Department of Medicine, Teikyo University School of Medicine, 2111 Kaga, Itabashiku., Tokyo 1738605, 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
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Abstract |
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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
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Introduction |
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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.
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Subjects and methods |
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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 (06 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 acidSchiff (PAS) and periodic acidsilver 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. 2 analysis was performed when appropriate.
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Results |
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Among urine samples from the AG-treated SHR, 55.4% manifested haematuria (Table 1). Haematuria occurred more frequently in AG-treated SHR than in untreated SHR (Table 1
). In some urine samples from AG-treated SHR, reddish sediments were noted (Figure 1A
), 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 1
). 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 1B
). In contrast, haematuria was rare in both untreated and AG-treated WKR (Table 1
); 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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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 3A) 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 3B
).
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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 2E). 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 4A
). The mixed and small size distributions appeared after incubation in 900 and 1050 mOsm/kg solutions, respectively (histogram not shown).
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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.
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Discussion |
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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. 2A, 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 2C
), the pattern was defined as glomerular. Mixed patterns incorporating glomerular and non-glomerular distributions have also been noted (Figure 2B
). 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 7572262 mOsm/kg. These results suggested that AG-induced haematuria is non-glomerular in origin.
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Acknowledgments |
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Notes |
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References |
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