Glomerular Microcirculation: Distinct Intracellular Mechanisms for Afferent and Efferent Arteriolar Tone

Glomerular Microcirculation: Distinct Intracellular Mechanisms for Afferent and Efferent Arteriolar Tone

Koichi Hayashi, Koichiro Homma, Shu Wakino, Tsuneo Takenaka, Hiroo Kumagai, and Takao Saruta

Summary. Angiotensin (ANG) II contributes importantly to the regulation of renal pre- and postglomerular arteriolar tone. The present study examined the subcellular signaling mechanisms for ANG II-induced afferent (AFF) and efferent arteriolar (EFF) constriction, using the isolated perfused hydronephrotic rat kidney. Angiotensin II-induced AFF and EFF constriction was abolished by an ANG II receptor antagonist (losartan). The pretreatment with N-ethylmaleimide (G

protein inhibitor) completely prevented the ANG II-induced constriction of EFF, but not AFF. Furthermore, signal interruption at the phospholipase C level by 2-nitro-4-carboxyphenyl-N,N-diphenyl- carbamate blocked the constriction of both arterioles. Next, ANG II-induced AFF constriction was completely inhibited by the blockade of inositol-1,4,5- trisphosphate (IP

) signaling by thapsigargin and L-type voltage-dependent calcium channel blockers, but relatively refractory to protein kinase C (PKC) inhibition (by chelerythrine). In contrast, EFF constriction was resistant to pranidipine, but partially responsive to thapsigargin and chelerythrine.

Finally, direct PKC activation by phorbol myristate acetate caused prominent EFF constriction, which was inhibited by manganese/free calcium medium, but not by pranidipine. Thus, PKC plays an obligatory role in ANG II-induced EFF constriction that requires extracellular calcium entry through non- selective cation channels. By contrast, ANG II-induced AFF constriction is mainly mediated by IP

and voltage-dependent calcium channel pathways.

Collectively, intracellular signaling mechanisms differ in AFF and EFF, which may determine the glomerular function.

Key words. Afferent arteriole, Efferent arteriole, Calcium channel, Inositol trisphosphate, Protein kinase C

225

Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi,

Shinjuku-ku, Tokyo 160-8582, Japan

Introduction

The kidney serves to function as an important organ to maintain body fluid homeostasis as well as electrolyte balance. Thus, both preglomerular afferent and postglomerular efferent arterioles play a pivotal role to govern glomeru- lar capillary pressure, and subsequently the glomerular filtration rate (GFR).

However, the regulation of afferent and efferent arteriolar tone should teleologically differ because parallel changes in the arteriolar resistance of these vessels would not alter GFR. Indeed, histological characteristics differ between afferent and efferent arterioles. Thus, vascular smooth muscles of afferent arterioles manifest spindle-shaped morphology, whereas those of efferent arterioles possess star-like morphology [1]. Similarly, myosin heavy chain isoform SM1 is reported to be present in both afferent and efferent arte- rioles, whereas SM2 is present only in the afferent arteriole [2]. Furthermore, although it is established that angiotensin (ANG) II is abundant in the kidney and represents an important intrarenal paracrine regulating the vascular tone of pre- (afferent) and post-glomerular (efferent) arterioles [3], the functional heterogeneity in the renal microvascular action of ANG II remains fully unde- termined. Furthermore, whether intracellular signaling pathways differ in afferent and efferent arterioles is not elucidated.

Direct Visualization of Renal Microcirculation

To clarify the different responsiveness of afferent and efferent arterioles to ANG II, we examined the arteriolar behavior, using the isolated perfused hydronephrotic rat kidney model that directly visualized the renal microves- sels in vitro [4–6]. The rat kidney was rendered hydronephrosis by ligation of the right ureter. After 6–8 weeks, renal tubular atrophy had progressed that allowed direct visualization of the renal microcirculation.

Figure 1 illustrates a schematic diagram of the apparatus used in the present study. The hydronephrotic kidney was placed on the stage of an inverted microscope modified to accommodate a heated chamber equipped with a thin glass viewing port on the bottom surface. Kidneys were per- fused with medium saturated with a gas mixture of O

/CO

within a pres- surized reservoir. The perfusion pressure was controlled by adjusting the back-pressure-type regulator. Images were obtained from an inverted micro- scope, and were captured by a computer equipped with a video acquisition board.

Using this experimental model, we found that the responsiveness of

afferent and efferent arterioles varied, depending on the underlying vaso-

constrictors used. Thus, as shown in Fig. 2, efferent arteriolar responses to

226 K. Hayashi et al.

these agents were blunted, as compared with those of afferent arterioles. In contrast, both ANG II and norepinephrine caused similar magnitude of vasoconstriction of afferent and efferent arterioles. Of note, it is demonstrated that similar magnitude of constriction of afferent and efferent arterioles results in an elevation in glomerular capillary pressure, leading to an increase in GFR [7].

Fig. 1. Apparatus used to study microvessels in the isolated perfused hydronephrotic kidney. The hydronephrotic kidney was placed on the stage of an inverted microscope modified to accommodate a heated chamber equipped with a thin glass viewing port on the bottom surface. Kidneys were perfused with medium saturated with a gas mixture of O

/CO

within a pressurized reservoir. The perfusion pressure was controlled by adjusting the back-pressure-type regulator. Images were obtained from an inverted microscope, and were captured by a computer equipped with a video acquisition board

Fig. 2. Distinct responsiveness of

afferent and efferent arterioles to

vasoconstrictor stimuli. Open bars,

afferent arterioles; filled bars,

efferent arterioles

Intracellular Signaling of ANG II

To delineate the cellular mechanism of the ANG II-induced constriction of afferent and efferent arterioles, the following signaling pathways have been examined. Thus, in general ANG II binds to its receptor, which couples with G proteins, and subsequently phospholipase C. This enzyme breaks down phosphatidylinositol 4,5-bisphosphate (PIP

) into inositol 1,4,5-trisphosphate (IP

) and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate then stimulates IP

receptors at the sarcoplasmic reticulum, releasing intracellular Ca from the sarcoplasmic reticulum. The elevated intracellular Ca would stimulate Cl channels and cause membrane depolarization, opening voltage-dependent Ca channels. On the other hand, the formation of DAG stimulates protein kinase C (PKC), and modifies smooth muscle contraction.

We therefore tested the above-mentioned hypothesis in the renal micro- circulation. Initially, AT1 receptor blockade by losartan completely inhibited the ANG II-induced constriction of both afferent and efferent arterioles in a dose-dependent manner [8]. These observations indicate that AT1 receptors constitute an important determinant that mediates the ANG II-induced con- striction of renal microvessels. Next, a G

protein inhibitor, N-ethylmaleimide, completely abolished the ANG II-induced efferent arteriolar constriction.

This agent however failed to alter the afferent arteriolar constriction.

Furthermore, a phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N- diphenyl carbamate (NCDC), completely blocked the ANG II-induced con- striction of both afferent and efferent arterioles [9]. Collectively, ANG II receptor stimulation would involve G protein and PLC in both afferent and efferent arterioles. However, at the G protein level, G

protein participates in the ANG II-induced efferent arteriolar tone, whereas other G protein, such as G

, may be responsible for afferent arteriolar constriction induced by ANG II.

Next, subcellular mechanisms for ANG II-induced constriction were evaluated, including IP

and DAG. In the presence of thapsigargin, which finally inhibits the IP

-mediated Ca release from the sarcoplasmic reticulum, the afferent arteriolar response to ANG II was completely abolished, whereas the efferent arteriolar response was diminished but not totally abolished [9]

(Fig. 3). Furthermore, a chloride channel blocker, IAA-94, reversed the ANG II-induced constriction of the afferent arteriole in a dose-dependent manner [10]. Similarly, a voltage-dependent Ca channel blocker, nifedipine, prominently reversed the ANG II-induced constriction of the afferent, but not efferent, arteriole [11].

In contrast, the pretreatment with staurosporine had modest effect on the

ANG II-induced constriction of the afferent arteriole, whereas the efferent

228 K. Hayashi et al.

arteriolar response was partially prevented by staurosporine [10] (Fig. 4). In concert, the above-mentioned observations on the afferent arteriole indicate that the IP

formed by phospholipase C stimulates Ca release from sarcoplas- mic reticulum (Fig. 5). The increased Ca would enhance Cl channel activity and the subsequent membrane depolarization. Such elevated membrane potential gates voltage-dependent Ca channels and finally induces Ca influx from this channel.

Next, we focus on the role of the intracellular Ca release mechanism in mediating the ANG II-induced efferent arteriolar constriction. As shown in Fig. 6, thapsigargin potently inhibited the ANG II-induced constriction [10]. Of note, the vasoconstrictor component was still retained in the pres- ence of thapsigargin. Similarly, ANG II-induced constriction was more sensi- tive to the inhibition of PKC by chelerythrine in the efferent than in the afferent arteriole [12], thus suggesting that the PKC-mediated vasoconstric- tor tone of the efferent arteriole was more greatly activated in the efferent arteriole.

Fig. 3. Role of intracellular Ca release in angiotensin II-induced constriction of renal microvessels. In the presence of thapsigargin, angiotensin II failed to alter afferent arteriolar diameter, but elicited constriction of efferent arterioles. *P < 0.05 vs baseline

Fig. 4. Protein kinase C contributes more greatly

to efferent than afferent arteriolar tone during

angiotensin II-induced stimulation. *P < 0.05 vs

baseline; **P < 0.01 vs baseline

Role of Intracellular Ca

When extracellular Ca is removed, afferent arterioles failed to constrict in response to ANG II. In the efferent arteriole, however, removing extracellular Ca or addition of manganese diminished, but not completely abolished the efferent arteriolar constriction [10].

To clarify whether extracellular Ca is involved in the PKC-mediated effer- ent arteriolar constriction, we examined the effect of removing extracellular Ca on phorbol myristate acetate (PMA)-induced constriction of the efferent arteriole. Thus, this manipulation completely inhibited the PMA-induced vasoconstriction of this vessel [12].

In concert, in the efferent arteriole, after binding with its receptors, ANG II-stimulated IP

would release Ca from sarcoplasmic reticulum, and could involve Ca influx from store-operated Ca channels (Fig. 7). In parallel with this mechanism, ANG II enhances PKC, which opens nonselective cation channels, or TRP channels, and subsequently elevates intracellular Ca concentration.

230 K. Hayashi et al.

Fig. 5. Mechanism for angiotensin II-induced constriction of the afferent arteriole. AT 1, angiotensin type 1 receptors; VDCC, voltage-dependent Ca channels; PLC, phospholipase C; DG, diacylglycerol; PKC, protein kinase C; MLCK, myosin light chain kinase; CaM, calmodulin

Fig. 6. Role of intracellular Ca release in

angiotensin II-induced efferent arteriolar

constriction. P < 0.05 vs baseline

Concluding Remarks

Although abundance of the intrarenal ANG II content clearly suggests an important role of this substance in the regulation of renal function as a deter- minant of glomerular hemodynamics, substantial evidence has accrued that ANG II elicits distinct activity on afferent and efferent arterioles. The IP

- mediated signaling pathway constitutes an important determinant of the afferent arteriolar response to ANG II (Table 1). In contrast, both IP

and DAG- PKC pathways contribute to the development of ANG II-induced constriction

Table 1. Summary showing different mechanisms for angiotensin II-induced constriction of renal microvessels

Afferent Efferent

1 . AT1 receptors =

2 . G protein ? (G

) G

3 . Phospholipase C =

4 . IP3/DAG-PKC IP3 >> PKC IP3 < PKC

5 . ECF Ca-dependency ++ +

6 . Ca influx pathway VDCC NSCC

AT1, angiotensin type 1 receptors; PKC, protein kinase C;

DAG, diacylglycerol; VDCC, voltage-dependent Ca channels;

NSCC, nonselective cation channels

Fig. 7. Mechanism for angiotensin II-induced constriction of efferent arterioles. AT 1,

angiotensin type 1 receptors; TRP, transient receptor protein potential; PLC, phospholi-

pase C; DAG, diacylglycerol; PKC, protein kinase C; MLCK, myosin light chain kinase; CaM,

calmodulin

of the efferent arteriole. In conclusion, the roles of intracellular signaling mechanisms, including G

protein, voltage-dependent calcium channels, IP

, and PKC, during ANG II stimulation differ in afferent and efferent arterioles, which may constitute segmental heterogeneity in the renal microvasculature, and therefore would serve to control the glomerular capillary pressure and subsequent renal injury.

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