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DOI: 10.1111/j.1549-8719.2009.00010.x

In Vivo Visualization of Glomerular Microcirculation and Hyperfiltration in Streptozotocin-Induced Diabetic Rats MINORU SATOH, SHINYA KOBAYASHI, ATSUNORI KUWABARA, NARUYA TOMITA, TAMAKI SASAKI, AND NAOKI KASHIHARA Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan

ABSTRACT Objectives: Knowledge of glomerular structural and hemodynamic changes in vivo is still limited under diabetic conditions. In this study, we examined the alterations in glomerular structure and permeability of macromolecules and the effects of telmisartan using a confocal laser microscope. Methods: Diabetes was induced by injecting streptozotocin. After 4 and 8 weeks, the filtration and permeability of differently sized compounds across the glomerular capillaries were visualized using a confocal laser microscope by injecting 500-kilodalton and 40-kilodalton dextran. At 7 weeks, some diabetic rats were treated with telmisartan for 1 week. The permeation of the 40-kilodalton dextran across the glomerular capillaries into Bowman’s space was quantified. Glomerular volume, diameters of the afferent and efferent arterioles, and glomerular permeability were compared. Results: Glomerular volume was significantly increased in the diabetic rats, and there was heterogeneity in the glomerular volumes. The diameter ratio of the afferent to efferent arterioles significantly increased, and there was increased glomerular permeability in the diabetic rats compared with the control rats. Telmisartan treatment reduced glomerular permeability without affecting glomerular volume. Conclusions: These data showed that glomerular hyperfiltration started from the early phase of diabetes, accompanied by dilatation of afferent arterioles and glomerular hypertrophy. Microcirculation (2010) 17, 103–112. doi: 10.1111 ⁄ j.1549-8719.2009.00010.x KEY WORDS: Albuminuria, angiotensin receptor blocker, endothelial permeability, glomerular hyperfiltration, glomerular morphology

INTRODUCTION

Diabetes mellitus (DM) is the main cause of endstage renal failure. In the early course of diabetes, several functional and structural alterations occur in the kidney. Some of these alterations include glomerular hyperperfusion ⁄ hyperfiltration [33], hypertrophy of the nephrons and gross renal enlargement [2], and changes in the mass and composition of the glomerular extracellular matrix. Both clinically and experimentally, early diabetes is characterized by an increase in glomerular filtration rate and renal plasma flow [34]. Glomerular hyperAddress for correspondence: Minoru Satoh, M.D., Ph.D., Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 7010192, Japan. E-mail: [email protected] Received 25 July 2009; accepted 19 October 2009. Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

filtration has been shown to result from elevations in glomerular capillary blood flow and glomerular capillary hydraulic pressure [1]. Glomerular microcirculation regulates glomerular filtration and renal hemodynamics by altering the vascular resistance of the afferent and efferent arterioles. Renal hemodynamics in early diabetes are characterized by preglomerular and postglomerular vasodilation and increased glomerular capillary pressure, leading to hyperfiltration. Knowledge of glomerular structural and hemodynamic changes in vivo is still limited under pathophysiologic conditions. Several experimental methods have been developed to study glomerular microcirculation directly. Hydronephrotic rats [30] can be used to investigate the effects of vasoactive substances only in nonfiltering glomeruli. However, this approach involves invasive manipulations that might alter the physiologic environment of general

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Glomerular filtration in STZ rats M. Satoh et al.

and ⁄ or local hemodynamics, particularly at the sensitive level of the microvasculature including the afferent and efferent arterioles. The pencil-probe charge coupled device videomicroscope can be used to study glomerular microcirculation in small animals directly under physiological conditions [36]. However, glomerular filtration of small molecules has not yet been examined by this method. Insight into the mechanisms underlying the early hemodynamic changes in the diabetic kidney is particularly important, because they are a risk factor for the subsequent development of microalbuminuria and overt diabetic nephropathy [7]. In the present study, we investigated the morphology of the glomeruli and the alteration of glomerular filtration in the early stage of DM by a novel in vivo technique using a confocal laser microscope. The reninangiotensin system (RAS) blockade has been shown to slow the progression of established diabetic nephropathy in DM [3,18]. Therefore, to further elucidate the role of angiotensin II (Ang II) in renal hemodynamics in diabetic rats, we examined in vivo the effects of a selective Ang II receptor antagonist, telmisartan, on renal hemodynamic changes. MATERIALS AND METHODS Experimental Protocol

The experimental protocols (No. 06-014) were approved in advance by the Ethics Review Committee for Animal Experimentation of the Kawasaki Medical School (Kurashiki, Japan). Male SpragueDawley rats (6–7 weeks of age) weighing 190 to 220 g were purchased from Charles River Japan Inc. (Kanagawa, Japan). Diabetes was induced by a single injection in the tail vein of streptozotocin (65 mg ⁄ kg body weight; Sigma-Aldrich Japan, Tokyo, Japan) diluted in citrate buffer, pH 4.5 (STZ-DM; n = 45). Age-matched non-diabetic control rats (Control; n = 30) were each injected with an equal volume of citrate buffer. Seven weeks after induction of DM, 15 rats were given drinking water containing telmisartan (30 mg ⁄ kg ⁄ day, STZDM + Telmisartan) over a period of 1 week. Physiological and Biochemical Measurements

At 4 and 8 weeks after induction of DM, the mean blood pressure was measured by the tail-cuff method (BP-98A; Softron, Tokyo, Japan). To collect urine samples, rats were placed in metabolic

cages for 24 hours and provided tap water but no food. Albumin concentrations in 24-hour urine samples were measured using an enzyme-linked immunosorbent assay kit (Exocell, Philadelphia, PA, USA). After urine was collected, blood samples were obtained from the tail vein; and serum creatinine, blood urea nitrogen, and fasting serum glucose levels were measured. Fluorescent Probes

Probes, including 500-kilodalton (kd) fluoresceindextran (anionic, excitation 494 nm, emission 518 nm), 40-kd Texas Red-dextran (neutral, excitation 595 nm, emission 615 nm), and 40-kd fluorescein-dextran (anionic, excitation 494 nm, emission 518 nm), were obtained from Invitrogen Japan (Tokyo, Japan) and stocked at 2 mg ⁄ mL in phosphate-buffered saline (pH 7.4). Alexa Fluor 594 conjugate isolectin Griffonia simplicifolia-IB4 (GS-IB4; excitation 590 nm, emission 617 nm) was also obtained from Invitrogen Japan, and a 0.25 mg ⁄ mL stock solution was prepared in phosphate-buffered saline (pH 7.4). Multiphoton Excitation Laser-Scanning Fluorescence Microscopy

The multiphoton microscope used in these studies was a Leica TCS SP2 AOBS MP confocal microscope system (Leica Microsystems Japan, Tokyo, Japan) with the following components: a Leica DM IRE2 inverted microscope powered by a wideband, fully automated, infrared (710–920 nm) combined photo-diode pump laser and modelocked titanium: sapphire laser (Mai-Tai, Spectra-Physics, Mountain View, CA, USA). Images were collected in time (xyt) series (0.5–5 Hz) with the Leica Confocal Software (LCS 2.61.1537) and analyzed with the LCS 3D, Process and Quantify packages. The excitation laser power on the sample was attenuated to between 2 and 28 mW using neutral-density filters. In Vivo Imaging of Intact Kidney

A catheter was inserted into the left external jugular vein of the rats under sevoflurane -induced anesthesia for infusion of the dye. Subsequently, a 15- to 20-mm dorsal incision was made under sterile conditions, and the left kidney was exteriorized. To image glomerular microcirculation, a cortical slice that was less than 1 mm in thickness was removed Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

Glomerular filtration in STZ rats M. Satoh et al.

to allow imaging of the most superficial glomeruli. Bleeding was minimal and stopped spontaneously within 2 minutes. During all procedures and imaging, the rat’s core body temperature was maintained at 37C with a homeothermic table. A 0.5-mL volume of 500-kd fluorescein-dextran solution was infused through the jugular venous catheter immediately before microscopic imaging. Next, for analysis of glomerular permeability, a 0.5-mL volume of 40-kd Texas Red-dextran solution was infused. Visualization of the glomerular endothelial glycocalyx was also accomplished by confocal microscopy in some rats. Alexa Fluor 594 conjugate isolectin GS-IB4 was infused through the jugular venous catheter. After 30 minutes, a 0.5-mL volume of 40-kd fluorescein-dextran solution was infused. Statistical Analysis

Values were expressed as mean ± SD. All parameters were evaluated with the two-tailed unpaired Student’s t-test, Welch’s t-test, or Mann–Whitney’s U-test. A P-value less than 0.05 denoted the presence of a significant difference. RESULTS Physiological data

Table 1 and 2 show physiological dates at 4 and 8 weeks. There are statistically decreases in body weight and serum creatinine levels, and increases in kidney weight, blood glucose level, blood urea nitrogen, amounts of albuminuria, and creatinine clearance in STZ-DM rats compared with Control Table 1. Physiologic data at 4 weeks

Control n BW (g) LKW ⁄ BW 100 g BG (mg ⁄ dL) SBP (mmHg) S-Cre (mg ⁄ dL) BUN (mg ⁄ dL) UAE (mg ⁄ day) Ccr (mL ⁄ min ⁄ BW 100 g)

15 369 0.41 154 112 0.21 18.7 13.4 1.00

± ± ± ± ± ± ± ±

27 0.03 16 13 0.02 2.4 1.3 0.11

STZ-DM 15 267 0.64 464 130 0.17 25.7 69.0 1.28

± ± ± ± ± ± ± ±

59a 0.09a 68a 14 0.02a 6.3a 17.6a 0.15a

Data are expressed as mean ± SD. BW, body weight; LKW, left kidney weight; BG, blood glucose; SBP, systolic blood pressure; S-Cre, serum creatinine; BUN, blood urea nitrogen; UAE, urinary albumin excretion; Ccr, creatinine clearance. a P < 0.05 vs Control. Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

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rats. Administrated with telmisartan at 8 weeks (STZ-DM + Telmisartan rats), there are statistically decreases in urinary albumin excretion compared with STZ-DM rats, but no differences in blood glucose levels, systolic blood pressure and renal function (blood urea nitrogen and creatinine clearance). Morphological Changes in Early Diabetic Glomeruli

Representative three-dimensional images of glomeruli are shown in Figure 1. Glomerular capillaries and arterioles were visualized using 500-kd FITC-labeled dextran and observed by confocal microscopy. The images were rebuilt for threedimensional structure (Supplemental Movie S1). Glomerular capillaries could be observed in detail, and efferent and afferent arteries were clearly visualized. Glomerular volume in each group was measured from the still image (Figure 2A). The average glomerular volume was significantly increased in the STZ-DM rats at 4 weeks, and was also increased in the STZ-DM rats and STZDM + Telmisartan rats compared with the Control rats at 8 weeks. There was no reduction in glomerular volume in the STZ-DM + Telmisartan rats compared with the STZ-DM rats. Notably, the number of glomeruli with larger volumes was increased and the distribution of volumes was expanded in the STZ-DM rats compared with the Control rats (Figure 2B). Heterogeneity in glomerular volume was shown in the early stage of STZDM. The distribution was also expanded in the STZ-DM + Telmisartan rats compared with the Control rats. The diameters of the afferent and efferent arteries are dependent on glomerular size, so the diameter ratio of afferent to efferent arterioles was compared in the Control, STZ-DM, and STZ-DM + Telmisartan rats (Figure 2C). The diameter ratios of afferent to efferent arterioles were significantly increased in the STZ-DM rats compared with the Control rats at 8 weeks. Telmisartan treatment restored the ratios of the afferent to efferent arterioles to those of the Control rats. It was also clear that there was an increased diameter ratio of the afferent to efferent arterioles in STZDM and this was restored by short-term use of an angiotensin receptor blocker. Permeability Changes in Early Diabetic Glomeruli

Glomerular hyperfiltration of macromolecules was examined by intravenous injection of 40-kd dex-

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Table 2. Physiologic data at 8 weeks

Control n BW (g) LKW ⁄ BW 100 g BG (mg ⁄ dL) SBP (mmHg) S-Cre (mg ⁄ dL) BUN (mg ⁄ dL) UAE (mg ⁄ day) Ccr (mL ⁄ min ⁄ BW 100 g)

15 385 0.54 152 119 0.21 18.7 20.2 1.03

± ± ± ± ± ± ± ±

STZ-DM

25 0.07 36 15 0.02 2.4 7.3 0.22

15 245 0.74 578 126 0.16 27.2 156.6 1.31

± ± ± ± ± ± ± ±

48a 0.11a 79a 13 0.03a 6.6a 57.5a 0.30a

STZ-DM + Telmisartan 15 229 ± 0.70 ± 570 ± 127 ± 0.16 ± 29.6 ± 89.3 ± 1.36 ±

21a 0.14a 113a 16 0.03a 6.6a 51.5b 0.34a

Data are expressed as mean ± SD. BW, body weight; LKW, left kidney weight; BG, blood glucose; SBP, systolic blood pressure; S-Cre, serum creatinine; BUN, blood urea nitrogen; UAE, urinary albumin excretion; Ccr, creatinine clearance. a P < 0.05 vs Control, bP < 0.05 vs STZ-DM.

Control

STZ-DM + Telmisartan

STZ-DM

Af Ef

Af

Ef

Ef

Figure 1. Representative three-dimensional image of glomeruli. Glomerular capillaries and arterioles were visualized by perfusion of 500-kd FITC-labeled dextran. Af, Afferent arteriole, Ef, Efferent arteriole. Scale bar = 50 lm.

tran conjugated with rhodamine (Figure 3). Glomerular capillaries and arterioles were visualized in advance by 500-kd FITC-labeled dextran perfusion. Filtered 40-kd rhodamine-labeled dextran was observed in Bowman’s capsule space in the STZ-DM rats (Figure 3, middle, Supplemental Movie S2), but in almost none of the Control rats (Figure 3, upper, Supplemental Movie S3). There was no obvious leakage of 40-kd rhodaminelabeled dextran in the glomeruli of the telmisartantreated rats (Figure 3, lower, Supplemental Movie S4). The time course of change in rhodamine intensity in the glomerular tufts and Bowman’s space was measured to evaluate the filtration ratio (Figure 4). In the STZ-DM rats, the filtration ratio was significantly increased compared with the Control rats at 4 weeks after DM induction (Figure 5A). At 8 weeks after DM induction, the ratio was much increased, whereas the STZ-DM + Telmisartan rats showed a significantly lower filtration ratio than the STZ-DM rats. The correlation between filtra-

tion ratio and urinary albumin excretion is shown in Figure 5B,C. Increased filtration ratios were observed in some glomeruli of the STZ-DM rats in spite of a lower level of urinary albumin excretion even at 4 weeks after DM induction, which indicates that glomerular hyperfiltration developed ahead of the appearance of urinary albuminuria during the early stages of diabetes in these rats. At 8 weeks after DM induction, telmisartan-treated rats showed both lower filtration ratios and lower urinary albumin excretion than DM rats. Glycocalyx Changes in Early Diabetic Glomeruli

Glomerular endothelial cells are covered with glycocalyx, which is an important contributor to the regulation of vascular permeability for macromolecules [21,26]. Therefore, we examined glomerular glycocalyx changes by perfusion of Alexa Fluor 594 conjugate isolectin GS-IB4, which binds to hyaluronic acid and heparin sulfates of the endothelial glycocalyx [19]. The isolectin GS-IB4 staining in the Control rats was uniform in the glomeruli, but was weak and nonuniform in the STZ-DM rats (Figure 6). The staining score was significantly decreased in the STZ-DM rats compared with the Control rats (1.6 ± 0.4 and 3.5 ± 0.2, respectively, P < 0.05). Telmisartan treatment partially restored the glomerular staining by isolectin GS-IB4 (staining score 2.7 ± 0.3, P < 0.05 vs. STZ-DM). DISCUSSION

We established an in vivo method to visualize the microcirculation and glomerular permeability of macromolecules using a two-photon laser microMicrocirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

Glomerular filtration in STZ rats M. Satoh et al.

C 15 *

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Figure 2. Measurement of glomerular morphology. (A) Glomerular volume in Control, STZ-DM, and STZDM + Telmisartan rats at 4 and 8 weeks after induction of diabetes. Fifty glomeruli were measured in each group. Data were expressed as mean ± SD. *P < 0.05 vs Control. (B) Distribution of glomerular volume at 4 and 8 weeks after induction of diabetes. (C) Altered diameter ratio of Afferent (Af) ⁄ Efferent (Ef) arterioles in Control, STZ-DM, and STZ-DM + Telmisartan rats at 8 weeks after induction of diabetes (n = 10 in each group). Data were expressed as mean ± SD. *P < 0.05 vs Control. †P < 0.05 vs STZ-DM.

Figure 3. Representative series of images showing hyperfiltration in glomeruli. Green color; 500-kd FITC-labeled dextran. Red color; 40-kd Rhodamine-labeled dextran. Scale bar = 50 lm.

scope. The results of this study indicate that glomerular hyperfiltration started during the early phase of diabetes and was accompanied by dilatation of the afferent arterioles and glomerular hypertrophy. Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

Examining the mechanisms of renal microvascular alterations in DM and the ramifications for overall organ function has remained a challenge because of the complexity of the renal microvascular system.

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A

Inner glomerular tuft

Fluorescence intensity

B

a

Time Bowman’s capsule space

Fluorescence intensity

C

b

Time

Figure 4. Evaluation method for filtration ratio. The blue area (a) in B indicates the amount of 40-kd dextran that reached the point in a glomerular tuft (blue circle in A). The red area (b) in C indicates the amount of the same dextran filtered to the point in a Bowman’s capsule space (red circle in A). The equation for filtration ratio is as follows: Filtration ratio = Average of area (b) ⁄ Average of area (a) from each triplicate point.

Several types of experimental techniques have been developed to evaluate renal microcirculation. Although each method possesses excellent and unique characteristics, they all require substantial manipulation that might alter the renal microvascular responsiveness. With the recent invention of the two-photon laser scanning fluorescence microscope, we can directly observe various dynamic cellular processes in the tissues of living organisms. A pencil lens-probe charge coupled device intravital videomicroscopic system is used to evaluate both systemic hemodynamics and renal microcirculation [35]. Furthermore, real-time images of the afferent and efferent arterioles as well as the glomeruli can

be continuously assessed, facilitating the functional characterization of these microvessels in vivo [36]. Two-photon fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution and has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, tumors, and kidney [28]. More importantly, the dynamic processes and multiple functions of an intact organ can be visualized and quantified in real-time using noninvasive methods [15]. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in realtime without fixation artifacts. In the present study, we used two-photon microscopy, which can achieve a level of resolution previously unattainable in intravital microscopy, to perform kinetic analyses and physiological studies of the diabetic kidney in living animals. We acquired optical sections of glomerular images with the two-photon microscope, and the sequential images were reconstructed into three-dimensional images. We confirmed that the glomeruli were enlarged in the STZ-DM rats, which is a well recognized feature of DM and has been observed at both early (microalbuminuric) and late (proteinuric) stages of nephropathy [10]. Early changes in glomerular hemodynamics have been implicated in the pathogenesis of diabetic glomerulopathy [12] including glomerular enlargement. It has been suggested that the observed loss of autoregulation results in increased glomerular capillary pressure, hyperfiltration, and glomerular hypertrophy secondary to increases in capillary length and surface area. The mechanisms of the loss of autoregulation in the STZ-induced diabetic kidney is thought to involve attenuation of pressure-induced afferent arteriolar vasoconstriction [11]. These changes take place within 4 days in the diabetic rat [22]. Loss of autoregulation at the onset of diabetes is also a feature of patients with diabetes [8,24]. Impaired autoregulation histologically appears as dilation of the afferent arterioles. We confirmed by in vivo renal imaging that the afferent artery was extended compared with the efferent artery in the early stage of diabetic nephropathy in rats. This result is in line with previous work reported by Li et al., who found that the afferent ⁄ efferent artery ratio was slightly increased in diabetic rats. In our study, treatment with a selective Ang II receptor antagonist for 1 week did not change the glomerular size in the Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

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Figure 5. Filtration ratio and urinary albumin excretion. (A) Filtration ratios in Control (n = 10) and STZ-DM (n = 11) at 4 weeks, and in Control (n = 15), STZ-DM (n = 13) and STZ-DM + Telmisartan (n = 9) rats at 8 weeks after induction of diabetes. Data were expressed as mean ± SD. *P < 0.05 vs Control. †P < 0.05 vs STZDM. (B, C) Correlation between filtration ratio and urinary albumin excretion at 4 (B) and 8 weeks (C) after induction of diabetes.

STZ-DM + Telmisartan rats. However, treatment improved the afferent ⁄ efferent artery ratio, which may have caused attenuation of the glomerular hyperfiltration. Indeed, treatment with the selective an Ang II receptor antagonist improved glomerulus hemodynamics as a short-term effect. Further studies are needed to clarify whether long-term treatment might influence the glomerular enlargement. We demonstrated that the distribution of the glomerular volumes in the STZ-DM rats was larger than in the Control rats. Indeed, all glomeruli are not enlarged in diabetic nephropathy, and our result is compatible with a previous report that the heterogeneity of glomerular volume distribution in diabetic nephropathy varies widely [32]. The authors of that study found that the Otsuka Long Evans Tokushima Fatty rat showed significantly higher heterogeneity in glomerular volume distribution than the age-matched Long-Evans Tokushima Otsuka rat. On the other hand, we did not confirm a relationship between glomerular volume and gloMicrocirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

merular permeability (data not shown). In insulindependent DM, glomerular enlargement can predict the development of overt nephropathy [5,23,29]. However, when we observed glomeruli in vivo, the permeability was not aggravated as the glomerulus grew larger. Using our method, we could observe only the glomeruli of the outer layer in the kidney. In spontaneously hypertensive rats, glomerular injury first appeared predominantly in the juxtamedullary nephrons and then extended toward more superficial nephrons [13]; thus, future research will be necessary to determine whether the results we observed were influenced by the location of the glomeruli. It has been reported that the permeability of albumin in the glomerulus increased immediately after the onset of DM, although there are few studies that examined the glomerular permeability of macromolecules including albumin in vivo. We showed that the glomerular permeability of macromolecules was increased in the STZ-DM rats. Indeed, glomer-

Glomerular filtration in STZ rats M. Satoh et al.

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3 2

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Figure 6. WGA-lectin staining in glomeruli at 8 weeks after induction of diabetes. Glomerular endothelial lectin was detected by perfusion of rhodamine-labeled WGA. Scale bar = 50 lm. Staining score was calculated as described in the Methods section (n = 10 in each group). Data were expressed as mean ± SD. *P < 0.05 vs Control. †P < 0.05 vs STZ-DM.

ular hyperfiltration occurred in the early stage of DM. Hyperfiltration as a transient disorder of glomerular function at the early stage of kidney disease may appear before albuminuria [6]. However, there is a poor correlation between urinary albumin excretion and glomerular filtration rate in patients with microalbuminuric insulin-dependent DM [4]. We also did not detect a relationship between glomerular permeability and urinary albumin excretion. Some 90% of normally filtered albumin has been shown to undergo proximal tubular reabsorption, and it has been shown that albumin reabsorption by the renal tubules deteriorates in early diabetic nephropathy [25,31]. We also showed that the hyperfiltration of the glomeruli had already occurred in the diabetic rat before it exhibited albuminuria. Moreover, albumin excretion was decreased when the glomerular hyperfiltration was reduced by treatment with a selective Ang II receptor antagonist. These results suggest that the hyperfiltration of the glomeruli leads to urinary albuminuria and that glomerular hyperfiltration may precede the decline in renal tubule function.

In the STZ-DM rats, the glomerular glycocalyx detected by GS-IB4 staining was decreased compared with the Control rats. The glycocalyx that covers the luminal surface of the endothelial cells and fills the fenestrae may be an important determinant of glomerular permeability [14]. We confirmed that hyperfiltration of macromolecules occurred in the glomerulus when deterioration of the glycocalyx was observed by in vivo imaging (data not shown). Treatment with a selective Ang II receptor antagonist reduced the loss of glycocalyx and also improved the hyperfiltration of macromolecules. It has been reported that the glycocalyx was decreased by high glucose [37], reactive oxygen species [16], or Ang II [17]. Earlier, we reported that treatment with a selective Ang II receptor antagonist ameliorated glomerular oxidative stress in renal diseases [9,27]. Hence, treatment with a selective Ang II receptor antagonist could inhibit the decrease in glycocalyx by reducing reactive oxygen species. Telmisartan has been reported to have PPARgamma agonistic activity, thus there is a possibility that this PPAR-gamma agonistic activity might contribute to renoprotection. We have previously reported that the PPAR-gamma agonist pioglitazone is effective in preventing obesity-related renal injury via blockade of NAD(P)H oxidase [20]. The PPAR-gamma activity of telmisartan might, therefore, cause reduction of ROS production in conjunction with the AT1R blockage activity of telmisartan in the STZ-DM glomeruli. CONCLUSIONS

In conclusion, we established an in vivo method to visualize microcirculation using a two-photon laser microscope. We have explored the glomerular structure and the presence of glomerular hyperfiltration and found that diabetic rats already show hyperfiltration of macromolecules in the early stage of DM. Treatment with a selective Ang II receptor antagonist may normalize glomerular hyperfiltration partially by reducing glomerular hypertension and by maintaining the glycocalyx. ACKNOWLEDGEMENTS

This work was supported by the KAKENHI (19590969 to NK), the Kawasaki Foundation for Medical Science and Medical Welfare (to MS), and the Okayama Medical Foundation (to MS). Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

Glomerular filtration in STZ rats M. Satoh et al.

Telmisartan was kindly supplied by Astellas Pharma Inc. (Tokyo, Japan). We are also grateful to Ms. Satomi Hanada and Ms. Etsuko Yorimasa for animal care and excellent technical assistance.

13.

Declaration of interest: There are no conflicts of interest for all authors.

14.

REFERENCES 1. Anderson S, Vora JP. (1995). Current concepts of renal hemodynamics in diabetes. J Diabetes Complications 9:304–307. 2. Bak M, Thomsen K, Christiansen T, Flyvbjerg A. (2000). Renal enlargement precedes renal hyperfiltration in early experimental diabetes in rats. J Am Soc Nephrol 11:1287–1292. 3. Bakris G, Burgess E, Weir M, Davidai G, Koval S. (2008). Telmisartan is more effective than losartan in reducing proteinuria in patients with diabetic nephropathy. Kidney Int 74:364–369. 4. Bangstad HJ, Osterby R, Rudberg S, Hartmann A, Brabrand K, Hanssen KF. (2002). Kidney function and glomerulopathy over 8 years in young patients with Type I (insulin-dependent) diabetes mellitus and microalbuminuria. Diabetologia 45:253–261. 5. Bilous RW, Mauer SM, Sutherland DE, Steffes MW. (1989). Mean glomerular volume and rate of development of diabetic nephropathy. Diabetes 38:1142– 1147. 6. Bouhanick B, Gallois Y, Hadjadj S, Boux de Casson F, Limal JM, Marre M. (1999). Relationship between glomerular hyperfiltration and ACE insertion ⁄ deletion polymorphism in type 1 diabetic children and adolescents. Diabetes Care 22:618–622. 7. Brenner BM, Lawler EV, Mackenzie HS. (1996). The hyperfiltration theory: a paradigm shift in nephrology. Kidney Int 49:1774–1777. 8. Christensen PK, Hansen HP, Parving HH. (1997). Impaired autoregulation of GFR in hypertensive non-insulin dependent diabetic patients. Kidney Int 52:1369–1374. 9. Fujimoto S, Satoh M, Horike H, et al. (2008). Olmesartan ameliorates progressive glomerular injury in subtotal nephrectomized rats through suppression of superoxide production. Hypertens Res 31:305–313. 10. Gundersen HJ, Osterby R. (1977). Glomerular size and structure in diabetes mellitus. II. Late abnormalities. Diabetologia 13:43–48. 11. Hayashi K, Epstein M, Loutzenhiser R, Forster H. (1992). Impaired myogenic responsiveness of the afferent arteriole in streptozotocin-induced diabetic rats: role of eicosanoid derangements. J Am Soc Nephrol 2:1578–1586. 12. Hostetter TH, Rennke HG, Brenner BM. (1982). The case for intrarenal hypertension in the initiation Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

111

and progression of diabetic and other glomerulopathies. Am J Med 72:375–380. Iversen BM, Amann K, Kvam FI, Wang X, Ofstad J. (1998). Increased glomerular capillary pressure and size mediate glomerulosclerosis in SHR juxtamedullary cortex. Am J Physiol 274:F365–373. Jeansson M, Haraldsson B. (2006). Morphological and functional evidence for an important role of the endothelial cell glycocalyx in the glomerular barrier. Am J Physiol Renal Physiol 290:F111–116. Kang JJ, Toma I, Sipos A, McCulloch F, Peti-Peterdi J. (2006). Quantitative imaging of basic functions in renal (patho)physiology. Am J Physiol Renal Physiol 291:F495–502. Kashihara N, Watanabe Y, Makino H, Wallner EI, Kanwar YS. (1992). Selective decreased de novo synthesis of glomerular proteoglycans under the influence of reactive oxygen species. Proc Natl Acad Sci USA 89:6309–6313. Koppel H, Yard BA, Christ M, Wehling M, van der Woude FJ. (2003). Modulation of angiotensin IImediated signalling by heparan sulphate glycosaminoglycans. Nephrol Dial Transplant 18:2240– 2247. Makino H, Haneda M, Babazono T, et al. (2008). Microalbuminuria reduction with telmisartan in normotensive and hypertensive Japanese patients with type 2 diabetes: a post-hoc analysis of The Incipient to Overt: Angiotensin II Blocker, Telmisartan, Investigation on Type 2 Diabetic Nephropathy (INNOVATION) study. Hypertens Res 31:657–664. Megens RT, Reitsma S, Schiffers PH, et al. (2007). Two-photon microscopy of vital murine elastic and muscular arteries. Combined structural and functional imaging with subcellular resolution. J Vasc Res 44:87–98. Namikoshi T, Satoh M, Tomita N, et al. (2007). Pioglitazone ameliorates endothelial dysfunction in obese rats with nephropathy. Biochem Biophys Res Commun 361:835–840. Nieuwdorp M, Mooij HL, Kroon J, et al. (2006). Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes 55:1127–1132. Osterby R, Gundersen HJ. (1980). Fast accumulation of basement membrane material and the rate of morphological changes in acute experimental diabetic glomerular hypertrophy. Diabetologia 18:493– 500. Osterby R, Gundersen HJ. (1975). Glomerular size and structure in diabetes mellitus. I. Early abnormalities. Diabetologia 11:225–229. Parving HH, Kastrup H, Smidt UM, Andersen AR, Feldt-Rasmussen B, Christiansen JS. (1984). Impaired autoregulation of glomerular filtration rate in type 1 (insulin-dependent) diabetic patients with nephropathy. Diabetologia 27:547–552.

112

Glomerular filtration in STZ rats M. Satoh et al.

25. Russo LM, Sandoval RM, Campos SB, Molitoris BA, Comper WD, Brown D. (2009). Impaired tubular uptake explains albuminuria in early diabetic nephropathy. J Am Soc Nephrol 20:489–494. 26. Satchell SC, Tooke JE. (2008). What is the mechanism of microalbuminuria in diabetes: a role for the glomerular endothelium? Diabetologia 51:714– 725. 27. Satoh M, Fujimoto S, Arakawa S, et al. (2008). Angiotensin II type 1 receptor blocker ameliorates uncoupled endothelial nitric oxide synthase in rats with experimental diabetic nephropathy. Nephrol Dial Transplant 23:3806–3813. 28. Sipos A, Toma I, Kang JJ, Rosivall L, Peti-Peterdi J. (2007). Advances in renal (patho)physiology using multiphoton microscopy. Kidney Int 72:1188–1191. 29. Steffes MW, Osterby R, Chavers B, Mauer SM. (1989). Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients. Diabetes 38:1077–1081. 30. Steinhausen M, Snoei H, Parekh N, Baker R, Johnson PC. (1983). Hydronephrosis: a new method to visualize vas afferens, efferens, and glomerular network. Kidney Int 23:794–806. 31. Tojo A, Onozato ML, Ha H, et al. (2001). Reduced albumin reabsorption in the proximal tubule of early-stage diabetic rats. Histochem Cell Biol 116:269–276. 32. Toyota E, Ogasawara Y, Fujimoto K, et al. (2004). Global heterogeneity of glomerular volume distribution in early diabetic nephropathy. Kidney Int 66:855–861. 33. Tucker BJ, Collins RC, Ziegler MG, Blantz RC. (1991). Disassociation between glomerular hyperfiltration and extracellular volume in diabetic rats. Kidney Int 39:1176–1183. 34. Veelken R, Hilgers KF, Hartner A, Haas A, Bohmer KP, Sterzel RB. (2000). Nitric oxide synthase isoforms and glomerular hyperfiltration in early diabetic nephropathy. J Am Soc Nephrol 11:71–79. 35. Yamamoto T, Hayashi K, Matsuda H, et al. (2000). Direct in vivo visualization of glomerular microcir-

culation by intravital pencil lens-probe CCD videomicroscopy. Clin Hemorheol Microcirc 23:103–108. 36. Yamamoto T, Tomura Y, Tanaka H, Kajiya F. (2001). In vivo visualization of characteristics of renal microcirculation in hypertensive and diabetic rats. Am J Physiol Renal Physiol 281:F571–577. 37. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C. (2005). Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. J Appl Physiol 99:1471–1476. SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article (http://www.microcirculationjournal.com, click on View content online): Movie S1. A three-dimensional image of rat glomerulus. Green color; 500-kd FITC-labeled dextran. Movie S2. Glomerular hyperfiltration of macromoleculesn in the STZ-DM rats. Green color; 500-kd FITC-labeled dextran. Red color; 40-kd Rhodamine-labeled dextran. Movie S3. Glomerular hyperfiltration of macromoleculesn in the Control rats. Movie S4. Glomerular hyperfiltration of macromoleculesn in the STZ-DM+Telmisartan rats. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Microcirculation 2010, 17, 103–112  2010 Blackwell Publishing Ltd

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