1

1

AJP- Regulatory, Integrative and Comparative Physiology.

MS # R-00645-2006.R1

Renal angiotensin II concentration and interstitial infiltration of immune

cells are correlated with blood pressure levels in salt-sensitive

hypertension.

Martha Franco1, Flavio Martínez2, Yasmir Quiroz 3, Othir Galicia 2,

Rocio Bautista 1, Richard J. Johnson 4, Bernardo Rodríguez-Iturbe 3.

1 Department of Nephrology, Instituto Nacional de Cardiología Ignacio Chávez Mexico

City, Mexico; 2 Department of Pharmacology, UASLP, Mexico; 3 Hospital

Universitario Universidad del Zulia and Centro de Investigaciones Biomédicas, IVIC-

Zulia, Maracaibo, Venezuela and 4 Division of Nephrology, Hypertension and

Transplantation, University of Florida, Gainesville FL

Correspondence: Bernardo Rodríguez-Iturbe, Nephrology, Hospital Universitario,

Ave. Goajira s/n, Maracaibo, Estado Zulia, Venezuela. Email: bernardori@telcel.net.ve

Short Title: Renal angiotensin and salt-sensitive hypertension

Page 1 of 26Articles in PresS. Am J Physiol Regul Integr Comp Physiol (May 2, 2007). doi:10.1152/ajpregu.00645.2006

Copyright © 2007 by the American Physiological Society.

2

2

Abstract. (226 words)

Renal immune cell infiltration and cells expressing angiotensin II (AII) in

tubulointerstitial areas of the kidney are features of experimental models of salt

sensitive hypertension (SSHTN). A high salt intake tends to suppress circulating AII

levels but intrarenal concentrations of AII have not been investigated in SSHTN. This

study explored the relationship between these features to gain insight in the

pathophysiology of SSHTN. Plasma AII and renal interstitial AII (microdialysis

technique), and the infiltration of macrophages, lymphocytes and AII positive cells were

determined in SSHTN induced by 5 weeks of a high salt diet (HSD) after short-term

infusion of angiotensin in rats with (n=10) and without (n=11) treatment with

mycophenolate mofetil (MMF) and in control rats fed a high (n=7) and normal (n=11)

salt diet. As in previous studies, MMF did not affect AII-associated hypertension but

reduced the interstitial inflammation and the SSHTN in the post AII-period. During the

HSD period, the AII group untreated with MMF had low plasma (2.4±SD1.4 pg/ml) and

high interstitial AII concentration (1310±208 pg/ml); MMF treatment resulted in

significantly lower interstitial AII (454 ±128 pg/ml). Renal AII concentration and the

number of tubulointerstitial AII positive cells were correlated. Blood pressure

correlated positively with interstitial AII and negatively with plasma AII, thus giving

compelling evidence of the paramount role of the AII within the kidney in the AII-

induced model of salt-driven hypertension.

Key words: lymphocytes, macrophages, interstitial inflammation, immune cells, renal

hypertension.

Page 2 of 26

3

3

INTRODUCTION

A series of investigations have documented that infiltration of immune cells, oxidative

stress and increased numbers of infiltrating inflammatory cells and proximal tubular

cells that express angiotensin II (AII positive cells), are present in the kidney in

experimental models of salt sensitive hypertension (SSHTN). The functional relevance

of these findings is indicated by a series of experiments that demonstrate that

suppression of the inflammatory infiltrate or the oxidative stress result in amelioration

or prevention of hypertension (reviewed in 17, 26). It is well known that inflammatory

reactivity is inextricably linked with oxidative stress as these conditions induce and

maintain one another (13); however, the participation of intrarenal AII activity as an

integral part of the tubulointerstitial inflammatory reactivity in the pathophysiology of

SSHTN has not been investigated. Angiotensin II positive cells have been identified in

the models of salt sensitive hypertension and double staining studies have shown that

the cells staining positive for AII are both proximal tubular cells and infiltrating

immune cells (1, 12, 14). Based on this evidence, we have postulated that in models of

SSHTN, the tendency to salt retention is driven by the intrarenal inflammation,

oxidative stress and AII activity (13,17) and results from other laboratories have

confirmed that intrarenal inflammation plays a role in the pathogenesis of hypertension

in several experimental conditions (5, 8, 10, 24).

The relevance of the intrarenal AII activity in the pathogenesis of hypertension

has been emphasized in the experiments of Navar and his associates (reviewed in 7)

who have elegantly shown that intrarenal AII functions as a separate compartment that

is not physiologically modulated by influences, such as plasma volume expansion, that

reduce plasma AII levels (9). Investigations from our group in the cellophane-wrapped

Page kidney model (25) demonstrated the critical role of interstitial inflammation and

Page 3 of 26

4

4

intrarenal, rather than plasma, angiotensin II activity in the long-term maintenance of

hypertension and more recently, micropuncture studies (3) showed that interstitial

inflammation is likely responsible for the cortical vasoconstriction that is present in salt-

sensitive hypertension. While the accumulated evidence is compelling, a demonstration

that increased concentration of AII in the kidney is, in fact, correlated with the severity

of hypertension, with the increased numbers of AII positive cells and with immune cell

infiltration has been lacking. Furthermore, concomitant evaluation of the renal and the

plasma AII concentration in relation to the blood pressure levels has not been made in

experimental models of SSHTN.

Therefore, the present work was done to define the following questions: 1)

What is the relationship between blood pressure, renal AII and plasma AII

concentrations in the salt-driven hypertension that follows AII infusion?; 2) Is there an

association between the concentration of AII in the kidney, the number of AII positive

cells and the severity of the inflammatory infiltrate?

Our studies demonstrate that blood pressure levels are negatively correlated with

plasma AII and positively correlated with renal AII concentrations. In addition, we

found that renal AII concentration is correlated with the number of AII positive cells

and infiltrating immune cells in tubulointerstitial areas of the kidney in this

experimental model.

METHODS

Male Sprague-Dawley rats from the animal facilities of the Instituto Nacional de

Cardiología (Mexico, D.F) weighing (340-360g) were used in the experiments. The

animals were housed and handled in accordance with institutional guidelines of animal

care. The following groups of rats were studied:

Page 4 of 26

5

5

Post-AII infusion salt sensitive hypertension (AII-SSHTN group, n=10). These rats were

studied following a protocol used in previous publications from our group (4, 14).

Briefly, AII (Sigma, St. Louis, MO) was infused during 2 weeks by subcutaneously

implanted osmotic pumps (Alzet model 2002, Alza Corp. Palo Alto CA) at a rate of 435

ng/kg/min. At the end of 2 weeks, pumps were withdrawn and after a period a washout

of 5 days on a normal sodium diet (0.4% NaCl) they were placed in a high salt diet (4%

NaCl, Harlan Co.) for 5 weeks. At the end of this period, the rats were prepared for the

studies of interstitial AII concentration by microdialysis as described afterwards and

when those studies were concluded kidneys were harvested for immunohistology (see

later).

Mycophenolate mofetil (MMF) treated AII-infused rats (AII-MMF group, n=11).

These rats received the AII infusion as described in the AII-SSHTN group above and, in

addition received MMF (30 mg/kg/day by gastric gavage) during the 2 week period of

AII infusion as described in earlier studies (4, 14). The rest of the experiment was

similar to the AII group described above including the washout period and the 5 weeks

of high salt diet.

Control rats with a high salt diet (c-HSD group n=10). These rats were housed in

similar conditions and were fed a 4% salt rat diet for 5 weeks

Control rats with normal salt diet (c-NSD group, n=11). These rats were housed in

similar conditions and were given a normal (0.4%) salt diet for 5 weeks prior to the

studies.

Additional experiments were done to determine AII renal and plasma

concentrations after the AII infusion was discontinued and before the induction of

SSHTN with the administration of a HSD. These experiments were done 5 days after

Page 5 of 26

6

6

removal of the osmotic minipumps in 10 additional rats of the AII -SSHTN group and in

9 additional rats of the AII-MMF group. Control studies were done in 8 rats with NSD.

Blood pressure. Systolic blood pressure (SBP) measurements were performed at

baseline and every two weeks in conscious, restrained rats by tail-cuff plethysmography

(Narco Biosystems, Austin, TX). Prior to the studies, all rats were conditioned to the

procedure as described in previous communications (4, 14). Determinations of the SBP

at the time when the studies were done were used in the correlation analyses.

Plasma and Renal AII determinations. For determinations of renal AII, rats were

prepared as for micropuncture studies as described in previous communications (3, 4,

21). Rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p., and

supplementary doses were administered as required). The rats were placed on a thermo-

regulated table, and the temperature was maintained at 37°C. The left kidney was

exposed, placed in a Lucite holder and mean arterial pressure was continuously

monitored with a pressure transducer placed in the femoral artery.

Renal AII concentration was determined in the renal cortex by a microdialysis

technique, as described by Nishiyama et al. (9) and Fox et al. (2) and Siragy et al (23)

and reported by our group in a previous communication (3). Preliminary studies were

done to test the microdialysis probe in vitro, testing AII concentrations from 2 to 10

nMol, and correlation coefficient was r2= 0.94. At a perfusion rate of 1, 5 µl/min, the

relative equilibrium rate was 66 ± 2%, which did not deteriorate with time. In vivo

calibrations were performed from 0 to 180 min to test the stability of the AII

concentrations according with the method described by Nishiyama et al (9). Thirty

minutes after implantation of the probe, the concentrations of AII reach a steady state

and remain stable up to 180 min. Previous studies have determined that AII derived

from tubular fluid does not contaminate the dialysate (9).

Page 6 of 26

7

7

For determination of AII, a 10-mm polysulfone microdialysis probe (Fresenius

Medical Care, USA) with a 33,000-D transmembrane diffusion cut-off was implanted

into the renal superficial cortex. Steel needles were inserted in both ends of the fiber.

The inflow of the probe was connected with a polyethylene tubing (PE-10) to a

microinfusion pump (Harvard apparatus, Holliston, MA). The probe was perfused with

Ringer´s solution containing 1% bovine serum albumin (pH 7.4), at a rate of 1.5 µl/min.

The dialysate was collected directly from the outflow steel tubing into a tube containing

a solution of inhibitors (30 µl of 500 mM EDTA, 15 µl of 1mM enalaprilat and 30 µl of

125 M o-phenantrolene and 0.2 mM pepstatin A in 95 % ethanol), to allow the

dialysate effluent to be immediately mixed with the inhibitors. After 90 min of the

microdialysis probe implantation, three 60-min sample collections were taken.

Immediately thereafter, each sample was vortex-mixed, and 100 µl of the solution was

transferred to a tube containing 1 ml chilled 100% methanol. Blood was collected at the

end of the experiment into a prechilled syringe containing the inhibitors solution. After

centrifugation at 4°C plasma was separated.

Angiotensin II Assay. Determination of plasma and interstitial fluid AII by enzyme-

linked competitive immunoassay using a commercial Kit (SPI Bio, Bertin Group,

France), following the procedure recommended by the manufacturer. All assays were

carried out by one researcher in order to avoid inter-observer variability.

The dialysate samples were reconstituted with 100 µl of 0.9% saline solution

and extracted with methanol. Methanol was evaporated to dryness with nitrogen;

samples were reconstituted in assay buffer, extracted in phenyl cartridges and assayed in

duplicate using an 8-point standard curve (125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.625

pg/ml, 7.81 pg/ml, 3.9 pg/ml, 1.95 pg/ml, and 0.98 pg/ml). Absorbance at 415 nm was

determined in a microplate reader (Bio-Rad Inc.; Hercules, CA, USA.). When AII

Page 7 of 26

8

8

concentration levels of the sample were above 125 pg/ml in the angiotensin standard

curve, dilutions (1:5, 1:10 and 1:25) were done in order to obtain lectures between 7.81

and 62.5 pg / ml.

Cross-reactivity with the following angiotensin peptides was determined:

angiotensin I (AI (1-9); Bachem, H-5038.0025), Angiotensin II (AII; Bachem, H-

1705.0025), , Angiotensin I -7 (A I/II (1-7); Bachem, H-1715.0025) and Angiotensin 3-

8 (A I/II (3-8); Bachem, H-8125.0025). A 100 pg/ml standard was prepared for each

peptide. Readings were done at 415 nm. Cross reactivity results were as follows:

Angiotensin I = <0.01%; Angiotensin II= 99.9%; Angiotensin 1-7= <0.01%,

Angiotensin 3-8= 34.2%. Cross-reactivity with Angiotensin III (H-Arg-Val-Tyr-Ile-

His-Pro-Phe-OH) was not tested because reagent was unavailable but is assumed to be

100% (Bachem Online Catalog) Results obtained in our laboratory shown above were

similar or better than those reported by the manufacturers of the commercial kit used in

these studies (SPI Bio, Bertin Group, France).

Recovery using 3[H]-AII was 88 ± 16 % in 6 samples (range from 82.16 to

94.23 %). The intra- and inter-assay variations (three different assays each one in

duplicate) were 3.93±0.56 and 10.7±0.58, respectively. The minimal detectable

concentration was 0.99 pg/ml.

Histological studies.

Coronal sections were used for histological studies. The fragments were fixed in

10% formalin and embedded in paraffin. Immunoperoxidase methodology was used to

identify lymphocytes (CD5 positive cells), macrophages (ED1 positive cells) as detailed

previously (11). Cellular infiltration was evaluated separately in the glomeruli and in

tubulointerstitial areas and expressed as positive cells per glomerular cross section (gcs)

or positive cells per mm2, respectively.

Page 8 of 26

9

9

The primary antibodies used were anti-CD5 (mouse monoclonal anti-rat

thymocytes and lymphocytes) and anti ED-1 (mouse monoclonal anti-rat monocytes

and macrophages), purchased from Biosource International (Camarillo, CA). Rabbit

anti-human angiotensin II antiserum with cross-reactivity to rat angiotensin II

(Peninsula Laboratories) was used to identify angiotensin II-positive cells. Specificity

of the AII staining was validated by the demonstration that staining was prevented by

pre-incubating the antibody with human angiotensin II. as described in a previous

communication (15). Secondary biotin-conjugated affinity-pure antibodies with

minimal reactivity to rat serum proteins were purchased from Accurate Chemical and

Scientific Co. Westbury, NY. Non-relevant antibodies were used for negative control

studies. Imunohistological techniques have been reported previously (1, 12, 15, 16).

Immunohistology was evaluated with an Olympus BX51 System Microscope

and DP70 microscope Digital camera, with Image Analysis software of Sigma Pro,

Leesburgh, VA, as described in previous communications (1, 15, 16).

Immunohistological analyses were done without previous knowledge of the

experimental group of the biopsy or the results of other determinations

Statistical Analysis. Differences between groups were examined with multigroup

analysis of variance and Tukey Kramer post-tests. Correlations between variables were

analyzed with Person’s tests. Data is shown as mean ± SD. Two-tailed p values <0.05

were considered statistically significant.

RESULTS.

The mean values of plasma AII, renal AII concentration, cellular immune

infiltration in tubulointerstitial areas and the SBP in the experimental groups are shown

in Table 1. CD5+ cells and ED1+ cells were rare (0-2/gcs) and AII+ cells were not

found in the glomeruli in all the experimental groups that were studied.

Page 9 of 26

10

10

As shown in Table 1, the experimental groups covered a range of blood pressure

that went from mean values of 160 mmHg in the SSHTN group to 118 mmHg in the c-

NSD group and all the experimental and control groups had mean blood pressure levels

significantly different from one another. Correspondingly, the mean values of plasma

and renal AII were significantly different from one another in all experimental groups.

The infiltrating CD5 (lymphocytes) and ED1 (macrophages) cells and AII positive cells

in tubulointerstitial areas were similar in the AII-MMF group and the c-HDS groups

who had blood pressures of 140 mmHg and 129 mmHg, respectively and significantly

different (p<0.001) in the SSHTN group and c-NSD groups between themselves and

from the rest (Table 1).

The plasma levels of AII were negatively correlated (p<0.0001) with the blood

pressure (Figure 1A) in sharp contrast with the positive correlation (p<0.0001) that

existed between renal AII and blood pressure (Figure 1B). Positive correlations also

existed between CD5 positive cells and blood pressure (Figure 2A) and ED1 positive

cells and blood pressure (Figure 2B). Renal AII concentration was correlated with the

intensity of the inflammatory ED1 infiltration (Figure 3A) and with the number of

tubulointerstitial cells that stained positive for AII (Figure 3B). All these correlations

were highly significant (p<0.0001). A representative microphotograph of AII positive

cells in the biopsy of a rat in the SSHTN group is shown in Figure 4. As shown in the

figure, both infiltrating cells and proximal tubular cells stain positive for AII in a pattern

that includes cell membrane and cytoplasm.

Table 2 shows SBP, plasma and interstitial AII, 5 days after AII infusion was

discontinued (osmotic minipumps removed) and before the induction of SSHTN with a

high salt diet. At this time, SBP and plasma AII had returned to normal while renal AII

Page 10 of 26

11

11

remains high in the AII-SSHTN group. In contrast, the AII-MMF group presents renal

AII concentration that is not significantly different from the control group.

DISCUSSION.

We had previously shown that experimental salt-sensitive hypertension is

associated with inflammatory infiltration and increased numbers of AII positive cells in

tubulointerstitial areas of the kidney (1, 3, 12, 14, 16). As in previous studies, we did

not find AII positive cells within the glomerular tuft, despite the fact that AII may be

produced in the glomerulus, albeit in lesser quantity than in tubulointerstitium. The

present studies demonstrated a relationship between increased intrarenal AII

concentration, the number of tubulointerstitial AII positive cells and the severity of the

inflammatory infiltration in the post-AII SSHTN model. The relationship between AII

positive cells and AII activity in interstitial fluid raises the possibility that they these

cells are potential sources of interstitial AII. Since both proximal tubular cells and

immune cells are capable of producing AII, it is conceivable that that AII produced by

infiltrating cells and by proximal tubular cells may be released in the interstitial fluid.

This possibility needs to be addressed in subsequent studies. However, it should be

noted that AII concentration in the kidney is modulated by physiologic mechanisms

independent of interstitial inflammation and, therefore, conclusions based on the

correlation with the immune cell accumulation are applicable only to the post-AII model

of SSHTN

Determinations of interstitial AII concentrations in the rats after the infusion of

angiotensin confirmed previous studies that showed that renal AII is increased after

exogenous angiotensin administration (27, 29). In fact, the values found 5 days after

AII infusion was stopped (Table 2) are higher than the values found 5 weeks later,

Page 11 of 26

12

12

during the period of SSHTN (Table 1). From these data is apparent that normal blood

pressure and high concentrations of renal AII coexist in the post-angiotensin infusion

period. Increased renal angiotensin concentration appears to be relevant to hypertension

specifically in the context of a high salt diet when a tendency to sodium retention would

result in SSHTN. Relevant to this issue, Kobori et al. (6) showed that intrarenal

angiotensinogen production is markedly increased in Dahl salt-sensitive hypertensive

rats, suggesting that augmentation of renal angiotensin activity may be involved in salt-

dependent hypertension. Of note, MMF treatment during the administration of

exogenous angiotensin prevented the increment in renal AII concentration induced by

exogenous AII (Table 2). The present studies do not allow discerning whether MMF is

suppressing AII production directly or as a result of the drug’s anti-inflammatory effects.

In order to study a wide range of values we choose to study experimental groups

in which we knew from previous studies (4, 14) that the immune cells infiltration, the

number of AII positive and blood pressure were significantly increased (AII infusion

model) and that all these characteristics were significantly reduced by MMF treatment

administered during AII infusion, prior to the SSHTN phase. In this manner, potential

direct effects of MMF on the experimental variables were unlikely since the drug was

stopped more than 5 weeks before the studies were done. Other investigators have

shown that AII infusion results in increased renal AII (20, 27, 28) and, in fact, 5 days

after the AII infusion was discontinued there was increased AII concentration in the

kidney (Table 2). It is undefined for how long the intrarenal AII remains elevated after

AII administration in the absence of a high salt diet but since AII infusion had been

stopped 40 days before the SSHTN studies were conducted (5 days of washout period

plus 5 weeks of high salt diet) it seems unlikely that intrarenal AII levels would be

solely due to a carried-over effect of exogenous angiotensin administration. MMF-

Page 12 of 26

13

13

treated rats were exposed to similar amounts of exogenous AII but had significantly less

intrarenal AII prior to and after SSHTN developed. Consequently, the findings in our

studies are likely independent of both exogenous AII and MMF.

The observation that plasma AII was suppressed whereas intrarenal AII was high

in SSHTN does not completely rule out the possibility that plasma AII is not

participating in this model, as it may not be suppressed as much as it should be from the

effects of the high salt diet and elevated blood pressure. Indeed, Sealey et al (22) have

proposed that intrarenal microvascular disease (which occurs in this model) may lead to

uneven renal perfusion (nephron heterogeneity) that may result in some nephrons to

overproduce renin whereas others may show an opposing effect. While we did not

measure renin levels, the observation that rats with SSHTN had lower (and not higher)

plasma AII than c-HSD rats suggests that plasma AII was even more suppressed in

SSHTN. Furthermore, the linear negative correlation between plasma AII and blood

pressure (Figure 1) would also argue against a pro-hypertensive role of incomplete

suppression of plasma AII since if such were the case, one would expect that the plasma

AII and blood pressure levels in the SSHTN rats would positively correlated..

The results of the present studies are in agreement with previous investigations

from our group, mentioned before, and others (5, 8, 10, 24), showing an association

between immune cell infiltration and the development of SSHTN. In fact, a recent

authoritative review suggests that the beneficial effects of AII blockers may be related

in part to anti-inflammatory effects (19). To be noted, the associations of blood

pressure levels with the intensity of the inflammatory infiltrate and the numbers of AII

positive cells are not evident in the middle range of the findings. As shown in Table 1,

the AII-MMF group and the c-HSD group do not differ significantly in the number of

infiltrating cells or in the number of AII positive cells and, nevertheless, these groups

Page 13 of 26

14

14

have significantly different mean blood pressure levels (140±8.4 mmHg and 129±4.7

mmHg, respectively, p<0.001 in Table 1). Consequently, other factors are also playing

a role in the development of SSHTN and it is reasonable to consider that these factors

are also responsible for driving the immune infiltrate from the low levels found in c-

NSD to the high levels found in the SSHTN group (Table 1). Among these conditions a

critical role is likely played by oxidative stress that is known to be inextricably

associated with both local inflammation and blood pressure (17, 26). Determination of

oxidative stress was not done in these experiments but in previous investigations we

have shown that antioxidant treatment reduces renal inflammatory infiltrate and

ameliorates hypertension (18).

Notwithstanding the findings in the middle range values shown in Table

1 and discussed above, the highly significant correlation between AII positive cells and

renal AII concentration (Figure 3B) is important and indicates that AII positive cells, if

overabundant, are likely associated with increased renal AII activity. To be sure, the

correlations found in these studies do not prove a causal relationship between

inflammatory infiltration and interstitial AII activity but this possibility is in line with

recent observations from our group in the same experimental model of SSHTN,

showing that blockade of angiotensin II type 1 receptor with candesartan and MMF-

induced reduction of interstitial inflammation have similar effects on glomerular

hemodynamics and blood pressure (3).

Finally, this work clearly demonstrates that plasma AII and renal AII

concentrations move in opposite directions in post-AII SSHTN and renal AII

concentrations have a direct and exquisite relationship with blood pressure levels in this

model of salt driven hypertension.

Page 14 of 26

15

15

Acknowledgements: This study was supported by grants 40943-M (M. Franco) from

the National Council of Science and Technology (CONACYT), Mexico, from NIH

grants HL68607 and DK52121 (R Johnson), and from FONACIT Grant 2005-000283,

Venezuela (B. Rodriguez-Iturbe). Dr Johnson is a consultant for and Nephromics Inc.

Page 15 of 26

16

16

REFERENCES.

1. Alvarez V, Quiroz Y, Nava M, Pons H, and Rodriguez-Iturbe B. Overload

proteinuria is followed by salt-sensitive hypertension caused by renal infiltration of

immune cells. Am J Physiol Renal Physiol. 283;F1132-141, 2002..

2. Fox J, Guan S, Hymel AA, and Navar LG. Dietary Na and ACE inhibition

effects on renal tissue angiotensin I and II and ACE activity in rats. Am J

Physiol Renal Phys 262:F902-F909, 1992..

3. Franco M, Martinez F, Rodriguez-Iturbe B, Johnson RJ, Santamaria J,

Montoya A, Nepomuceno T, Bautista R, Tapia E, and Herrera-Acosta J.

Angiotensin II, interstitial inflamation, and the pathogenesis of salt-sensitive

hypertension. Am J Physiol Renal Physiol 291; F1281-F1287

4. Franco M, Tapia E, Santamaría J, Zafra I, García-Torres R, Rodríguez-

Iturbe B, Gordon Kl, Johnson RJ, and Herrera-Acosta J. Renal cortical

vasoconstriction contributes to the development of salt-sensitive hypertension

after Angiotensin II exposure. J Amer Soc Nephrol. 12: 2263-2271, 2001.

5. Gu JW, Tian N, Shparago M, Tan W, Bailey AP, and Manning Jr RD.

Renal NF{kappa}B Activation and TNF{alpha} up-regulation correlate with

salt-sensitive hypertension in Dahl Salt-sensitive (SS) Rats. Am J Physiol Regul

Integr Comp Physiol. 291: R1817-R1824, 2006

6. Kobori H, Nishiyama A, Abe Y, and Navar LG. Enhancement of intrarenal

angiotensinogen in Dahl salt sensitive rats on high salt diet. Hypertension 41:

592-597, 2003.

7. Kobori H, Ozawa Y, Suzaki Y, Prieto-Carrasquero MC, Nishiyama A,

Shoji T, Cohen EP, and Navar LG. Young Scholars Award Lecture:

Page 16 of 26

17

17

Intratubular angiotensinogen in hypertension and kidney diseases. Am J

Hypertens 19:541-550, 2006.

8. Mattson Dl, James L, Berdan EA, and Meister CJ. Immune suppression

attenuates hypertension and renal disease in the Dahl salt-sensitive rat.

Hypertension 48: 149-156, 2006

9. Nishiyama A, Seth DM, and Navar LG. Renal interstitial fluid concentrations

of angiotensins I and II in anesthetized rats. Hypertension. 39:129-34, 2002.

10. Ozawa Y, Kobori H, Suzaki Y, and Navar LG. Sustained Renal Interstitial

Macrophage Infiltration Following Chronic Angiotensin II Infusions.

Am J Physiol Renal Physiol. 292: F330-F339, 2006.

11. Parra G, Moreno P, and Rodríguez-Iturbe B. Glomerular proliferative activity and

T lymphocyte infiltration in acute serum sickness. Clin Immunol Immunopathol 82:

299-302, 1997.

12. Quiroz Y, Pons H, Gordon Kl, Rincón J, Chávez M, Parra G, Herrera-Acosta J,

Gómez-Garre D, Largo R, Egido J, Johnson RJ, and Rodríguez-Iturbe B.

Mycophenolate mofetil prevents the salt-sensitive hypertension resulting from short-

term nitric oxide síntesis inhibition. Am J Physiol Renal Physiol 281:F38-F47, 2001.

13. Rodriguez-Iturbe B, and Johnson RJ. The role inflammatory cells in the

kidney in the induction and maintenance of hypertension. Nephrol Dial

Transplant 21: 260-263, 2006

14. Rodríguez-Iturbe B, Pons H, Quiroz Y, Gordon K, Rincón J, Chávez M, Parra G,

Herrera-Acosta J, Gomez-Garre D, Largo R, Egido J, and Johnson RJ.

Mycophenolate mofetil prevents salt-sensitive hypertension resulting from angiotensin

II exposure. Kidney Int 59: 2222-2232, 2001.

Page 17 of 26

18

18

15. Rodriguez-Iturbe B, Quiroz Y, Ferrebuz A, Parra G, and Vaziri ND.

Evolution of renal interstitial inflammation and NF-κB activation in

spontaneously hypertensive rats. Am J Nephrol 24: 587-594, 2004.

16. Rodriguez-Iturbe B, Quiroz Y, Nava M, Bonet L, Chavez M,. Herrera-Acosta J,

Johnson RJ, and Pons HA. Reduction of renal immune cell infiltration results in

blood pressure control in genetically hypertensive rats. Am J Physiol Renal Physiol.

282:F191-F201, 2002

17. Rodriguez-Iturbe B, Vaziri ND, Herrera-Acosta J, and Johnson RJ.

Oxidative stress, renal infiltration of immune cells and salt-sensitive

hypertension: All for one and one for all. Am J Physiol Renal Physiol 286:

F606-F616, 2004

18. Rodríguez-Iturbe B, Zhan Chang-De, Quiroz Y, Sindhu RK, and Vaziri ND.

Antioxidant-rich diet improves hypertension and reduces renal immune infiltration in

spontaneously hypertensive rats. Hypertension 41:341-346, 2003.

19. Ruiz-Ortega M, Esteban V, Ruperez M, Sanchez-Lopez E, Rodriguez-Vita

J, Carvajal G, and Egido J. Renal and vascular hypertension-induced

inflammation: role of angiotensin II. Curr Opin Nephrol Hypertens. 15: 159-

166, 2006.

20. Sadjadi J, Kramer Gl, Yu CH, Welborn MB 3rd, and Modrall JG.

Angiotensin II exerts positive feedback on the intrarenal renin-angiotensin

system by an angiotensin converting enzyme-dependent mechanism. J Surg Res.

129:272-277, 2005.

21. Sanchez-Lozada LG, Tapia E, Avila-Casado C, Soto V, Franco M,

Santamaria J, Nakagawa T, Rodriguez-Iturbe B, Johnson RJ, and Herrera-

Acosta J. Mild hyperuricemia induces glomerular hypertension in normal rats.

Am J Physiol Renal Physiol 283:F1105-1110, 2002.

Page 18 of 26

19

19

22. Sealey JE, Blumenfeld JD, Bell GM, Pecker MS, Sommers SC, and Laragh

JH. On the renal basis for essential hypertension: nephron heterogeneity with

discordant renin secretion and sodium excretion causing a hypertensive

vasoconstriction-volume relationship. J Hypertens 6: 763-777, 1988

23. Siragy HM, Howell NL, Ragsdale NV, and Carey RM. Renal interstitial fluid

angiotensin. Modulation by anesthesia, epinephrine, sodium depletion and renin

inhibition. Hypertension 25: 1021-1024, 1995.

24. Stewart T, Jung FF, Manning J, and Vehaskari VM. Kidney immune cell

infiltration and oxidative stress contribute to prenatally programmed

hypertension. Kidney Int 68: 2180-2188, 2005.

25. Vanegas V, Ferrebuz A, Quiroz Y, and Rodriguez-Iturbe B. Hypertension in

Page (cellophane wrapped) kidney is due to interstitial nephritis. Kidney Int 68:

1161-1170, 2005.

26. Vaziri ND, and Rodriguez-Iturbe B. Mechanisms of Disease: oxidative stress

and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol

2: 582-593, 2006.

27. Von Thun AM, Veri R, El Dahr SS, and Navar LG. Augmentation of

intrarenal angiotensin II levels by chronic angiotensin II infusion. Am J Physiol

Renal Physiol 266: F120-F128, 1994..

28. Wang CT, Navar LG, and Mitchell KD. Proximal tubular fluid angiotensin II

levels in angiotensin II-induced hypertensive rats. J Hypertens 21:353-360,

2003.

29. Zhou LX, Hymel A, Imig ID, and Navar LG. Renal accumulation of

circulating angiotensin II in angiotensin II-infused rats. Hypertension 27: 658-

662, 1996

Page 19 of 26

20

20

FIGURE LEGENDS.

Figure 1. Negative correlation between plasma AII (log 2 scale) and systolic blood

pressure in salt sensitive hypertension (1A) contrasting with the positive correlation

present between blood pressure and renal AII concentration (log 2 scale).

Figure 2. Systolic blood pressure is positively correlated with lymphocyte infiltration

(CD5 + cells, 2A) and macrophage infiltration (ED1 + cells, 2B) in tubulointerstitial

areas.

Figure 3. Tubulointerstitial inflammation, as represented by macrophage infiltration

(ED1 + cells) is correlated with renal AII concentration (3A). Correlation between the

renal AII concentration and the number of cells staining positive for AII in

tubulointerstitial areas.

Figure 4. Microphotographs showing angiotensin positive cells that are both proximal

tubular cells (arrow heads) as well as infiltrating interstitial cells (arrows). Positive

staining is observed in cell membranes and cytoplasm. Increased numbers of AII

positive cells are present in a biopsy from the SSHTN group (4A) in contrast with the

near absence of these cells in a biopsy of a rats from the NSD group. (imunoperoxidase

technique, original magnification x400)

Page 20 of 26

TABLE 1.

SSHTN group

(n=11)

AII-MMF

group (n=10)

c-HSD group

(n=10)

c-NSD group

(n11)

SBP (mmHg) *** 160±7.24 140±8.36 129±4.74 118±5.64

Plasma AII (pg/ml) α 2.37±1.38 4.20±1.94 40.74±31.12 114.4±54.4

Renal AII (pg/ml) § 1310±208.8 454±128.2 154.95±91.0 121±30.0

AII+ cells/mm2 ω 51.9±7.78 25.7±4.80 25.49±4. 22 10.9±2.17

CD5+ cells/mm2 γ 61.4±9.43 30.2±4.31 37.37±4.07 17.9±3.09

ED1+ cells/mm2 γ 34.6±5.73 18.9±2.86 24.18±3.64 10.7±1.40

SBP= Systolic Blood Pressure; AII+ cells, lymphocytes (CD5+ cells) and macrophages

(ED1+ cells) are given as positive cells for mm2 in cortical tubulointerstitial areas.

Plasma AII determinations were done in 9 rats from the AII group, 8 rats from the AII-

MMF group, 6 rats from the c-HSD group and 9 rats from the c-NSD group.

** experimental groups are p<0.01 or p<0.001 vs one another α experimental groups are p<0.01 to p<0.001 vs one another § experimental groups are p<0.001 vs one another except for HSD and NSD ω experimental groups are p<0.001 vs one another except for AII-MMF and HSD.γ experimental groups are p<0.001 vs one another except for AII-MMf vs HSD that are p<0.05

Page 21 of 26

Table 2. Systolic Blood Pressure and angiotensin II levels five days after angiotensin II

infusion and before the induction of salt-sensitive hypertension.

AII group

(n=10 )

AII+MMF group

(n=9)

cNSD group

(n=8)

SBP (mmHg) 138.1±20.8 126.7±12.9 126.0±7.35

Plasma AII (pg/ml) 115.1±48.4 (9) 111.8±23.4 116.3±42.0

Renal AII (pg/ml) 1637±345.0 (8)*** 359.7±153.2 (8)

147.8±107.2

***P<0.001 vs. AII+MMF and cNSD. Renal AII in the AII group is also higher

(p<0.05) than the value found at the end of the SSHTN period shown in Table 1.

Numbers in parenthesis indicate the number of animals studied when it was different

from that shown for the total group.

Page 22 of 26

0.25 0.5 1 2 4 8 16 32 64 128 256

100110120130140150160170180

HSDNSDSSHTNAII-MMF

r= 0.869p<0.0001

Plasma AII (pg/ml)

Sys

tolic

Blo

od P

ress

ure

mm

Hg)

64 128 256 512 1024 2048

100110120130140150160170180

r=0.887p<0.0001

HSDLSDAIIMMF

Renal AII (pg/ml)

Sys

tolic

Blo

od P

ress

ure

(mm

Hg)

Figure 1

1A

1B

Page 23 of 26

0 10 20 30 40 50 60 70 80100110120130140150160170180

HSDNSDSSHTNAII-MMF

r =0.818p<0.0001

CD5 + cells /mm2

Sys

tolic

Blo

od P

ress

ure

(mm

Hg)

0 10 20 30 40 50

100110120130140150160170180

HSD

r= 0.803p<0.001

NSDSSHTNAII-MMF

ED1+ cells/mm2

Sys

tolic

Blo

od P

ress

ure

(mm

Hg)

Figure 2

2A

2B

Page 24 of 26

0 10 20 30 40 50

0

500

1000

1500

2000HSD

r=0.750p<0.001

NSDSSHTNAII-MMF

ED1+ cells /mm2R

enal

AII

(pg/

ml)

64 128 256 512 1024 2048

010203040506070

HSDNSDSSHTNAII-MMF

r= 0.861p<0.0001

Renal AII (pg/ml)

Tub

uloi

nter

stiti

al

AII

+ c

ells

/mm

2

Figure 3

3A

3B

Page 25 of 26

Figure 4

4A

4B

Page 26 of 26