AT1 blockade during lactation as a model of chronic nephropathy: mechanisms of renal injury

AT-1 BLOCKADE DURING LACTATION AS A MODEL OF CHRONIC

NEPHROPATHY: MECHANISMS OF RENAL INJURY

Flavia Gomes Machado, Elizabete Pereira Barros Poppi, Camilla Fanelli, Denise

Maria Avancini Costa Malheiros, Roberto Zatz and Clarice Kazue Fujihara

Laboratory of Renal Pathophysiology (LIM-16), Renal Division, Department of

Clinical Medicine, Faculty of Medicine, University of São Paulo, São Paulo, Brazil.

Mailing address:Roberto Zatz, MD, Ph.D.Laboratório de Fisiopatologia RenalAv. Dr. Arnaldo, 455, 3-s/334201246-903 São Paulo SPBrazilTel No. 55-11-3068-9428Fax. No. 55-11-3083-1693E-mail address: [email protected]

Running head: Neonatal losartan as a model of chronic kidney disease

of 40Articles in PresS. Am J Physiol Renal Physiol (April 9, 2008). doi:10.1152/ajprenal.00020.2008

Copyright © 2008 by the American Physiological Society.

ABSTRACT

Suppression of the renin-angiotensin system during lactation causes

irreversible renal structural changes. In this study we investigated 1) the time

course and the mechanisms underlying the chronic kidney disease caused by

administration of the AT1 receptor blocker, losartan, during lactation, and 2)

whether this untoward effect can be used to engender a new model of chronic

kidney disease. Male Munich-Wistar pups were divided into two Groups: C, whose

mothers were untreated; and LLact, whose mothers received oral Losartan, 250

mg/kg/day, during the first 20 days after delivery. At 3 months of life, both nephron

number and the GFR were reduced in LLact rats, whereas glomerular pressure was

elevated. Unselective proteinuria and decreased expression of the ZO-1 protein

were also observed, along with modest glomerulosclerosis, significant interstitial

expansion and inflammation, and wide glomerular volume variation, with a stable

subpopulation of exceedingly small glomeruli. In addition, the urine osmolality was

persistently lower in LLact rats. At 10 months of age, LLact rats exhibited systemic

hypertension, heavy albuminuria, substantial glomerulosclerosis, severe renal

interstitial expansion and inflammation, and creatinine retention. Conclusions: 1)

Oral losartan during lactation can be used as a simple and easily reproducible

model of chronic kidney disease in adult life, associated with low mortality and no

arterial hypertension until advanced stages; 2) The mechanisms involved in the

progression of renal injury in this model include glomerular hypertension,

glomerular hypertrophy, podocyte injury and interstitial inflammation.

Keywords: Chronic kidney disease; nephrogenesis; angiotensin II

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INTRODUCTION

Stimulation of AT-1 receptors (AT-1R) by angiotensin II (AII) has long been

known to exert diverse important biological effects in adult mammals. Depending

on which intracellular downstream system is activated, AII binding to AT-1R can

promote renal and systemic vasoconstriction, blood pressure elevation and sodium

conservation, on one hand, or enhance the immune response and inflammatory

events, favoring the development of chronic kidney disease, on the other (21;28).

Accordingly, suppression of the renin-angiotensin system (RAS) with ACE

inhibitors and/or AT-1R blockers has become a mainstay of treatment of chronic

progressive nephropathies.

More recently, it has been shown that, beside its hemodynamic and

proinflammatory effects, AII and AT-1R play an important role in the final phases of

nephrogenesis, participating both in the process of nephron maturation and in the

formation of the ureters (11). In rodents, nephrogenesis is completed in the first

two weeks after birth (32). During this period, ACE activity, AII concentration and

the density of AT1-R are all increased in the renal tissue (8). Accordingly, several

research groups have shown that suppression of the RAS at this phase causes

irreversible renal structural and functional changes in adult life, affecting the

maturation of nephrons and renal vessels and leading to a substantial reduction in

the nephron number (8;32;35).

The aim of the present study was to investigate 1) the time course, the long

term outcome and the pathogenesis of the chronic kidney disease that develops in

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rats given AT-1R blockers during the final phase of nephrogenesis; 2) the

possibility that such treatment be employed to construct an experimental model of

chronic kidney disease, that could mimic more closely than current models the

development and outcome of the corresponding human disease.

MATERIALS AND METHODS

Experimental groups

21 adult female Munich-Wistar rats, 10-12 weeks of age and weighing 160

to 200g, obtained from a local facility at University of São Paulo, were housed in

individual plastic boxes after mating, fed regular rodent chow containing 0.5 Na

and 22% protein (Nuvital Labs, Curitiba, Brazil), and given tap water. Rats were

maintained at 23±1 °C and 60±5% relative air humidity, under an artificial 12-12

hour light/dark cycle. After delivery, 6 pups were kept with each dam until weaning.

During the first 20 days of lactation, the dams received the AT-1R antagonist,

Losartan, 250 mg/kg/day dissolved in the drinking water. In preliminary

experiments, this was found to be the highest dose not causing death or severe

growth stunting of the pups. After being weaned on Day 25 after birth, the male

offspring were housed in cages in groups of no more than four and given

unrestrained access to food and water. Only male pups were utilized in this study,

since a marked gender difference exists regarding the effect of angiotensin II

receptor blockers and angiotensin converting enzyme inhibitors, females being

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much less susceptible (17). Thirty-three male offspring from Losartan-treated dams

(Group LLact) and thirty-three age-matched control rats never exposed to Losartan

(Group C) were utilized in these studies. To avoid biasing, no group received more

than 2 pups from the same litter. In this manner, groups were homogeneous

regarding the origin of the animals. All experimental procedures were approved by

the local Research Ethics Committee (CAPPesq, process nº 0166/07), and

developed in strict conformity with our institutional guidelines and with international

standards for manipulation and care of laboratory animals.

Functional studies

At 3 months of age, 5 C and 5 LLact were anesthetized with Inactin, 100

mg/Kg ip, and prepared for renal functional studies, as described earlier (3), to

measure mean arterial pressure (MAP), glomerular filtration rate (GFR), renal

plasma flow (RPF), and renal vascular resistance (RVR) using conventional

clearance techniques (3). The glomerular hydraulic pressure (PGC) was determined

by performing direct micropuncture measurements on superficial glomeruli (3).

Glomerular counting

At 3 months of age, 5 rats from each group were anesthetized with i.p.

ketamine (50 mg/kg) and xylazine (10 mg/kg) and the kidneys were rapidly

perfused retrogradely through the abdominal aorta with a 20% India ink solution as

described previously (5). Thereafter, the right kidney was gently decapsulated,

sliced and kept in a 50% hydrochloric acid solution at 37oC for 1 hour, then in

saline solution at 4oC overnight. After the material was ressuspended in 15 mL of

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saline, a 1-mL aliquot was examined under a stereomicroscope at 50×

magnification for glomerular counting. Counting was performed independently by

two observers (FGM and CF). The glomerular count attributed to each rat was the

average of the two countings thus obtained. The procedure was repeated if a

discrepancy of more than 10% between the two observers occurred.

Renal morphology: short term studies

Ten rats from each group were anesthetized with ketamine, 50 mg/kg, and a

300-µL blood sample was collected from the abdominal aorta for measurement of

the circulating concentrations of creatinine (SCreat), sodium (PNa) and potassium

(PK). The kidneys were then retrogradely perfusion-fixed through the abdominal

aorta with Dubosq-Brazil solution after a brief washout with saline to remove blood

from the renal microcirculation. After weighing, two midcoronal renal slices were

postfixed in buffered 4% formaldehyde, then embedded in paraffin using

conventional sequential techniques, and 4-µm-thick sections were examined for

assessment of glomerular size, the extent of glomerular injury and the fractional

interstitial area, as well as for immunohistochemical detection of macrophages, AII

positive cells and glomerular zonula occludens-1 (ZO-1) protein.

Renal morphology and time course of renal disease: long term studies

Thirteen rats from each group were followed from 3 to 10 months of age

with determination of tail-cuff pressure (TCP) and 24-h urine albumin excretion rate

(UalbV) at 3, 6 and 10 months of age. TCP was assessed using a commercially

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available (Visitech Systems, Apex, NC) automated method (13), under light

restraining and after light warming. To avoid any interference of stress, all rats

were preconditioned to the procedure, and were invariably calm at the time of TCP

determination. In addition, TCP values were always taken as the average of at

least three consecutive measurements that varied by no more than 2 mmHg, that

is, after stabilization of blood pressure.

At the end of the study period, rats were anesthetized and blood and renal

tissue were obtained and processed as described for the short-term experiments

Histomorphometric analysis

All morphometric evaluations were performed in 4-µm-thick sections by a

single observer (DMACM) blinded to the groups. For each rat, the extent of

glomerular injury (GS) was estimated, in sections stained by the periodic acid-

Schiff (PAS) reaction, by attributing to each glomerulus a score corresponding to

the sclerosed fraction of the tuft area, and deriving a glomerulosclerosis index

(GSI) for each rat as described previously (3). At least 120 glomeruli were

examined per rat. The percentage of the renal cortical area occupied by interstitial

tissue (%INT), used as a measure of the degree of interstitial expansion, was

estimated in Masson-stained sections by a point counting technique (12).

Mean glomerular random cross sectional area (AG) was determined by

averaging individual values for 50 consecutive glomerular tuft profiles in a section

from the left kidney of each rat by counting points falling within the tuft profiles.

Glomeruli were examined at 200× under a 144-point grid, covering a 64,900-µm2

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area. The average glomerular tuft volume (VG) for each rat was then calculated as

VG = 1.25 (AG)3/2 (10).

Immunohistochemical analysis

Immunohistochemistry was performed on 4-µm-thick sections, mounted on

glass slides coated with 2% silane. Sections were deparaffinized and rehydrated

by conventional techniques, then heated in citrate buffer for antigen retrieval.

Incubation with the primary antibody was carried out overnight at 4oC, and omitted

in the negative control experiments.

For ED-1 detection, sections were preincubated with 5% normal rabbit

serum to prevent nonspecific binding, then incubated overnight at 4oC with a

monoclonal mouse anti-rat ED-1 antibody (Serotec, Oxford, United Kingdom)

diluted in bovine serum albumin (BSA) at 0.5%. After being rinsed with Tris-

buffered saline (TBS), sections were incubated with a 2% solution of rabbit anti-

mouse immunoglobulin (Dako, Glostrup, Denmark) in BSA, then with an alkaline

phosphatase anti-alkaline phosphatase (APAAP) complex (Dako, Glostrup,

Denmark). Sections were then developed with a fast-red dye solution (Sigma-

Aldrich, Saint Louis, MO), counterstained with Mayer’s hemalaum, and covered

with a glycerin-gelatin mixture.

To detect cells staining positively for AII, an indirect streptavidin-biotin

alkaline phosphatase (SBAP) technique was employed. Sections were

preincubated with avidin and biotin solutions (Biotin Blocking System, Dako, CA) to

block nonspecific binding of these compounds, and with horse serum diluted at 2%

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in a 2% solution of nonfat milk in TBS to avoid nonspecific protein binding.

Sections were then incubated overnight at 4oC with a polyclonal rabbit anti-human

AII antibody (Peninsula Lab., San Carlos, CA) diluted at 0.25% in BSA. Thereafter,

sections were incubated during 45 minutes at room temperature with biotinylated

anti-rabbit IgG (Vector Labs, Burlingame, CA) diluted at 0.1% in BSA, then with a

SBAP complex (Dako, Glostrup, Denmark) for an additional 30 minutes, and finally

developed, counterstained and mounted as described above.

The ZO-1 molecule was detected using a rabbit polyclonal antibody (Zymed

Laboratories, San Francisco, CA) and a commercially available kit (Novocastra

Labs, UK). Sections were otherwise processed as described above.

The extent of renal infiltration by macrophages or AII-positive cells (in

cells/mm2) was evaluated in a blinded manner at ×200 magnification. For each

section, 25 microscopic fields (corresponding to a total area of 1.6 mm2) were

examined. To estimate the intensity of the glomerular expression of ZO-1, the

fraction of the tuft area staining positively for this molecule was determined by an

adapted point-counting technique (12) in 50 consecutive glomerular profiles,

examined at 200× under a 144-point graticulate.

Analytical techniques

UalbV was determined by radial immunodiffusion (18). Screat, PNa, PK and

urine osmolality (Uosm) were assessed by conventional laboratory techniques.

Fractionation of urinary proteins was performed by SDS-PAGE electrophoresis,

utilizing an adaptation of the discontinuous system described by Laemmli (15). A

selectivity index (SI) was calculated as the ratio of the 180 kD to the 68 kD band.

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Statistical analysis

Statistical differences were assessed by two-way ANOVA, with treatment

and time as intervening factors and with pairwise posttest comparisons according

to the Bonferroni method (34). Student’s unpaired t test was used in the functional

studies and in nephron number analysis, which involved only simple comparisons

between Groups LLact and C. Since Screat, UalbV, GSI and the density of interstitial

AII-positive cells exhibited non-Gaussian distributions, the corresponding data

were subjected to log transformation prior to statistical analysis. To assess

deviations from Gaussian distribution of AG, the Kolmogorov-Smirnov test was

performed using GraphPad Prism version 4.00 for Windows, GraphPad Software,

San Diego CA, www.graphpad.com. Results are expressed as Mean ± SE.

RESULTS

Functional studies

Functional parameters measured at 3 months of age are shown in Table 1.

No difference in BW was observed between groups. In agreement with earlier

reports (35), the number of nephrons was reduced by nearly 33% in Group LLact

compared to C (p<0.05). MAP was similar between LLact and C. However, GFR and

RPF were reduced by nearly 1/3 in Group LLact, whereas RVR was increased by

46%. Although LLact rats were normotensive, PGC was 10 mmHg above control in

this group (p<0.05).

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Long term studies

Renal and functional parameters obtained at 3 and 10 months of age are

represented in Table 2. BW was similar between C and LLact at 3 months, and also

at 10 months of age. Left kidney weight (LKW) did not differ between groups, but

increased with time in C. No differences between groups or effect of time were

observed regarding PNa or PK. Screat was similar between groups at 3 months of

age, but rose significantly at 10 months in Group LLact only. No statistically

significant difference was observed regarding urine flow at 3 months of age. Urine

flow was markedly elevated in LLact rats at 10 months of age, attaining a mean

value over twice as high as in age-matched controls (p<0.05). Accordingly, urine

osmolality was 30% lower in LLact than in control at 3 months, and reduced by more

than 50% compared to control at 10 months of age (p<0.05 for both differences).

The time course of tail-cuff pressure is described in Fig. 1A. As in the

functional studies, LLact rats were normotensive at 3 months of age. TCP remained

at normal levels at 6 months of age. However, TCP rose by almost 40 mmHg in

Group LLact at 10 months of age, while little variation was observed in controls

(P<0.05 LLact vs. C). Fig. 1B depicts the time course of urine albumin excretion rate.

Albuminuria was always higher in Group LLact, and exhibited a clearly progressive

nature, reaching values ninefold higher than control at 10 months of age.

Data on the integrity of the glomerular barrier are given in Fig. 2. The SI was

markedly increased in LLact at 3 months of age, denoting loss of the ability of the

glomerular barrier to retain high-molecular-weight proteins. This difference

persisted at 10 months of age, although the difference between values for 3 and 10

months of age in LLact did not reach statistical significance. The quantitative

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immunohistochemical expression of the ZO-1 molecule was lower than control in

Group LLact at 3 months. This difference persisted at 10 months, with a further

decrease of this parameter in Group LLact, while the control value remained stable.

As described previously (7), rats in Group LLact exhibited variable degrees of

papillary atrophy under macroscopic examination. The analysis of renal structural

injury and inflammation is detailed in Figs. 3 and 4. Glomerular segmental sclerotic

lesions, which were infrequent in LLact rats at 3 months of age, were abundant at 10

months of age (Figs. 3A and 4A). Expansion and inflammation of the interstitial

tissue were already observed at 3 months in LLact rats, and were drastically

increased at 10 months of age (Figs 3B and 4B). Infiltration of the renal interstitium

by macrophages (Figs. 3C and 4C) and by cells staining positively for AII (Figs. 3D

and 4D) followed a similar pattern, with intensity roughly proportional to that of

interstitial expansion.

A statistical analysis of the glomerular dimensions is shown in Fig. 5. At 3

months of age, glomerular areas followed a pattern not significantly different from a

Gaussian distribution in normal controls (Figs. 5A and 5B), the calculated

glomerular volume averaging 0.90±0.03 x 106 um3. In Group LLact, a large number

of unusually small glomerular profiles were observed. The appearance of these

glomeruli was inconspicuous (Fig. 5A), whereas in several of them the hilum was

clearly visible (inset in Fig. 5A), ruling out the possibility that these profiles

represented solely glomeruli sectioned close to a pole. The presence of these

small glomeruli skewed the frequency distribution of glomerular areas to the left,

promoting a significant deviation from normality. Accordingly, the percentage of

profiles with areas under 3,000 µm2 was 18±3% in Group LLact rats, compared to

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only 6±1% in control (p<0.05). The mean glomerular volume in Group LLact at 3

months of age was 1.18±0.06×106 µm3, (p<0.05 vs. control). When the profiles with

areas under 3,000 µm2 were excluded from the calculation, the average glomerular

volume for Group LLact was even higher (1.50±0.08×106 µm3), further underlining

the occurrence of glomerular compensatory growth in Group LLact. In agreement

with this concept, the fraction of glomerular profiles with areas exceeding 15,000

µm2 was 17±3% in Group LLact, compared with only 1±1% in controls (p<0.05). At

10 months of age, the distribution of glomerular areas in normal controls was

skewed to the right, indicating that a substantial number of glomeruli underwent

hypertrophy. Indeed, the mean glomerular volume (excluding the profiles

measuring less than 3,000 µm2) was increased at this time in C (1.56±0.05×106

µm3) compared to the value obtained at 3 months of age (p<0.05), whereas the

percentage of glomerular profiles with areas greater than 15,000 µm2 was 15±2

(p<0.05 vs. the corresponding 3-month value). In Group LLact, the distribution of

areas was also shifted to the right, whereas the mean glomerular volume (again

excluding profiles with areas below 3,000 µm2) was 2.03±0.08×106 µm3 (p<0.05 vs.

10-month C and 3-month LLact) and the proportion of profiles larger than 15,000

µm2 was 35±2 % (p<0.05 vs. 10-month C and 3-month LLact), indicating additional

glomerular hypertrophy in these rats. The percentage of small glomeruli (profile

area < 3,000 µm2) remained constant with time in Group LLact (18±2%, p>0.05 vs.

the 3-month value), suggesting that this population of hypotrophic glomeruli is

relatively stable.

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DISCUSSION

As described previously, blockade of the AT-1R during lactation resulted in a

30% reduction in the number of nephrons, confirming previous reports (29;32;35),

although some discordant findings have been described (19), and corroborating

the concept that the presence of AII is essential for adequate nephron maturation

(8;32). The molecular mediators and the cellular signaling pathways that may be

responsible for this salutary action of AII on nephrogenesis are presently unclear,

although evidence has been reported that activation of insulin-like growth factor

(IGF) and of its receptor may play a crucial role (24).

Confirming previous reports (2;7;9;32;35), LLact rats developed severe and

progressive renal functional and structural changes, that were nevertheless

associated with zero mortality, making it possible to follow these rats until very

advanced stages. This behavior was remarkably homogeneous: at 3 months of

age all rats in Group LLact were diseased, and low coefficients of variation were

observed for parameters such as albuminuria and tail-cuff pressure It must be

emphasized that in the present study the teratogenic effect of losartan was

obtained by offering the drug to the dams in the drinking water, taking advantage of

the fact that losartan crosses easily the blood-milk barrier (31). It was thus possible

to obviate the need for individual intraperitoneal injections, utilized in many

previous studies.

At 3 months of age, GFR was diminished by 30% in rats that received

losartan during lactation, compared to controls. This finding is in agreement with

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most previous reports (7;8;29;35), despite a few discordant observations (17).

Since the reduction in GFR and in RPF was proportional to the reduction in the

number of nephrons, it is tempting to conclude that no compensatory

hyperperfusion or hyperfiltration, such as observed in rats with uninephrectomy or

5/6 renal ablation (3;33), occurs in this model. However, it must be noted that a

substantial fraction of glomeruli in LLact rats was found to be hypotrophic, a

proportion likely underestimated since, unlike normal glomeruli, hypotrophic

glomeruli are easily overlooked if sectioned close to one of the poles. Since these

hypotrophic glomeruli, which probably result from nephron malformation, are

expected to contribute little to the overall GFR and may even have lost their

connection with the respective tubules (4), some compensatory hyperfiltration must

have occurred in the remaining nephrons. On the other hand, the finding that a

substantial number of glomeruli in LLact rats exhibited mean cross-sectional areas

in excess of 15,000 µm2 (the frequency of such glomeruli was negligible in

controls) indicates that compensatory glomerular hypertrophy also occurred in

these animals.

The intimate mechanisms underlying the compensatory increase in single

nephron GFR in LLact rats are not entirely clear. Part of the compensatory response

likely involved an elevation of PGC, at least at 3 months of age (unfortunately, the

severe deformation of the renal surface due to interstitial expansion/inflammation

precluded the determination of PGC by micropuncture at 10 months of age). Since

systemic hypertension was absent in these rats at the time the functional studies

were performed, glomerular hypertension, at least at this phase, must have

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resulted from afferent arteriolar dilatation and/or efferent arteriolar vasoconstriction.

It is unclear whether this arteriolar dysfunction is merely part of a compensatory

response or whether arteriolar malfunctioning in adult life constitutes another ill

effect of losartan administration during nephrogenesis. It should be noted that the

number of litters (21) and the number of functional observations (5 per group) were

both relatively small and, although differences between groups were almost

invariably statistically significant, the present functional measurements inevitably

carry some degree of uncertainty. Thus, the results of the present functional

studies should be interpreted with caution, and definitive conclusions in this regard

will have to await confirmation by future studies.

LLact rats developed several aspects of chronic glomerular structural and

functional injury. Albuminuria was already evident at 3 months of age and exhibited

a clearly progressive nature, reaching levels one order of magnitude higher than in

controls at 10 months of age. Albuminuria was associated with limitation of the

size-selective properties of the glomerular walls, as assessed by the increase in a

selectivity index, already observed at 3 months of age. However, since the

selectivity index was not further increased at 10 months of age, the aggravation of

albuminuria observed at this time must have resulted from factors in addition to a

size defect, such as rarefaction of the negative surface charge of the glomerular

walls. The reduced expression of the ZO-1 molecule suggests that podocyte injury

may explain at least in part the development of albuminuria in LLact rats. Since

podocytes are responsible for the synthesis of part of the negatively charged

molecules that contribute to limit the passage of polyanions through the glomerular

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walls, podocyte damage can explain the development of proteinuria by a combined

defect in the size- and charge-selective properties of the glomerular wall.

Although albuminuria was already present at 3 months of age, glomerular

injury at this time was only mild, though significant, in LLact rats compared to

controls. Glomerulosclerosis was much more pronounced at 10 months of age, the

GSI reaching levels over fiftyfold higher, whereas serum creatinine was

significantly elevated, compared to age-matched controls. Although the exact

mechanisms leading to the development of glomerular injury in this model are not

entirely clear, an obvious possibility is that the maladaptive glomerular

hypertension and hypertrophy represented important pathogenic factors.

Glomerular hypertension and/or hypertrophy have been postulated to initiate and

maintain progressive glomerular injury in several experimental models (22;36) by

promoting mechanical stretch to the glomerular walls according to La Place’s law

(26) and, as a consequence, podocyte injury with formation of synechiae with

Bowman’s capsule (20;30), mesangial expansion (25) and infiltration by

inflammatory cells (27). This process tends to acquire a progressive nature as a

growing number of glomeruli drop out due to severe sclerosis, and additional

burden is imposed to the remaining nephrons. Absence of early renal

hemodynamic changes may be one of the reasons why female rats fail to develop

progressive nephropathy after neonatal exposure to angiotensin II receptor

blockers or angiotensin converting enzyme inhibitors (17)

Progressive increase of the fractional cortical interstitial area was, along with

glomerular injury, a prominent feature of the nephropathy that developed in the

LLact group. Interstitial expansion was associated with clear signs of chronic

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inflammation, such as interstitial fibrosis and infiltration by mononuclear cells, a

large fraction of which were macrophages. Curiously, many of these inflammatory

cells stained positively for AII, suggesting that, although its presence during

nephrogenesis is essential for adequate nephron maturation, AII exerts in this

model a similar proinflammatory role as in other experimental models of

progressive nephropathy (6). The pathogenesis of interstitial

expansion/inflammation in this model is unclear. Interstitial injury could be a direct

consequence of glomerular injury due to propagation of tuft inflammation through

synechiae with Bowman’s capsule and direct passage of circulating inflammatory

mediators to the periglomerular interstitium (14). In addition, the exaggerated

filtration of proteins due to impairment of the glomerular barrier might induce the

synthesis of cytokines, chemokines and growth factors by proximal tubular cells in

association with augmented protein endocytosis (1). However, interstitial

expansion and inflammation were already evident in LLact rats at 3 months of age,

when albuminuria and glomerular injury were still mild. Therefore, the development

of marked interstitial injury in LLact rats cannot have resulted exclusively from

glomerular injury, and must have been influenced by other factors. One speculative

explanation could be that the tubular cells associated with hypotrophic glomeruli

might, as postulated for other experimental models, undergo epithelial-

mesenchymal transformation and originate myofibroblasts, which are known to

actively synthesize components of the interstitial matrix (23).

In addition to the severe structural changes observed at the glomeruli and

the interstitium, which led to GFR reduction and creatinine retention, LLact rats

exhibited evidence of tubular dysfunction. At 3 months of age, when albuminuria,

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interstitial expansion and glomerulosclerosis were still mild, urine osmolality was

nearly 25% lower than in controls, while urine flow was correspondingly increased.

This abnormality was aggravated at 10 months of age, when urine flow more than

doubled, and urine osmolality was reduced to less than 50%, compared with age-

matched controls. These findings, which corroborate previous reports (9), suggest

that the concentrating ability of LLact rats is impaired in disproportion to renal

structural injury. The reasons for this abnormality are unclear. One possible

explanation is that the delicate operation of the countercurrent mechanism is

exquisitely dependent on the complex anatomical arrangement of tubular and

vascular structures at the renal medulla, and might be disrupted by papillary

atrophy or even by mild expansion of the renal interstitium (9). The renal

concentrating ability could be further compromised by depletion of aquaporin 2,

which has been described in rats treated with an ACE inhibitor during lactation (9).

Depletion of the Na-K-2Cl cotransporter at the thick ascending limb of Henle has

also been described in these rats (16), and might lead to a salt-losing state, with

polyuria and diminished concentrating ability.

It is noteworthy that, even in the presence of a chronic, progressive

nephropathy, with substantial reduction of the nephron number and of the GFR,

systemic hypertension did not develop in LLact rats until after 6 months of life, when

renal injury had already attained an advanced state. The reasons for this

unexpected finding are not immediately apparent. However, it must be noted that,

as discussed earlier, tubular function may be impaired in these rats, which may be

prone to salt loss (7). Such abnormality would compensate for the inevitable trend

toward salt retention imposed by the development of chronic kidney injury, and

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prevent blood pressure elevation until nephron loss and interstitial inflammation

were severely aggravated by progression of the disease.

In view of the characteristics and the time course of the ensuing

nephropathy, administration of losartan during lactation constitutes a suitable

model of chronic progressive nephropathy, easily reproducible and requiring no

surgery or special preparation. An additional advantage of this model is its low

mortality, likely associated with the absence of hypertension during its initial

stages, allowing the progression of the nephropathy at a relatively slow rate and,

consequently, the development of progressive renal insufficiency and of the

adaptations and maladaptations that characterize human chronic nephropathies.

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ACKNOWLEDGMENTS

Preliminary results of this study were presented at the American Society of

Nephrology Meeting, San Diego, CA, November 14-19, 2006, and published in

abstract form (J. Am. Soc. Nephrol. 17: 655A, 2006). We thank Bianca Helena

Fernandes, Claudia Ramos Sena and Luciana Faria de Carvalho for expert

technical assistance.

Supported by grant 05/56066-3 from the State of São Paulo Foundation for

Research Support (FAPESP). CKF and RZ are recipients of Research Awards

(Nos. 304039/2006-3 and 326.429/81, respectively) from the Brazilian Council of

Scientific and Technologic Development (CNPq).

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FIGURE LEGENDS

Figure 1 – Time course of tail-cuff pressure (TCP, A) and urine albumin excretion

rate (UalbV, B) in control rats (C, n=13) and in rats treated with losartan

during lactation (LLact, n=13). a p<0.05 vs. respective control value; b

p<0.05 10-month vs. respective 3-month value ; c p<0.05 10-month vs.

respective 6-month value.

Figure 2 – Selectivity index of proteinuria (SI, left panel) and expression of the ZO-

1 protein (right panel) in control (clear bars) and LLact (filled bars). a

p<0.05 LLact vs C.

Figure 3 – Representative micrographs of the renal tissue in C and LLact at 3 and

10 months of age, showing glomerulosclerosis (A); renal cortical

interstitial expansion/inflammation (B); and renal cortical infiltration by

macrophages (C) and cells staining positively for AII (D). To enhance

the visibility of cells staining positively for ED-1 or AII (both originally in

red), the images shown in Figures 3C and 3D were processed using

Adobe® PhotoShop, version 5.0, to dim the structures staining in blue

(basically nuclei of cells negative for the relevant molecules), then

converted to grayscale.

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Figure 4 – Bargraph representation of the intensity of glomerulosclerosis (A); renal

cortical interstitial expansion/inflammation (B); and renal cortical

infiltration by macrophages (C) and cells staining positively for AII (D) in

control (clear bars, n=10 at 3 months and n=13 at 10 months of age)

and LLact (filled bars, n=10 at 3 months and n=13 at 10 months of age). a

p<0.05 vs. respective control value; b p<0.05 10-month vs. respective 3-

month value.

Figure 5 – A) Representative micrographs of renal tissue showing the variability of

glomerular areas in C and LLact at 3 and 10 months of age. A population

of exceedingly small glomeruli is seen in LLact. That these are indeed

small glomeruli and not normal glomeruli sectioned close to a pole is

shown in the inset, in which the hilum of one of these glomeruli can be

easily seen. B) Frequency distribution of glomerular areas in C (upper

panels) and LLact (lower panels) at 3 months (left) and 10 months (right).

Distribution was strongly non-Gaussian in LLact, showing a subpopulation

of small glomeruli at both 3 (n=10/group) and 10 (n=13/group) months of

age. C) Bargraph showing the proportion of small, medium-sized and

large glomerular areas in C and LLact. Both glomerular tuft profiles with

small areas (indicating hypotrophy) and with large areas (indicating

compensatory hypertrophy) were much more numerous in LLact than in C

(p<0.05).

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Table 1 – Renal function study at 3 months of age

BW NN MAP GFR RPF RVR PGC

g ×103 mmHg ml/min mmHg/ml/min mmHg

C ( n=5) 323±18 27±1 108±2 1.42±0.07 4.13±0.24 14.3±0.7 53±1

LLact (n=5) 285±14 18±1a 104±1 0.94±0.06a 2.77±0.27a 20.9±2.3a 63±2a

Results expressed as mean ± SE. BW, body weight; NN, nephron number (per one

kidney); MAP, mean arterial pressure; GFR, glomerular filtration rate; RPF, renal

plasma flow; RVR, renal vascular resistance; PGC, glomerular hydraulic pressure.

ap<0.05 vs. C.

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Table 2 - Renal functional and systemic parameters at 3 and 10 months of age

3 months 10 months

C

(n=10)

LLact

(n=10)

C

(n=13)

LLact

(n=13)

BW (g) 278±9 264±10 417±7b 393±11b

LKW (g) 1.49±0.06 1.67±0.05 1.97±0.05 b 1.82±0.06

PNa (mmol/L) 139±2 141±2 142±2 140±1

PK (mmol/L) 3.7±0.1 3.9±0.2 4.3±0.2 4.3±0.2

Screat (mg/dL) 0.64±0.11 0.70±0.09 0.68±0.04 0.94±0.06ab

VU24h (ml/24h) 23±2.2 31±1.5 29±1.7 64±7.1ab

Uosm (mOsm/Kg) 1210±116 921±45a 1111±83 543±58ab

Results expressed as mean ± SE. BW, body weight; LKW, left kidney weight; PNa,

plasma sodium concentration; PK, plasma potassium concentration; Screat, serum

creatinine concentration; VU24h, daily urinary volume; Uosm, urine osmolality.

ap<0.05 vs. C, bp<0.05 vs. respective 3-month value.

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Tail-cuff pressure

0 2 4 6 8 10100

110

120

130

140

150

160

170

180

months

LLact

C

abc

b

mm

Hg

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Urinary albumin excretion

0 2 4 6 8 100

25

50

75

100

125

150

175

months

LLact

C

abc

ab

abc

mg/

24h

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0

10

20

30

40

50

60

70

a

a

CLLact LLactC3 mo 10 mo

ZO-1

%0.00

0.05

0.10

0.15

0.20

0.25

0.30

a

a

CLLact LLactC3 mo 10 mo

SI

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C

D

C 3mo LLact 3mo LLact 10moC 10mo

B

A

100 μm

100 μm

100 μm

100 μm

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% cortical interstitial area

0

1

2

3

4

5

6

7

8

9

10

a

ab

C 3mo LLact 10moLLact 3mo C 10mo

%

Cortical interstitial infiltration by macrophages

0

50

100

150

200

250

ab

a


cells

/mm

2

Cortical interstitial infiltration by AII + cells

0

5

10

15

20

25

30

35

ab


cells

/mm

2

a

Glomerulosclerosis index

0

10

20

30

40

50

60

70ab


a

GS

I

A B

DC

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C 3mo LLact3mo LLact10moC 10mo

100 μm

50 μm

of 40

0

5

10

15

20

25

0-1500

1501-3000

3001-4500

4501-6000

6001-7500

7501-9000

9001-10500

10501-12000

12001-13500

13501-15000

15001-16500

16501-18000

18001-19500

19501-21000

21001-22500

22501-24000

24001-25500

25501-27000

27001-28500

28501-30000

30001-31500

31501-33000

33001-34500

34501-36000

Section area

Glo

mer

uli,

%

0

5

10

15

20

25

0-1500

1501-3000

3001-4500

4501-6000

6001-7500

7501-9000

9001-10500

10501-12000

12001-13500

13501-15000

15001-16500

16501-18000

18001-19500

19501-21000

21001-22500

22501-24000

24001-25500

25501-27000

27001-28500

28501-30000

30001-31500

31501-33000

33001-34500

34501-36000

Glo

mer

uli,

%

Section area

0

5

10

15

20

25

0-1500

1501-3000

3001-4500

4501-6000

6001-7500

7501-9000

9001-10500

10501-12000

12001-13500

13501-15000

15001-16500

16501-18000

18001-19500

19501-21000

21001-22500

22501-24000

24001-25500

25501-27000

27001-28500

28501-30000

30001-31500

31501-33000

33001-34500

34501-36000

Glo

mer

uli,

%

Section area

0

5

10

15

20

25

0-1500

1501-3000

3001-4500

4501-6000

6001-7500

7501-9000

9001-10500

10501-12000

12001-13500

13501-15000

15001-16500

16501-18000

18001-19500

19501-21000

21001-22500

22501-24000

24001-25500

25501-27000

27001-28500

28501-30000

30001-31500

31501-33000

33001-34500

34501-36000

Glo

mer

uli,

%

Section area

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0%10%20%

30%40%50%60%70%

80%90%

100%

C 3mo C 10mo LNB 3 mo LNB 10 mo

>15000>3000 e <15000<3000

LLact 3mo LLact 10mo

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AT1 blockade during lactation as a model of chronic nephropathy: mechanisms of renal injury

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