Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration

Kidney International, Vol. 43 (1993), pp. 324—333

Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration CARMEN GARCIA—ESCRIBANO, MARIA LuIsA D1EZ-MARQUES, MERCEDES GONZALEZ-RUBIO, MANUEL RODR1GUEZ-PUYOL, and DIEGO RODR1GUEZ-PUYOL Department of Physiology and Pharmacology and Medicine and Section of Nephrology, University of Alcald de Henares and Hospital PrIncipe de Asturias, Madrid, Spain

Somatostatin antagonizes angiotensin II effects on mesangial cell contraction and glomerular filtration. The effects of somatostatin (ST) on the regulation of the glomerular filtration rate have not been extensively studied. The present experiments were designed to analyze this possible relationship. ST alone did not modify the planar cell surface area (PCSA) of cultured rat mesangial cells (CRMC), but it prevented and reversed the reduction in PCSA induced by 10 nri angiotensin II (Ang

of ST in the cat reduces renal vascular resistance [9] but

H) in a dose- and time-dependent manner. ST (1 sM) completely

ST or even with intrarenal administration. For instance, in addition to its intrinsic renal actions, ST could modulate the systemic synthesis of some vasoactive substances such as

prevented and reversed the increase in the myosin light chain phosphorylation induced by 10 flM Ang II. Incubation with pertussis toxin (PT, 0.5 sg/ml) inhibited the effect of ST on the Ang Il-dependent changes in PCSA, but this effect was not inhibited by the blockade of the vasodilatatory prostaglandins (indomethacin, 10 sM) or nitric oxide

increases it in the dog [10]. In the human, it seems to decrease renal perfusion [11] besides inducing a transient increase in blood pressure and a reduction in splanchnic blood flow [12]. This lack of definitive conclusions could be due to the difficult interpretation of the results obtained with systemic infusion of

glucagon or GH [13, 14], thus interfering with the interpretation

of the renal results. In addition, the effects of ST on the

(L-N-methyl-arginine, 0.2 mM) synthesis. 2' ,5'-dideoxyadenosine regulation of GFR and RPF may not be the same in different (DDA, 0.1 mM), an adenylate cyclase blocker, and methylene blue

physiological conditions. It is possible that the renal actions of ST could differ in basal conditions or under renin-angiotensin system or vasopressin stimulation. In fact, it has been described that ST antagonizes angiotensin II effects on the adrenal gland area of isolated rat glomeruli, also in a dose- and time-dependent [1], and it would not be surprising that the ST inhibitory effect on vasopressin could also occur in other non-tubular structures. fashion. Finally, intravenous administration of ST (200 nglkg body wt as (MB, 30 LM), a soluble guanylate cyclase blocker, did not interfere with the ST inhibitory effect on the Ang LI-dependent reduction in PCSA of rat mesangial cells. ST also blocked the reduction in PCSA induced by phorbol myristate acetate (PMA, 300 nM). ST was also able to prevent and revert the Ang II dependent reduction in glomerular cross-sectional

a bolus plus a continuous injection of 25 ng/min/kg body wt) partially blocked the reduction in GFR (measured as C1,,) and RPF (measured as CPAH) and the increase in filtration fraction induced by the intravenous administration of Ang 11(1.7 g/minfkg body wt) in anesthetized rats. In

summary, these results suggest that ST could antagonize the renal actions of Ang II, increasing the GFR and RPF decreased by Ang II, and this effect could be dependent, at least partially, on a direct relaxing effect of ST on mesangial cells.

Thus, the present experiments were designed in order to clarify, in an approach including in vitro and in vivo experiments, the possible role of ST as a modulator of glomerular filtration, mainly in the presence of an Ang II excess, in an attempt to explain the intrinsic mechanisms of action of this hormone. Methods

Materials Collagenase type IA, from Clostridium hisriolyticum, angiotensin II, phorbol myristate acetate, pertussis toxin, indomethaci L-N-methyl-arginine, methylene blue, L-glutamine, myosin light chain standard, and myosin light chain monoclonal antibody (specific against 20 kD myosin light chains) were and toad urinary bladder [3—5]. It seems that this effect could be purchased from Sigma (St. Louis, Missouri, USA). Alkaline mediated by a direct modulation of adenylate cyclase activity phosphatase-linked rabbit anti-mouse IgG serum and 5-bromo[6, 7]. Moreover, ST may inhibit the diuretic-induced renin 4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/ release [8]. On the other hand, the information concerning the NBT) were from Bio-Rad (Richmond, California, USA). Penipossible regulation of glomerular filtration rate (GFR) or renal cillin was obtained from Laboratorios Level SA (Barcelona, plasma flow (RPF) by ST is controversial. Intravenous infusion Spain). Streptomycin sulfate was obtained from Antibioticos SA (Madrid, Spain). RPMI 1640, Hanks balanced salt solution and fetal calf serum were obtained from Flow Laboratories (Woodcock Hill, UK). 2',5'-dideoxyadenosine and SDS-PAGE Received for publication February 18, 1992 and in revised form August 18, 1992 electrophoresis standards for molecular weight were from PharAccepted for publication August 18, 1992 macia (Uppsala, Sweden). [32P] orthophosphoric acid (HCI free) was from Amersham International (Buckinghamshire, © 1993 by the International Society of Nephrology The information available at the present moment about the renal actions of somatostatin (ST) is scarce [1]. Specific ST binding sites have been found in renal cortex and medulla [2] and it is usually accepted that ST antagonizes the effects of arginine vasopressin on water permeability in collecting ducts

324

GarcIa-Escribano et al: Somatostatin effects on mesangial cells

325

A 110

Fig. 1. Somatostatin prevents the changes induced by angiotensin II in planar cell surface area (PCSA) of cultured rat mesangial cells. Results are expressed as percent of the PCSA at time 0 and are the mean SEM of 5 experiments. Abbreviations are: C, cells with buffer; Ang II, cells with angiotensin II; ST, cells with somatostatin. A. ST + Ang II: cells were preincubated for 10 minutes with 1 /LM somatostatin (ST) and then (time 0)10 nM angiotensin II (Ang II) added. *D < 0.05 vs. C, ST and ST + Ang II. B. Cells were preincubated for 10 minutes with different ST concentrations and then 10 nM Ang II for 30 minutes added. *P < 0.05 vs. Ang II.

C

100

ST

ST + Mg 90 C,)

00

Glomerular isolation and mesangial cell culture

80

Renal glomeruli were isolated from Wistar rats weighing 150

to 200 g. Kidneys were removed under ether anesthesia and glomeruli isolated by successive mechanical sieving (105 and 75

70

m) [15, 16]. The final preparation consisted of glomeruli

Ang II

60 — 0

—10

10

30

20

40

Time, minutes

without Bowman's capsule and afferent or efferent arterioles. Tubular contamination was less than 5%. Buffer A (Tris 20 m, NaC1 130 mt, KCI 5 m, sodium acetate 10 m, glucose 5 mM, pH 7.45) was used in all the steps of the isolation procedure. Isolated glomeruli obtained by a similar mechanical sieving procedure (150 and 50 m) from rats weighing 100 to 150 g were

treated with collagenase, plated in plastic culture flasks and incubated as previously described [17, 18]. The culture medium

consisted of RPM! 1640 supplemented with 10% fetal calf serum, L-glutamine (1 mM), penicillin (0.66 tg/ml), streptomycm sulfate (60 g/ml), and buffered with Hepes, pH 7.2. Culture media were changed every two days. Studies were performed on day 21 or 22, at which time epithelial cells were no longer

B 110

*

detected in the culture flasks. The identity of the cells was

*

confirmed by morphological and functional criteria [17, 18].

100

C

buffer A containing 2.5 mri Ca2 and maintained at room

90 (0 a.

Incubation procedures for microphotograph experiments In every experiment, cells or glomeruli were washed twice, discarding the culture or isolation media and placed in fresh temperature. After 15 minutes in these conditions, experiments were started.

In the first set of experiments, cells or glomeruli were

0

preincubated for 10 minutes with buffer or 1 tM somatostatin

80

(ST) and, after this time, 10 flM Ang II was added to the incubation media. Microphotographs were taken just before Ang II addition and after 30 minutes of incubation. Control cells

70

or glomeruli, incubated only with buffer, were used in each case. Thereafter, the same experiments were performed but

Ang

using different final concentrations of ST (10— 10 to 106 M).

In the second set of experiments, cells or glomeruli were 60

i

a

a

a

a

10

9

8

a

7

a

a

6

—Log [ST], M

UK). Somatostatin (cyclic 14-28) was a gift from Serono S.A.

(Madrid, Spain). All the other reagents were of the highest commercially available grade.

incubated for 30 minutes with buffer or 10 flM Ang II. Then, ST was added to the incubation media, at a final concentration of 1

tM and microphotographs were taken just before the addition of buffer or Ang II (time 0), immediately before the ST (time 30)

and 30 minutes after the ST (time 60). Incubations performed only with buffer, ST or Ang II were used as controls. The same experiments were again performed in the presence of different concentrations of ST (final concentrations 10b0 to lO_6 M). Microphotographs were also performed in the presence of some pharmacological modulators, always with the same experimental design. After 15 minutes of incubation with a specific drug, 1 M ST was added to the incubation media for 10

326

GarcIa-Escribano et at: Somatostatin effects on mesangial cells

A

110 -

Fig. 2. Somatostatin reverses the changes induced by angiotensin II in planar cell surface area (PCSA) of cultured rat mesangial cell. Results are expressed as percent of the PCSA at time 0 and they are the mean SEM of 5 experiments. Abbreviations are: C, cells with buffer; Ang II, cells with angiotensin II; ST, cells with somatostatin. A. Ang II + ST: Cells were incubated for 30 minutes with 10 nM angiotensin II (Ang II) and then 1 LM somatostatin (ST) added for 30 minutes. *D < 0.05 vs. C and ST. **P < 0.05 vs. C, ST and Ang II -I- ST. B. Cells incubated for 30 minutes with 10 nM Ang II and then different ST concentrations

C 100

added for 30 minutes. < 0.05 vs. Ang II.

minutes and then 10 nM Ang II was also added. Microphotographs were taken just before Ang II addition and 30 minutes later. The drugs and their final concentrations were as follows: indomethacin (I, 10 p.M), L-N-methyl-arginine (LNMA, 0.2 mM), 2',S'-dideoxyadenosine (DDA, 0.1 mM) and methylene blue (MB, 30 JiM). Pertussis toxin (PT) was also used in some experiments, but the preincubation time was 18 hours, and the PT concentration was 0.5 Jig/mI. Finally, cells were preincubated for 10 minutes with buffer or

ST

90 (I)

0 C.)

80 **

70 Ang II

1 JiM somatostatin (ST) and then either 300 nM phorbol myristate acetate (PMA) or 100 JiM H202 was added to the incubation media. Microphotographs were taken just before PMA or H202 addition and after 30 minutes of incubation. Control cells, incubated only with buffer, were used in each

60 —

I

I

I

I

I

I

I

—10

0

10

20

30

40

50

60

70

Time, minutes

case. The ability of cells to exclude the trypan blue dye was tested in the different experimental conditions mentioned above.

Determination of planar cell surface area (PCSA) and glomerular cross-sectional area (GCSA) While incubations were performed, mesangial cells grown in

B

110 -

conventional plastic culture flasks, or samples of 100 d of *

glomerular suspensions on excavated glass slides, maintained at

2°C), were observed under phase room temperature (22 contrast with an inverted photomicroscope Olympus IMT 2

*

100

Phosphorylation of myosin light chain Measurement of myosin light chain (MLC) phosphorylation was performed as described [19], with minor modifications [20].

Rat mesangial cells grown on standard culture flasks were preincubated for three hours at 37°C in Tyrode's solution

C

*

(Shibuya-Hu, Tokyo, Japan) with a 150 magnification [15—18].

Serial photographs were taken under the experimental conditions cited above. Seven to 16 cells and 30 to 50 glomeruli were analyzed per photograph. No specific selection criteria existed: every cell or glomeruli which could be measured was analyzed. PCSA and GCSA were determined by computer-aided planimetric techniques. Measurements were performed by two different investigators in a blind fashion. The intraobserver and interobserver variations for cells and glomeruli were 2 and 5% (cells) and 1 and 3% (glomeruli), respectively.

*

90 (I)

0 0

80

70

60

Ang

I

I

I

I

I

10

9

8

7

6

I

I

containing 50 sCi/ml of carrier-free [32P] orthophosphoric acid (HC1 free). Afterwards, cells maintained at room temperature —Log [ST], M were incubated with 1 JiM ST for 10 minutes, followed by 10 nM Ang II for 30 minutes. In the other group of experiments, cells labeled with [32P] orthophosphoric acid were incubated with 10 nM Ang II for 30 minutes at room temperature; ST (1 JiM, 30 incubations, the medium was siphoned off and the total protein mm) was then added. Control cells were incubated in Tyrode's was precipitated directly on the culture flask by adding I ml of solution under the same experimental conditions. Following the 2% TCA. This was followed by washes with 1 ml of 2% TCA

327

GarcIa-Escribano et al: Somatostatin effects on mesangial cells

A

B

C

Fig. 3. Morphological changes in cultured rat mesangial cells. A. Cells in basal conditions. B. Cells after 30 minutes of incubation with 10 nsi Ang II. C. The same cells as in B but after 30 minutes of incubation with I M ST. Notice especially the cells marked with the arrows.

and 1 ml of water containing 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.1 j.tg/ml phenylmethylsulfonyl fluoride (PMSF). Precipitated protein was dissolved in 200 d of gel

Studies of renal function in anesthetized rats

Clearance studies were performed in 36 male Wistar rats

electrophoresis buffer [2% SDS, 10% (vol/vol) glycerol, 5% weighing 300 to 350 g. Animals were anesthetized with sodium 2-mercaptoethanol, and 0.002% bromophenol blue, pH 8.8]. pentobarbital (40 mg/kg body wt) and surgically prepared for The samples were heated for three minutes in boiling water and clearance studies [22] by inserting polyethylene catheters (PE 50, Vygon, France) in the femoral artery and vein and in the electrophoresed. Protein concentrations of each sample were

bladder, after tracheostomy. All the surgical wounds were covered with warm, saline moistened gauzes. The femoral electrophoresed: lactalbumin, 14,400; trypsin inhibitor, 20,100; artery catheter was connected to a pressure transducer (EM carbonic anhydrase, 30,000; ovalbumin, 43,000; albumin, 750, Lectromed, UK) and the pressure was continuously reg67,000; and phosphorylase b, 94,000. In addition, a MLC istered with a polygraph (Uni-Graph 1000-506, Letica). An also determined [21]. The following marker proteins were

standard was also electrophoresed. isotonic saline infusion containing [metoxi-14C] inulin and [3H] Samples of 30 pA were electrophoresed on 5 to 30% gradient para-aminohippuric acid was started at 1.2 ml/hr through the polyacrylamide gels using continuous buffer system (150 V, 3 to venous catheter to allow clearance determinations. After 45 4 hours). Gels were stained for three hours in a solution minutes of equilibration, three 20-minute urine collections were containing 0.5% copper sulfate, 1.5% Coomassie blue (R-250), performed into preweighed plastic vials containing 0.25 ml 10% acetic acid, 23% methanol. The gels were destained in a water-stabilized mineral oil, taking 100 pA blood samples at the 0.5% copper sulfate, 10% acetic acid, 25% methanol solution. beginning and the end of each 20 minute period in heparinized The phosphorylated 20,000 dalton protein which comigrates microhematocrit tubes. Then, rats were divided in three groups with the MLC standard was located. A slice containing all of the of 12 animals each: the first one received just the 1.2 mllmin radioactivity was cut from the gel and solubilized in 30% H202 saline infusion, the second one was continuously injected with at 70°C for one hour. The radioactivity was quantitated by 1.7 tgImin/kg body wt Ang II in addition to the saline injection, liquid scintillation spectrometry and corrected for the total and the third one received the same amount of saline infusion and Ang II; after 40 minutes, the third one was treated with a protein electrophoresed. To confirm the results obtained, labeled cellular proteins, 200 ng/kg body wt ST bolus plus a continuous injection of 25 prepared and separated as described above (SDS-PAGE), were ng!min/kg body wt ST. In these conditions, three additional transferred onto nitrocellulose filters in a buffer containing 25 20-minute clearance periods were performed. After measuring mM Tris, 192 mivi glycine, 20% vollvol methanol, pH 8.3, with blood hematocrit, plasma and urine concentrations of [metoxiapplication of 250 mA for two hours at 4°C. The nitrocellulose '4C] inulin and [3H] para-aminohippuric acid were measured paper was incubated for two hours with the myosin light chain with a two-channel scintillation counter, which corrects crossmonoclonal antibody, diluted 1/200, followed by incubation talk between isotopes. with alkaline phosphatase-linked rabbit anti-mouse IgG serum. The immunostained bands were visualized after incubation with Statistical methods BCIP/NBT. Autoradiograms were obtained of the bands specifically immunostained. Then, these bands were cut (dupliResults are expressed as X SEM and the number of cates), the nitrocellulose was dissolved with formic acid and the experiments is shown in every case. Comparisons were perradioactivity was measured, correcting the value for the total formed by the paired or unpaired Student's t-test, one- or protein content. two-way analysis of variance, Scheffe's multiple comparison

328

Garcia-Escribano et a!: Somatostatin effects on mesangial cells

Table 1. Percentage of contracted cultured rat mesangial cells under

different experimental conditions

Reversion

Prevention

30 mm

30 mm

C Ang II

ST

ST-Ang II

5±1 6±3

46 4

84

C Ang II

ST

4±2

44 3 7±3 45 3

60 mm

150

7±4

45

3a

10±4

14 3 Ang lI-ST Data are expressed in % of contracted cells with respect to the total

cells measured and are the mean

SEM of 5 experiments. A

contraction was considered significant when the changes in PCSA were over 8%. Prevention: ST was added before Ang II, Reversion: ST was added after Ang II. Abbreviations are: C, cells with buffer only; Ang II, angiotensin 1110 nM; ST, somatostatin I /LM; ST-Ang II, cells preincu-

100

bated for 10 minutes with ST I LM and then Ang 1110 n was added; Ang Il-ST. cells incubated for 30 minutes with Ang 1110 nM and then ST

1 tM was added. a P < 0.05 vs. the other groups, at the same time

50

test and Friedman test as needed. A P < 0.05 was considered statistically significant.

0

Results Figure 1 shows the consequences of the ST pretreatment on the changes in PCSA induced by Ang II. ST 1 /LM alone did not

C

Ang II

ST

ST-Ang II

C

Ang II

ST

Ang Il-ST

modify the PCSA of cultured mesangial cells but completely blocked the 25 to 30% reduction in PCSA induced by 10 nM Ang II (Fig. 1A). As shown in Figure 1B, this inhibition seemed to be dose-dependent, with a threshold effect at a ST concentration of 10 nM and a maximum effect of 1 M. Results were completely

comparable when ST was added to the cell incubation media

after 30 minutes with 10 tiM Ang II: 1 M ST completely reversed the Ang II dependent changes in PCSA (Fig. 2A) and

this inhibition was also dose-dependent, with a significant

150

blockade from 10 nM ST (Fig. 2B). This ability of ST to reverse the Ang 11-induced mesangial cell contraction is further documented in Figure 3. The left panel (A) of this figure shows the mesangial cells in basal conditions. Notice particularly the cells marked with the arrows. After 30 minutes with 10 flM Ang II,

the PCSA of these cells decreased significantly (B, central panel), whereas after 30 minutes of the addition of 1 /LM ST PCSA increased again and the cells tended to recover their original form (C, right panel). Table 1 performs a similar analysis but considering the percentage of contracted cells. As the interobserver variability in the cell measurement was about 5%, it was considered that a cell showed a contraction when the change in PCSA with respect to the basal situation was over

Fig. 4. Modulation of myosin light chain (MLC) phosphorylation

(MLCP) by angiotensin II and somatostatin in cultured rat mesangial cells. Results are expressed as percent of the control cell MLCP from the same day of the study and are the mean SEM of 4 experiments. Abbreviations are: C, cells with buffer; Ang II, cells with angiotensin II; B. ST, cells with somatostatin. A. ST-Ang II: Cells preincubated for 10 minutes with 1 tLM somatostatin (ST) and then 10 flM angiotensin II

(Ang II) added for 30 minutes. Ang II + ST: Cells incubated for 30 minutes with 10 nM angiotensin II (Ang II) and then I M somatostatin < 0.05 vs. the other 3 groups. **D < 0.05 (ST) added for 30 minutes.

vs. C.

0

100

50

0

8%. With this criterium, Ang 11(10 nM) contracted between 40 to 57% of the cells, and this effect was prevented and reversed by ST (1 ILM).

After finishing these morphological experiments, more than 94% of the cells excluded the trypan blue dye.

329

GarcIa-Escribano et a!: Somatostatin effects on mesangial cells Immunoblot

-r 1

1

2

-

3

Fig. 5. Proteins of 32P labeled mesangial cells were separated in SDS-PAGE and then transferred onto nitrocellulose. The immunostaining of these proteins with a monoclonal antimyosin light chain antibody gave a well defined band in the nitrocellulose (A). The autoradiogram of these bands (B) showed that Ang II increased the 32P incorporation in myosin light chain (Lanes 2: Ang Ill nM. Lanes 3: Ang 1110 nM) with respect to the control cells (Lanes I), whereas the preincubation with 1 M ST completely blocked this effect (Lanes 4).

4

4

- — W ___

2

3

Autoradiography

s-S —— -

—

110 -

Table 2. Changes in planar cell surface area (PCSA) of cultured rat mesangial cells in different experimental conditions Time minutes C

AngIl

ST ST-Ang II

PT+ ST-AngH 1+ ST-AngII LNMA + ST-Ang II

DDA+ ST-AngII MB+ ST-AngH

ST

0

30

100 100 100 100 100 100 100 100 100

101±2 78±5a 102±1 98

C 100

ST + PMA

3

81

102±3 98

2

97±3 98±4

C,) C-)

a-

90

Results are expressed as percent of values at time 0 and are the mean SEM of 5 experiments. Abbreviations are: C, cells with buffer only; Ang H, angiotensin 1110 flM, ST, somatostatin 1 tM; ST-Ang II, cells

PMA

preincubated for 10 minutes with ST I M and then Ang 1110 n was added; PT, preincubation for 18 hours with pertussis toxin 0.5 g/ml; I, preincubation for 10 minutes with indomethacin 10 M; LNMA, preincubation for 10 minutes with L-N-methyl-arginine 0.2 mM; DDA,

80 —

preincubation for 10 minutes with 2', 5'-dideoxyadenosine 0.1 ms, MB,

preincubation for 10 minutes with methylene blue 30 tM. For more

—10

details, see Methods. a P < 0.05 vs. time 0 and vs. the other groups.

0

10

20

30

40

Time, mm

6. Somatostatin prevented the changes induced by phorbol myristate acetate in planar cell surface area (PCSA) of cultured rat Fig.

mesangial cells. Results are expressed as percent of the PCSA at time

Figure 4 shows the interactions between Ang II and ST with 0 and they are the mean SEM of 5 experiments. Abbreviations are: ST respect to the 32P incorporation in a protein comigrating with + PMA, cells preincubated for 10 minutes with I jiM somatostatin (ST) myosin light chain. As shown in both panels, Ang II increased and then (time 0) 300 nM phorbol myristate acetate (PMA) added; C, and ST decreased 32P incorporation to this myosin light chain- cells with buffer; PMA, cells with PMA; ST. cells with somatostatin. <0.05 vs. C, ST and ST + PMA.

like protein. Moreover, preincubation with ST (1 jiM, upper panel) before the Ang II treatment as well as the addition of the same ST concentration to cells previously incubated with Ang II (lower panel) completely blocked the Ang TI-dependent increased phosphorylation. Figure 5A shows the typical pattern of immunostaining with the MLC antibody, with a single band in SDS-PAGE. Lanes 1, 2, 3 and 4 represent, respectively, control cells, cells incubated with 1 flM Ang 11(30 mm), cells

between Ang II and ST in the presence of different pharmacological inhibitors. As it can be observed, the complete inhibition induced by ST on the Ang TI-related reduction of PCSA in cultured rat mesangial cells was completely blocked by prein-

cubation with pertussis toxin, but it was independent of the blockade of prostanoid or nitric oxide synthesis with indo(30 mm). As shown in the autoradiogram (Fig. 5B), ST com- methacin and LNMA, respectively. Moreover, when cAMP pletely blocked the effect of Ang II on P incorporation in the synthesis was blocked with DDA, or when the soluble guanylMLC. When considering the radioactivity in these immuno- ate cyclase was inhibited with MB, the inhibitory effect of ST incubated with 10 nM Ang 11(30 mm), and cells preincubated for 10 minutes with 1 jiM ST (10 mm) and then added Ang 1110 flM

stained bands, corrected for proteins, 10 nM Ang II increased it by 89 10% with respect to the control values (N = 3, P < 0.05), whereas the respective increase for the cells preincubated with 1 jiM ST and then added 10 nii Ang II was only 9 7% (N = 3, not statistically different from the control values).

Table 2 includes the results concerning the interactions

was still observed. Figure 6 shows the results of the incubation of mesangial cells with PMA, with or without ST, demonstrating that this peptide also completely blocked the reduction in PCSA induced by PMA. H202 also induced a significant reduction of mesangial cell PCSA (PCSA with respect to initial PCSA after 30 mm, 81 3%, N = 5, P < 0.05) which was completely

330

GarcIa-Escribano et a!: Somalostatin effects on mesangial cells

A 110

Fig. 7. Somatostatin prevents the changes induced by angiotensin 11 in

a glomerular cross sectional area (GCSA) of isolated rat gloineruli. Results are expressed as percent of the GCSA at time 0 and they are the

mean SEM of 5 experiments. Abbreviations are: C, glomeruli with buffer; Ang IL, glomeruli with angiotensin II; ST, glomeruli with somatostatin. A. ST + Ang II: Glomeruli preincubated for 10 minutes

with 1 jiM somatostatin (ST) and then (time 0) added 10 nM aflgiOteflsifl H (Ang II). Symbols are: closed bars, time 0; hatched bars, time 30. *D

100

< 0.05 vs. time 0. B. glomeruli preincubated for 10 minutes with different ST concentrations and then 10 nM Ang II added for 30 minutes. *D < 0.05 vs. Ang II.

(1)

0

blocked by ST (PCSA with respect to initial PCSA after 30 mm, 102

90

0

2%, N = 5).

When the analysis of the changes in cross sectional areas was

performed on isolated glomeruli, results were comparable to those of cells: the Ang II induced reduction in GCSA was

80

completely blocked by previous incubation with ST (Fig. 7A) or by treatment of the Ang IT-contracted glomeruli with ST (Fig.

8A). In both cases, the effect was dose-dependent, with a significant effect of ST at 10—8 M and a complete inhibition at

70

i0 M (Figs. 7 and 8B). Table 3 includes the clearance studies in anesthetized rats.

C

No differences could be detected in the mean values of the three basal experimental periods of the three groups of rats (Control group: C1,, 0,61 0.21 mllmin, CPAH 1.87 0.41 ml/min, FF32 3%, hematocrit value 51 3%. Ang II group: C1, 0.70 0.31 0.42 ml/min, FF 28 3%, hematocrit mi/mm, CPAH 2.13

Ang II

ST

ST-Ang II

B

value 48 4%. ST group: C1 0.65 0.22 mi/mm, CPAH 2.01 0.44 mllmin, FF 27 4%, hematocrit value 50 3%). Param-

110

eters of renal function did not change in control rats throughout the 60 minutes after the basal period. However, Ang II infusion induced a progressive decrease of GFR, RPF and RBF and an 100

increased FF which was detected from the first experimental period after starting the infusion. ST infusion partially blocked the Ang TI-dependent changes in renal function and, as it can be observed by analyzing the data of experimental period 3, GFR increased by about 25%, RPF and RBF increased by about 35%

and FF decreased by about 20% when ST was administered during the continuous infusion of Ang II.

(I)

00

90

Discussion

The present results clearly demonstrate that ST inhibits the 80 reduction in PCSA and the increased MLC phosphorylation induced by Ang IT in cultured rat mesangial cells. In other words, ST relaxes these cells when they are contracted with Ang II, since not only does it block the effects on PCSA, but also the changes in MLC phosphorylation. This parallel exper70 8 imental approach, analyzing both morphological and biochem10 9 ical criteria of contraction, is very important in order to assess —Log [STI, M the vasoactive properties of any potential modulator of mesangial cell contraction, as morphological changes could not provide correct information [23, 24]. In this sense, the studies of MLC phosphorylation with ST alone support the importance of biochemical studies. ST reduced 32P incorporation in MLC of performed when trying to assess the relaxant properties of any mesangial cells, supporting a direct relaxing effect of the substance on cultured mesangial cells. Two characteristics of the ST-dependent relaxation must be peptide, but was unable to induce any significant change in PCSA in these cells maintained in plastic flasks, perhaps stressed. First, the effect of ST was evident both when the cells because under these culture conditions cells are unable to were preincubated with the hormone and when ST was added to increase their PCSA. Thus, biochemical studies must always be the incubation media after Ang II incubation, suggesting a

76

GarcIa-Escribano et a!: Somatostatin effects on mesangial cells

Table 3. Clearance studies in anesthetized rats: Interactions between angiotensin II and somatostatin

A 110

B

GFR C Ang II Ang II + ST RPF C Ang II Ang II + ST RBF C Ang II Ang II + ST

100

U)

0

331

90

80

70

C

ST

Ang II

Ang Il-ST

3

100 100 100

103±4

100 100 100

104

100 100 100

100±4

100 100

105±3

103±5

100±7

134

146 144

137

FF C Ang II Ang II + ST

2

1

100

83 78 62 73

102±2

102±5

85 3&

70 8

6ab 5ab

84 7ab

7

99 3

7ab

57 51

62 7'

72 7& 12w'

109 I3

1l'

6ab

46 9'

100 7

34 gab

67 5

93±7

98±4

69

33

10ab

52 8'

14ab

7ab

9ab

66 8

9ab

117 6

Results are expressed as percent of basal values (B) and are the mean + SEM of 12 rats in each experimental group. Abbreviations are: GFR, glomerular filtration rate measured as CIN; RPF, renal plasma flow measured as CPAH; RBF, renal blood flow; FF, filtration fraction; B, mean of the first two 20-minute clearance periods; C, control rats; Ang II, after B rats received 1.7 sg/min/kg body wt angiotensin II; Ang II + ST, after B rats received 1.7 sg/min/kg body wt of angiotensin H and after the 20-minute experimental periods 1 and 2, they received 200 ng/kg body wt as a direct injection and then 25 ng/min/kg body wt of somatostatin.

a < 0.05 vs. B b P < 0.05 vs. C

P < 0.05 vs. C and Ang II

B 110

preventing and a reversing role for ST on the Ang II effects, respectively. Second, in both cases, the ST inhibitory effect was dose-dependent, with an effective blocking concentration of about 10 flM and over. With respect to these concentrations, it is very difficult to extrapolate to the in vivo situation. First,

100

the incubation of mesangial cells in plastic culture flasks with a

U)

0a

selected, not completely physiological medium and at room temperature differs significantly from the in vivo glomeruli. Secondly, the Ang II concentration was chosen because of its proven efficacy, and it is possible that the dose-response curve of ST would be different with another Ang II concentration. In any case, there was a clear inhibitory relationship between 10 flM Ang II and 10 nM ST. both at rather low concentrations, which supports the possibility that the effect of ST may not be

90

80

only pharmacological.

To the best of our knowledge, this is the first report in the literature describing the effects of ST on cultured rat mesangial 70

10

9

8

7

6

—Log [ST], M

cells. Thus, some experimental protocols were designed in order to start to clarify the mechanisms responsible for the observed effects in mesangial cells. The first question to analyze

would be the presence of specific membrane receptors in

mesangial cells. Radioligand studies with labeled ST were not performed, but Roca, Arilla and Prieto demonstrated the presglomerular cross sectional area (GCSA) of isolated rat glomeruli. Results are expressed as percent of the GCSA at time 0 and are the ence of ST receptors on the renal cortex [21, supporting the Fig. 8. Somatostatin reverses the changes induced by angiotensin H in

mean SEM of 5 experiments. Abbreviations are: C, cells with buffer; Ang II, cells with angiotensin II; ST, cells with somatostatin. A. Ang I1-ST: Glomeruli incubated for 30 minutes with 10 nM angiotensin II

(Ang IL) and then I

somatostatin (ST) added for 30 minutes.

Symbols are: closed bars, time 0; hatched bars, time 30; open bars, time 60. *U < 0.05 vs. time 0. B. Glomerulj incubated for 30 minutes with 10 flM Ang II and then different ST concentrations added for 30 minutes. *D < 0.05 vs. Ang II.

possibility that at least part of these receptors would be in

mesangial cells. The second question to answer was the possible dependence of the observed effect of ST on the presence of a specific G protein in the cell membranes. As shown in the Results section, pertussis toxin blocked the relaxing effect of ST

on mesangial cells, supporting the existence of this kind of protein. Similar proteins have been described as mediators of

332

GarcIa-Escribano et a!: Somatostatin effects on mesangial cells

ST effects in other cellular systems such as GH4C1 cells [251. It the mesangial cell. Moreover, ST also blocked the reduction in is thus possible that they also mediate the hormone effect in GFR and RPF induced by Ang II, thus giving further support to mesangial cells. The third question to answer was whether the the relaxant properties of this peptide in the presence of an effect of ST was the consequence of a direct interaction with the excess of vasoconstrictor substances. However, it is not pos-

cells or whether it depended on a secondary release of other sible to establish a direct correlation between in vivo and cell vasoactive metabolites. Mesangial cells synthesize PGE2 [261 effects, since clearance studies were performed only to assess and nitric oxide (NO) [271, both of which are relaxant mediators the ST inhibitory effect, without testing different infusion rates [28, 29]. Thus, it could be the case that ST induce an increased of both Ang II and ST, and also because micropuncture studies production of these metabolites which would be directly re- focusing on the different determinants of GFR were not carried sponsible for the relaxation. When PGE2 and NO synthesis out. In summary, these results demonstrate the relaxing effect of were respectively inhibited by blocking cyclooxygenase with indomethacin [301 or NO synthetase with LNMA [311, ST was ST in cultured rat mesangial cells treated with Ang II. This

still able to block the Ang Il-dependent contraction, thus effect seems to depend on the presence of a G-protein in the cell suggesting the independence of the ST relaxing effects of POE2 and NO synthesis. With respect to the intracellular mechanisms of action of ST in cultured rat mesangial cells, some preliminary information may be obtained from the present results. ST-dependent relaxation was observed both in the presence and in the absence of

membrane, being independent of the secondary production of other relaxing metabolites as PGE2 or NO. These actions were not only evident in cells, but similar results were also obtained

DDA. Since this metabolite blocks cAMP production [30], it can be proposed that the relaxing effect of ST must not be mediated by this cyclic nucleotide. In addition, the effect of ST was not blocked by MB, an inhibitor of the soluble guanylate cyclase [32], pointing to an intracellular mechanism independent of this enzymatic system. Although rather improbable, another possibility to account for the ST inhibition of the Ang II effect would be a displacement of the latter from its membrane receptors. No specific radioligand studies have been performed to confirm this aspect, but different evidences argue against this possibility. First, ST also blocked the contractile effect of hydrogen peroxide on

situations characterized by an excess of vasoconstrictors. If we

cultured rat mesangial cells, an effect which seems to be dependent on platelet-activating factor [20], and it does not

in isolated glomeruli and in vivo clearance studies, which highlights the possible physiological or pathophysiological im-

portance of this peptide as a modulator of renal filtration in

remember the demonstration by Kurokawa et al [36] of the presence of ST in glomerular structures, we can propose a role for ST as a local regulatory hormone in the kidney. Acknowledgments The present work was financed in part by Serono S.A. Partial aspects

of the work were presented at the XXX Meeting of the American Society of Nephrology and were published in abstract form in Kidney International [37]. M. Gonzalez Rubio is a fellow of the Comunidad Autdnoma de Madrid. We would like to thank S. Lamas and F. Gago for their critical revision of the manuscript. Reprint requests to Diego Rodr(guez-Puyol, M.D., Seccion de Nefrologla, Hospital Universitario Principe de Asturias, Alcalá de Henares,

seem probable that both Ang II and platelet-activating factor act 28880 Madrid, Spain. through the same membrane receptor. Second, ST also blocked

the PMA contractile effect, and this phorbol ester obviously acts by a mechanism independent of the Ang II membrane

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