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Urinary excretion of epidermal growth factor and Tamm–Horsfall protein in three rat models with increased renal excretion of urine
Regulatory Peptides 72 (1997) 179–186
Urinary excretion of epidermal growth factor and Tamm–Horsfall protein in three rat models with increased renal excretion of urine a, b c b a Jesper Thulesen *, Per Erik Jørgensen , Ole Torffvit , Ebba Nexø , Steen Seier Poulsen a
Institute of Medical Anatomy, Department B, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark b Department of Clinical Biochemistry, KH University Hospital of Aarhus, Aarhus, Denmark c Department of Internal Medicine, University Hospital of Lund, Lund, Sweden Received 10 March 1997; received in revised form 9 October 1997; accepted 10 October 1997
Abstract Epidermal growth factor (EGF) and Tamm–Horsfall protein (THP) are synthesized in the kidneys by the distal tubular cells and excreted into urine. The urinary excretion of these peptides has been suggested as a potential index for distal tubular function. The urinary excretion rates of EGF and THP were examined in three groups of rats with increased renal excretion of urine: uninephrectomy, non-osmotic polyuria and diabetic osmotic polyuria. Twenty-four hour urine samples were obtained after 7, 14 and 21 days. The urinary volume per kidney was doubled in uninephrectomy when compared to controls. There was a seven-fold increase in urinary volume in rats with non-osmotic polyuria and diabetic osmotic polyuria, as compared to controls. Uninephrectomy, non-osmotic polyuria and diabetes all affected the urinary excretion of EGF and THP differently. The EGF excretion in uninephrectomized rats was 60–80% of that of the controls, whereas THP excretion was unchanged, indicating that EGF excretion varied with renal tissue mass. Non-osmotic polyuria caused a five-fold increase in THP excretion but no change in EGF excretion. THP excretion in the diabetic rats was increased three-fold after 21 days when compared to controls, whereas EGF excretion was decreased when expressed per kidney weight. Immunohistochemistry demonstrated that EGF and THP were colocalized in the thick ascending limbs of Henle’s loops and distal tubules in all five groups of rats. In conclusion, the EGF excretion appears to follow renal tissue mass and seems independent of urinary volume, whereas THP excretion is dependent mainly on urinary volume. This has implications for the use of EGF and / or THP excretion rates as an indicator for distal tubular function. 1997 Elsevier Science B.V. Keywords: Diabetes mellitus; Epidermal growth factor; Nephrectomy; Polyuria; Rats; Tamm–Horsfall protein
1. Introduction Epidermal growth factor (EGF) and Tamm–Horsfall protein (THP) are both synthesized in the kidneys by cells in the thick ascending limbs of Henle’s loops and in the early part of the distal convoluted tubules [1–3]. Both peptides are present in high amounts in urine, but their functional significance is unknown. EGF is a mitogenic peptide with a molecular weight of approximately 6 kDa, and THP is an extremely carbohydrate-rich glycopeptide with a molecular weight of approximately 70 000 kDa *Corresponding author: Tel.: 1 45 35327260; fax 1 45 35369612; e-mail: [email protected] 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-0115( 97 )01058-6
[2,4]. EGF may conduct reparative and regulatory processes in the kidney and participate in maintaining the integrity of the urothelium in the urinary tract [5]. THP is the most abundant glycoprotein of renal origin present in normal urine [2], and it provides the matrix for urinary cast formation [6]. It has been suggested that THP is involved in the generation of the hypotonic fluid delivered to the distal nephron, possibly by rendering the thick ascending limb of Henle’s loop relatively impermeable to water [7]. The contribution of EGF and THP from plasma to urine is negligible, since the excretion of EGF from plasma accounts for less than 10% of the urinary excretion [8–10], and THP cannot be filtrated from plasma because of its high molecular weight. Especially THP, but also EGF,
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have been used as markers of distal tubular function of the kidney in studies concerned with various renal diseases [11–15]. The present study was undertaken to examine and compare the renal excretion rates of EGF and THP in three experimental rat models with increased renal excretion of urine: uninephrectomized rats, rats with non-osmotic polyuria and untreated diabetic rats with osmotic polyuria. Our goal was to dissociate the effects of producing larger-thannormal diuresis on the urinary excretion of EGF and THP from those of pathologic diuresis in diabetes and of compensatory renal processes in uninephrectomy.
glucose concentration was subsequently measured every morning and showed values above 20 mmol l 21 . (5) Insulin-treated diabetes, n 5 6 (served as controls for the untreated diabetic group). Experimental diabetes was induced and blood glucose concentration measured as described for the untreated diabetic rats. The rats were treated daily to normoglycaemia with a long-acting heattreated insulin preparation (Ultralente, pH 5.5, Novo-Nordisk, Denmark) in accordance with recommended treatment of diabetic rats [17]. (6) Control, n 5 6. Control animals were injected intraperitoneally with 50 mM citrate buffer (pH 4.0) in a volume equivalent to the volume injected in diabetesinduced animals.
2. Materials and methods
2.1. Experimental groups
2.2. Experimental procedure
The animal studies were approved by the local animal committee of Copenhagen, Denmark. Eight-week-old female Wistar rats, weighing 200–220 g (Panum Institute, Copenhagen, Denmark), were randomly allocated into the following groups: (1) Unilateral nephrectomy, n 5 6. Nephrectomy was performed under barbiturate anaesthesia (Brietal, methohexital, 50 mg kg 21 i.p., Eli Lilly, USA) five days before the rats were placed in metabolic cages. A small flank incision was made, the pedicle of the right kidney was tied, and the kidney was removed. Care was taken not to damage the adrenal gland. The muscle and skin were then sutured. Analgesics (Temgesic, 0.1 mg kg 21 s.c., buprenorfin, Reckitt & Colman Pharmaceuticals, UK) were injected before the operative procedure and then postoperatively twice daily for two days. (2) Sham-operated rats, n 5 6. The pre- and post-operative procedures were as described for the uninephrectomized group. The right kidney was lightly manipulated manually, and the muscle and skin were then sutured. (3) Non-osmotic polyuria, n 5 6. Five percent sucrose was added to the drinking water, and the availability of rat chow was restricted to 8 g day 21 five days before the rats were placed in the metabolic cages. Sucrose water and food restriction result in non-osmotic polyuria caused by a high water intake [16]. Urine samples were tested for glucose (BM-Test-5L, Boehringer Mannheim, Germany) to exclude glucosuria. (4) Streptozotocin-induced experimental diabetes, n 5 6. A single intraperitoneal injection of 60 mg kg 21 streptozotocin (Sigma Chemical Co., USA) dissolved immediately before administration in freshly prepared 50 mM citrate buffer (pH 4.0) was administered five days before the rats were placed in metabolic cages. Blood glucose concentration was measured 48 h later by a One Touch II instrument (Lifescan, USA) using the glucose oxidase method, and only rats with a blood glucose concentration exceeding 20 mmol l 21 were included in the study. Blood
The rats were kept in individual metabolic cages (Techniplast, model 1700, Italy) for the following periods: days 5–7, 12–14, and 19–21. The 24 h urine samples were obtained on days 7, 14, and 21 after initial habituation of the rats to the metabolic cages. The temperature (218C) and relative humidity (55%) of the room were controlled, with a 12 h light–dark cycle. The rats were allowed free access to drinking water and rat chow (Altromin No. 1314, Altromin International, Germany), unless otherwise stated. Urine samples were stored at 2 208C until analysed.
2.3. Immunohistochemistry On day 21, the rats were anaesthetized with barbiturate (Brietal, Methohexital, Eli Lilly, USA) and the kidneys were removed and weighed. The right kidney was immediately fixed by immersion into ice-cold, freshly prepared, buffered 4% paraformaldehyde. The fixed tissue samples were embedded in paraffin and cut into 10 mm sections using a microtome and then placed on gelatine-coated glass slides. The sections were pre-embedded in 10% porcine serum in 0.1 mM phosphate buffer, pH 8.0, for 30 min. The primary antisera were rabbit anti-rat urinary EGF (90001) diluted 1:3200 and rabbit anti-rat THP diluted 1:3200. The immunoreactions were visualized by means of the streptavidin / biotin system using biotinylated swine anti-rabbit immunoglobulins (code No. E353, DAKO, Denmark) as the second layer and StreptABComplex / Horseradish peroxidase (code No. 377, DAKO, Denmark) as the third layer, with diaminobenzidine for staining. Sections were counterstained with haematoxylin. The sections were examined by means of a Zeiss Axiophot microscope. The examiner of the histological sections was not aware of the origin of the specimens when evaluating the stained sections. Photographs (Agfa-pan APX25 film) were used for comparison of neighbouring sections stained for EGF and THP, respectively.
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2.4. Tissue samples for biochemical analyses The left kidney from control and diabetic rats was homogenized at 08C in 10 ml homogenization buffer (10 mM piperazine-N,N9-bis (2-ethanesulphonic acid), 1 mg EGTA, 3 mM magnesium chloride, 400 mM sodium chloride, 5 N NaOH, final pH 7.4) per g tissue. The homogenate was centrifuged (20 000g, 40 min, 48C). The resulting supernatant (supernatant 1) was stored at 2 208C. The pellet was resuspended in the homogenization buffer (10 ml / g tissue) with 2% triton X-100 to make the membrane bound forms of EGF soluble from the renal tissue. The resuspended pellet was incubated overnight at 48C prior to centrifugation (20 000g, 40 min, 48C). The resultant supernatant, supernatant 2, was stored at 2 208C. The total contents of EGF and protein were calculated as the sum of the contents in the two supernatants, and corrected with the factor obtained by dividing kidney wet tissue weight with the homogenized amount of renal tissue.
2.5. Analytical techniques EGF was measured by an enzyme-linked immunoassay (ELISA) [9]. In brief, the IgG fractions from two rabbit antisera raised against rat-EGF were used as catching and detecting antibodies, respectively, and EGF purified from rat urine was used as a calibrator [9]. The two supernatants from renal tissue samples were diluted 1:2 in 0.1 M sodium phosphate, 0.1% albumin, pH 8.0, prior to trypsinization as previously described [18]. THP was analysed with a simplified ELISA as previously described [19]. In brief, antibodies against both rat and human urinary THP were raised in rabbits, and THP was purified from rat urine and used as calibrator. Protein was measured employing the Pierce BCA method. Creatinine was measured with automatic equipment (SMAC, Technicon, USA).
2.6. Statistics Results are shown as the median and range. Comparison between groups was performed using the Kruskal–Wallis one-way analysis by ranks, and if significant difference was found, followed by the non-parametric Mann–Whitney U-test (two-tailed). Probability values of P , 0.05 were considered significant.
3. Results The 24 h volume of urine (Fig. 1A) was approximately 20 ml from the control and the sham-operated control groups on days 7, 14 and 21. It was increased approximately seven-fold on these days in the diabetic and sucrose-fed rats, whereas the volume of urine from uni-
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nephrectomized and insulin-treated diabetic rats was comparable to that of controls. The 24 h urinary excretion of EGF (Fig. 1B) remained at a constant level of approximately 2 nmol in the control and the sham-operated control groups. It was reduced in the uninephrectomized group on days 7, 14 and 21 to 75%, 60% and 84%, respectively, of the level in the shamoperated control group. The excretion of EGF from diabetic, insulin-treated diabetic and sucrose-fed rats was comparable to that of the controls, even though diabetic rats tended to excrete less EGF than controls on day 14. The 24 h urinary excretion of THP (Fig. 1C) from the control and the sham-operated control groups also remained at a constant level, approximately 100 mg, on days 7, 14 and 21, whereas the sucrose-fed rats had a four- to six-fold increase in their urinary excretion of THP on the days measured. The excretion of THP from the diabetic group was comparable to that of the controls on day 7 and gradually increased on days 14 and 21, but never to the level of the sucrose-fed rats. The excretion of THP from uninephrectomized and insulin-treated diabetic rats was comparable to that of the control and the sham-operated control rats on the days measured. The excretion of creatinine and of total protein (Fig. 1D,E) on days 7, 14 and 21 from the control group was expressed as 100% in order to compensate for the increased excretion during the study. The group of diabetic rats had approximately doubled their urinary excretion of creatinine, and the urinary excretion of total protein from this group was increased five- to seven-fold when compared to the controls on the days measured. The excretion of creatinine and total protein from insulin-treated diabetic, sucrose-fed and uninephrectomized rats was comparable to that of control and sham-operated control rats. The median kidney weight on day 21 (Fig. 2A) was 1.15 g from the control group and 1.14 g from the shamoperated control group. The uninephrectomized and the diabetic groups both had increased kidney weight, 40% and 33%, respectively, when compared to the appropriate controls. The kidney weight of the insulin-treated diabetic and sucrose-fed rats was comparable to that of controls. The urinary excretion of EGF and THP on day 21 was expressed as the ratios of EGF to renal tissue mass and THP to renal tissue mass (Fig. 2B,C). The diabetic group had a reduced ratio of EGF to renal tissue mass, whereas the ratio of THP to renal tissue mass from the diabetic and sucrose-fed groups was increased when compared to that of the controls. The ratios of EGF to renal tissue mass and THP to renal tissue mass from the insulin-treated diabetic and uninephrectomized rats were comparable to those of the control and sham-operated control rats. Histological examination of the kidney sections from the control group on day 21 (Fig. 3A,B) demonstrated immunoreactivity of EGF predominately localized to the luminal membranes of the thick ascending limbs of Henle’s loops and distal tubules, whereas the immuno-
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Fig. 1. The volume of urine and the urinary excretion of EGF, THP, creatinine and total protein in 24 h urine samples obtained on days 7, 14 and 21 in three rat models with increased excretion of urine. (A) 24 h volume of urine, (B) 24 h urinary excretion of EGF, (C) 24 h urinary excretion of THP, (D) 24 h urinary excretion of creatinine and (E) of total protein. CTRL, control; NEPH, uninephrectomized; SHAM, sham-operated control; DIA, diabetic; INS-DIA, insulin-treated diabetic; SUCR, non-osmotic polyuria. * P , 0.05 vs. appropriate controls. ** P , 0.01 vs. appropriate controls.
reactivity of THP was localized intracellularly in the same cells. Deposits of glycogen were present in the cells of the thick ascending limbs of Henle’s loops and distal tubules in the kidneys of the diabetic group (Fig. 3C,D). The intensity of EGF- and THP-immunoreactivity (Fig. 3E–H) appeared weaker in the kidneys of the diabetic rats when compared to the kidneys of the control group. In the other groups, the histological appearance and intensity of EGF-
and THP-immunoreactivity were comparable to those of the controls. The immunoreactive concentration of EGF was measured in the kidneys of control and diabetic rats on day 21 (Table 1). The concentration of EGF in the kidneys of diabetic rats was reduced to approximately 55% of the level in the kidneys of the control rats when expressed per mg renal tissue and per mg protein.
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Fig. 2. Wet tissue weight of kidneys and the ratios of EGF excretion and THP excretion to kidney weight on day 21. (A) Wet tissue weight of kidneys, (B) ratio of EGF excretion to kidney weight, and (C) ratio of THP excretion to kidney weight. CTRL, control; NEPH, uninephrectomized; SHAM, sham-operated control; DIA, diabetic; INS-DIA, insulin-treated diabetic; SUCR, non-osmotic polyuria. * P , 0.05 vs. appropriate controls.
4. Discussion In the present study, we compare the urinary excretion rates of EGF and THP in three rat models with increased renal excretion of urine. EGF and THP have both been proposed or used as markers of distal tubular function of the kidneys in studies with renal diseases [11–15]. Both peptides are synthesized by the cells in the thick ascending limb of Henle’s loop and in the early part of the distal convoluted tubule [1–3], both peptides are excreted in urine, and the glomerular filtration of both peptides from plasma is negligible [8–10]. Because of that, it could be expected that the two peptides would behave alike. However, we report that the excretion rates of EGF and THP are affected differently in the three models employed. The uninephrectomized rats excreted the same urinary volume as the control group. Expressed per kidney, the volume of urine produced by the remaining kidney was therefore twice as high in the uninephrectomized rats as in the controls. If the excretion rates of EGF and THP were unaffected by compensatory processes occurring in the remaining kidney after uninephrectomy one would expect the excretion rates of the two peptides to be halved.
However, the 24 h urinary excretion of EGF from the uninephrectomized rats was reduced to only about 70% of the excretion from the sham-operated control rats. Interestingly, the excretion of EGF seems to increase in accordance with the increment in the weight of the kidney at three weeks after uninephrectomy, as judged from the ratio of EGF excretion to renal tissue mass. This finding of a compensatory increase in renal EGF excretion rate after uninephrectomy is in agreement with findings in healthy human kidney donors [20]. A previous study on uninephrectomized rats did not find a compensatory increased urinary EGF excretion rate [21]. However, the latter result was observed in rats which also underwent duodenectomy and submandibulectomy [21], which might have affected the physiological condition of the animals. In contrast to EGF, the 24 h excretion of THP measured in the present study remained unchanged after uninephrectomy, and was comparable to that of the sham-operated control rats on all days measured. The remaining kidney therefore fully compensated for the excretion of THP and thus did not reflect a reduction in renal tissue mass. We compared two groups of rats with polyuria in order to distinguish alterations in the urinary excretion of EGF
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Fig. 3. EGF- and THP-immunoreactivity (IR) in kidneys obtained on day 21 from control rats and rats with streptozotocin-induced diabetes. (A) EGF-IR and (B) THP-IR in serial sections from the control group; (C) EGF-IR and (D) THP-IR in serial sections from the diabetic group; (E) and (F) EGF-IR in control and diabetic rats, respectively; (G) and (H) THP-IR in control and diabetic rats, respectively. Immunohistochemical examination revealed colocalization of EGF-IR and of THP-IR in the thick ascending limb of Henle’s loop and in the distal convoluted tubule. The distal tubular cells of the kidneys from the diabetic rats contained intracellular accumulations of glycogen (arrowheads) and the intensity of EGF- (arrows) and THP-IR appeared weaker than observed in controls. These photographs are representative of multiple sections examined from the groups. Magnification 3 200 (A,B,E–H), 3 300 (C,D).
J. Thulesen et al. / Regulatory Peptides 72 (1997) 179 – 186 Table 1 The immunoreactive concentration of EGF and the content of EGF expressed as pmol EGF per mg protein in renal tissue on day 21
Control rats Diabetic rats
pmol EGF / g renal tissue
pmol EGF / mg protein
99.4 (73.1–146.9) 56.8 (41.8–110.3)*
0.96 (0.56–1.42) 0.56 (0.39–1.07)*
The values are shown as median and range. *P , 0.01 vs. control rats.
and THP produced by processing larger-than-normal volumes of urine from those induced by metabolic effects of diabetes. The polyuria control group was obtained by addition of sucrose to the drinking water of normal rats which also were food restricted to encourage them to drink more fluid [16]. The increased diuresis in streptozotocininduced diabetic rats is osmotic and caused by a high plasma glucose level that exceeds the threshold of the kidney for resorption. The urinary excretion of EGF was not changed by polyuria, although it tended to be lower in the diabetic rats on day 14 when compared to the controls. Thus, the excretion of EGF from the diabetic rats was unchanged, even though these rats were severely catabolic. Previous reports have measured increased urinary excretion of EGF from diabetic rats [22,23]. In these studies, however, the diabetic rats with high body-weight loss and thereby the rats with most severe diabetes, were excluded [22,23], which might have affected the results. The unchanged EGF excretion from the sucrose-fed group is in accordance with a previous study on rats [24]. In the present study, the kidneys of diabetic rats appeared with a reduced intensity of EGF-immunoreactivity on day 21. In agreement with this finding, the immunoreactive concentration of EGF in renal tissue from the diabetic rats on day 21 was reduced. However, SayedAhmed et al. [25] have previously reported an increased intensity of EGF-immunoreactivity in the kidneys from untreated diabetic rats up to 30 days after the induction of diabetes. This increment was preceded by a rise in EGF mRNA and an initial increased intensity of EGF-receptorimmunoreactivity [25]. The reasons for the discrepancies between the results of Sayed-Ahmed et al. [25] and the present study are not known but differences in the experimental procedures, for example tissue fixation and types of antibodies, may account. In contrast to EGF, the urinary excretion of THP followed the increased diuresis and increased in both groups of rats with polyuria. However, the increment in the excretion of THP from diabetic rats was not so high as in the sucrose-fed rats. In a previous study with diabetic rats, which were insulin-treated but still hyperglycemic, the urinary excretion of THP was increased 10 and 50 days after the induction of diabetes, whereas the intensity of renal THP mRNA and of THP-immunoreactivity in the kidneys were decreased [19]. In a previous study on humans, the THP excretion was reduced in patients who had diabetes for less than 10 years, whereas it was
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increased in patients who suffered from it for more than 10 years [12]. An anatomical glomerulotubular imbalance of the diabetic kidney, with increased length of the proximal and distal tubules, has been described in conjunction with the appearance of abnormal distal tubular cells with accumulations of intracellular glycogen [26]. The latter might affect the functional state of these cells. In the present study, the ratios of EGF and THP excretion per renal tissue mass were calculated, to obtain a parameter for distal tubular function. The uninephrectomized rats had unchanged ratios of both EGF and THP excretion to renal tissue mass. The diabetic rats had a decreased ratio of EGF excretion to renal tissue mass but increased ratio of THP excretion to renal tissue mass, whereas the rats with non-osmotic polyuria had unchanged EGF excretion to renal tissue mass but also increased ratio of THP excretion to renal tissue mass. These data indicate that the excretion of EGF reflects the distal tubular function of protein synthesis better than THP, since EGF excretion to renal tissue mass was unaffected in uninephrectomy but reduced in the pathologic kidneys of the diabetic rats. The excretion of THP is more dependent on the volume of urine, although the excretion of THP was reduced in diabetic rats as compared to the sucrose-fed group. In conclusion, the present study demonstrates that the excretion rates of EGF and THP are affected differently by increased renal excretion of urine, even though they are both excreted in urine after local renal synthesis in the distal tubular cells. This implies that results should be interpreted cautiously if the urinary excretion rate of a peptide synthesized in the distal part of the nephron is used as a parameter for the protein-synthesizing ability of these tubular cells. Further studies are needed in order to find the best candidate among the possible peptides.
Acknowledgements This study was supported by grants from The Danish Biotechnology Center for Signal-peptide Research, The Danish Medical Research Council, The Novo-Nordisk Foundation, Lund and Tore Nilsson Foundation and Royal Physiographic Society. The authors gratefully acknowledge the technical assistance of Jette Hansen, Inger Marie Jensen and Jette Schousboe, and the photographic assistance of Grazyna Poulsen.
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[3] Salido EC, Lakshmanan J, Fischer DA, Shapiro LJ, Barajas L. Expression of epidermal growth factor in the rat kidney, an immunohistochemical and in situ hybridization study. Histochemistry 1991;96:65–72. [4] Carpenter G, Wahl M. The epidermal growth factor family. In: Sporn MB, Roberts AB, editors. Handbook of experimental pharmacology, vol. 1. Berlin: Springer, 1995:69–171. [5] Fisher DA, Salido EC, Barajas L. Epidermal growth factor and the kidney. Annu Rev Physiol 1989;51:67–80. [6] McQueen EG. The nature of urinary casts. J Clin Pathol 1962;15:367–73. [7] Hoyer JR, Sisson SP, Vernier RL. Tamm Horsfall glycoprotein. Ultrastructural immunoperoxidase localization in rat kidney. Lab Invest 1979;41:168–73. [8] Nexø E, Jørgensen PE, Hansen MR. Human epidermal growth factor – On molecular forms present in urine and blood. Regul Pept 1992;42:75–84. [9] Jørgensen PE, Rasmussen TN, Olsen PS, Raaberg L, Poulsen SS, Nexø E. Renal uptake of epidermal growth factor from plasma in the rat. Regul Pept 1990;28:273–80. [10] Jørgensen PE, Hilchey SD, Nexø E, Poulsen SS, Quilley CP. Urinary epidermal growth factor is excreted from the rat isolated perfused kidney in the absence of plasma. J Endocrinol 1993;193:227–34. [11] Josefsberg Z, Ross SA, Lev-Ran A, Hwang DL. Effects of enalapril and nitrendipine on the excretion of epidermal growth factor and albumin in hypertensive NIDDM patients. Diabetes Care 1995;18:690–3. [12] Zimmerhackl LB, Pleiderer S, Kinne R, Manz F, Schuler G, Brandis M. Tamm–Horsfall-protein excretion as a marker of ascending limb transport indicates early renal tubular damage in diabetes mellitus type I. J Diabetes Complications 1991;3:112–4. [13] McKenzie JK, Patel R, McQueen EG. The excretion of Tamm– Horsfall urinary mucoprotein in normals and in patients with renal disease. Australas Ann Med 1964;13:32–9. [14] Pfleiderer S, Zimmerhackl LB, Kinne R, Manz F, Schuler G, Brandis M. Renal proximal and distal tubular function is attenuated in diabetes mellitus type 1 as determined by the renal excretion of alfa1-microglobulin and Tamm–Horsfall protein. Clin Invest 1993;71:972–7.
[15] Thornley C, Dawnay A, Cattell WR. Human Tamm–Horsfall glycoprotein: Urinary and plasma levels in normal subjects and patients with renal disease determined by a fully validated radioimmunoassay. Clin Sci 1985;68:529–35. [16] Carpenter FG. Impairment and restoration of rat urinary bladder responsiveness following distension. Am J Physiol 1983;244:R106– 13. [17] Rasch R. Control of blood glucose levels in the streptozotocin diabetic rat using a long-acting heat-treated insulin. Diabetologia 1979;16:185–90. [18] Jørgensen PE, Vinter-Jensen L, Nexø E. An immunoassay designed to quantitate different molecular forms of rat urinary epidermal growth factor with equimolar potency: Application on fresh rat urine. Scand J Clin Lab Invest 1996;56:25–36. [19] Rasch R, Torffvit O, Bachmann S, Jensen PK, Jacobsen NO. Tamm–Horsfall glycoprotein in streptozotocin diabetic rats: A study of kidney in situ hybridization, immunohistochemistry, and urinary excretion. Diabetologia 1995;38:525–35. [20] Jørgensen PE, Kamper AL, Munck O, Strandgaard S, Nexø E. Urinary excretion of epidermal growth factor in living human kidney donors and their recipients. Eur J Clin Invest 1995;25:442–6. [21] Olsen PS, Nexø E, Poulsen SS, Hansen F, Kirkegaard P. Renal origin of rat urinary epidermal growth factor. Regul Pept 1984;10:37–45. [22] Hwang DL, Lev-Ran A, Tay YC, Dev N. Epidermal growth factor excretion and receptor binding in diabetic rats. Life Sci 1989;44:407–16. [23] Guh JY, Lai YH, Shin SJ, Chuang LY, Tsai JH. Epidermal growth factor in renal hypertrophy in streptozotocin-diabetic rats. Nephron 1991;59:641–7. [24] Jørgensen PE, Poulsen SS, Nexø E, Christensen S. Effect of water deprivation, desmopressin (DDVAP) infusion, and oral loads of water, Na 1 and NH 1 4 on urinary excretion of epidermal growth factor in the rat. Regul Pept 1993;44:17–24. [25] Sayed-Ahmed N, Besbas N, Mundy J, Muchaneta-Kubara E, Cope G, Pearson C, El Nahas M. Upregulation of epidermal growth factor and its receptor in the kidneys of rats with streptozotocin-induced diabetes. Exp Nephrol 1996;4:330–9. [26] Rasch R. Tubular lesions in streptozotocin-diabetic rats. Diabetologia 1984;27:32–7.
Urinary excretion of epidermal growth factor and Tamm–Horsfall protein in three rat models with increased renal excretion of urine a, b c b a Jesper Thulesen *, Per Erik Jørgensen , Ole Torffvit , Ebba Nexø , Steen Seier Poulsen a
Institute of Medical Anatomy, Department B, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark b Department of Clinical Biochemistry, KH University Hospital of Aarhus, Aarhus, Denmark c Department of Internal Medicine, University Hospital of Lund, Lund, Sweden Received 10 March 1997; received in revised form 9 October 1997; accepted 10 October 1997
Abstract Epidermal growth factor (EGF) and Tamm–Horsfall protein (THP) are synthesized in the kidneys by the distal tubular cells and excreted into urine. The urinary excretion of these peptides has been suggested as a potential index for distal tubular function. The urinary excretion rates of EGF and THP were examined in three groups of rats with increased renal excretion of urine: uninephrectomy, non-osmotic polyuria and diabetic osmotic polyuria. Twenty-four hour urine samples were obtained after 7, 14 and 21 days. The urinary volume per kidney was doubled in uninephrectomy when compared to controls. There was a seven-fold increase in urinary volume in rats with non-osmotic polyuria and diabetic osmotic polyuria, as compared to controls. Uninephrectomy, non-osmotic polyuria and diabetes all affected the urinary excretion of EGF and THP differently. The EGF excretion in uninephrectomized rats was 60–80% of that of the controls, whereas THP excretion was unchanged, indicating that EGF excretion varied with renal tissue mass. Non-osmotic polyuria caused a five-fold increase in THP excretion but no change in EGF excretion. THP excretion in the diabetic rats was increased three-fold after 21 days when compared to controls, whereas EGF excretion was decreased when expressed per kidney weight. Immunohistochemistry demonstrated that EGF and THP were colocalized in the thick ascending limbs of Henle’s loops and distal tubules in all five groups of rats. In conclusion, the EGF excretion appears to follow renal tissue mass and seems independent of urinary volume, whereas THP excretion is dependent mainly on urinary volume. This has implications for the use of EGF and / or THP excretion rates as an indicator for distal tubular function. 1997 Elsevier Science B.V. Keywords: Diabetes mellitus; Epidermal growth factor; Nephrectomy; Polyuria; Rats; Tamm–Horsfall protein
1. Introduction Epidermal growth factor (EGF) and Tamm–Horsfall protein (THP) are both synthesized in the kidneys by cells in the thick ascending limbs of Henle’s loops and in the early part of the distal convoluted tubules [1–3]. Both peptides are present in high amounts in urine, but their functional significance is unknown. EGF is a mitogenic peptide with a molecular weight of approximately 6 kDa, and THP is an extremely carbohydrate-rich glycopeptide with a molecular weight of approximately 70 000 kDa *Corresponding author: Tel.: 1 45 35327260; fax 1 45 35369612; e-mail: [email protected] 0167-0115 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved. PII S0167-0115( 97 )01058-6
[2,4]. EGF may conduct reparative and regulatory processes in the kidney and participate in maintaining the integrity of the urothelium in the urinary tract [5]. THP is the most abundant glycoprotein of renal origin present in normal urine [2], and it provides the matrix for urinary cast formation [6]. It has been suggested that THP is involved in the generation of the hypotonic fluid delivered to the distal nephron, possibly by rendering the thick ascending limb of Henle’s loop relatively impermeable to water [7]. The contribution of EGF and THP from plasma to urine is negligible, since the excretion of EGF from plasma accounts for less than 10% of the urinary excretion [8–10], and THP cannot be filtrated from plasma because of its high molecular weight. Especially THP, but also EGF,
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have been used as markers of distal tubular function of the kidney in studies concerned with various renal diseases [11–15]. The present study was undertaken to examine and compare the renal excretion rates of EGF and THP in three experimental rat models with increased renal excretion of urine: uninephrectomized rats, rats with non-osmotic polyuria and untreated diabetic rats with osmotic polyuria. Our goal was to dissociate the effects of producing larger-thannormal diuresis on the urinary excretion of EGF and THP from those of pathologic diuresis in diabetes and of compensatory renal processes in uninephrectomy.
glucose concentration was subsequently measured every morning and showed values above 20 mmol l 21 . (5) Insulin-treated diabetes, n 5 6 (served as controls for the untreated diabetic group). Experimental diabetes was induced and blood glucose concentration measured as described for the untreated diabetic rats. The rats were treated daily to normoglycaemia with a long-acting heattreated insulin preparation (Ultralente, pH 5.5, Novo-Nordisk, Denmark) in accordance with recommended treatment of diabetic rats [17]. (6) Control, n 5 6. Control animals were injected intraperitoneally with 50 mM citrate buffer (pH 4.0) in a volume equivalent to the volume injected in diabetesinduced animals.
2. Materials and methods
2.1. Experimental groups
2.2. Experimental procedure
The animal studies were approved by the local animal committee of Copenhagen, Denmark. Eight-week-old female Wistar rats, weighing 200–220 g (Panum Institute, Copenhagen, Denmark), were randomly allocated into the following groups: (1) Unilateral nephrectomy, n 5 6. Nephrectomy was performed under barbiturate anaesthesia (Brietal, methohexital, 50 mg kg 21 i.p., Eli Lilly, USA) five days before the rats were placed in metabolic cages. A small flank incision was made, the pedicle of the right kidney was tied, and the kidney was removed. Care was taken not to damage the adrenal gland. The muscle and skin were then sutured. Analgesics (Temgesic, 0.1 mg kg 21 s.c., buprenorfin, Reckitt & Colman Pharmaceuticals, UK) were injected before the operative procedure and then postoperatively twice daily for two days. (2) Sham-operated rats, n 5 6. The pre- and post-operative procedures were as described for the uninephrectomized group. The right kidney was lightly manipulated manually, and the muscle and skin were then sutured. (3) Non-osmotic polyuria, n 5 6. Five percent sucrose was added to the drinking water, and the availability of rat chow was restricted to 8 g day 21 five days before the rats were placed in the metabolic cages. Sucrose water and food restriction result in non-osmotic polyuria caused by a high water intake [16]. Urine samples were tested for glucose (BM-Test-5L, Boehringer Mannheim, Germany) to exclude glucosuria. (4) Streptozotocin-induced experimental diabetes, n 5 6. A single intraperitoneal injection of 60 mg kg 21 streptozotocin (Sigma Chemical Co., USA) dissolved immediately before administration in freshly prepared 50 mM citrate buffer (pH 4.0) was administered five days before the rats were placed in metabolic cages. Blood glucose concentration was measured 48 h later by a One Touch II instrument (Lifescan, USA) using the glucose oxidase method, and only rats with a blood glucose concentration exceeding 20 mmol l 21 were included in the study. Blood
The rats were kept in individual metabolic cages (Techniplast, model 1700, Italy) for the following periods: days 5–7, 12–14, and 19–21. The 24 h urine samples were obtained on days 7, 14, and 21 after initial habituation of the rats to the metabolic cages. The temperature (218C) and relative humidity (55%) of the room were controlled, with a 12 h light–dark cycle. The rats were allowed free access to drinking water and rat chow (Altromin No. 1314, Altromin International, Germany), unless otherwise stated. Urine samples were stored at 2 208C until analysed.
2.3. Immunohistochemistry On day 21, the rats were anaesthetized with barbiturate (Brietal, Methohexital, Eli Lilly, USA) and the kidneys were removed and weighed. The right kidney was immediately fixed by immersion into ice-cold, freshly prepared, buffered 4% paraformaldehyde. The fixed tissue samples were embedded in paraffin and cut into 10 mm sections using a microtome and then placed on gelatine-coated glass slides. The sections were pre-embedded in 10% porcine serum in 0.1 mM phosphate buffer, pH 8.0, for 30 min. The primary antisera were rabbit anti-rat urinary EGF (90001) diluted 1:3200 and rabbit anti-rat THP diluted 1:3200. The immunoreactions were visualized by means of the streptavidin / biotin system using biotinylated swine anti-rabbit immunoglobulins (code No. E353, DAKO, Denmark) as the second layer and StreptABComplex / Horseradish peroxidase (code No. 377, DAKO, Denmark) as the third layer, with diaminobenzidine for staining. Sections were counterstained with haematoxylin. The sections were examined by means of a Zeiss Axiophot microscope. The examiner of the histological sections was not aware of the origin of the specimens when evaluating the stained sections. Photographs (Agfa-pan APX25 film) were used for comparison of neighbouring sections stained for EGF and THP, respectively.
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2.4. Tissue samples for biochemical analyses The left kidney from control and diabetic rats was homogenized at 08C in 10 ml homogenization buffer (10 mM piperazine-N,N9-bis (2-ethanesulphonic acid), 1 mg EGTA, 3 mM magnesium chloride, 400 mM sodium chloride, 5 N NaOH, final pH 7.4) per g tissue. The homogenate was centrifuged (20 000g, 40 min, 48C). The resulting supernatant (supernatant 1) was stored at 2 208C. The pellet was resuspended in the homogenization buffer (10 ml / g tissue) with 2% triton X-100 to make the membrane bound forms of EGF soluble from the renal tissue. The resuspended pellet was incubated overnight at 48C prior to centrifugation (20 000g, 40 min, 48C). The resultant supernatant, supernatant 2, was stored at 2 208C. The total contents of EGF and protein were calculated as the sum of the contents in the two supernatants, and corrected with the factor obtained by dividing kidney wet tissue weight with the homogenized amount of renal tissue.
2.5. Analytical techniques EGF was measured by an enzyme-linked immunoassay (ELISA) [9]. In brief, the IgG fractions from two rabbit antisera raised against rat-EGF were used as catching and detecting antibodies, respectively, and EGF purified from rat urine was used as a calibrator [9]. The two supernatants from renal tissue samples were diluted 1:2 in 0.1 M sodium phosphate, 0.1% albumin, pH 8.0, prior to trypsinization as previously described [18]. THP was analysed with a simplified ELISA as previously described [19]. In brief, antibodies against both rat and human urinary THP were raised in rabbits, and THP was purified from rat urine and used as calibrator. Protein was measured employing the Pierce BCA method. Creatinine was measured with automatic equipment (SMAC, Technicon, USA).
2.6. Statistics Results are shown as the median and range. Comparison between groups was performed using the Kruskal–Wallis one-way analysis by ranks, and if significant difference was found, followed by the non-parametric Mann–Whitney U-test (two-tailed). Probability values of P , 0.05 were considered significant.
3. Results The 24 h volume of urine (Fig. 1A) was approximately 20 ml from the control and the sham-operated control groups on days 7, 14 and 21. It was increased approximately seven-fold on these days in the diabetic and sucrose-fed rats, whereas the volume of urine from uni-
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nephrectomized and insulin-treated diabetic rats was comparable to that of controls. The 24 h urinary excretion of EGF (Fig. 1B) remained at a constant level of approximately 2 nmol in the control and the sham-operated control groups. It was reduced in the uninephrectomized group on days 7, 14 and 21 to 75%, 60% and 84%, respectively, of the level in the shamoperated control group. The excretion of EGF from diabetic, insulin-treated diabetic and sucrose-fed rats was comparable to that of the controls, even though diabetic rats tended to excrete less EGF than controls on day 14. The 24 h urinary excretion of THP (Fig. 1C) from the control and the sham-operated control groups also remained at a constant level, approximately 100 mg, on days 7, 14 and 21, whereas the sucrose-fed rats had a four- to six-fold increase in their urinary excretion of THP on the days measured. The excretion of THP from the diabetic group was comparable to that of the controls on day 7 and gradually increased on days 14 and 21, but never to the level of the sucrose-fed rats. The excretion of THP from uninephrectomized and insulin-treated diabetic rats was comparable to that of the control and the sham-operated control rats on the days measured. The excretion of creatinine and of total protein (Fig. 1D,E) on days 7, 14 and 21 from the control group was expressed as 100% in order to compensate for the increased excretion during the study. The group of diabetic rats had approximately doubled their urinary excretion of creatinine, and the urinary excretion of total protein from this group was increased five- to seven-fold when compared to the controls on the days measured. The excretion of creatinine and total protein from insulin-treated diabetic, sucrose-fed and uninephrectomized rats was comparable to that of control and sham-operated control rats. The median kidney weight on day 21 (Fig. 2A) was 1.15 g from the control group and 1.14 g from the shamoperated control group. The uninephrectomized and the diabetic groups both had increased kidney weight, 40% and 33%, respectively, when compared to the appropriate controls. The kidney weight of the insulin-treated diabetic and sucrose-fed rats was comparable to that of controls. The urinary excretion of EGF and THP on day 21 was expressed as the ratios of EGF to renal tissue mass and THP to renal tissue mass (Fig. 2B,C). The diabetic group had a reduced ratio of EGF to renal tissue mass, whereas the ratio of THP to renal tissue mass from the diabetic and sucrose-fed groups was increased when compared to that of the controls. The ratios of EGF to renal tissue mass and THP to renal tissue mass from the insulin-treated diabetic and uninephrectomized rats were comparable to those of the control and sham-operated control rats. Histological examination of the kidney sections from the control group on day 21 (Fig. 3A,B) demonstrated immunoreactivity of EGF predominately localized to the luminal membranes of the thick ascending limbs of Henle’s loops and distal tubules, whereas the immuno-
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Fig. 1. The volume of urine and the urinary excretion of EGF, THP, creatinine and total protein in 24 h urine samples obtained on days 7, 14 and 21 in three rat models with increased excretion of urine. (A) 24 h volume of urine, (B) 24 h urinary excretion of EGF, (C) 24 h urinary excretion of THP, (D) 24 h urinary excretion of creatinine and (E) of total protein. CTRL, control; NEPH, uninephrectomized; SHAM, sham-operated control; DIA, diabetic; INS-DIA, insulin-treated diabetic; SUCR, non-osmotic polyuria. * P , 0.05 vs. appropriate controls. ** P , 0.01 vs. appropriate controls.
reactivity of THP was localized intracellularly in the same cells. Deposits of glycogen were present in the cells of the thick ascending limbs of Henle’s loops and distal tubules in the kidneys of the diabetic group (Fig. 3C,D). The intensity of EGF- and THP-immunoreactivity (Fig. 3E–H) appeared weaker in the kidneys of the diabetic rats when compared to the kidneys of the control group. In the other groups, the histological appearance and intensity of EGF-
and THP-immunoreactivity were comparable to those of the controls. The immunoreactive concentration of EGF was measured in the kidneys of control and diabetic rats on day 21 (Table 1). The concentration of EGF in the kidneys of diabetic rats was reduced to approximately 55% of the level in the kidneys of the control rats when expressed per mg renal tissue and per mg protein.
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Fig. 2. Wet tissue weight of kidneys and the ratios of EGF excretion and THP excretion to kidney weight on day 21. (A) Wet tissue weight of kidneys, (B) ratio of EGF excretion to kidney weight, and (C) ratio of THP excretion to kidney weight. CTRL, control; NEPH, uninephrectomized; SHAM, sham-operated control; DIA, diabetic; INS-DIA, insulin-treated diabetic; SUCR, non-osmotic polyuria. * P , 0.05 vs. appropriate controls.
4. Discussion In the present study, we compare the urinary excretion rates of EGF and THP in three rat models with increased renal excretion of urine. EGF and THP have both been proposed or used as markers of distal tubular function of the kidneys in studies with renal diseases [11–15]. Both peptides are synthesized by the cells in the thick ascending limb of Henle’s loop and in the early part of the distal convoluted tubule [1–3], both peptides are excreted in urine, and the glomerular filtration of both peptides from plasma is negligible [8–10]. Because of that, it could be expected that the two peptides would behave alike. However, we report that the excretion rates of EGF and THP are affected differently in the three models employed. The uninephrectomized rats excreted the same urinary volume as the control group. Expressed per kidney, the volume of urine produced by the remaining kidney was therefore twice as high in the uninephrectomized rats as in the controls. If the excretion rates of EGF and THP were unaffected by compensatory processes occurring in the remaining kidney after uninephrectomy one would expect the excretion rates of the two peptides to be halved.
However, the 24 h urinary excretion of EGF from the uninephrectomized rats was reduced to only about 70% of the excretion from the sham-operated control rats. Interestingly, the excretion of EGF seems to increase in accordance with the increment in the weight of the kidney at three weeks after uninephrectomy, as judged from the ratio of EGF excretion to renal tissue mass. This finding of a compensatory increase in renal EGF excretion rate after uninephrectomy is in agreement with findings in healthy human kidney donors [20]. A previous study on uninephrectomized rats did not find a compensatory increased urinary EGF excretion rate [21]. However, the latter result was observed in rats which also underwent duodenectomy and submandibulectomy [21], which might have affected the physiological condition of the animals. In contrast to EGF, the 24 h excretion of THP measured in the present study remained unchanged after uninephrectomy, and was comparable to that of the sham-operated control rats on all days measured. The remaining kidney therefore fully compensated for the excretion of THP and thus did not reflect a reduction in renal tissue mass. We compared two groups of rats with polyuria in order to distinguish alterations in the urinary excretion of EGF
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Fig. 3. EGF- and THP-immunoreactivity (IR) in kidneys obtained on day 21 from control rats and rats with streptozotocin-induced diabetes. (A) EGF-IR and (B) THP-IR in serial sections from the control group; (C) EGF-IR and (D) THP-IR in serial sections from the diabetic group; (E) and (F) EGF-IR in control and diabetic rats, respectively; (G) and (H) THP-IR in control and diabetic rats, respectively. Immunohistochemical examination revealed colocalization of EGF-IR and of THP-IR in the thick ascending limb of Henle’s loop and in the distal convoluted tubule. The distal tubular cells of the kidneys from the diabetic rats contained intracellular accumulations of glycogen (arrowheads) and the intensity of EGF- (arrows) and THP-IR appeared weaker than observed in controls. These photographs are representative of multiple sections examined from the groups. Magnification 3 200 (A,B,E–H), 3 300 (C,D).
J. Thulesen et al. / Regulatory Peptides 72 (1997) 179 – 186 Table 1 The immunoreactive concentration of EGF and the content of EGF expressed as pmol EGF per mg protein in renal tissue on day 21
Control rats Diabetic rats
pmol EGF / g renal tissue
pmol EGF / mg protein
99.4 (73.1–146.9) 56.8 (41.8–110.3)*
0.96 (0.56–1.42) 0.56 (0.39–1.07)*
The values are shown as median and range. *P , 0.01 vs. control rats.
and THP produced by processing larger-than-normal volumes of urine from those induced by metabolic effects of diabetes. The polyuria control group was obtained by addition of sucrose to the drinking water of normal rats which also were food restricted to encourage them to drink more fluid [16]. The increased diuresis in streptozotocininduced diabetic rats is osmotic and caused by a high plasma glucose level that exceeds the threshold of the kidney for resorption. The urinary excretion of EGF was not changed by polyuria, although it tended to be lower in the diabetic rats on day 14 when compared to the controls. Thus, the excretion of EGF from the diabetic rats was unchanged, even though these rats were severely catabolic. Previous reports have measured increased urinary excretion of EGF from diabetic rats [22,23]. In these studies, however, the diabetic rats with high body-weight loss and thereby the rats with most severe diabetes, were excluded [22,23], which might have affected the results. The unchanged EGF excretion from the sucrose-fed group is in accordance with a previous study on rats [24]. In the present study, the kidneys of diabetic rats appeared with a reduced intensity of EGF-immunoreactivity on day 21. In agreement with this finding, the immunoreactive concentration of EGF in renal tissue from the diabetic rats on day 21 was reduced. However, SayedAhmed et al. [25] have previously reported an increased intensity of EGF-immunoreactivity in the kidneys from untreated diabetic rats up to 30 days after the induction of diabetes. This increment was preceded by a rise in EGF mRNA and an initial increased intensity of EGF-receptorimmunoreactivity [25]. The reasons for the discrepancies between the results of Sayed-Ahmed et al. [25] and the present study are not known but differences in the experimental procedures, for example tissue fixation and types of antibodies, may account. In contrast to EGF, the urinary excretion of THP followed the increased diuresis and increased in both groups of rats with polyuria. However, the increment in the excretion of THP from diabetic rats was not so high as in the sucrose-fed rats. In a previous study with diabetic rats, which were insulin-treated but still hyperglycemic, the urinary excretion of THP was increased 10 and 50 days after the induction of diabetes, whereas the intensity of renal THP mRNA and of THP-immunoreactivity in the kidneys were decreased [19]. In a previous study on humans, the THP excretion was reduced in patients who had diabetes for less than 10 years, whereas it was
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increased in patients who suffered from it for more than 10 years [12]. An anatomical glomerulotubular imbalance of the diabetic kidney, with increased length of the proximal and distal tubules, has been described in conjunction with the appearance of abnormal distal tubular cells with accumulations of intracellular glycogen [26]. The latter might affect the functional state of these cells. In the present study, the ratios of EGF and THP excretion per renal tissue mass were calculated, to obtain a parameter for distal tubular function. The uninephrectomized rats had unchanged ratios of both EGF and THP excretion to renal tissue mass. The diabetic rats had a decreased ratio of EGF excretion to renal tissue mass but increased ratio of THP excretion to renal tissue mass, whereas the rats with non-osmotic polyuria had unchanged EGF excretion to renal tissue mass but also increased ratio of THP excretion to renal tissue mass. These data indicate that the excretion of EGF reflects the distal tubular function of protein synthesis better than THP, since EGF excretion to renal tissue mass was unaffected in uninephrectomy but reduced in the pathologic kidneys of the diabetic rats. The excretion of THP is more dependent on the volume of urine, although the excretion of THP was reduced in diabetic rats as compared to the sucrose-fed group. In conclusion, the present study demonstrates that the excretion rates of EGF and THP are affected differently by increased renal excretion of urine, even though they are both excreted in urine after local renal synthesis in the distal tubular cells. This implies that results should be interpreted cautiously if the urinary excretion rate of a peptide synthesized in the distal part of the nephron is used as a parameter for the protein-synthesizing ability of these tubular cells. Further studies are needed in order to find the best candidate among the possible peptides.
Acknowledgements This study was supported by grants from The Danish Biotechnology Center for Signal-peptide Research, The Danish Medical Research Council, The Novo-Nordisk Foundation, Lund and Tore Nilsson Foundation and Royal Physiographic Society. The authors gratefully acknowledge the technical assistance of Jette Hansen, Inger Marie Jensen and Jette Schousboe, and the photographic assistance of Grazyna Poulsen.
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