret the LDL enhancement of [Ca21i increase by AVP in mesangial cells of SHR. LDL may have other action site to mobilize [Ca2)i in glomerular mesangial cells derived from SHR. For instance, LDL may affect extracellular calcium entry in addition to intracellular calcium release. In addition, the basal levels of [Ca2]i were similar in cells from SHR and WKY, but the basal levels of MAP kinase and [3Hthymidine incorporation were significantly higher in SHR than WKY. These differences may also influence no additive effect of LDL on the AVP-activated MAP kinase and [3Hlthymidine incorporation in cells from SHR. Central to the development of atherosclerosis is the involvement of lipid deposition in the wall of arteries, particularly LDL [34]. Recent works suggest that the oxidation of LDL in vessel walls is a critical step in the pathogenesis of atherosclerosis [351. In glomerulus, focal glomerulosclerosis is an analogous condition of atherosclerosis, and oxidized LDL is involved in the pathogenesis of focal glomeruloscierosis [36). The present finding that LDL augmented the AVP-induced cellular signaling and growth seems

unlikely to he directly related to sclerotic disorder, because the present study was determined by only a short exposure to LDL,

0 Control

10 ig!ml LDL

Fig. 8. A 24 hour exposure to 10 pg/mI LDL enhances the I >< io M

endothelin-1 -induced increase in /Ca2Ji in cultured glomerular mesangial cells derived from WKY Symbols are: () the basal levels; () the I X lO M endothelin-1-induced [Ca21i. Values are means SEM, N = 6.

[Ca2]i in Figure 4, hut it was not so evident in Figure 3. Also, the AVP-induced change in pHi, MAP kinase activation and [3H]thy-

midine incorporation were not altered by LDL in glomerular mesangial cells of SHR. Since the AVP-induced cellular alkalin-

and the effect of LDL was distinct from that of oxidized LDL [15, 37].

In summary, we demonstrated that cellular signaling of AVP was markedly enhanced in glomerular mesangial cells of SHR, as

compared to that in WKY. This difference was based on an increase in AVP receptor capacity in cells of SHR. The LDL pretreatment augmented the AVP-induced cellular signaling and proliferation in cells of WKY, but not in cells of SHR. A lack of the effect of LDL seems likely to be due to the marked participation of the genetic factor of increased AVP receptor numbers in glomerular mesangial cells of SHR. The present results indicate that an increase in AVP receptor capacity has a profound effect on

1513

Ishikawa et a!: AVP action in SHR mesangium

A

60

B

T

P<0.05

50 40 30 C

20 Fig. 9. Modulation by LDL of the endothelin-1and PDGF-BB-activated M4P kinase in cultured glomerular mesangial cells of WKY. The LDL

10

0 Control

10 gIml LDL

Control

the AVP-induced cellular signaling and proliferation, and that LDL has a little alteration on the action of AVP in glomerular mesangial cells derived from SHR. Acknowledgments The present study was presented at 10th International Congress of Endocrinology in San Francisco, California June 12—15, 1996. The present

study was supported by grants from the Ministry of Education, Science and Culture of Japan. We thank Mrs. Midori Tokita for her technical assistance.

Reprint requests to San-e Ishikawa, M.D., Division of Endocrinology and

Metabolism, Department of Medicine, Jichi Medical School, 3311-1 Yakushiji Minamikawachi, Tochigi 329-04, Japan.

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