Aminoguanidine induces haematuria of non-glomerular origin in spontaneously hypertensive rats

Takami Arai, Kengo Morimoto1, Megumi Oka, Tomoyuki Hikita, Kentaro Arai, Kiyoshi Umezawa2, Mitsumasa Nagase and Tatsuo Yamamoto3

Department of Medicine, Teikyo University School of Medicine, 2–11–1 Kaga, Itabashiku., Tokyo 173–8605, 1 Teikyo University School of Medicine, Mizonokuchi, Takatsuku, Kawasakisi, 2 Sysmex, Co. Ltd, Kobe and 3 Seirei Hamamatsu Hospital, Department of Internal Medicine, Division of Nephrology, Sumiyoshi, Hamamatsu, Shizuoka, Japan



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Administration of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase (NOS), induces glomerulosclerosis in spontaneously hypertensive rats (SHR). We investigated the effects of administering aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS), on glomerular histology, serum creatinine concentration, albuminuria and haematuria in SHR.

Methods. SHR and Wistar Kyoto rats (WKR) (age, 7 weeks) were given a daily water supply with or without 0.1% AG. Every 4 weeks, 24 h urine samples were collected and checked for haematuria by a dipstick method, and systolic blood pressure was measured. After 16 weeks, serum creatinine, albuminuria and glomerulosclerosis indices (GSI) were evaluated, and the size of urinary erythrocytes in AG-treated SHR was measured by flow cytometry. Glomeruli were observed by transmission and scanning electron microscopy. Some AG-treated SHR received a furosemide injection and then urinary erythrocyte size was determined.

Results. Systolic blood pressure, serum creatinine, albuminuria and GSI were similar between the untreated and AG-treated groups in both strains. However, AG treatment induced significant haematuria in SHR, but not in WKR. Electron microscopy did not provide any evidence for glomerular bleeding sites in AG-treated SHR. In urine with osmolalities exceeding 750 mOsm/kg, haematuria of AG-treated SHR consisted of erythrocytes smaller in size than venous erythrocytes. After furosemide injection leading to near isotonic urine, the size of urinary erythrocytes was similar to that of venous erythrocytes.

Conclusions. The absence of morphological evidence for glomerular bleeding sites and similar intrinsic size between urinary and venous erythrocytes suggest that AG induces a non-glomerular type of haematuria in SHR.

Keywords: aminoguanidine; flow cytometry; haematuria; spontaneously hypertensive rats



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Nitric oxide is important in both progression and amelioration of renal disease (reviewed in [1]). Two distinct nitric oxide synthase isoforms, constitutive (cNOS) and inducible (iNOS), have been identified [1]. Administration of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, alters the course of some renal diseases [1]. Segmental glomerular hyalinosis and sclerosis as well as afferent arteriolar fibrinoid necrosis appeared in spontaneously hypertensive rats (SHR) following 3 weeks of L-NAME administration [2]. iNOS was described originally in activated macrophages, but has also been demonstrated in glomerular endothelial [3] and mesangial cells [4] after stimulation with cytokines. In addition, Tojo et al. [5] have demonstrated recently that iNOS can be expressed constitutively in the terminal glomerular afferent arterioles. Thus, inhibition of iNOS in glomeruli and afferent arterioles may induce pathological lesions in SHR. Aminoguanidine (AG) is generally accepted as a specific inhibitor of iNOS [6]. The aim of this study was to evaluate the effects of AG administration on renal function and histology in SHR.

In the present study, we demonstrated that AG administration induced haematuria in SHR, with the absence of morphological evidence for glomerular bleeding. We also examined whether AG-induced haematuria in SHR is of glomerular or non-glomerular origin by measuring the size of urinary erythrocytes.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Male SHR and Wistar Kyoto rats (WKR), weighting ~200 g, were purchased at the age of 7 weeks from Charles River (Tokyo, Japan). WKR served as controls for comparison with SHR. After collecting 24 h urine samples using metabolic cages, each rat strain was subdivided into two groups, specifically: untreated WKR (n=22), AG-treated WKR (n=24), untreated SHR (n=22) and AG-treated SHR (n=22). The AG-treated SHR and WKR were given 0.1% aminoguanidine sulfate (Wako, Osaka, Japan) in drinking water available ad libitum. Every 4 weeks, 24 h urine samples were collected and haematuria was assessed semiquantitatively by a dipstick method (Hema-Combistix, Ames Byer, Tokyo, Japan). Haematuria was graded as -, ±, 1+, 2+ or 3+ by comparing colour reaction dipsticks with a colour chart. To confirm the presence of haematuria, urine samples were examined by light microscopy (x400). Body weight, 24 h water and food intake, and 24 h urinary albumin excretion were measured every 4 weeks. Albumin excretion was determined by an immunodiffusion method described previously [6]. Systolic blood pressure was measured in the tail artery with a manometer–tachometer (KN-210–1, Nature Co., Tokyo, Japan). After collecting 24 h urine samples at 16 weeks, 3 h urine samples were collected subsequently to measure the size and number of urinary erythrocytes by flow cytometry as described below, after which some rats received an intraperitoneal injection of furosemide, as also described below.

Size of urinary and venous erythrocytes estimated by automated flow cytometry
We measured the size of urinary and venous erythrocytes with an automated flow cytometer (UF-100, Sysmex Co., Kobe, Japan) [7], because of the proposal that the origin of urinary erythrocytes in human subjects can be differentiated by measuring their size: small erythrocytes are said to originate from glomeruli, while those similar in size to venous erythrocytes apparently have a non-glomerular origin [8,9]. Since urine osmotic changes lead to alterations in erythrocyte shape [10,11], we also evaluated osmotic effects on the size of erythrocytes. Blood samples (0.1 ml) obtained from the tail vein with a heparinized syringe were diluted with a standard solution (Kindaly Solution AF-2, Fusou Co., Osaka, Japan): pH 7.4; 300 mOsm/kg; Na+, 140 mEq/l; K+, 2.0 mEq/l; Ca2+, 3.0 mEq/l; Mg2+, 1.0 mEq/l; Cl-, 110 mEq/l; CH3COO-, 8 mEq/l; and glucose, 100 mg/dl. Three graded concentrations of blood suspension (0.001, 0.01 and 0.1%) were prepared, and then incubated in solutions with osmolalities varying between 130 and 3000 mOsm/kg that had been prepared by increasing or decreasing the final volume of the standard solution. The effects of pH and albumin concentrations on the size of erythrocytes were also examined. The blood suspension (0.1%) was incubated for 1 h in solutions with pHs ranging from 3 to 12, prepared by adjusting the standard solution with 10 M HCl or NaOH. Blood suspensions were incubated for 1 h in standard solution supplemented with graded concentrations of albumin (0–6 g/dl). The relative size of erythrocytes after incubation in each solution was expressed as a percentage of the size after incubation in the standard solution.

To investigate whether size reduction of erythrocytes induced by high osmolality solutions is reversible, venous erythrocytes were transferred to standard solution after a 30 min incubation in a 1200 mOsm/kg solution, and were incubated at the lower osmolality for 120 min before their size was assessed.

In the 3 h urine samples, the size and number of urinary erythrocytes were determined with the UF-100 system. If the volume of the 3 h urine samples did not reach 0.8 ml, the 200 mOsm/kg solution was added to the urine samples to reach the 0.8 ml minimum volume required for analysis with the UF-100 equipment (dilution of a blood suspension with the 200 mOsm/kg solution did not affect the size of erythrocytes, as described below). Urine osmolality was measured with an osmometer (Fiske, Norwood, MA).

Furosemide injection
To investigate whether or not size reduction in urinary erythrocytes is induced by high urine osmolality, certain rats were injected with furosemide solution (Nippon Hoechst Marion Roussel, Tokyo, Japan) at a concentration of 0.5 mg/ml in 0.9% NaCl, as outlined below. After collecting 3 h urine samples after 16 weeks of AG administration, some AG-treated SHR showing 3+ haematuria by dipstick in 3 h urine samples received an intraperitoneal injection of furosemide solution (0.4 ml/100 g). Urine samples were collected every 15 min until 60 min after the furosemide injection. The size and number of urinary erythrocytes were determined in the first urine sample after furosemide injection with an osmolality <750 mOsm/kg.

Histology and blood samples
After 24 or 3 h urine samples were collected at 16 weeks of AG treatment or observation with no treatment, rats from each of the four groups were killed. Pentobarbital sodium (0.1 ml/100 g; Nembutal, Abbott Laboratories, North Chicago, IL) was injected intraperitoneally. By a flank incision, the right kidney was removed and processed for morphological evaluation by light, immunofluorescence and electron microscopy. Blood samples were obtained from the aorta to measure serum creatinine, urea nitrogen, total protein and albumin concentrations. Coronal sections of the right kidney were fixed overnight in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer, pH 7.2. Sections at a 2 mm thickness were stained with haematoxylin and eosin, periodic acid–Schiff (PAS) and periodic acid–silver methenamine. A glomerular sclerosis index was estimated by point counting methods as described previously [6]. The frequency of glomerular lesions was determined by examination of 50 glomeruli in each rat. An arteriolar injury score was obtained as described by Ono et al. [2]. The frequency of arteriolar lesions was determined by examination of arteriolar profiles at vascular poles of 30 glomeruli. For transmission electron microscopy (TEM), the renal cortex was cut into small pieces and fixed in 2% glutaraldehyde for 2 h, followed by post-fixation in 4% OsO4 for 2 h. The specimens were treated with graded concentrations of ethanol, embedded in Epon and evaluated using a transmission electron microscope (JEM-1200EX, Nihondenshi, Tokyo, Japan).

Evaluation of the glomerular basement membrane by scanning electron microscopy
The glomerular basement membrane (GBM) was observed with a scanning electron microscope (JSM-25S3III, Nihondenshi). Three AG-treated SHR with 2+ or 3+ haematuria in the 3 h urine samples and three untreated WKR without haematuria during the experimental period were selected. The left kidney was removed and the cortex was processed for evaluation by scanning electron microscopy (SEM) as described previously [12].

Statistics
Results are expressed as the mean±SEM. Student's t-test was employed following analysis of variance (ANOVA). A P-value <0.05 was considered statistically significant. {chi}2 analysis was performed when appropriate.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Body weight at each time point was similar in the four groups during the experimental period (data not shown). Serum creatinine concentrations after 16 weeks of AG or no treatment were similar in the four groups (untreated WKR, 0.32±0.01 mg/dl, n=16; AG-treated WKR, 0.37±0.03 mg/dl, n=16; untreated SHR, 0.34±0.01 mg/dl, n=16; and AG-treated SHR, 0.34±0.01 mg/dl, n=16, NS, ANOVA). Although 24 h urinary albumin excretion was greater in both SHR groups than in both WKR groups, no difference was noted between untreated and AG-treated groups in either strain (untreated WKR, 1.9±0.2 mg/day, n=22; AG-treated WKR, 1.5±0.2 mg/day, n=24; untreated SHR, 3.5±0.5 mg/day, n=22; AG-treated SHR, 4.9±0.8 mg/day, n=22, P<0.01, ANOVA).

Among urine samples from the AG-treated SHR, 55.4% manifested haematuria (Table 1Go). Haematuria occurred more frequently in AG-treated SHR than in untreated SHR (Table 1Go). In some urine samples from AG-treated SHR, reddish sediments were noted (Figure 1AGo), corresponding to the presence of numerous erythrocytes by light microscopy; these samples showed 3+ haematuria by dipstick. We defined urine samples with reddish sediment as macroscopic haematuria. Macroscopic haematuria was more frequent in AG-treated SHR than in untreated SHR (Table 1Go). No haematuria was detected in the four groups at the beginning of the experiment. Haematuria was increased from 4 weeks onward in both untreated and AG-treated SHR groups (Figure 1BGo). In contrast, haematuria was rare in both untreated and AG-treated WKR (Table 1Go); no difference was noted in prevalence of haematuria between untreated and AG-treated WKR. Twenty-four hour urine volume and water intake tended to be greater in untreated WKR than in the other three groups, but were similar between untreated and AG-treated SHR groups at each time point (data not shown). These results indicate that AG treatment induced significant haematuria in SHR, but not in WKR.


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Table 1. Haematuria in SHR and WKR with or without AG administration

 


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Fig. 1. Macroscopic haematuria (A) and the frequency of haematuria at each time point (B). (A) Urine sample 1 showed reddish sediment, unlike urine sample 2. A 24 h urine sample with the reddish sediment was defined as macroscopic haematuria. (B) The frequency of haematuria at each time point was shown in four groups. Filled bars, macroscopic haematuria; striped bars, microscopic haematuria 2+ or 3+; open bars, microscopic haematuria±or 1+. W-, untreated WKR; W+, AG-treated WKR; S-, untreated SHR; S+, AG-treated SHR.

 
Profiles of the 3 h urine samples from AG-treated SHR are presented in Table 2Go. The UF-100 system was used to measure the size of urinary erythrocytes, calculate their mean size and generate a size distribution plot [7] (Figure 2Go). The mean size of urinary erythrocytes in 3 h urine samples ranged from 71.5 to 113.9 ‘channels’, 21 channels being equal to 1 µm [7]. Three patterns of size distribution were noted. Sample number 11 in Table 2Go showed a single peak at a size similar to that observed for venous erythrocytes, as described below (Figure 2AGo; normal pattern). In contrast, sample number 10 showed two peaks, one at a size identical to that of venous erythrocytes, the other one smaller (Figure 2BGo; mixed pattern). Six of 11 samples from the AG-treated SHR showing haematuria exhibited this pattern (Table 2Go). Sample number 7 (small pattern) had a single peak at a smaller size (Figure 2CGoFigure 2BGo). Four of 11 samples showed this pattern (Table 2Go). Urine osmolalities ranged from 757 to 2262 mOsm/kg (Table 2Go).


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Table 2. Profiles of samples with haematuria in AG-treated SHR

 


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Fig. 2. An automated flow cytometer, UF-100, yielded the size distribution of urinary erythrocytes. Vertical and horizontal lines in each panel represent relative numbers of erythrocytes and erythrocyte ‘channel’ sizes, respectively, 21 channels representing 1 µm. (A) Sample 11 in Table 2Go shows a normal size distribution of urinary erythrocytes, resembling the venous erythrocytes shown in (E). (B) Sample 10 in Table 2Go shows a mixed pattern with two peaks, one at a size identical to that of venous erythrocytes and another representing a smaller size. (C) Sample 7 in Table 2Go shows a small size pattern, with a single peak smaller than that of venous erythrocytes. (D) Furosemide injection in rat number 10 increased the size of urinary erythrocytes, resulting in a normal size distribution with a reduction of urine osmolalities from 1668 to 292 mOsm/kg. (E) A size distribution of venous erythrocytes (normal pattern) was obtained after incubation for 1 h in the standard buffer (300 mOsm/kg).

 
We next examined whether a high urine osmolality leads to erythrocyte size reduction. Rats number 7, 8, 10 and 11 (Table 2Go) manifested 3+ haematuria and were injected with furosemide solution. The osmolality of the sample from rat number 10 decreased from 1668 to 292 mOsm/kg, and the erythrocyte size shifted upward, producing a peak identical in size to that of venous erythrocytes (Figure 2DGo). The pattern of sample number 11 after furosemide injection remained normal, with a mean erythrocyte size of 119.2 channels. The urine osmolality decreased from 876 to 358 mOsm/kg. Evaluation of the size distribution of samples 7 and 8 was not performed after furosemide injection, because the urine samples showed no haematuria after the injection, presumably due to dilution.

Morphological quantitative analysis revealed the GSI to be similar between untreated and AG-treated groups (untreated WKR, 15.4±0.9%, n=8; AG-treated WKR, 15.6±0.7%, n=8; untreated SHR, 17.3±1.2%, n=8; and AG-treated SHR, 17.2±1.1%, n=8, NS, ANOVA). Afferent arteriolar hyalinosis was observed very rarely, if at all, in AG-treated SHR (Figure 3AGo) or in the other three groups (not shown). TEM observation did not reveal any glomerular lesion responsible for bleeding, such as thinning of the GBM. By SEM observation, no gap formation was detected in the GBM of either untreated WKR or AG-treated SHR (Figure 3BGo).



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Fig. 3. (A) Light microscopic observation of glomerular and arteriolar findings from an AG-treated SHR. No abnormal arteriolar lesions such as fibrinoid necrosis were observed in arterioles (PAS stain; scale bar=4 µm). (B) An AG-treated SHR with 3+ haematuria in 3 h urine samples at 16 weeks of AG treatment was selected and the GBM was observed by SEM. No gap formation was noted in the GBM (scale bar=10 µm).

 
Both SHR groups had higher blood pressures than both WKR groups, but no differences were noted at any time point between untreated and AG-treated groups in either strain (data not shown).

Although venous erythrocytes of both WKR groups were larger than those of both SHR groups (P<0.01), no differences were noted between untreated and AG-treated groups in either strain (untreated WKR, 124.0±0.4 channels, n=10; AG-treated WKR, 124.4±0.4 channels, n=10; untreated SHR, 119.7±0.4 channels, n=10; and AG-treated SHR, 119.2±0.3 channels, n=10, P<0.01, ANOVA).

Incubation of venous erythrocytes from AG-treated SHR in a 300 mOsm/kg solution for 1 h resulted in a normal size distribution (Figure 2EGo). Erythrocyte size was decreased by exposure to solutions with an osmolality >750 mOsm/kg relative to erythrocytes in a solution of 300 mOsm/kg (Figure 4AGo). The mixed and small size distributions appeared after incubation in 900 and 1050 mOsm/kg solutions, respectively (histogram not shown).



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Fig. 4. (A) Osmotic effects on the size of venous erythrocytes. Solutions with the indicated osmolalities were prepared as described in Materials and methods. Three graded concentrations of blood suspension were incubated for 1 h in the solutions. The relative sizes of erythrocytes after incubation in each solution are expressed as a percentage of the size in the standard solution (300 mOsm/kg). {square}, 0.1% blood suspension; {circ}, 0.01%; {triangleup}, 0.001%. (B) Restoration of the size of erythrocytes after exposure to high osmolality solutions. Venous erythrocytes (0.1% suspension) were incubated for the times indicated in a 300 (•) or 1200 mOsm/kg ({circ}) solution. After the first 30 min of incubation in the 1200 mOsm/kg solution, the erythrocytes were transferred to the 300-mOsm/kg solution, and were incubated further ({square}). The relative size of erythrocytes after incubation for the time indicated in each solution was expressed as a percentage of the size after 30 s in the standard solution.

 
A reduction in the size of erythrocytes occurred in a 1200 mOsm/kg solution within 30 s (Figure 4BGo), and this change was not reversible upon resuspension in a 300 mOsm/kg solution (Figure 4BGo).

No size alteration occurred with respect to pH between pH 4 and 11. Albumin concentrations up to 6 g/100 ml did not significantly affect the numbers or sizes of erythrocytes.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The present study showed that haematuria occurred more frequently in AG-treated than untreated SHR groups, with the absence of morphological evidence for glomerular bleeding sites, such as thinning or gap formation in the GBM [12–14]. We and other investigators have demonstrated that SEM observation is more reliable than TEM for detecting GBM gap formation [12,15]. In the present study, however, neither TEM nor SEM revealed GBM gap formation in AG-treated SHR.

Haematuria has been classified into three types based on the size distribution pattern for urinary erythrocytes [8,9]. A size distribution resembling that shown in Figure. 2AGo, with a peak at the same or a larger size than venous erythrocytes, was defined as ‘non-glomerular’. When the distribution was shifted to the left, indicating smaller cells (e.g. Figure 2CGo), the pattern was defined as ‘glomerular’. ‘Mixed’ patterns incorporating glomerular and non-glomerular distributions have also been noted (Figure 2BGo). Based on clinical and laboratory findings including data from renal biopsy specimens, small urinary erythrocytes are likely to originate from glomeruli and larger erythrocytes from other sources [8,9]. However, these criteria do not take urine osmolality into consideration. In the present study, urine osmolalities in AG-treated SHR exceeded 750 mOsm/kg, and solutions with osmolalities greater than this induce smaller erythrocytes. Thus, urine samples showing normal or mixed cell size pattern in haematuria included erythrocytes similar in size to venous erythrocytes, but smaller cells may be induced by high urine osmolalities. In fact, urine osmotic reduction from 1668 to 292 mOsm/kg by furosemide injection restored urinary erythrocyte size to that of venous erythrocytes. Thus, urinary erythrocytes associated with AG treatment in SHR were of essentially the same size as venous erythrocytes, but varied with changes in osmotic environment. Taken together with morphological findings that showed no evidence of glomerular lesions that would allow leakage of erythrocytes, our results suggest that AG-induced haematuria in SHR is of non-glomerular origin.

AG administration ameliorates diabetic complications such as diabetic nephropathy by inhibiting generation of advanced glycation end-products [16]. No adverse effect such as haematuria in streptozotocin-induced diabetic rats has been reported so far. Susceptibility of rats to AG-related haematuria may be strain dependent. Alternatively, hypertension may contribute to haematuria and may have complicated AG treatment in our animals. Clinical trial of AG treatment on diabetic patients has been stopped recently because of major side effects (detailed information not available) in human patients which have not been seen in previous rat studies [17], necessitating observation of whether excessive episodes of haematuria occur in AG-treated diabetic patients, especially those with hypertension.

In addition to inhibiting iNOS and the formation of advanced glycation end-products, AG has been reported to cause aggregation of leukocytes, inhibit diamine oxidase, alter the renal response to insulin-like growth factor I, and decrease urine flow and sodium excretion [18]. It remains unknown, however, whether such effects are related to AG-induced haematuria in SHR. The mechanism of AG-induced haematuria in SHR remains to be determined.

In summary, administration of AG in the drinking water for 16 weeks increased haematuria in SHR. SEM and TEM did not reveal glomerular lesions, and serum creatinine concentration, albuminuria and GSI did not differ between AG-treated and untreated animals in both WKR and SHR strains. The intrinsic urinary erythrocyte size of AG-induced haematuria in SHR was similar to that of venous erythrocytes, although urinary erythrocyte size decreased at the relatively high urine osmolality of 757–2262 mOsm/kg. These results suggested that AG-induced haematuria is non-glomerular in origin.



   Acknowledgments
 
The authors gratefully acknowledge the technical assistance of Ryo Iwasaka, Youko Sakurai and Masako Yamazaki.



   Notes
 
Correspondence and offprint requests to: Mitsumasa Nagase MD, Department of Medicine, Teikyo University School of Medicine, 2–11–1, Kaga, Itabashiku, Tokyo 173–8605, Japan. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 24. 2.99
Revision received 4. 2.00.



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