11

Chapter

General introduction

Marjon Roos

William van Rodijnen

Piet ter Wee

Geert Jan Tangelder

Chapter 1

12

Introduction

The kidneys have various

functions, of which the excretion from

the body of excess water and water-

soluble waste products is one. To this

end fluid from the blood is filtered and

processed by the two kidneys. Each

kidney contains a million

microscopically sized functional units

called nephrons, see figure 1.1. A

nephron is composed of a filtering

unit, the glomerulus, from where the

filtered fluid flows into a tubule (see

arrows), which contains different

segments. In the regulation of

glomerular filtration small blood

vessels are important, which are

positioned at the inflow and outflow

sites of each filtering unit. They are

called afferent and efferent arteriole,

respectively (figure 1.1). The filtration

function of the kidney can be harmed

during the course of various illnesses,

one of them being diabetes mellitus

(45). This disease, which at present

increases dramatically in the world is

subdivided in two main forms (see

below). The second designated type 2,

is often preceded by overweight or

obesity (13, 42).

In the present thesis obesity

and in particular diabetes mellitus

have been simulated in rats, in order to investigate microvascular determinants

that are involved in damaging the small filter-units in each kidney. We were

particularly interested in recognizing alterations in the molecular mechanisms

possibly related to changes in functioning and reactivity of the small feeding and

draining arterioles of the glomeruli (figure 1.1). We studied, among others, the

reactions of these vessels to changes in local blood pressure, designated the

Afferent

arteriole

Efferent

arteriole

Bowman’s

capsuleGlomerulus Tubular network

Loop of Henle with

capillary network

Renal vein

Figure 1.1. Schematic illustration of a nephron (upper panel), the actual functional unit of which each kidney contains about a million. The beginning of each nephron is the glomerulus, where fluid is filtered from the blood into the nephron. Lower panel, example of a light microscopic picture of a perfused glomerulus from a hydronephrotic kidney, with its feeding afferent (right side) and draining efferent arteriole (left side).

General introduction

13

myogenic response, to depolarization of the cell-membrane and to the

circulating hormone angiotensin II, with special attention to the modulating

influence of alterations in signaling molecules generated within the so-called

cyclo-oxygenase (COX) pathway.

General aspects of diabetes mellitus

In the 2nd century AD, the word diabetes, meaning “siphon”, i.e. a bent

tube through which liquid can flow away from a vessel, was first used by the

Greek physician Aretaeus to describe patients with great thirst and excessive

urination. In the 17th century it was noticed that the urine of many of these

patients had a sweet smell and taste, so the word mellitus, meaning “like

honey,” was added to the name of the disease (2). Diabetes mellitus is seen as

one of the main threats to human health in the 21st century (35, 62). When

untreated it is characterized by chronic hyperglycemia, i.e. high glucose levels in

the blood (62). Increased concentrations of glucose in the blood and body fluids

alter, among others, the structure and function of various proteins. There are

two main forms of diabetes mellitus.

Type 1 diabetes, also called insulin-dependent diabetes mellitus, is due

primarily to autoimmune-mediated destruction of beta cells in the pancreatic

islets, resulting in absolute insulin deficiency (62). In the Netherlands 74,000

people are affected by type 1 diabetes (1). In addition, an increase of 23% was

found between 1980-1990 in a study that compared diabetes incidence in the

Netherlands among children younger than 20 years of age (46). This comparison

suggests an increase in the number of type 1 diabetic patients also in the near

future.

Type 2 diabetes is characterized by insulin resistance, which is defined

by a relative insensitivity of the target tissues for insulin, resulting in a reduced

glucose uptake (62). Of all diabetic patients 85-90% is affected by type 2

diabetes. This means that in The Netherlands alone 526,000 people, or 3.3% of

the total population, suffer from this disease. It is predicted that in 20 years

time the number of type 2 diabetic individuals will increase by 35% (1). A risk

factor for its development is obesity. One out of every 3 people with obesitas will

develop type 2 diabetes mellitus (13, 42).

General aspects of obesity

The prevalence of obesity and of people with overweight in The

Netherlands has been reported to be 1.6 million individuals, which is 10% of the

population (3). Obesity occurs when the number of calories consumed exceeds

the number that is metabolized, the remainder being stored in adipose (fat)

tissue.

Chapter 1

14

The kidney

In addition to the excretion of excess water, waste products and foreign

substances, the kidneys regulate extracellular fluid volume and osmolarity,

maintain ion balance, regulate pH, and participate in endocrine pathways. The

various segments of the functional unit, i.e. the nephron (see figure 1.1, upper

panel), execute these functions in collaboration with the surrounding blood

vessels. Arterial blood arriving at a nephron has passed via successively smaller

interlobular arteries, and finally the distal interlobular artery, into the afferent

arteriole. From there it runs into the glomerulus, where plasma without proteins

filters out from the capillary loops into the capsule of Bowman. From there the

filtrate flows into the first and subsequent tubular segments of the nephron

(arrows figure 1.1). Blood leaves the glomerulus via the efferent arteriole, and

from there it flows first through microvessels surrounding the tubular segments

of that same nephron before finally leaving the kidney via the renal vein.

The resistance of the pre- and postglomerular microvessels determines

glomerular filtration rate and regulates glomerular capillary pressure.

Determinants of filtration rate as measured in a single nephron are: single

nephron plasma flow, glomerular capillary pressure, capillary ultrafiltration

coefficient and systemic oncotic pressure (7). Increases in the first 3 and/or a

decrease in the last one may increase filtration rate. Subsequent analyses

Number of diabetic patients Number of diabetic patients with nephropathy

1985 1995 2005 2015 2025

0

50

100

150

200

250

300

350

Year

Dia

beti

c p

ati

ents

(m

illions)

Figure 1.2. Current and expected prevalence of diabetes mellitus, type 1 and type 2 combined (dashed line), and the concomitant (predicted) rise in diabetic nephropathy (solid line). Adapted from King et al. (32).

General introduction

15

revealed that glomerular capillary pressure was the main determinant of

glomerular damage (7). Glomerular capillary pressure is mainly determined by

the resistance of the immediate preglomerular arterioles and the efferent

arteriole (40). When a glomerulus is damaged it can not properly filter anymore.

When this happens to many glomeruli, nephropathy or kidney failure will

develop.

Diabetic nephropathy

The four main complications of diabetes mellitus are: nephropathy,

cardiovascular disease, retinopathy and neuropathy. The latter three are beyond

the scope of the present thesis. Diabetic nephropathy is initially characterized by

an increased loss of albumin in the urine. This is thought to be preceded by

hyperfiltration, which is often found in diabetic patients with normoalbuminuria.

Clinically, more albumin in the urine is divided into micro- and

macroalbuminuria. A slow loss of glomerular filtration capacity is thought to start

in the microalbuminuric phase, which is characterized as an urinary albumin

excretion between 20 and 200 µg/min. Microalbuminuria usually proceeds into

macroalbuminuria which is defined as a urinary albumin excretion above 200

µg/min. This phase is associated with a more prominent decline in glomerular

filtration rate.

Figure 1.2 shows the expected number of patients with diabetic

nephropathy in the near future (solid line) based on the estimated increase of

both types of diabetes mellitus combined (dashed line). It is known that 20-40%

of the patients with type 1 diabetes mellitus will develop diabetic nephropathy,

despite an improved glycemic control (32). As mentioned above, diabetic

nephropathy is often preceded by a phase of hyperfiltration (e.g. 14, 17, 18, 20,

57). In addition to possible alterations within the glomerulus itself, e.g. of the

capillary wall, the pre- and postglomerular arterioles also may play a role in the

development of hyperfiltration (4, 59). A decrease in preglomerular resistance is

seen as an important determinant, while postglomerular resistance often is

maintained (9, 61). This combination results in a heightened glomerular pressure

causing hyperfiltration, and with longer duration, damage to the glomerular

capillaries. A decreased preglomerular resistance can be caused by a decreased

sensitivity for vasoactive stimuli inducing constriction, and/or an increased

sensitivity to vasodilatory substances.

Nephropathy in obese patients

Not only diabetes, but also obesity can change normal renal physiology.

Even without hyperglycemia it is already associated, among others, with an

increase in glomerular filtration rate, increased renal plasma flow and an

Chapter 1

16

increased urinary albumin excretion (12). The latter is considered a first sign of

endothelial and/or podocyte dysfunction in the glomerulus (5), while

hyperfiltration provides a link to glomerulosclerosis (21). The pathogenetic

Ca2+SR

K+

DEPOLARIZATION

Ca2+

Ca2+↑

Rho Kinase

?

-

+

Myosin

Myosin-P

MLCK

Ca42+,CaM

MLCK

VASOCONSTRICTION

CaM

Ca2+ MLC-Phosphatase-P

(Inactive)

MLC-

Phosphatase(Active)

Many vasoconstrictor

pathways(see text)

Receptor

DAG

PKC

IP3R

G

AngII

PLC

PIP2 IP3

↑ Pressure

Figure 1.3. Schematic overview of molecular mechanisms in vascular smooth muscle cells leading to vasoconstriction, and of angiotensin II, pressure and membrane depolarization induced signaling. Membrane depolarization can be evoked, among others, by inhibition of potassium-efflux, as happens with various vasoconstrictors (see text). SR sarcoplasmic reticulum; MLC myosin light chain; MLCK MLC kinase; CaM calmodulin; PLC phospholipase C; PIP2 phosphatidylinositol 4,5-biphosphate; IP3 inositol 1,4,5-

triphosphate; IP3R IP3 receptor; Ca2+ calcium; DAG 1,2-diacylglycerol and PKC protein kinase C.

General introduction

17

mechanisms involved, however, probably differ between obesity and diabetes

mellitus (12).

Renal microvascular constriction or vasodilatation, and alterations in diabetic

nephropathy

Changes in resistance of the kidney and its glomeruli can be regulated by

vasoconstriction or vasodilatation. This is brought about by the vascular smooth

muscle cells in the wall of the renal arterioles, which interact with surrounding

cells such as the endothelium, mesangial cells in the glomerulus and tubular

epithelium. Depolarization of the vascular smooth muscle cell membrane, or the

response of these cells to a change in transmural pressure (myogenic response),

or their responsiveness to the circulating signaling molecule angiotensin II or to

locally produced products of the COX pathway are all important determinants of

their functioning. First, basic aspects of vascular smooth muscle cell contraction

will be shortly described. Thereafter, the role of each stimulus mentioned above

in maintaining glomerular pressure will be succinctly introduced, followed for

each stimulus by a paragraph discussing its involvement in diabetic nephropathy.

Vascular smooth muscle cell contraction

Vasoconstriction is brought about by contraction of vascular smooth

muscle cells (VSMCs). For the latter a rise in the intracellular free calcium-ion

concentration is important, as is shown in figure 1.3 (start at Ca2+ in the black

box). Calcium then binds to calmodulin, and the calcium-calmodulin complex

binds in turn to MLC-kinase (MLCK) thereby stimulating phosphorylation of myosin

(myosin-P). Myosin-P is able to interact with actin filaments resulting in VSMC-

contraction and vasoconstriction. Alternatively, when rho-kinase is activated (see

also figure 1.3, lower right), it sustains vasoconstriction by inhibiting the

dephosphorylation of myosin-P. It does so by converting the active myosin light

chain (MLC) phosphatase to its inactive form (53).

Membrane depolarization

Membrane depolarization of VSMCs is a physiological component of many

vasoconstrictor pathways, as is also schematically shown in figure 1.3. It can be

evoked, among others, by inhibition of potassium-efflux by closure of potassium

channels. Signaling pathways which lead to such closure operate either via

activation of protein kinase C (PKC) or rho-kinase, or via inhibition of cAMP or

cGMP. Both pathways are used by vasoconstrictors (30, 41). Membrane

depolarization is able to cause a rise in intracellular calcium due to opening of

voltage-gated calcium channels in the outer VSMC membrane (34, 48, 47).

Chapter 1

18

Interestingly, this may lead to rho-kinase activation via an as yet unknown

PLA2

Peroxidase Cox

1/2

Prostaglandin H2

PGE

synthase

PGE2

PGD

synthase

PGD2

Prostacyclin

synthase

PGI2

Thromboxane

synthase

TxA2

dilatation constriction

Vessel

tonus

AA

GQ

DAGPKC

IP3

AngII

Receptor

PLCPIP

2

Ca2+SRIP

3R

Ca2+

PGF

synthase

PGF2

Prostaglandin G2

Glucose

1

2

Figure 1.4. Schematic overview of cyclo-oxygenase (COX) pathways in vascular smooth muscle cells, and how angiotensin II (AngII), mark numbered 1, or hyperglycemia, mark numbered 2, can influence this balance. Hyperglycemia has other effects as well which are not shown (e.g. glycosilation of proteins and generation of reactive oxygen species (51)). Two isoforms of COX, designated 1 or 2 (COX 1/2), are present in the kidney. PLC phospholipase C; PIP2 phosphatidylinositol 4,5-biphosphate; IP3 inositol 1,4,5-triphosphate; IP3R IP3 receptor; Ca2+ calcium; DAG 1,2-diacylglycerol and PKC protein kinase C; PLA2 phospholipase A2; AA arachidonic acid; PG prostaglandin (for I2, D2 and E2).

General introduction

19

Interestingly, this may lead to rho-kinase activation via an as yet unknown

mechanism (see figure 1.3) (34, 47, 48).

In arterioles of diabetic rats the responses of potassium channels were

found to be increased, as well as their functional availability, thereby promoting

dilatation of these microvessels (27). The same investigators also found a

decreased responsiveness in these rats to membrane depolarization, as evoked by

increasing the extracellular potassium chloride concentration. This indicates that

afferent arteriolar contractile and calcium responses are diminished in diabetes

mellitus (10, 11).

Myogenic response

The myogenic response is the acute constriction of a blood vessel in

reaction to an increase in transmural pressure, and vasodilatation when pressure

decreases. In the kidney, it is an important mechanism to protect the glomerulus

from changes in systemic blood pressure (36). As transmural pressure was

increased, vascular smooth muscle cells of renal arteries exhibited a linear

depolarization from an average resting potential of -57±2 mV at 20 mm Hg to

-38±2.4 mV at 120 mm Hg (22). This indicates that VSMC membrane

depolarization is involved in the myogenic response, as is also depicted in figure

1.3. In renal arterioles the subsequent rise in intracellular calcium could be

inhibited by L-type calcium channel blockers, suggesting that voltage-gated

calcium entry mechanisms are responsible for the rise in intracellular calcium

with an elevated transmural pressure (22, 25). How the vascular wall translates

changes in transmural pressure into changes in VSMC membrane potential is at

present unknown (15).

In afferent arterioles of diabetic rats an impaired myogenic responsiveness was

found, which could be restored by a high dose of the aselective COX-inhibitor

ibuprofen (26). Therefore, an alteration in the synthesis of one or more products

within the different COX-pathways (see below) might play a role in the reduced

reactivity to pressure as found in diabetic rats (26).

Angiotensin II

Angiotensin II is a circulating hormone that also can be formed in the

kidney. It is an important regulator of the diameters of pre- and postglomerular

arterioles, and hence, of glomerular hemodynamics (40). It can elevate

intracellular calcium-levels in VSMCs by stimulating calcium mobilization from

intracellular stores, and secondly by activating calcium entry mechanisms. When

angiotensin II binds to its AT1-receptor phospholipase C (PLC) is activated, as is

shown in figure 1.3. Activated PLC hydrolyses phosphatidylinositol 4,5-

Chapter 1

20

biphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol

(DAG). IP3 binds to receptors on the sarcoplasmic reticulum where calcium

channels are opened and stored calcium is released into the cytoplasm. DAG in

turn, activates PKC which, among others, is thought to increase the sensitivity of

the contractile apparatus for calcium (56).

In diabetic pre- and postglomerular microvessels various changes in the

effects of angiotensin II have been found: an increase in reactivity (10), no effect

of the diabetes (10, 49) or a decreased reactivity (28, 29). The study that

observed in afferent arterioles an increased responsiveness to angiotensin II due

to diabetes mellitus did not investigate any underlying mechanisms (10); by

contrast, in efferent arterioles no change was seen. One of the studies that

reported a normal afferent arteriolar reaction to angiotensin II found that this

could occur thanks to a modulation of superoxide dismutase that suppressed an

altered influence of endogenous nitric oxide (NO) caused by the diabetes mellitus

(49). A decreased reactivity to angiotensin II due to diabetes could be restored

by feeding the rats a diet enriched with 1% myo-inositol (29). Adding myo-inositol

to the diet resulted in a increased incorporation of myo-inositol into the

membrane which restored the vasoconstrictor capacity to angiotensin II (29),

myo-inositol is a precursor of IP3 (see figure 1.3). In another study the decreased

reaction to angiotensin II could be partially restored by the aselective COX-

inhibitor indomethacin (28), again indicating an alteration in the synthesis of one

or more products within the COX pathway (see below) due to diabetes mellitus.

COX-pathway

Table 1.1. Overview of prostanoids active in the kidney and their receptors with the main vasoactive effect.

Prostanoid Constrictive Dilatory

Thromboxane A2 TP

PGE2 EP1, EP3 EP2, EP4

PGI2 IP

PGF2α FP

PGD DP

Partially adapted from (8). PG prostaglandin (for E2, I2, F2α and D)

Products of the COX-pathway, called prostanoids, are important

mediators of renal hemodynamics (8, 23, 24, 43). As is shown schematically in

figure 1.4, they are formed when phospholipase A2 (PLA2) is activated by a rise in

intracellular calcium and/or by PKC. PLA2 then liberates arachidonic acid from

membrane phospholipids. Arachidonic acid is then bound by a complex of one of

the COX enzymes and peroxidase. It is known that there are two COX isoforms (1

and 2) present in the kidney (60), of which COX-1 is the most abundant. The

General introduction

21

complex of COX-1 or –2 with peroxidase converts arachidonic acid into PGG2 and

PGH2, which in turn are rapidly transformed into more stable prostanoids by cell-

specific synthases (60). Prostanoids can alter vessel tone depending on which

type is produced, as is shown in figure 1.4. For example, production of

thromboxane A2 leads in the kidney to vasoconstriction (50) and of prostaglandin

I2 (PGI2) to vasodilatation (19).

Table 1.1 gives a schematic overview of the different prostanoid

receptors present in the kidney and their main vasoactive action. Only

prostaglandin E2 (PGE2) has both vasoconstrictive and vasodilatory properties.

Angiotensin II can stimulate the synthesis and release of various

prostanoids (39, 50). As is shown in figure 1.4 (see mark numbered as 1), binding

of angiotensin II to its receptor activates PLC, which leads to a subsequent

increase in cytosolic calcium levels via IP3. This, and the activation of PKC can

both activate PLA2 (50). Via the latter pathway also hyperglycemia can stimulate

PLA2 activity (16), as is indicated in figure 1.4 by number 2. Hyperglycemia has

also other effects which are not shown in the figure such as alterations in

metabolism, glycosylation of proteins and generation of free oxygen radicals

(51).

As mentioned already, in diabetic renal microvessels the aselective COX-

inhibitor indomethacin was capable of partially restoring an attenuated reaction

to angiotensin II (28). However, which of the two COX-isoforms is involved, and

to which extent is presently unknown. In renal microvessels a possible

relationship between diabetes and COX-2 has not been investigated so far.

Diabetes and obesity models used

Type 1 diabetes mellitus was induced in male Sprague-Dawley rats by

injection of streptozotocin (STZ). This naturally occurring antibiotic, which is

produced by the fungus Streptomyces achromogenes, is in sufficient dose toxic

for the beta cells in the pancreatic islets. The glucose moiety of STZ is the

essential component that specifically directs STZ to the beta cell. Once inside

the beta cell it causes destruction of DNA and apoptosis. Subsequently, there are

not enough beta cells left to produce insulin, which will result in hyperglycemia.

The STZ-injection is often combined with subsequent partial insulin substitution,

as was also done in most of our experiments, to prevent a too high level of

hyperglycemia.

As a prediabetic obesity model the genetically obese Zucker rat was used

(33). These rats develop obesity because of leptin resistance due to a missense

mutation in its hypothalamic receptor (55, 58). Lean control rats are genetically

identical except for this mutation. The obese rat displays many of the health

Chapter 1

22

problems seen in human obesity such as hyperlipidemia, late-onset hypertension,

hypercholesterolemia, and hyperinsulinemia (6, 31, 38, 44, 52). Moreover, with

increasing age the obese Zucker rat develops proteinuria and focal segmental

glomerulosclerosis, ultimately leading to kidney failure (31, 38).

Visualization of microvessels in the hydronephrotic kidney

The isolated perfused hydronephrotic kidney was used to directly

visualize that part of the renal microvasculature that is predominantly important

for the regulation of glomerular capillary pressure, i.e. the distal interlobular

arteries, afferent and efferent arterioles. Chronic hydronephrosis was induced by

ligating the left ureter 6-8 weeks before the experiment. This results in a

complete atrophy of the tubular elements with minimal effects on the pre- and

postglomerular microvasculature (54). Since there are no tubular structures

present, the tubulo-glomerular feedback mechanism is absent in this model. This

allows for direct assessment of vascular reactivity of the microvessels just

mentioned, without the need to account for possible indirect effects mediated

via the tubulo-glomerular feedback mechanism. On the day of an experiment

hydronephrotic kidneys were isolated as is extensively described in the materials

and methods sections of chapters 2-6. Figure 1.5 shows schematically the set-up

used for in vitro perfusion of hydronephrotic rat kidneys, as well as a picture of a

hydronephrotic kidney in the actual set up. Perfusion flow through the kidney

was measured with a flow-probe in the catheter between the pressurized

reservoir and the renal artery.

In this thesis we mainly studied the responsiveness to angiotensin II,

added directly to the perfusion fluid, which is described in chapters 3, 4, 5 and

6. Simultaneous measurements of distal interlobular arteries, afferent and

efferent arterioles could often be performed. Membrane depolarization was

evoked not only by increasing the extracellular potassium concentration

(chapters 4 and 6), but also with barium chloride (chapter 6). The latter

selectively blocks the inward rectifier potassium channel and could be

conveniently used to obtain concentration-response curves (41).

A unique property of the hydronephrotic kidney is the possibility to study

myogenic reactivity, as was done in chapters 2 and 6. Since the individual vessels

in this model can be visualized without exposure to surgical trauma, ischemia,

hypoperfusion or hypoxia, the myogenic response is always present (54).

However, since only preglomerular vessels are myogenically active (25),

pressure-induced diameter changes were determined for distal interlobular

arteries and afferent arterioles only. Two important characteristics of the

myogenic response in the hydronephrotic kidney model are: a) it is quite rapid

General introduction

23

with the steady state reached within seconds, and b) the degree of

vasoconstriction is proportional to the elevation in perfusion pressure for the

range of arterial pressures seen in vivo (37).

Figure 1.5. Set-up used for in vitro perfusion of hydronephrotic rat kidneys and microscopic observation of the periglomerular microvessels (upper panel, adapted from (37)). Lower panel shows an example of a hydronephrotic kidney placed in this set up.

Chapter 1

24

Outline of this thesis

Part I: diabetes mellitus

Why 20-40% of the diabetic patients develop nephropathy, and the others

do not is currently unknown. We addressed the hypothesis that a loss of

responsiveness of pre- and/or postglomerular arterioles to certain stimuli might

be involved. To this end, we combined the aforementioned STZ-model of

diabetes mellitus type 1 with hydronephrosis and measured, among others,

changes in diameter of these microvessels.

In chapter 2, the responsiveness to pressure was investigated in control

and diabetic rats coming from two different suppliers, as well as a possible

difference in sensitivity to the vasodilator prostanoid PGE2 of control rats.

In chapter 3, a study is presented on the effects in diabetes of a new

class of drugs: selective COX-2 inhibitors. The responsiveness to angiotensin II

was investigated and the involvement of thromboxane A2 here-in was

determined.

In chapter 4, we investigated whether aspecific inhibition of COX could

restore a decreased responsiveness to angiotensin II in diabetic rats. In addition,

a possible involvement of the vasodilatory prostanoid PGE2 was studied.

Part II: Obesity

In chapter 5, the influence of obesity on renal microvascular reactivity

was studied. Obesity in humans is associated with proteinuria and an increased

glomerular filtration, possibly related to an increase in glomerular capillary

pressure. We investigated in obese Zucker rats which exhibited proteinuria, and

in their lean controls, whether this is related to a chronic alteration in the

diameter of pre- and/or postglomerular microvessels. We also investigated a

possible change in their reactivity to angiotensin II.

Part III: rho-kinase

It is clear from the above that vascular tone of the smallest microvessels

is important for proper regulation of renal hemodynamics (40). An important

determinant of vascular tone is the contraction of vascular smooth muscle. This

is determined not only by levels of intracellular free calcium, but also by the

sensitivity of its contractile apparatus (53). A potential modulator of the latter is

rho-kinase. In chapter 6, we addressed the question of a possible central role for

rho-kinase in the regulation of periglomerular microvascular tone.

General introduction

25

General discussion and summary

Chapter 7 presents a general discussion about the findings described in

the preceding chapters as well as some new supplemental data.

The thesis ends with a summary of the results obtained and the

conclusions drawn.

Chapter 1

26

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