ARTICLE |
Correspondence to: Shyi J. Shin, Div. of Endocrinology and Metabolism, Kaohsiung Medical University Hospital, 100 Shih-Chuan 1st Rd, Kaohsiung 80708, Taiwan. E-mail: d680032@kmu.edu.tw
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Summary |
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Increased intrarenal atrial natriuretic peptide (ANP) mRNA expression has been reported in several disorders. To further investigate the action of renal ANP, we need to elucidate the exact site of its alteration in diseased kidneys. ANP mRNA and ANP were detected by in situ hybridization and immunohistochemistry in the kidneys from five normal and five diabetic rats. Renal ANP mRNA in eight normal and nine diabetic rats was measured by RT-PCR with Southern blot hybridization. In normal and diabetic rats, the distribution of ANP mRNA and ANP-like peptide was mainly located in proximal, distal, and collecting tubules. However, diabetic rats had significant enhancement of ANP mRNA and ANP-immunoreactive staining in the proximal straight tubules, medullary thick ascending limbs, and medullary collecting ducts. ANP mRNA in the outer and inner medulla of nine diabetic rats increased 5.5-fold and 3.5-fold, but only 1.8-fold in the renal cortex. This preliminary study showed that ANP mRNA and ANP immunoreactivity in proximal straight tubules, medullary thick ascending limb, and medullary collecting ducts apparently increased in diabetic kidneys. These findings imply that ANP synthesis in these nephrons may involve in adaptations of renal function in diabetes. (J Histochem Cytochem 50:15011507, 2002)
Key Words: atrial natriuretic peptide, proximal straight tubule, medullary thick ascending limb, collecting duct, distal convoluted tubule, diabetic kidney, angiotensin II, vasopressin
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Introduction |
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ALTHOUGH the kidney is a principal target for atrial natriuretic peptide (ANP), several studies have implicated this organ as a source of ANP (
In addition to prostaglandin (
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Materials and Methods |
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Animal Experiments
Male Wistar rats (purchased from the animal center of National Cheng Kung University, Taiwan) weighing 230260 g were individually housed in metabolic cages. Diabetes was induced by a single peritoneal injection of 55 mg/kg streptozotocin (Sigma Chemical; St Louis, MO). Twenty-four hours later, induction of diabetes was confirmed by measuring their tail blood glucose levels (Accutrend Glucose; Boehringer Mannheim, Indianapolis, IN). Rats with blood glucose levels >19.4 mmol/liter were included. Diabetic rats received ultralente insulin (Novo-Nordisk; Copenhagen, Denmark) designed to achieve blood glucose levels between 19.4 and 33.3 mmol/liter. Thirteen body weight- and age-matched rats were studied as normal controls. In experiment 1, five normal and five diabetic rats on day 42 of the study were anesthetized with sodium pentobarbital (Abbott Laboratories; Chicago, IL) and infused with normal saline via the left cardiac ventricle, and then with phosphate buffer solution (pH 7.4) containing 4% paraformaldehyde for 15 min. Kidneys were removed and immersed in 4% paraformaldehyde for 2 hr. Thin kidney slices containing cortex, outer medulla, and inner medulla of normal and diabetic rats were embedded in the same paraffin block and cut into 4-µm sections for in situ mRNA hybridization and immunohistochemical study. In experiment 2, eight normal and nine diabetic rats were sacrificed to collect blood to measure plasma ANP, angiotensin II (Ang II), arginine vasopressin, sodium, and osmolality on day 42 after streptozotocin or citric buffer injection. Kidneys were immediately removed and separated into cortex, outer medulla, and inner medulla for ANP and ß-actin mRNA analysis. Urine ANP, sodium, glucose, and osmolality were measured on day 42. This study was approved by the Animal Care and Treatment Committee of Kaohsiung Medical University.
In Situ Hybridization
Sections were pretreated with microwave heating as described in our previous study (
Digoxigenin-labeled RNA hybrids were detected using an ELISA kit (Roche Molecular Biochemicals). After immersion in 1.5% blocking solution, the slides were exposed to anti-digoxigeninalkaline phosphatase conjugate diluted 1:1000 for 30 min. The hybrids were visualized as purple/black precipitates by subsequent alkaline phosphatasechloro-3-indolyl phosphate and nitroblue tetrazolium. We compared the relative intensity of ANP mRNA in normal and diabetic rats on the same slide to avoid discrepancies due to varying thickness and different processing times. The ANP mRNA staining intensities were graded according to the following scale: 0 = very weak staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The intensity was determined in 10 glomeruli and 10 tubule segments per animal and was expressed as the average intensity per tubules and glomeruli. The intensity and distribution of ANP mRNA were evaluated by two nephrologists without prior knowledge of the source of the sections.
Immunohistochemistry
Sections were pretreated with microwave heating as described above. After microwave treatment, sections were washed in PBS and incubated with 1% bovine serum albumin for 30 min to block nonspecific staining. Sections were drained and incubated for 3 h at RT in a humidity chamber with the respective rabbit anti-rat primary antibody (Phoenix Pharmaceuticals; Belmont, CA) for ANP (1:1000) diluted with antibody diluent (DAKO; Glostrup, Denmark). After washing in PBS, endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 20 min, followed by sequential 10-min incubations with biotinylated link antibody and peroxidase-labeled streptavidin (DAKO). Staining was completed after incubation with Vector VIP substratechromogen solution, then counterstaining with methyl green (Vector Laboratories; Burlingame, CA), and then mounted in aqueous mounting medium. Negative control studies were performed with primary antibody being replaced by normal rabbit serum and counterstaining with hematoxylin. The ANP immunostaining intensities were graded according to the following scale: 0 = very weak staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining.
RNA Isolation and Reverse Transcription
Total RNA was extracted from the renal cortex, outer medulla, and inner medulla using a modified guanidium isothiocyanate method. The integrity of the RNA was assessed by formaldehydeagarose gel electrophoresis followed by ethidium bromide staining, and the quality was determined by absorbance at 260 nm. Two micrograms of total RNA from renal cortex, outer medulla, and inner medulla were reverse-transcribed by incubation with 20 µl reverse transcription mixture containing 20 pmole oligo (dT)18 primer, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 30 U RNase inhibitor, 0.5 mM dNTPs, and 50 U MMLV reverse transcriptase (Stratagene Laboratories; Palo Alto, CA) at 37C for 2 h. The reverse transcriptase was inactivated by heating for 5 min at 94C.
Polymerase Chain Reaction Amplification and Southern Blot Hybridization
PCR reaction was performed at a final concentration of 1 x PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM dNTPs, 0.4 µM sense and antisense oligos, 2.0 U Taq DNA polymerase (Boehringer Mannheim) to a total volume of 50 µl. The amplifications were performed for 45 sec at 94C, 45 sec at 60C, and 90 sec at 72C in a PerkinElmer Cetus 9600 thermocycler (PerkinElmer Cetus; Norwalk, CT). Sense primers for ANP were 5'-GGCTCCTTCTCCATCACCAA-3', corresponding to bp 423, and antisense primers were 5'-TGTTATCTTCGGTACCG-3', corresponding to bp 445461, which yielded a 458-bp PCR product. Sense primers for ß-actin were 5'-CGTAAAGACCTCTATGCCAA-3', corresponding to bp 27482767, and antisense were 5'-AGCCATGCCAAATGTCTCAT-3', corresponding to bp 32013222, which yielded a 349-bp PCR product. The amplification cycles of the PCR procedure were evaluated for ANP and ß-actin mRNA. The amplification cycles of all following PCR analysis were selected only during the exponential phase. The amplification cycles were repeated 30 and 22 times for ANP and ß-actin mRNA, respectively, in the renal cortex, outer medulla, and inner medulla.
From each PCR production, the amplified products were electrophoresed on 1.5% agarose gels and transferred to nylon membranes (Schleicher & Schuell; Dassel, Germany). The blots were hybridized with 32P-labeled, randomly primed rat ANP and ß-actin cDNA prepared by PCR cloning for 16 hr at 37 C, according to the standard technique. After each hybridization, the blots were washed twice in a solution containing 0.1% sodium dodecyl sulfate (SDS) and 2 x SSC (0.3 M NaCl, 30 mM sodium citrate) for 15 min at RT and then twice in 0.1% SDS and 0.1 x SSC at 65 C. Blots were exposed to Kodak BIOMAX-MR (Eastman Kodak; Rochester, NY) film at 70C. A radioisotope-labeled probe for ß-actin to be used as an internal control was also made using the primer extension method. After autoradiography, the X-ray film was scanned by a laser densitometer (Molecular Dynamics; Sunnyvale, CA) and the data were analyzed by MD ImageQuant software release version 3.22. To determine the relative changes in tissue ANP mRNA expression, the yield of ANP PCR products was normalized to the amount of ß-actin cDNA amplified from the same RT cDNA of tissue samples, a method that has been used in our previous studies (
Assay Methods
Plasma samples were extracted using Sep-Pak C18 cartridges (Water Associates; Milford, MA) that had been pre-wetted with 4 ml of 60% acetonitrile (ACN) in 0.1% trifluoacetic acid (TFA). The cartridges with the absorbed peptides were then washed with 6 ml of 0.1% TFA and eluted with 3 ml of 60% ACN in 0.1% TFA. These elutants were lyophilized and reconstituted for radioimmunoassay. Ang II, vasopressin, and ANP immunoreactivities from samples of plasma were determined by RIA methods after the lyophilisate was resuspended in RIA buffer. Concentrations of plasma and urinary sodium and osmolality were determined in an automatic analyzer (Nova Biochemical; Newton, MA).
Statistical Analysis
The data are expressed as mean ± SEM. To test the difference between the two groups, two-tailed unpaired Student's t-test was performed. A p value <0.05 was considered statistically significant.
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Results |
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Biochemical and physical data of normal and diabetic rats on the 42nd day of experiment 2 are shown in Table 1. In the diabetic group, the mean blood glucose level, the daily water intake, and the urine amount were markedly increased compared to the corresponding values of normal rats. Compared to the normal rats, the diabetic group showed a significant decrease in body weight but a significant increase in mean kidney weight:body weight ratio. In the untreated diabetic rats, plasma ANP, vasopressin, and osmolality levels, as well as urine ANP, sodium, glucose, and osmolality excretion rate, were significantly increased, whereas plasma sodium and Ang II concentrations showed no significant difference.
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We determined directly the distribution of ANP mRNA in renal sections from normal and diabetic rats by in situ hybridization using a specific antisense cRNA probe for ANP, as shown in Fig 1. In normal rats, ANP mRNA intensity was moderate in proximal convoluted tubule (PCT), proximal straight tubule (PST), medullary thick ascending limb (MTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). The glomeruli, blood vessels, and the thin limb of Henle's loop showed weak staining for ANP mRNA. We detected no signal using a sense cRNA probe (Fig 1D and Fig 1H). A similar but significantly increased distribution of ANP mRNA intensity was observed in diabetic rats. The upper panel of Fig 3 shows that the density of ANP mRNA hybridization increased in PST, MTAL, DCT, OMCD, and IMCD of diabetic kidneys compared to those corresponding segments in normal control rats.
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Fig 2 shows the results of immunohistochemical studies for ANP-like immunoreactivities in the renal cortex, outer medulla, and inner medulla from one representative normal and one diabetic rat. In normal rats, the signal of ANP-like immunoreactive staining was detected mainly in PCT, PST, MTAL, DCT, CCD, OMCD, and IMCD, although very weak expression was also detected in the glomeruli and thin limb of Henle's loop. Diabetic rats had a similar distribution pattern of ANP immunostaining intensity. There was, however, a considerable increase in intensity of ANP immunostaining in diabetic rats. The lower panel of Fig 3 shows the changes of ANP immunoreactive intensity in diabetic rats. The ANP immunoreactivity clearly increases in PST, MTAL, OMCD, and IMCD of the diabetic rats compared with normal control rats. No immunoreactive labeling was observed when the sections were incubated with pre-absorbed antibody or with rabbit normal serum in the absence of primary antibody (data not shown).
Fig 4 (upper panel) shows the autoradiographs of RT-PCR amplifications of ANP and ß-actin mRNA in the renal cortex, outer medulla, and inner medulla from three representative normal and three representative diabetic rats on day 42. The relative ratios of the densitometry measure of PCR products for ANP with those for ß-actin mRNA from eight control and nine diabetic rats are shown in the lower panel of Fig 4. The relative ratios of cDNA product for ANP mRNA with ß-actin mRNA in the renal cortex, outer medulla, and inner medulla were found to have increased by 1.8-, 5.5-, and 3.5-fold in diabetic rats compared to normal rats.
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Discussion |
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The present study demonstrates that ANP synthesis increased predominantly in PST, MTAL, and medullary collecting ducts in diabetic rats. This conclusion is based on the following findings: (a) the intensity of ANP mRNA, detected directly by in situ hybridization, was considerably increased in PST, MTAL, DCT, and MCD of diabetic kidneys; (b) the immunoreactive staining for ANP in PST, MTAL, and MCD of diabetic kidneys was also enhanced; (c) ANP mRNA expression in renal cortex, outer medulla, and inner medulla of diabetic kidneys showed 1.8-, 5.5-, and 3.5-fold increases compared to the corresponding data for normal rats.
The first report demonstrating that ANP prohormone-like peptide could be produced and secreted in the primary cultures of neonatal and adult rat kidney cells was made by
Using immunohistochemical studies for ANP-like immunoreactivity, several investigations have demonstrated the localization of ANP in the normal kidney. Most of these studies have reported that positive ANP-immunoreactive staining was localized to cortical distal tubule epithelial cells in rat and human kidneys (
Because the kidney is able to synthesize ANP, the alteration of renal-synthesized ANP has also been investigated in several disorders (
In the kidney, the tubules and vasa recta are arranged in a hairpin pattern to preserve osmotic gradients and enhance urinary concentration (
In summary, our results demonstrate that the cellular distribution of ANP mRNA and ANP-like immunoreativity in PST, MTAL, and medullary collecting ducts was clearly enhanced in diabetic rat kidneys. This study indicates that increases in ANP synthesis in these nephrons may participate in renal adaptation to hyperglycemia and hyperosmolality in diabetes.
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Acknowledgments |
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Supported by grants NSC-89-2314-B037-157 from the National Science Council and RE86M018C for Dr. Feng-Jie Lai from the National Health Research Institutes in Taiwan.
We are grateful to C.-L. Chen and S.-J. Lee for expert technical assistance.
Received for publication May 20, 2002; accepted June 5, 2002.
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