J Am Soc Nephrol 11: 497–506, 2000
Functional and Structural Correlates of Glomerulosclerosis
after Renal Mass Reduction in the Rat
KAREN A. GRIFFIN,* MARIA M. PICKEN,† MONIQUE CHURCHILL,‡
PAUL CHURCHILL,‡ and ANIL K. BIDANI*
Departments of *Medicine and †Pathology, Loyola University Medical Center/Hines VA Hospital, Maywood,
Illinois; and ‡Department of Physiology, Wayne State University, Detroit, Michigan.
Abstract. Previously, it was shown that 5/6 renal mass reduction by surgical excision (RK-NX) results in a marked reduction of glomerulosclerosis (GS) at 6 wk compared with the
conventional 5/6 renal ablation by infarction (RK-I) model. To
determine the pathogenetic correlates of the striking differences in GS, radiotelemetrically measured BP; single nephron
function; glomerular volume; the temporal expression of
mRNA for renin, transforming growth factor-b, and plateletderived growth factor-B; and plasma renin concentration were
compared between RK-NX, RK-I, and sham-operated control
rats. Hypertension only developed in the RK-I model, was
present at 3 d after infarction, and was correlated with both an
increased expression of renin mRNA by Northern analysis and
elevated plasma renin concentration. Structural (glomerular
volume) and functional (single nephron blood flow and GFR)
indices of the compensatory adaptive response were significantly but similarly increased in the RK-NX and RK-I rats
compared with sham-operated controls, indicating that these
adaptations per se are not responsible for the initiation of GS
after 5/6 renal mass reduction. Glomerular capillary pressure
(PGC) was also significantly increased in both RK-I (56 6 2
mmHg) and RK-NX rats (50 6 0.9 mmHg) compared with
controls (46 6 0.8 mmHg, P , 0.01), but the increase was
significantly greater in RK-I versus RK-NX rats (P , 0.05)
consistent with the higher BP in RK-I rats. These data indicate
that differences in renin probably account for the early divergence of BP (and PGC) responses between RK-I and RK-NX
models. Transforming growth factor-b and platelet-derived
growth factor-B mRNA expression in pooled RNA from kidneys from each group showed increases at 21 d along with
early evidence of glomerular injury in the RK-I group but not
in the RK-NX group, consistent with their postulated roles as
molecular mediators of GS, but only in rats with pathologic
glomerular hypertension.
The progressive nature of chronic renal disease has been extensively investigated using the 5/6 renal ablation model (right
uninephrectomy and infarction of two-thirds of the left kidney)
(1–5). These animals, over time, develop a syndrome of systemic and glomerular hypertension (HTN), proteinuria, and
progressive glomerulosclerosis (GS) of the initially normal
remnant nephrons. Data in both experimental and human glomerular disease have indicated that increased accumulation of
extracellular matrix may be the final common pathway in the
pathogenesis of GS (6,7). It has been postulated that increased
expression of the growth factors transforming growth factor-b
(TGF-b) and platelet-derived growth factor-B (PDGF-B), mediated by the growth effects of angiotensin II (AngII) on
glomerular mesangial cells, results in glomerular extracellular
matrix synthesis and eventual GS (8 –12). However, significant
controversy persists as to the mechanisms responsible for the
initiation of this pathogenetic sequence of events (1–5,13–16).
It has been suggested that the adaptations associated with the
compensatory hypertrophy response per se may be maladaptive and result in eventual GS (2–5,13–16). Some investigators
have ascribed a primary pathogenetic importance to the increased glomerular capillary pressures and flows associated
with the compensatory increases in function, the so-called
hemodynamic theory (2,3,15,16), while other investigators
have blamed the cellular events and increased growth factor
expression associated with the structural glomerular hypertrophy response (4,11–14). Since both glomerular HTN and glomerular hypertrophy coexist in the infarction model (RK-I) and
precede the development of GS, it has been difficult to separate
the individual contribution of these two pathogenetic mechanisms. Similarly, the beneficial effects of a low protein diet in
this model have not allowed the relative importance of the two
pathogenetic pathways to be ascertained, as both hemodynamic
and structural components of the compensatory hypertrophy
response are abrogated by a low protein diet (2–5,16 –19). The
beneficial effects of angiotensin-converting enzyme inhibition
have likewise been ascribed to its hemodynamic effects (reduction in PGC) or alternatively to the blockade of the growth
effects of AngII (3–5,11,12,14,16,20).
We have recently demonstrated that when equivalent approximately 5/6 renal mass reduction (RMR) is achieved by
surgical excision (RK-NX) instead of infarction, there is a
marked reduction in GS at 6 wk compared to the traditional
infarction (RK-I) model (21). The present studies were per-
Received August 19, 1998. Accepted August 19, 1999.
Correspondence to Dr. Karen A. Griffin, Loyola University Medical Center,
2160 South First Avenue, Maywood, IL 60153. Phone: 708-202-8387, extension 24120; Fax: 708-202-7978.
1046-6673/1103-0497
Journal of the American Society of Nephrology
Copyright © 2000 by the American Society of Nephrology
498
Journal of the American Society of Nephrology
formed to define the pathogenetic correlates of these striking
differences in GS between the two models. Radiotelemetric BP
monitoring, single nephron function, glomerular hypertrophy,
the temporal expression of renin, TGF-b and PDGF-B mRNA,
and plasma renin concentration were examined in these two
models of RMR and compared to sham-operated control rats.
Materials and Methods
Male Sprague Dawley rats (200 to 300 g body wt) were fed a
standard (24%) protein diet (Purina, St. Louis, MO) and synchronized
to a 12/12 h light (6 a.m. to 6 p.m.) and dark (6 p.m. to 6 a.m) cycle.
Animal experimentation was conducted in accord with the National
Institutes of Health Guidelines for the Care and Use of Laboratory
Animals. All rats received food and water ad libitum throughout the
study. After measurement of basal serum creatinine (SCr), all rats
underwent either sham surgery or approximately 5/6 renal ablation
(right nephrectomy and either infarction [RK-I] or surgical excision
[RK-NX] of 2/3 of the left kidney). SCr was again measured at 3 d to
stratify for RMR.
Four sets of studies were performed in separate sets of animals to:
(1) characterize the structural and functional adaptations at the whole
kidney level at 3 wk as well as for the assessment of morphologic
injury; (2) define the determinants of the hyperfiltration response at
the single nephron level at 3 to 4 wk; (3) define the temporal
expression of mRNA for renin and the growth factors TGF-b and
PDGF-B over the initial 3 wk after RMR; and (4) examine the
temporal changes in plasma renin concentration (PRC) after RMR.
Whole Kidney Studies
At the time of renal ablation or sham surgery, radio transmitters
(Data Sciences International, St. Paul, MN) were installed (21–24).
The rats were anesthetized with sodium pentobarbital (45 mg/kg,
intraperitoneally), and the sensor’s catheter was inserted into the aorta
below the level of the renal arteries. The radio frequency transmitter
was fixed to the peritoneum. The signals from the pressure sensor
were converted and temperature compensated and sent via the radio
frequency transmitter to the telemetry receiver. The receiver was
connected to a BCM-100 consolidation matrix that transmitted the
signals to the Dataquest IV acquisition system (Data Sciences International). Systolic BP in each animal was continuously recorded at
10-min intervals throughout the course of 3 wk, each reading representing the average BP during a 5-s sampling period. Tail vein blood
samples were obtained at 3 d for measurement of SCr as an index of
the degree of renal mass reduction. After 3 wk, tail vein SCr and 24-h
urine collections for protein excretion were obtained. The rats were
then anesthetized with intravenous sodium pentobarbital (40 mg/kg),
a tracheostomy was performed using polyethylene (PE-200) tubing,
and the rats were surgically prepared for measurement of inulin
clearance and renal blood flow (RBF) as described previously (21–
26). In brief, a carotid artery was cannulated with PE-50 tubing and
connected to a Windograf (model 40-8474; Gould, Glen Burnie, MD)
for continuous recording of mean arterial pressure. A femoral vein
was cannulated with PE-50 tubing and a priming dose of inulin in 150
mM NaCl was administered, followed by a continuous maintenance
infusion of 150 mM NaCl containing inulin at 0.055 ml/min to
maintain the plasma concentration of inulin at approximately 50 mg/dl
and for replacement of surgical and ongoing fluid losses. The left
ureter was then cannulated with polyethylene tubing for collection of
urine samples. A 1.0-mm R series flow probe (Transonic Systems,
Ithaca, NY) was placed around the left renal artery for measurement
of RBF by a flowmeter (Transonic Systems), as described previously
J Am Soc Nephrol 11: 497–506, 2000
(21–26). At the conclusion of the surgery, a 150 mM NaCl bolus equal
to 1% of body weight was administered. Two 20-min clearances of
inulin were obtained. Blood samples were obtained at the midpoint of
each urine collection. The kidneys were then perfusion-fixed at their
ambient BP and subsequently processed for morphologic and morphometric studies.
Morphologic Methods. Transverse sections of the kidney
through the papilla were fixed in situ by perfusion for 5 min at the
measured BP with 1.25% glutaraldehyde in 0.1 M cacodylate buffer.
Sections were cut at a thickness of 2 mm and stained with hematoxylin
and eosin and periodic acid-Schiff. Sections were evaluated systematically in each kidney for evidence of glomerular injury in a blinded
manner by standard morphologic methods (21–24). At least 100
glomeruli in each animal, and usually more, were evaluated for the
presence of lesions of segmental sclerosis (collapsed capillaries with
obliteration of the capillary lumina, frequently accompanied by a
fibrous adhesion between the glomerular tuft and Bowman’s capsule)
and/or necrosis (fibrinoid necrosis of part or all of the glomerular tuft
with loss of architecture often accompanied by capillary thrombosis,
karyorrhexis, fibrin leakage into the Bowman space, and proliferation
of the parietal epithelium of Bowman’s capsule). The severity of
glomerulosclerosis was expressed as the percentage of glomeruli
exhibiting such injury.
Morphometric Methods. Glomerular volume was measured by
area perimeter analysis (Bioquant System IV software; R&M Biometrics, Nashville, TN). The cross-sectional area (AG) of 75 consecutive
glomerular profiles contained in one kidney section for each animal
was measured using a digitizing pad as described previously
(21,23,24). The mean glomerular volume (VG) was then calculated
from the respective mean AG as VG 5 b/k (AG3/2), where b 5 1.38 is
the size distribution coefficient and k 5 1.1 is the shape coefficient for
glomeruli idealized as spheres (27,28).
Micropuncture Studies to Define the Determinants of
Single Nephron Hyperfiltration
These rats underwent sham or RMR surgery as described above,
but without the placement of radio transmitters. At 3 to 4 wk, the rats
were anesthetized with inactin (100 mg/kg, intraperitoneally) and
placed on a temperature-regulated (37°C) micropuncture table and
prepared for micropuncture studies as described previously (29). Left
kidney GFR, proximal tubular pressure (PT), stop flow pressures
(PSF), peritubular capillary (first order) pressures (PPC), and SNGFR
were determined using 3H inulin. Nephrons were mapped by injection
of 0.9% NaCl stained with fast green dye. Only nephrons clearly
within the normal remnant parenchyma and well away from scar areas
were selected for micropuncture. Three to five proximal tubular
collections were obtained in each rat. Similarly, 3 to 4 PSF measurements with a continuously recording Servonull system were made in
each rat. Simultaneous femoral arterial and renal vein collections were
obtained to determine inulin extraction and whole kidney filtration
fraction (17,30). Afferent arterial colloid oncotic pressure (pA) was
calculated using the femoral arterial plasma protein concentration and
the Landis–Pappenheimer equation. PGC was calculated as PSF 1 pA.
Other equations used were: glomerular plasma flow (QA) 5 SNGFR/
FF; glomerular blood flow (GBF) 5 QA/1 2 Hct; efferent arteriolar
blood flow (EABF) 5 GBF 2 SNGFR; afferent, efferent, and total
renal arteriolar resistance (RA5AP 2 PGC/GBF 3 (7.982 3 1010); RE
5 PGC 2 PPC/EABF 3 (7.982 3 1010); (RTA 5 RA 1 RE), respectively; net ultrafiltration pressure (PUF) 5 PGC 2 PT 2 pA; and
ultrafiltration coefficient (Kf) 5 SNGFR/PUF (29 –31).
J Am Soc Nephrol 11: 497–506, 2000
Temporal Expression of Growth Factors
These rats underwent sham or RMR surgery and installation of
radio transmitters for BP radiotelemetry as described for rats undergoing renal hemodynamic studies. Separate sets of sham (n 5 8),
RK-NX (n 5 9), and RK-I (n 5 9) rats each were sacrificed at 3, 7,
and 21 d, and the kidneys were harvested for Northern blot analysis
for renin, TGF-b, and PDGF-B mRNA expression. The infarcted
areas (scars) and the immediately adjacent areas were excised quickly,
and the remaining kidney tissue was placed into liquid nitrogen.
Northern Blot Analysis. RNA isolation was performed by a
modification of the method of Chirgwin et al. (32). One hundred
micrograms of this “non-scar” renal tissue was homogenized with
guanidinium isothiocyanate. The RNA was isolated by isopycnic
centrifugation over CsCl, and its concentration was measured spectrophotometrically at 260/280 nm using the molar extinction coefficient of nucleic acids. A standardized aliquot of RNA (20 mg) was
separated by electrophoresis on a formaldehyde agarose denaturing
gel and transferred to a Nytran® membrane by capillary transfer.
Lanes were loaded with pooled RNA from each of the three groups of
rats sacrificed at each of the three separate time points. The RNA was
immobilized to the membrane by ultraviolet cross-linking.
The membrane was successively hybridized with four parts (15 ml
of Formamide, 0.6 ml Denhardt’s solution, 1.5 ml of 1 M phosphate
buffer, 7.5 ml of 203 SSC, 1.5 ml of 205 SDS, 2.4 ml of diethylpyrocarbonate water, and 1.5 ml of salmon sperm DNA)/blot and one
part 50 dextran sulfate solution with 32P-dCTP-labeled cDNA probes
(renin [gift of Kevin R. Lynch, University of Virginia, Charlottesville,
VA], TGF-b, PDGF-B, and GAPDH, which was used as the housekeeping gene [American Type Culture Collection, Manassas, VA]).
The cDNA was radiolabeled using a random prime labeling kit
(Pharmacia, Piscataway, NJ). After each hybridization, the membrane
was washed and placed in an x-ray cassette for the requisite exposure
time. Autoradiograms were quantified using computerized densitometry (Bioquant Systems IV software; R&M Biometrics) and corrected
for protein loading. This was accomplished by factoring the relative
density of the various mRNA autoradiograms by the GAPDH mRNA
autoradiogram (the values in sham controls were arbitrarily assigned
a value of 1).
Temporal Changes in PRC
PRC measurements were performed at 3 and 21 d after RMR.
These rats underwent sham or RMR surgery and installation of radio
transmitters for BP radiotelemetry as described for rats undergoing
renal hemodynamic studies. For rats that were to undergo PRC measurements at 3 d, the right or left femoral artery was catheterized with
flexible Tygon® tubing at the time of RMR surgery. The distal end of
the catheter was tunneled through the subcutaneous tissue and exteriorized at the back of the neck. The catheter was flushed and filled
with a heparin dextrose solution and plugged with a straight blunted
pin. For the rats undergoing PRC measurements at 21 d, the placement
of the femoral artery catheter was performed 2 d earlier under sodium
pentobarbital (45 mg/kg, intravenously) anesthesia. Blood samples
were obtained from rats resting quietly in restrainers. Blood was
drawn into prechilled ice-cold heparinized plastic syringes (25,26).
Blood samples were centrifuged at 4°C, and the plasma was stored at
220°C until analyzed. The rats were sacrificed after blood samples
were obtained.
Laboratory Analyses
Urinary protein was measured by the quantitative sulfosalicylic
acid method with human serum albumin serving as standard. Serum
Mediators of Glomerulosclerosis
499
creatinine was measured using a creatinine analyzer (Beckman Instruments, Fullerton, CA) (21,24). Inulin in urine and plasma filtrates was
determined spectrophotometrically by the diphenylamine method as
described previously (21–26). GFR was calculated using standard
formulas. PRC was determined by incubating plasma with excess
homologous renin substrate, in the presence of inhibitors of converting enzyme and angiotensinase, and determining the amount of AngI
generated during the incubation period, as described previously
(25,26). PRC was expressed in units of nanograms of AngI per
milliliter plasma per hour incubation with substrate (i.e., ng AngI/ml
per h).
Statistical Analyses
All results are expressed as mean 6 SEM. Statistical analysis was
performed using ANOVA followed by Student-Newman-Keuls test or
by Kruskal-Wallis nonparametric ANOVA followed by Dunn multiple comparison test as appropriate (33). A P value of ,0.05 was
considered statistically significant.
Results
Whole Kidney Studies
Table 1 shows that the baseline body weight, SCr, and 24-h
urine protein excretion were not different between the three
groups. SCr at 3 d was significantly increased in the two groups
that had undergone RMR compared with the sham group, but
was not different between the RK-NX and the RK-I groups,
indicating comparable RMR. The final body weight was not
significantly different between the three groups. The RK-I rats
had significantly higher BP compared to both the sham and the
RK-NX rats, but the BP of the RK-NX rats was not significantly different from the sham control rats. By 3 wk, evidence
of glomerular injury, although still relatively mild, was only
present in the RK-I rats in the form of a modest but significant
increase in 24-h protein excretion rates as well as the percentage of glomeruli that exhibited histologic evidence of glomerular injury. These indices were not different between RK-NX
and sham control groups.
Figure 1, A and B, provides the data for the functional and
structural indices of the renal hypertrophy response at 3 wk
after RMR. Kidney weight and glomerular volume (Figure 1A)
were significantly increased in the two RMR groups compared
to the sham rats but were not significantly different from each
other. By 3 wk after RMR, the RBF and GFR of the remnant
left kidneys of both RMR groups had increased such that no
significant differences were seen between them and the intact
left kidney of the sham-operated controls (Figure 1B).
Single Nephron Studies
The body weights, SCr, and urine protein excretion data of
the rats undergoing micropuncture studies are presented in
Table 2 and were essentially similar to Table 1. Also provided
are the whole kidney RBF and GFR data for these groups.
Table 3 presents the renal functional data at the single nephron
level for the sham control and the two RMR groups. Both
single nephron plasma flow and GFR were significantly greater
in the two RMR groups compared with the sham controls but
were not significantly different from each other. Mean arterial
J Am Soc Nephrol 11: 497–506, 2000
b
a
RMR, renal mass reduction; SCr, serum creatinine; RK-NX, 5/6 renal mass reduction by surgical excision; RK-I, 5/6 renal ablation by infarction.
P , 0.01 compared to sham.
c
P , 0.05 compared to sham and RK-NX.
0.25 6 0.2
0.6 6 0.2
3.6 6 1.6c
0.006 6 0.0014
0.006 6 0.0006
0.011 6 0.006c
25.97 6 2.1
40.66 6 2.8b
44.83 6 1.8b
122.5 6 1.7
124.3 6 2.3
151.9 6 8.4*
358 6 12.3
346 6 15.6
345 6 10.2
32.05 6 4.9
73.4 6 3.80b
67.6 6 2.3b
34.24 6 1.82 0.003 6 0.0002
33.60 6 2.62 0.003 6 0.0007
27.8 6 2.5 0.002 6 0.0005
Sham (n 5 8)
RK-NX (n 5 10)
RK-I (n 5 7)
250 6 7.3
245 6 4.9
240 6 7.3
Body Weight
(g)
SCr (mmol/L)
Group
Body Weight
(g)
Initial
SCr (mmol/L)
3rd
Proteinuria (g/d)
Average Systolic
BP (mmHg)
Final (3 wk)
SCr (mmol/L)
Proteinuria (g/d)
Glomerular
Injury (%)
Journal of the American Society of Nephrology
Table 1. Basal and final data in rats that underwent whole kidney adaptation studies at 3 wk after RMRa
500
Figure 1. (A) Left kidney weight and glomerular volume for the sham
(M), 5/6 renal ablation by infarction (RK-I) (f), and 5/6 renal mass
reduction by surgical excision (RK-NX) (o) rats at 3 wk after sham
ablation or 5/6 renal ablation. *P , 0.01 versus sham. (B) Renal blood
flow (RBF) and GFR for the left kidney of the same three groups of
rats sacrificed at 21 d. There were no significant differences between
the groups. The mean arterial pressure under anesthesia for the three
groups was as follows: sham, 120.8 6 2.8 mmHg; RK-NX, 115.3 6
5.1 mmHg; RK-I, 132.9 6 4.9 mmHg. P , 0.05 RK-NX versus RK-I.
pressure (AP) under anesthesia was significantly greater in the
RK-I compared with the RK-NX and sham rats, consistent with
the average systolic BP recorded in the unanesthetized rats of
the three groups during the whole kidney studies. All components of renal vascular resistance (total, afferent, and efferent)
were reduced in both RMR groups compared to sham controls;
however, the reduction in RA in the RK-I group did not reach
statistical significance. However, it should be noted that the RA
in the RK-I group is expected to be somewhat elevated due to
the superimposition of autoregulatory afferent vasoconstriction, albeit impaired, in response to the significantly higher AP
in this group. The PGC and PUF were significantly greater in
both RMR groups compared to the sham controls, but the
increase was significantly greater in the RK-I compared to the
RK-NX group. By contrast, the increase in Kf in the two RMR
groups compared to the sham rats was statistically significant
only for the RK-NX rats.
Initial
Group
Sham (n 5 10)
RK-NX (n 5 10)
RK-I (n 5 10)
3rd
Final (3 to 4 wk)
Body Weight
(g)
SCr (mmol/L)
Proteinuria (g/d)
SCr (mmol/L)
Body Weight
(g)
Proteinuria (g/d)
RBFb
(ml/min per kg)
GFRb (ml/min per kg)
228 6 5.3
242 6 8.2
236 6 6.6
28.3 6 3.5
29.2 6 1.8
26.5 6 2.7
0.003 6 0.0006
0.003 6 0.0005
0.003 6 0.0005
30.9 6 3.5
74.3 6 5.3c
76.9 6 3.5c
404 6 11
370 6 10
329 6 17d
0.005 6 0.001
0.007 6 0.001
0.029 6 0.006e
32.6 6 2.5
28.9 6 4.2
23.5 6 2.1
3.9 6 0.3
2.7 6 0.2
2.2 6 0.3d
J Am Soc Nephrol 11: 497–506, 2000
Table 2. Basal and final data of rats that underwent single nephron function studies at 3 to 4 wk after RMRa
a
RBF, renal blood flow. Other abbreviations as in Table 1.
The measurements are for only the intact left kidneys of sham control rats and the remnant left kidney of RK-NX and RK-I rats.
c
P , 0.01 compared to sham.
d
P , 0.05 compared to sham.
e
P , 0.01 compared to sham and RK-NX.
b
Table 3. Single nephron function at 3 wk after RMRa
Group
AP (mmHg) QA (nl/min)
SNGFR
(nl/min)
RA 3 1010
(dyn-s-cm25)
RE 3 1010
(dyn-s-cm25)
PGC (mmHg)
DP (mmHg)
PUF (mmHg)
KF (nl/s per mmHg)
2.6 6 0.3
1.2 6 0.2b
1.6 6 0.2c
1.6 6 0.2
0.7 6 0.15b
1.0 6 0.16
1.0 6 0.1
0.5 6 0.08b
0.6 6 0.09c
46 6 0.8
50 6 0.9b
56 6 1.1b,d
34.5 6 0.8
39.1 6 1.1c
43.5 6 0.6c,d
16.7 6 0.4
21.7 6 0.4b
26.2 6 1b,d
0.04 6 0.002
0.06 6 0.008c
0.05 6 0.006
a
AP, mean arterial pressure; SNGFR, single-nephron GFR; RTA, total renal arterial resistance; PGC, glomerular capillary pressure; RA, afferent arteriolar resistance; RE, efferent
arteriolar resistance; QA, glomerular plasma flow; PUF, ultrafiltration pressure; KF, ultrafiltration coefficient.
b
P , 0.01 versus sham.
c
P , 0.05 versus sham.
d
P , 0.05 versus RK-NX.
e
P , 0.01 versus sham and RK-NX.
Mediators of Glomerulosclerosis
Sham (n 5 10)
104.6 6 2 183 6 18 38 6 1.8
RK-NX (n 5 10) 104.3 6 3 427 6 39b 78 6 9.7c
RK-I (n 5 10)
135.8 6 3e 440 6 64b 73 6 9.0c
RTA 3 1010
(dyn-s-cm25)
501
502
Journal of the American Society of Nephrology
J Am Soc Nephrol 11: 497–506, 2000
Studies of Temporal Expression of mRNA for Renin,
TGF-b, and PDGF-B
Figure 2 illustrates the average systolic BP in the rats that
underwent Northern blot analysis at each time point (3, 7, and
21 d). The RK-I rats had significantly increased systolic BP at
all time points compared to both the sham and RK-NX rats. By
contrast, no significant differences were seen in systolic BP
between the RK-NX and sham rats at any of the time points.
Northern blot analysis was used to measure the temporal expression of renin, TGF-b, and PDGF-B at 3, 7, and 21 d.
Figure 3 shows the relative density units/sham controls for the
mRNA expression of renin, TGF-b, and PDGF-B. Renin
mRNA expression was increased in the RK-I rats at 3 and 7 d
compared to the sham and RK-NX rats but was subsequently
suppressed at 21 d. By contrast, the RK-NX rats exhibited no
increase in renin mRNA expression at any time point, but
rather a temporary suppression on day 7. The temporal pattern
of TGF-b and PDGF-B mRNA expression was characterized
by a marked increase in both at 21 d in only the RK-I group in
whom glomerular injury had started to develop, whereas only
a modest increase in TGF-b but not PDGF-B mRNA expression was noted at all time points in the RK-NX rats.
Temporal Changes in PRC after RMR
Table 4 presents the radiotelemetrically measured average
systolic BP and the PRC data for the separate sets of sham,
RK-NX, and RK-I rats, each of whom underwent PRC measurements at 3 and 21 d. As can be noted, changes in PRC after
RMR were directionally similar to the changes in tissue renin
mRNA expression. PRC was significantly increased in the
RK-I rats at 3 d compared to both the sham and the RK-NX
rats. At 21 d, PRC of RK-I rats was not significantly different
than sham rats but was still significantly higher than that of the
RK-NX rats. Average systolic BP showed an excellent correlation with PRC at 3 d (Figure 4) (r 5 0.84, P , 0.0001), but
not at 21 d (r 5 0.36 P . 0.09).
Figure 3. Densitometric analysis of Northern blot. Relative densitometric values for each mRNA were corrected by dividing each value
by that for the GAPDH mRNA in each blot. The values for renin,
platelet-derived growth factor-B (PDGF-B), and transforming growth
factor-b (TGF-b) mRNA expression are shown as the relative density
units/controls (sham) at 3, 7, and 21 d after renal mass reduction in
sham (M), RK-NX (o), and RK-I rats (f). Pooled RNA obtained
from separate sets of sham (n 5 8), RK-NX (n 5 9), and RK-I (n 5
9) each, at the three time points after RMR, was used for the Northern
blot studies. Repeated Northern analysis using the same pooled samples yielded similar results.
Discussion
Figure 2. Average systolic BP (mmHg) for the sham (M), RK-I (f),
and RK-NX (o) sacrificed at each of the time points (3, 7, and 21 d).
*P , 0.01 versus sham and RK-NX rats.
The functional and structural adaptations after severe RMR
have been postulated to be maladaptive and responsible for the
proteinuria and progressive GS that subsequently develop in
these animals (2– 4,11–16). Support for these conclusions has
primarily been obtained by studies in the RK-I model, which
develops hypertension soon after RMR (1–5,20 –24). However,
reduction of an equivalent amount of renal parenchyma by
surgical excision (RK-NX model) instead of infarction does
not result in the development of hypertension at least during
J Am Soc Nephrol 11: 497–506, 2000
Mediators of Glomerulosclerosis
503
Table 4. Plasma renin concentration and average systolic BP at 3 and 21 d after RMRa
At ;21 Days
At 3 Days
Group
Sham
RK-NX
RK-I
n
PRC (ng AngI/ml per h)
SBP (mmHg)
n
PRC (ng AngI/ml per h)
SBP (mmHg)
7
7
8
18.8 6 1.9
12.8 6 1.5
59.9 6 9.6c
129.8 6 2.4
137.3 6 1.9
168.9 6 3.2c
7
7
7
17.9 6 0.9
9.3 6 1.3b
18.6 6 3.5
120.4 6 3.2
125.2 6 3.7
185.8 6 6.2c
a
PRC, plasma renin concentration; SBP, systolic blood pressure. Other abbreviations as in Table 1.
P , 0.05 compared to sham and RK-I.
c
P , 0.001 versus compared to sham and RK-NX.
b
Figure 4. Correlation between plasma renin concentration (ng
AngI/ml per h) at 3 d and radiotelemetrically measured average
systolic BP in sham (M), RK-NX (F), and RK-I rats (f) (r 5 0.84,
slope 0.59, P , 0.001).
the first 6 wk after RMR (21,26,34 –36). These data suggest
that mechanisms in addition to a severe reduction in nephron
number are responsible for the development of hypertension in
the infarction model. The increased expression of renin mRNA
at 3 and 7 d and the increased PRC at 3 d in the RK-I, but not
in the RK-NX rats, are consistent with the results of previous
studies and suggest an important role for the renin-angiotensin
system in the initial pathogenesis of hypertension in the RK-I
model (34 –39). The excellent correlation between PRC and
radiotelemetrically measured BP at 3 d provides strong support
for such an interpretation. It should be noted that given the
reduced renal mass present in the RK-I rats, the increase in
renin mRNA expression in the noninfarct remnant renal tissue
is probably insufficient to explain the increases in the PRC
observed in these rats. It is likely that both renin mRNA
expression and renin content were more markedly increased in
the nonsampled, peri-infarcted areas as has been demonstrated
previously (38). It is therefore likely that renin release is not
homogeneous in the remnant kidneys of RK-I rats, with the
zones close to the infarct area releasing disproportionately
more renin than more distant ones and contributing to the
increased PRC (and BP) in these rats as well as affecting the
function of other nephrons (38). However, the relative suppres-
sion of renin mRNA expression and the decline in the PRC by
21 d in the RK-I rats, which have also been noted in previous
studies (34,35,37–39), suggest that additional renin-independent mechanisms, possibly related to hyperaldosteronism
and/or volume expansion, play a role in maintaining the hypertension over time in RK-I rats (38,40). The lack of correlation between BP and PRC at 21 d is consistent with such an
interpretation. However, it is noteworthy that despite the increased BP and the possible volume expansion present by 3 wk
in the RK-I rats, the PRC was still not suppressed below that of
sham controls and was significantly greater than in RK-NX
rats. Such considerations suggest that despite the relative reduction in renin mRNA expression and the PRC, the reninangiotensin system may nevertheless be pathogenetically important for the maintenance of sustained hypertension in RK-I
rats.
Based on the initial micropuncture studies in the RK-I
model, it had been suggested that the compensatory increase in
single-nephron GFR (SNGFR) after RMR per se may be
maladaptive, eventually resulting in glomerular injury and
sclerosis (2,3). Subsequent studies have indicated that of the
individual hemodynamic determinants of this augmentation in
SNGFR, an increased PGC is the parameter of primary importance in the pathogenesis of the “hyperfiltration” glomerular
injury (3,15,16). The micropuncture data in the present study
indicate that even marked increases in single nephron glomerular blood flow and filtration rate after severe approximately
5/6 RMR may not per se be sufficient to result in GS. These
data additionally indicate that only modest increases in PGC, as
seen in the RK-NX rats, are sufficient to achieve substantial
single nephron hyperfiltration. No significant differences in
pre- and postglomerular resistances were observed between the
two groups. These data are therefore consistent with the interpretation that the greater PGC increase observed in the RK-I
rats reflects glomerular transmission of the higher systemic
pressures present in the RK-I rats, rather than being intrinsic to
the hemodynamic adaptations that follow RMR. Moreover, our
data indicate that such modest increases in PGC as seen in the
RK-NX rats may not be pathologic, at least over the short term.
These interpretations are additionally supported by our previous observations in the WKY strain of rats, which fail to
develop HTN even after 5/6 renal ablation by infarction (29).
These rats also exhibited modest elevations in PGC (approximately 5 mmHg) comparable to those observed in RK-NX rats
504
Journal of the American Society of Nephrology
in the present study and did not develop significant GS despite
substantial single nephron hyperfiltration for up to 16 wk.
Therefore, increases in PGC sufficient to be pathologic seem to
be a superimposed consequence of systemic hypertension on
the intrinsic adaptive changes of RMR. A similar conclusion
was reached by Meyer and Rennke (41) after more limited
RMR (41). Significantly greater systemic and glomerular HTN
was noted in rats that had undergone 40% RMR by segmental
renal infarction despite greater compensatory hyperfiltration in
the normotensive uninephrectomized rats (50% RMR).
Some investigators have suggested that structural glomerular
hypertrophy (and the associated cellular responses), rather than
glomerular hyperfiltration (and the associated hemodynamic
changes), may be the maladaptive process that eventually
causes GS (11–14). Our data do not support such a postulate.
Both RK-I and RK-NX rats exhibit comparable glomerular
hypertrophy, despite the differences in GS that are observed by
6 wk between the two groups (21). However, these data do not
exclude the potential of long-term adverse effects of glomerular hypertrophy because of the associated increases in glomerular capillary wall tension as predicted by the Laplace Law
(Tension 5 Pressure 3 Radius) (28 –30,42,43). Nevertheless,
these data do suggest that glomerular hypertrophy per se may
also be of limited pathologic import in the absence of pathologic glomerular hypertension. Additionally, the similarity of
glomerular hypertrophy responses between the RK-NX and
RK-I rats despite the early differences in renin mRNA expression and PRC do not support a primary role for AngII as a
determinant of the glomerular hypertrophy response after
RMR.
Our data similarly do not support a primary role for TGF-b
and PGDF-B in the renal hypertrophy response after RMR.
Although a modest increase in TGF-b mRNA was observed in
RK-NX rats at all time periods, its relationship to compensatory hypertrophy is not clear. Comparable renal and glomerular
hypertrophy was observed in the RK-I rats without an increase
in TGF-b at 3 and 7 d after RMR. The marked increase in
PDGF-B and TGF-b observed at 21 d in the present study in
the RK-I group in whom glomerular injury has started to
develop, but not in the RK-NX rats, suggests that such increased expression may be a consequence of glomerular hypertension and/or may be initiated by the early increases in
renin (and AngII) at 3 and 7 d in the RK-I rats. Significant in
vitro and in vivo evidence supports the potential of AngII to
directly cause an increase in the expression of PDGF-B and
TGF-b in the RK-I model through its “growth effects” on
mesangial cells analogous to those on vascular smooth muscle
cells (8 –12,14,44). AngII is thought to initiate a molecular
cascade by activating the immediate early genes c-fos and
c-myc, followed by increases in the growth factors PDGF-B
and TGF-b with resulting increases in extracellular matrix
accumulation and GS. However, the present study does not
allow a direct and independent evaluation of the role of increased AngII and/or aldosterone in the absence of glomerular
hypertension, because the mechanical stress generated by glomerular capillary hypertension per se has the potential to alter
glomerular mesangial and/or endothelial function and to initi-
J Am Soc Nephrol 11: 497–506, 2000
ate increased generation of cytokines, collagen synthesis, and
GS (40,44 – 47). It is also possible that infarction per se may
result in the generation of local factors other than AngII that
may lead to increased PDGF-B and TGF-b expression and
eventual GS in the absence of systemic and/or glomerular
HTN. However, the absence of such GS in normotensive WKY
rats despite infarction argues against such a possibility (29).
Therefore, although the present data are consistent with the
postulated roles of PDGF-B and TGF-b as mediators of glomerulosclerosis (6,7,10 –12,14), they do not provide evidence
that these growth factors are the primary initiators of the renal
hypertrophy and/or GS in the absence of pathologic glomerular
hypertension and/or increased AngII after 5/6 RMR. However,
such interpretations regarding the role of TGF-b and PDGF-B
in glomerular hypertrophy and injury are not definitive due to
the limitations of the methods used in our study. Only pooled
kidney tissue from rats from each group was used for the
message expression studies. Moreover, the message expression
was not localized within the kidney. Because the message from
the glomeruli is only a small part of the total message in the
whole kidney isolates, it is quite possible that specific timedependent patterns of glomerular expression of TGF-b and
PDGF-B after RMR may be different and therefore may not be
detected by the methods used in the present study. Nevertheless, the temporal pattern of TGF-b and PDGF-B mRNA
expression in the RK-I group with marked increases in both at
21 d is consistent with previous reports in the RK-I group
(9,11,45). Increased glomerular endothelial TGF expression
using in situ reverse transcription was observed by Lee et al. at
23 to 25 d, but not at 6 to 14 d after 5/6 renal ablation in rats
fed a standard protein diet (45). However, modest increases in
PDGF-B glomerular expression have been noted as early as 1
wk after 5/6 ablation by some investigators (9,11).
Collectively, these data support the hypothesis that pathologic glomerular hypertension is likely to be the primary initiator of intraglomerular cellular events such as increased
PDGF-B and/or TGF-b expression, which eventually culminate in glomerulosclerosis. Furthermore, these data support the
postulate that such pathologic glomerular hypertension is a
consequence of enhanced transmission of systemic hypertension and is not intrinsic to the glomerular hemodynamic adaptations that follow severe RMR. Renal autoregulatory mechanisms that normally serve to protect glomerular capillaries
from transmission of systemic hypertension have been demonstrated to be impaired in rats with remnant kidneys
(24,25,48,49), and although renal autoregulation is comparably
impaired in both RK-I and RK-NX rats (21), it should be noted
that recent studies using continuous radiotelemetry in awake
unanesthetized rats have demonstrated a marked exaggeration
in the amplitude of BP fluctuations in hypertensive RK-I rats
(22–24). Therefore, the impact of impaired autoregulation on
glomerular pressures is likely to be much greater in the hypertensive RK-I rats than the normotensive RK-NX rats, and the
relatively modest but significant differences in PGC observed
between RK-I and RK-NX rats under anesthesia are likely to
significantly underestimate the ambient glomerular capillary
exposure to hypertension in unanesthetized RK-I rats. The
J Am Soc Nephrol 11: 497–506, 2000
excellent correlation between radiotelemetrically measured BP
and GS in individual RK-I rats provides additional support for
the dominant role of hypertensive mechanisms in the pathogenesis of GS after RMR (21–24,48). Therefore, the striking
difference in the severity of glomerulosclerosis after comparable RMR by infarction or surgical excision appears largely to
be a consequence of the presence or absence of systemic
hypertension in these two models and is independent of the
compensatory functional and structural adaptations after RMR.
Acknowledgments
This work was supported in part by National Institutes of Health
Grant DK40426. The authors gratefully acknowledge the technical
assistance of Jue Ouyang, Lisa Kelly, and Wanda Plott and the
secretarial assistance of Martha Prado.
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