# Phlorizin Prevents Glomerular Hyperfiltration but  not Hypertrophy in Diabetic Rats

Authors: Slava Malatiali; Issam Francis; Mario Barac-Nieto
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

The relationships of renal and glomerular hypertrophies to development of hyperfiltration and proteinuria early in streptozotocin-induced diabetes were explored. Control, diabetic, phlorizin-treated controls, and diabetic male Fischer rats were used. Phlorizin (anNa+-glucose cotransport inhibitor) was given at a dose sufficient to normalize blood glucose.  Inulin clearance (Cinulin) and protein excretion rate (PER) were measured. For morphometry, kidney sections were stained with periodic acid Schiff.  At one week, diabetes PER increased 2.8-folds (P<.001), Cinulin increased 80% (P<.01). Kidney wet and dry weights increased 10%–12% (P<.05), and glomerular tuft area increased 9.3% (P<.001). Phlorizin prevented proteinuria, hyperfiltration, and kidney hypertrophy, but not glomerular hypertrophy. Thus, hyperfiltration, proteinuria, and whole kidney hypertrophy were related to hyperglycemia but not to glomerular growth. Diabetic glomerular hypertrophy constitutes an early event in the progression of glomerular pathology which occurs in the absence of mesangial expansion and persists even after changes in protein excretion and GFR are reversed through glycemic control.


---

## Body

## 1. Introduction

Nephropathy occurs in
about 35% of patients with diabetes mellitus and diabetic nephropathy is
responsible for the majority of kidney dialysis and transplants [1]. Renal
hyperfiltration and hypertrophy are early manifestations of diabetic
nephropathy and much research has been devoted to elaborate if these are major
contributors to development of overt renal disease.Glomerular growth has been seen as the initial renal event in diabetes
and found to precede tubular growth [2]. It was believed that glomerular growth
with the associated increase in surface area for filtration as observed in
diabetic animals and patients [3, 4] is a
major determinant of renal hyperfiltration [5].Others suggest instead that hyperfiltration
is the initial event and is thought to occur as a consequence of a decreased
afferent arteriolar tone in hyperglycemia, probably mediated by early
glycosylation products [6]. Excess filtration then would lead to glomerular
hypertension and hypertrophy of glomerular cells in response to excessive stretch
[7, 8], resulting eventually in glomerular sclerosis [9].Recently, it has been proposed that whole kidney growth precedes [10] and leads to
hyperfiltration [11, 12], with a major contribution of tubular rather than of glomerular
hypertrophy to the development of hyperfiltration through altered
tubuloglomerular feedback. It is proposed that in uncontrolled diabetes
there is an increased tubular Na+-glucose reabsorption [13], due to the
increase in filtered glucose load and to increased expression of SGLT1, SGLT2
and GLUT2 transporters in proximal tubule cells [14]. The decreased distal
delivery of Na+ will cause an increase in GFR through
tubuloglomerular feedback (TGF) [12, 15].Although renal (glomerular and tubular) growth and hyperfiltration may contribute
significantly to development of overt diabetic nephropathy [9], the
interrelationships between these early renal diabetic events are not clear.
Therefore, the aim of this study was to identify the importance of glomerular
and renal morphological changes to the development of hyperfiltration and
proteinuria in early experimental diabetes, and to study the effect of blocking
tubular Na+-glucose reabsorption on glomerular and renal
hypertrophic growth and on hyperfiltration and proteinuria.


## 2. Materials and Methods

### 2.1. Animals

Male Fischer rats (8 weeks old) were placed in a room with an 8:00–20:00 light, 20:00–8:00 dark cycle,
kept at22.3±0.3°C and 31.2±0.8% humidity. The rats had free access to water and standard
rat chow (801151, Special Diets Services, UK). All animals were cared for in
accordance with the Guide for the Care and Use of Laboratory Animals and
all experimental protocols used in this study were approved by the Research
Administration Committee at Kuwait University. Four groups
were used: control rats (C), diabetic rats (D), diabetic rats treated with
Phlorizin (DPLZ), and control (nondiabetic) rats treated with phlorizin (CPLZ).
This group was added to study any direct renal effects of phlorizin.


### 2.2. Induction of Diabetes

Diabetes was induced by intraperitoneal (i.p)
injection of 55 mg/kg Streptozotocin (STZ, S-0130, Sigma, USA),
dissolved in 50 mM trisodium-citrate buffer, pH = 4.5. Controls were given the
citrate buffer (vehicle) alone. After STZ injection, animals were placed in
metabolic cages and development of diabetes was confirmed 16 hours later if
pronounced glucosuria and polyuria developed.


### 2.3. Phlorizin Treatment

Diabetic (DPLZ) and control rats (CPLZ) were treated with phlorizin (P-3449, Sigma, USA)
starting 16 hours after STZ or citrate bufferIP injection.
Phlorizin was dissolved at room temperature in propylene glycol
(1,2-propanediol, 82282, Fluka, Switzerland). The first day the rats were given a total
of 400 mg/kg phlorizin s.c. split into doses of 200 mg/kg at 8:00 AM and at 8:00 PM. The second day the dose
was raised to 400 mg/kg twice daily and the treatment was continued for 6 days [16].Blood glucose levels were measured twice daily in the treatment group on
samples taken from the tail, using a glucometer (GLUCOTREND 2, Roche, Germany).
Nonfasting blood glucose levels were measured in all groups before sacrifice, 7
days after treatment.


### 2.4. Protein Excretion Rate

Urinary protein concentration was measured using a
modified Lowry assay [17]. Protein excretion rate (PER) was calculated from
urinary protein concentration (Up, mg/ml) and urine flow rate (V, ml/24hrs).
The urine samples were collected over twenty four hours from rats placed in
metabolic cages.To assess tubular proteinuria, the urinary excretion of beta2-microglobulin
was tested. The concentration of beta2-microglobulin in rat urine
was measured using beta2-Microglobulin PET Kit (K 0052, DAKO, Denmark).
Changes in absorbance read at 340 nm (Hitachi model
911, Boehringer Mannheim, Germany) were proportional to the
concentration of beta2-microglobulin in standards of known
concentrations. The urinary excretion rate of beta2-microglobulin
was calculated from its urinary concentration and the urine flow rate.


### 2.5. Renal Hemodynamics

Glomerular filtration rate
was estimated by measuring the renal plasma clearance of inulin (Cinulin).
Each rat was weighed and anaesthetized with Inactin (Thiobutabarbital sodium C10H15N2NaO2S,
T-133, Sigma, UK) at a dose of 100 mg/kg IP. The
left femoral artery was catheterized for continuous monitoring of arterial
blood pressure by a pressure transducer. Blood samples were collected from the
arterial catheter. Tracheostomy was performed to insure proper ventilation. The
femoral vein was catheterized and an infusion with sterile Ringer's solution (154 mM
NaCl, 5.61 mM KCl, 2.16 mM CaCl2, 5.95 mM NaHCO3, 5.55 mM glucose) was started at a rate of 0.06 ml/min using a syringe pump
(Harvard Apparatus Ltd, UK). The bladder was cannulated with a stainless steel
cannula for urine collection. The cannula was connected to a flexible tube of
the appropriate size for urine collection. For estimation of urine flow rate,
the tube was carefully removed, the urine emptied in a tared Eppendorf tube and
weighed.After a 45–60 mininutes period of stabilization, an infusion containing inulin (from
Chicory root, I-2255, Sigma, USA) was started. The perfusion solution contained 36 mg/mL of inulin in Ringer's, and
was infused at a rate of 0.06 mL/min. Priming
dose of 160 mg/kg was given intravenously. After a 30 minutes equilibration
period, three or four timed samples of urine
were collected (15–20 minutes each). At the midpoint of each urine collection
period, an arterial blood sample of 300μl
was collected in dry heparinzed tubes, and centrifuged at 2000 g for 10
minutes. Plasma and urine samples were stored at −70°C for later
analysis of inulin concentrations.Concentration of inulin was measured in urine and plasma samples [18] after precipitation
with 10% TCA. Clearance values were expressed in mL/min per 100 g of initial
body weight.


### 2.6. Morphometry

Seven days after STZ or vehicle treatment, rats were anesthetized; the right kidney was removed,
weighed, dried, and reweighed. The left kidney was perfusion-fixed with 10%
neutral-phosphate buffered formalin. After 15 minutes perfusion, the left
kidney was removed and a 3 mm thick transverse cross-section was cut and placed
in formalin. Twenty four hours later sections were embedded in wax, cut into 4
micron sections, and mounted on APES (3-aminopropyltriethoxysilane, Sigma,
A-3648) coated slides for morphological studies. Periodic acid-Schiff (PAS)
stain was used for measurements of total glomerular tuft and mesangial matrix
areas [19]. The sections were hydrated and then placed in 0.5% periodic acid
(5–10 minutes). The slides were washed, successively dehydrated in 70%, 95%,
100% ethanol, and 100% xylene, protected with a cover slip using DPX, and
allowed to dry overnight. A total of 15 to 20
completely round glomeruli showing their tuft attached at the hilum were
selected randomly from all renal cortical zones in each animal. Morphometry of glomeruli was done with
a CAS 200 cell analysis system (Becton & Dickinson Image Cytometry Systems,
USA). Total glomerular tuft area (GA, mm2) was calculated after manual tracing of each tuft contour. Glomerular
tuft volume GTV was estimated fromGTV=b/k×(GA)3/2[20], where b=1.38
is a shape coefficient for a sphere and k=1.1 a size distribution
coefficient.Mesangial matrix area in each glomerular tuft was measured using a threshold method that
selectively highlights all PAS-positive areas within the tuft.


### 2.7. Statistical Analysis

All results were expressed
as mean ± SEM and were analyzed by one way ANOVA to establish differences
between groups. WhenF was significant, comparisons between any two groups were
further tested using unpaired two-tailed students t-test for equal or
unequal variances according to the equality of variance test. Correlation
studies were done using Pearson’s test. The software used was SPSS 11 for
Windows. For all statistical tests, a P value of less than 0.05 was
considered significant.


## 2.1. Animals

Male Fischer rats (8 weeks old) were placed in a room with an 8:00–20:00 light, 20:00–8:00 dark cycle,
kept at22.3±0.3°C and 31.2±0.8% humidity. The rats had free access to water and standard
rat chow (801151, Special Diets Services, UK). All animals were cared for in
accordance with the Guide for the Care and Use of Laboratory Animals and
all experimental protocols used in this study were approved by the Research
Administration Committee at Kuwait University. Four groups
were used: control rats (C), diabetic rats (D), diabetic rats treated with
Phlorizin (DPLZ), and control (nondiabetic) rats treated with phlorizin (CPLZ).
This group was added to study any direct renal effects of phlorizin.


## 2.2. Induction of Diabetes

Diabetes was induced by intraperitoneal (i.p)
injection of 55 mg/kg Streptozotocin (STZ, S-0130, Sigma, USA),
dissolved in 50 mM trisodium-citrate buffer, pH = 4.5. Controls were given the
citrate buffer (vehicle) alone. After STZ injection, animals were placed in
metabolic cages and development of diabetes was confirmed 16 hours later if
pronounced glucosuria and polyuria developed.


## 2.3. Phlorizin Treatment

Diabetic (DPLZ) and control rats (CPLZ) were treated with phlorizin (P-3449, Sigma, USA)
starting 16 hours after STZ or citrate bufferIP injection.
Phlorizin was dissolved at room temperature in propylene glycol
(1,2-propanediol, 82282, Fluka, Switzerland). The first day the rats were given a total
of 400 mg/kg phlorizin s.c. split into doses of 200 mg/kg at 8:00 AM and at 8:00 PM. The second day the dose
was raised to 400 mg/kg twice daily and the treatment was continued for 6 days [16].Blood glucose levels were measured twice daily in the treatment group on
samples taken from the tail, using a glucometer (GLUCOTREND 2, Roche, Germany).
Nonfasting blood glucose levels were measured in all groups before sacrifice, 7
days after treatment.


## 2.4. Protein Excretion Rate

Urinary protein concentration was measured using a
modified Lowry assay [17]. Protein excretion rate (PER) was calculated from
urinary protein concentration (Up, mg/ml) and urine flow rate (V, ml/24hrs).
The urine samples were collected over twenty four hours from rats placed in
metabolic cages.To assess tubular proteinuria, the urinary excretion of beta2-microglobulin
was tested. The concentration of beta2-microglobulin in rat urine
was measured using beta2-Microglobulin PET Kit (K 0052, DAKO, Denmark).
Changes in absorbance read at 340 nm (Hitachi model
911, Boehringer Mannheim, Germany) were proportional to the
concentration of beta2-microglobulin in standards of known
concentrations. The urinary excretion rate of beta2-microglobulin
was calculated from its urinary concentration and the urine flow rate.


## 2.5. Renal Hemodynamics

Glomerular filtration rate
was estimated by measuring the renal plasma clearance of inulin (Cinulin).
Each rat was weighed and anaesthetized with Inactin (Thiobutabarbital sodium C10H15N2NaO2S,
T-133, Sigma, UK) at a dose of 100 mg/kg IP. The
left femoral artery was catheterized for continuous monitoring of arterial
blood pressure by a pressure transducer. Blood samples were collected from the
arterial catheter. Tracheostomy was performed to insure proper ventilation. The
femoral vein was catheterized and an infusion with sterile Ringer's solution (154 mM
NaCl, 5.61 mM KCl, 2.16 mM CaCl2, 5.95 mM NaHCO3, 5.55 mM glucose) was started at a rate of 0.06 ml/min using a syringe pump
(Harvard Apparatus Ltd, UK). The bladder was cannulated with a stainless steel
cannula for urine collection. The cannula was connected to a flexible tube of
the appropriate size for urine collection. For estimation of urine flow rate,
the tube was carefully removed, the urine emptied in a tared Eppendorf tube and
weighed.After a 45–60 mininutes period of stabilization, an infusion containing inulin (from
Chicory root, I-2255, Sigma, USA) was started. The perfusion solution contained 36 mg/mL of inulin in Ringer's, and
was infused at a rate of 0.06 mL/min. Priming
dose of 160 mg/kg was given intravenously. After a 30 minutes equilibration
period, three or four timed samples of urine
were collected (15–20 minutes each). At the midpoint of each urine collection
period, an arterial blood sample of 300μl
was collected in dry heparinzed tubes, and centrifuged at 2000 g for 10
minutes. Plasma and urine samples were stored at −70°C for later
analysis of inulin concentrations.Concentration of inulin was measured in urine and plasma samples [18] after precipitation
with 10% TCA. Clearance values were expressed in mL/min per 100 g of initial
body weight.


## 2.6. Morphometry

Seven days after STZ or vehicle treatment, rats were anesthetized; the right kidney was removed,
weighed, dried, and reweighed. The left kidney was perfusion-fixed with 10%
neutral-phosphate buffered formalin. After 15 minutes perfusion, the left
kidney was removed and a 3 mm thick transverse cross-section was cut and placed
in formalin. Twenty four hours later sections were embedded in wax, cut into 4
micron sections, and mounted on APES (3-aminopropyltriethoxysilane, Sigma,
A-3648) coated slides for morphological studies. Periodic acid-Schiff (PAS)
stain was used for measurements of total glomerular tuft and mesangial matrix
areas [19]. The sections were hydrated and then placed in 0.5% periodic acid
(5–10 minutes). The slides were washed, successively dehydrated in 70%, 95%,
100% ethanol, and 100% xylene, protected with a cover slip using DPX, and
allowed to dry overnight. A total of 15 to 20
completely round glomeruli showing their tuft attached at the hilum were
selected randomly from all renal cortical zones in each animal. Morphometry of glomeruli was done with
a CAS 200 cell analysis system (Becton & Dickinson Image Cytometry Systems,
USA). Total glomerular tuft area (GA, mm2) was calculated after manual tracing of each tuft contour. Glomerular
tuft volume GTV was estimated fromGTV=b/k×(GA)3/2[20], where b=1.38
is a shape coefficient for a sphere and k=1.1 a size distribution
coefficient.Mesangial matrix area in each glomerular tuft was measured using a threshold method that
selectively highlights all PAS-positive areas within the tuft.


## 2.7. Statistical Analysis

All results were expressed
as mean ± SEM and were analyzed by one way ANOVA to establish differences
between groups. WhenF was significant, comparisons between any two groups were
further tested using unpaired two-tailed students t-test for equal or
unequal variances according to the equality of variance test. Correlation
studies were done using Pearson’s test. The software used was SPSS 11 for
Windows. For all statistical tests, a P value of less than 0.05 was
considered significant.


## 3. Results

### 3.1. Blood Glucose, Body, and Kidney Weights, Urine Flow and Protein Excretion Rates in Phlorizin-Treated
and Untreated Diabetic Rats

Streptozotocin-induced
diabetes caused significant increases in blood glucose concentration, urine
flow rate, and PER and a significant decrease in body weight
(Table1).Table 1
Body and kidney weights, blood and urinary changes in Fischer rats at
one-week diabetes.
Bwt0=
Initial body weight, Bwtf= final body weight, BG = blood glucose, V= urine flow rate measured over 24 hours from rats placed in metabolic cages,
PER = protein excretion rate, Wt = weight, C= control,
D= diabetic, DPLZ = phlorizin-treated diabetic, CPLZ =
phlorizin-treated
controls. Results are expressed as mean
± SE and compared using unpaired two-tailed student's t-test.
Bwt0BwtfBGVPERKidney wetKidney dry(g)(g)(mM/l)(ml/24hrs)(mg/24hrs)wt/Bwt0%wt/Bwt0%C196.5±3.2208±6.55.6±0.36.5±1.048.2±1.50.39±0.010.085±0.003n=14D192±2.0177.8±4.9**27.7±1.4##63.4±6.4##23.2±5.6#0.43±0.01#0.095±0.001#n=15DPLZ199.6±4.2175.7±2.6**5.9±1.147.5±4##14.3±2.6#0.39±0.020.078±0.005n=9CPLZ200±2.7187.6±4.2*φ5.1±0.221.3±2.6##φφφ14±1.8#0.4±0.020.082±0.004n=5=*P<.05, =**P<.001 compared to initial weight using
paired two-tailed student's t-test. =#P<.05, =##P<.001 compared to control, =φP<.05, =φφφP<.001 CPLZ versus DPLZ.Seven-day
treatment of diabetic rats with phlorizin reduced the blood glucose levels to
normal values (Table1) within 60 hours (Figure 1); however, there was still
significant diuresis and decrease in body weight (Table 1). Diuresis and body weight loss were also
observed in phlorizin-treated control rats (Table 1).Figure 1
Blood glucose concentrations in phlorizin-treated
diabetic rats (n=5).One week of diabetes led
to small (10–12%) but significant (P<.05) increases in the kidney wet
and dry weights expressed as percentages of initial body weights (Table 1).
Phlorizin treatment prevented the mild renal growth observed in diabetic rats.
Phlorizin had no effect on renal wet or dry kidney weights in control rats.


### 3.2. Glomerular Morphology and Proteinuria in Phlorizin-Treated and Untreated Diabetic Rats

At one-week diabetes, glomerular capillary tuft area and
volume are increased by 9.3% and 14.6%, respectively, with no significant change in
PAS-positive mesangial matrix area (Table2, Figure 2). Phlorizin treatment did
not prevent glomerular growth (Figure 2, Table 2) nor altered mesangial matrix area.
However, phlorizin prevented diabetic proteinuria since PER in
PLZ-treated diabetic rats was not significantly different from that in
PLZ-treated controls. The 1.7 fold higher PER observed in PLZ-treated control
rats compared with untreated controls is consistent with an independent effect
of phlorizin on the renal handling of proteins.Table 2
Glomerular morphometry in Fischer
rats at one-week diabetes:n= number of rats, N=
total number of observations, GTV = glomerular tuft volume. Tuft A= glomerular
tuft area, MMA = mesangial matrix area. All variables were analyzed using one
way ANOVA, and then between group differences were compared using unpaired
two-tailed student's t-test.
ControlDiabeticDPLZn=5, N=72n=6, N=89n=5, N=70GTV486±11557±11***621±153***μm3×103Tuft A5293.9±117.85796.7±74.4***6225.1±102.9***μm2MMA163.3±6159.1±4.5164.6±6.6μm2MMA/tuftA%3.07±0.12.77±0.12.65±0.1**=**P<.01,
=***P<.001, compared to control.Table 3
Inulin clearances (GFR) in the experimental groups:C= control, D= diabetic,
DPLZ = phlorizin-treated diabetic rats, CPLZ = phlorizin-treated controls. Cinulin= clearance of
inulin, Bwt0= initial body weight, V= urine flow rate during
clearance experiment, MBP = mean blood pressure. Clearance values were
normalized to 100 g of initial body weight. Results are expressed as mean ± SEM and compared using
student's t-test.
Control (n=7)Diabetic (n=5)DPLZ (n=5)CPLZ (n=5)Cinulin  (ml·min-1·100 g-1)0.47±0.020.84±0.1**0.43±0.05#0.61±0.07Bwt0   (g)203.2±5.6196.2±1.5200.2±3.8200±2.7V  (μl·min-1·100 g-1)5.6±0.59.6±1.3*13.±3.1*6±0.7MBP (mmHg)126.6±3.3123.9±4.2121.8±2.2123.0±2.3=*P<.05, =**P<.01 compared to control. =#P<.01 compared to diabetic.Glomerular
morphological changes due to one-week diabetes in Fischer rats. Glomerular tuft
area significantly increased after one week of diabetes. PAS positive area
exhibited no change in one-week diabetics (D) when compared to controls (C).
Phlorizin treatment (DPLZ) did not prevent glomerular growth. (4μm, paraffin sections, periodic acid-Schiff
(PAS) stain, ×400).
(a)(b)(c)Excretion of beta2-microglobulin was measured to assess
tubular protein reabsorption [20]. There were no traces of beta2-microglobulin
in urine of control, and diabetic rats. However, there were significant amounts
of beta2-microglobulin in the urine of phlorizin-treated control and
diabetic rats. Urinary excretions of beta2-microglobulin were 0.018±0.01
and 0.023±0.01 mg/24h in PLZ-treated controls and PLZ-treated diabetic rats, respectively (P=0.7).
Thus, while one-week diabetes does not alter the renal handling of beta2-microglobulin,
phlorizin has an inhibitory effect on the tubular reabsorption of beta2-microglobulin
in control and diabetic rats.The early 3-4 fold diabetes-associated increase in PER
was not found to correlate with glomerular tuft or mesangial matrix areas or
volumes, but instead was correlated withCinulin (Figure 3, r2=0.6, P<.01), suggesting that is mostly related to functional rather than
to gross morphological glomerular changes.Figure 3
Correlation of protein excretion rate (PER) with inulin clearance (P<.001)
in control, diabetic, and phlorizin-treated control and diabetic Fischer rats.
Inulin clearance was normalized to 100 g of initial body weight.


### 3.3. Glomerular Hemodynamic Changes in Phlorizin-Treated and Untreated Diabetic Rats

Mean arterial blood pressure was similar in all the
groups studied (Table2). One-week diabetic rats had an 80% higher (P<.01)
Cinulin than vehicle-treated nondiabetic time controls (Table 2).
Phlorizin treatment prevented the diabetes-associated increase in Cinulin.
Phlorizin had no significant hemodynamic effect in control rats.The effects of phlorizin on diabetic-induced changes are summarized in Figure4. These
results show that in diabetes, phlorizin, an inhibitor of proximal tubule sodium-glucose
reabsorption, prevents glomerular hyperfiltration, proteinuria and whole kidney
(tubular) growth but not glomerular tuft growth.Figure 4
Percent
changes from control of kidney dry weight to initial body weight ratio (KDW/BW0), glomerular tuft area (GTA),
protein excretion rate (PER), and glomerular filtration rate (GFR) in diabetic
(D) and phlorizin-treated diabetic (DPLZ) rats. Phlorizin treatment prevented renal growth, proteinuria and
hyperfiltration, but did not prevent glomerular hypertrophy in diabetic rats. Absolute
values and statistics are presented in Tables 1, 2, and 3.


## 3.1. Blood Glucose, Body, and Kidney Weights, Urine Flow and Protein Excretion Rates in Phlorizin-Treated
and Untreated Diabetic Rats

Streptozotocin-induced
diabetes caused significant increases in blood glucose concentration, urine
flow rate, and PER and a significant decrease in body weight
(Table1).Table 1
Body and kidney weights, blood and urinary changes in Fischer rats at
one-week diabetes.
Bwt0=
Initial body weight, Bwtf= final body weight, BG = blood glucose, V= urine flow rate measured over 24 hours from rats placed in metabolic cages,
PER = protein excretion rate, Wt = weight, C= control,
D= diabetic, DPLZ = phlorizin-treated diabetic, CPLZ =
phlorizin-treated
controls. Results are expressed as mean
± SE and compared using unpaired two-tailed student's t-test.
Bwt0BwtfBGVPERKidney wetKidney dry(g)(g)(mM/l)(ml/24hrs)(mg/24hrs)wt/Bwt0%wt/Bwt0%C196.5±3.2208±6.55.6±0.36.5±1.048.2±1.50.39±0.010.085±0.003n=14D192±2.0177.8±4.9**27.7±1.4##63.4±6.4##23.2±5.6#0.43±0.01#0.095±0.001#n=15DPLZ199.6±4.2175.7±2.6**5.9±1.147.5±4##14.3±2.6#0.39±0.020.078±0.005n=9CPLZ200±2.7187.6±4.2*φ5.1±0.221.3±2.6##φφφ14±1.8#0.4±0.020.082±0.004n=5=*P<.05, =**P<.001 compared to initial weight using
paired two-tailed student's t-test. =#P<.05, =##P<.001 compared to control, =φP<.05, =φφφP<.001 CPLZ versus DPLZ.Seven-day
treatment of diabetic rats with phlorizin reduced the blood glucose levels to
normal values (Table1) within 60 hours (Figure 1); however, there was still
significant diuresis and decrease in body weight (Table 1). Diuresis and body weight loss were also
observed in phlorizin-treated control rats (Table 1).Figure 1
Blood glucose concentrations in phlorizin-treated
diabetic rats (n=5).One week of diabetes led
to small (10–12%) but significant (P<.05) increases in the kidney wet
and dry weights expressed as percentages of initial body weights (Table 1).
Phlorizin treatment prevented the mild renal growth observed in diabetic rats.
Phlorizin had no effect on renal wet or dry kidney weights in control rats.


## 3.2. Glomerular Morphology and Proteinuria in Phlorizin-Treated and Untreated Diabetic Rats

At one-week diabetes, glomerular capillary tuft area and
volume are increased by 9.3% and 14.6%, respectively, with no significant change in
PAS-positive mesangial matrix area (Table2, Figure 2). Phlorizin treatment did
not prevent glomerular growth (Figure 2, Table 2) nor altered mesangial matrix area.
However, phlorizin prevented diabetic proteinuria since PER in
PLZ-treated diabetic rats was not significantly different from that in
PLZ-treated controls. The 1.7 fold higher PER observed in PLZ-treated control
rats compared with untreated controls is consistent with an independent effect
of phlorizin on the renal handling of proteins.Table 2
Glomerular morphometry in Fischer
rats at one-week diabetes:n= number of rats, N=
total number of observations, GTV = glomerular tuft volume. Tuft A= glomerular
tuft area, MMA = mesangial matrix area. All variables were analyzed using one
way ANOVA, and then between group differences were compared using unpaired
two-tailed student's t-test.
ControlDiabeticDPLZn=5, N=72n=6, N=89n=5, N=70GTV486±11557±11***621±153***μm3×103Tuft A5293.9±117.85796.7±74.4***6225.1±102.9***μm2MMA163.3±6159.1±4.5164.6±6.6μm2MMA/tuftA%3.07±0.12.77±0.12.65±0.1**=**P<.01,
=***P<.001, compared to control.Table 3
Inulin clearances (GFR) in the experimental groups:C= control, D= diabetic,
DPLZ = phlorizin-treated diabetic rats, CPLZ = phlorizin-treated controls. Cinulin= clearance of
inulin, Bwt0= initial body weight, V= urine flow rate during
clearance experiment, MBP = mean blood pressure. Clearance values were
normalized to 100 g of initial body weight. Results are expressed as mean ± SEM and compared using
student's t-test.
Control (n=7)Diabetic (n=5)DPLZ (n=5)CPLZ (n=5)Cinulin  (ml·min-1·100 g-1)0.47±0.020.84±0.1**0.43±0.05#0.61±0.07Bwt0   (g)203.2±5.6196.2±1.5200.2±3.8200±2.7V  (μl·min-1·100 g-1)5.6±0.59.6±1.3*13.±3.1*6±0.7MBP (mmHg)126.6±3.3123.9±4.2121.8±2.2123.0±2.3=*P<.05, =**P<.01 compared to control. =#P<.01 compared to diabetic.Glomerular
morphological changes due to one-week diabetes in Fischer rats. Glomerular tuft
area significantly increased after one week of diabetes. PAS positive area
exhibited no change in one-week diabetics (D) when compared to controls (C).
Phlorizin treatment (DPLZ) did not prevent glomerular growth. (4μm, paraffin sections, periodic acid-Schiff
(PAS) stain, ×400).
(a)(b)(c)Excretion of beta2-microglobulin was measured to assess
tubular protein reabsorption [20]. There were no traces of beta2-microglobulin
in urine of control, and diabetic rats. However, there were significant amounts
of beta2-microglobulin in the urine of phlorizin-treated control and
diabetic rats. Urinary excretions of beta2-microglobulin were 0.018±0.01
and 0.023±0.01 mg/24h in PLZ-treated controls and PLZ-treated diabetic rats, respectively (P=0.7).
Thus, while one-week diabetes does not alter the renal handling of beta2-microglobulin,
phlorizin has an inhibitory effect on the tubular reabsorption of beta2-microglobulin
in control and diabetic rats.The early 3-4 fold diabetes-associated increase in PER
was not found to correlate with glomerular tuft or mesangial matrix areas or
volumes, but instead was correlated withCinulin (Figure 3, r2=0.6, P<.01), suggesting that is mostly related to functional rather than
to gross morphological glomerular changes.Figure 3
Correlation of protein excretion rate (PER) with inulin clearance (P<.001)
in control, diabetic, and phlorizin-treated control and diabetic Fischer rats.
Inulin clearance was normalized to 100 g of initial body weight.


## 3.3. Glomerular Hemodynamic Changes in Phlorizin-Treated and Untreated Diabetic Rats

Mean arterial blood pressure was similar in all the
groups studied (Table2). One-week diabetic rats had an 80% higher (P<.01)
Cinulin than vehicle-treated nondiabetic time controls (Table 2).
Phlorizin treatment prevented the diabetes-associated increase in Cinulin.
Phlorizin had no significant hemodynamic effect in control rats.The effects of phlorizin on diabetic-induced changes are summarized in Figure4. These
results show that in diabetes, phlorizin, an inhibitor of proximal tubule sodium-glucose
reabsorption, prevents glomerular hyperfiltration, proteinuria and whole kidney
(tubular) growth but not glomerular tuft growth.Figure 4
Percent
changes from control of kidney dry weight to initial body weight ratio (KDW/BW0), glomerular tuft area (GTA),
protein excretion rate (PER), and glomerular filtration rate (GFR) in diabetic
(D) and phlorizin-treated diabetic (DPLZ) rats. Phlorizin treatment prevented renal growth, proteinuria and
hyperfiltration, but did not prevent glomerular hypertrophy in diabetic rats. Absolute
values and statistics are presented in Tables 1, 2, and 3.


## 4. Discussion

One week after induction of streptozotocin diabetes,
renal and glomerular hypertrophy and hyperfiltration were observed in Fischer
rats. Renal hypertrophy, although small, was significant and occurred
independently of body weight changes. Hyperfiltration was evident by a
significant increase inCinulin similar to that reported earlier in
SD rats [21].One week of phlorizin
treatment prevented renal whole kidney growth and hyperfiltration; however, it
did not prevent glomerular tuft growth. The sustained glomerular growth with
phlorizin treatment is probably related to the fact that hyperglycemia
persisted for at least 60 hours after PLZ and STZ treatments. Hyperglycemia
induces the release of growth factors such as VEGF [22, 23], TGF-β [24], and
Angiotensin II [25] by resident glomerular cells and by infiltrating
macrophages [26] that invade the glomeruli as early as one day after STZ
treatment [26]. Once induced, glomerular growth persists even when glycemia was
normalized with PLZ for at least three days, which indicates that glomerular
growth can persist independently of the rates of Na+-glucose
cotransport, and of hyperglycemia. Trophic effects of phlorizin per se on the
glomerulus have not been reported. Podocytes [27] and glomerular mesangial
cells [28] show SGLT 1 expression indicating that Na+-glucose
cotransport is active in these cells. Blocking this pathway with phlorizin will
reduce glucose entry [28] and decrease their size [29] rather than induce their
growth or proliferation.By contrast, diabetic renal tubular growth, manifested by increases in total wet and dry
kidney weights [30], was totally prevented by phlorizin treatment. This
indicates that in STZ diabetes, hypertrophy of renal tubular cells depends on
hyperglycemia and the associated increase in tubular Na+-glucose
cotransport, and is rapidly reversed by normalizing blood glucose concentration.
Thus, diabetic renal tubular and glomerular hypertrophies show different
dynamics upon normalization of blood glucose and probably involve different
growth pathways.The diabetes-associated
increase in GFR detected in this study is independent of glomerular growth,
since with phlorizin treatment GFR was not elevated despite sustained
glomerular growth. Furthermore in diabetes, while the increase in GFR was of
80%, that of the glomerular tuft cross-sectional area was only 14%. Since the flow
is proportional to the square of the cross-sectional area, most of the increase
in GFR was likely due to changes in pre-or postglomerular resistances rather
than in glomerular cross-sectional area.Similar to the findings in this study, phlorizin had no hemodynamic
effects in control rats [31]. In control rats, Na+-glucose
cotransport represents at most 5% of total Na+ transport in the
nephron [31]. However, in diabetic rats, proximal Na+-dependent glucose
reabsorption increases [13, 32] with increases in filtered glucose load and in expression
of glucose transporters (GLUT 2) [14, 32]. Enhanced proximal reabsorption
decreases distal sodium delivery leading to preglomerular dilatation and
hyperfiltration through tubuloglomerular feedback (TGF) [12]. Acute infusion of
phlorizin, at a dose that does not normalize the blood glucose level, was shown
to decrease GFR [13]. Blocking the diabetes-enhanced sodium glucose cotransport
with PLZ decreases expression of GLUT2 [33], greatly decreases proximal
reabsorption of sodium, increases its distal delivery, and leads to normalization
in GFR, possibly through reversal of TGF [12]. The effect of phlorizin in
preventing hyperfiltration in the current study was similar to that reported
earlier [13] but occurred with a higher PLZ dose which resulted in
normoglycemia and a normal filtered glucose load within 60 hours of PLZ
treatment. Therefore, in our experiments, the effect of phlorizin on GFR could
be direct, due to inhibition of tubular reabsorption of glucose and indirect,
through normalization of the blood glucose level and of processes such as
enhanced proximal sodium-glucose cotransport that are dependent on
hyperglycemia. The prevention of hyperfiltration with
either low or high doses of PLZ suggests that activation of TGF is the main
mechanism by which hyperglycemia increases GFR in diabetes and that its other
effects on renal vessels have little influence on early diabetic
hyperfiltration.The reduction inCinulin with PLZ cannot be accounted for by
an effect of PLZ on extracellular fluid volume (ECFV). Phlorizin-treated
diabetic rats had twenty-four-hour urine flow rate and diuretic response to Ringer’s infusion similar to diabetic rats indicating
similar state of hydration in both groups. Therefore, PLZ had no significant
effect on the ECFV of these animals. Phlorizin treatment reduced body weight in
both DPLZ and CPLZ rats, but reduced Cinulin only in DPLZ animals.
Thus, the change in body weight associated with PLZ treatment has no direct
effect on Cinulin.Hyperfiltration was correlated with mild proteinuria in
this model of early diabetes. However, since a correlation does not necessarily
indicate a causal relationship, the current data do not allow us to define
whether diabetes leads to mild proteinuria only through hyperfiltration or if
additional effects are involved. Early diabetic proteinuria was totally
prevented by one week phlorizin treatment indicating that it, as well as
hyperfiltration, is due to processes dependent on hyperglycemia and independent
of glomerular tuft growth which was not reversed when glycemia was normalized
with phlorizin treatment. The absence of excretion of beta2-microglobulin,
a marker of tubular protein transport [20], observed in diabetic rats indicates
that there is no early effect of diabetes on tubular protein reabsorption.In this study we observed persistent glomerular hypertrophy in the absence of proteinuria,
hyperfiltration, or whole kidney growth when the glycemia was controlled. We
speculate that persistent glomerular growth may be a precursor of glomerular
sclerosis and of renal insufficiency (low GRF) that occur in the absence of
proteinuria or of kidney hypertrophy in about 20% of patients with diabetic
nephropathy [34–36].


## 5. Conclusion

This study shows that early in diabetes renal tubular and glomerular hypertrophy, mild
proteinuria and hyperfiltration occur in Fischer rats. The data indicate that
the renal functional changes observed early in diabetes (hyperfiltration and
proteinuria) cannot be accounted for by the observed glomerular hypertrophy
and, in contrast to glomerular growth, are readily reversed by phlorizin-induced
normalization of blood glucose. Glomerular hypertrophy is an early event in the
development of STZ-diabetes-induced glomerular pathology, occurred in the
absence of mesangial matrix expansion and persisted after short-term
normalization of the glycemia. The alleviation of early renal functional and
some structural (tubule hypertrophy) changes in diabetes with phlorizin, as it
does in the retina [29], supports its potential therapeutic
role in minimizing diabetic microvascular disease. Further studies are needed to investigate if
diabetes-induced glomerular hypertrophy leads to glomerulosclerosis and how it
can be prevented or reversed.



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*Source: 305403-2008-08-26.xml*