Aldosterone Rapidly Enhances Levels of the Striatin and Caveolin-1 Proteins in Rat Kidney: The Role of the Mineralocorticoid Receptor

BACKGROUND: Striatin and caveolin-1 (cav-1) are scaffolding/regulating proteins that are associated with salt-sensitive high blood pressure and promote renal sodium and water reabsorption, respectively. The mineralocorticoid receptor (MR) interacts with striatin and cav-1, while aldosterone increases striatin and cav-1 levels. However, no in vivo data have been reported for the levels of these proteins in the kidney. METHODS: Male Wistar rats were intraperitoneally injected with normal saline solution, aldosterone alone (Aldo: 150 µg/kg body weight), or aldosterone after pretreatment with eplerenone, an MR blocker, 30 minutes before the aldosterone injection (eplerenone [Ep.]+Aldo). Thirty minutes after the aldosterone injection, the amount and localization of striatin and cav-1 were determined by Western blot analysis and immunohistochemistry, respectively. RESULTS: Aldosterone increased striatin levels by 150% (P<0.05), and cav-1 levels by 200% (P<0.001). Eplerenone had no significant effect on striatin levels, but partially blocked the aldosterone-induced increase in cav-1 levels. Aldosterone stimulated striatin and cav-1 immunoreactivity in both the cortex and medulla. Eplerenone reduced cav-1 immunostaining in both areas; however, striatin intensity was reduced in the cortex, but increased in the medulla. CONCLUSION: This is the first in vivo study demonstrating that aldosterone rapidly enhances renal levels of striatin and cav-1. Aldosterone increases striatin levels via an MR-independent pathway, whereas cav-1 is partially regulated through MR.

Comparison of amphotericin B lipid complex (Abelcet) and liposomal amphotericin B (Ambisome) on rat kidney: a morphological evaluation / Comparasión de la anfotericina B complejo lipídico (Abelcet) y la anfotericina B liposomal (AmBisome) en el riñón de rata: una evaluación morfológica

The aim of our study was to compare the nephrotoxic effects of liposomal amphotericin B (Ambisome) and amphotericin B lipid complex (Abelcet) on rat kidneys at short (14 days) and long term (28 days) treatment applications. Thirty-six male Wistar rats were included and divided into six groups (n=6). Groups 1 and 4 are composed as control groups by administrating intraperitoneal (ip) 0, 9 molar Serum physiologic for a period of 14 and 28 days respectively. Group 2 and 3 are treated with 5 mg/kg Ambisome and 5 mg/kg Abelcet for 14 days respectively, group 5 and 6 are treated with same agents for 28 days respectively. Then, the rats were transcardially perfused, samples were taken from cortex and medulla regions of kidneys. The micrographs of group 1 and 4 were seen as normal. For short term treatment, some morphological changes were seen in proximal tubule cells in group 3 whereas in group 2 the graphs were observed as normal. However, after long term drug using in group 5 and 6 there were vacuolization, increased lysosomal structures and deep basal folding's into tubular cells lumen. These experiments establish that renal damage were seen in short and long term use of Abelcet and long term use of Ambisome.

El objetivo de nuestro estudio fue comparar los efectos nefrotóxicos de la anfotericina B liposomal (AmBisome) y anfotericina B en complejo lipídico (Abelcet) sobre riñones de ratas, en el tratamiento de aplicación a corto (14 días) y largo plazo (28 días). Fueron incluidas en el estudio 36 ratas Wistar machos, divididas en seis grupos (n = 6). Los Grupos 1 y 4 fueron grupos de control mediante la administración intraperitoneal (ip) de 0, 9 molar de suero fisiológico durante un periodo de 14 y 28 días respectivamente. Los Grupos 2 y 3 fueron tratados con 5 mg/kg de ambisome y 5 mg/kg abelcet durante 14 días respectivamente, y finalmente los grupos Grupos 5 y 6 tratados con los mismos agentes durante 28 días, respectivamente. Luego, las ratas fueron perfundidas vía transcardíaca, y se tomaron muestras de la corteza y la médula renal. Las micrografías de los grupos 1 y 4 se observaron normal. En el tratamiento a corto plazo, algunos cambios morfológicos se observaron en las células del túbulo proximal en el grupo 3, mientras que en el grupo 2 los gráficos se observaron normales. Sin embargo, después de utilizar la droga a largo plazo en los grupos 5 y 6 hubo vacuolización, aumento de las estructuras lisosomales y un profundo plegamiento basal de las células del lumen tubular. Estos experimentos establecen que el daño renal se produce en el uso a corto y largo plazo de Abelcet, y largo plazo de Ambisome.

Immunocytochemical Study of Calbindin D(28k) Positive Cells in the Developing Rat Kidney / 대한해부학회지

Calbindin D(28k),a calcium binding protein,is found in various tissues,including some cells in the distal nephron.It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D(28k) in adult rat kidney.However,the exact time of expression during differentiation in the embryonic kidney is not known.During development,intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms.However,the reason for the different modes of cell death is not known.As calbindin is reported to protect cells against apoptosis,we examined the expression of calbindin D(28k) in the developing rat kidney.Kidneys from 16-,17-,18-and 20-day-old fetuses and 1-,3-,5-,7-,14-and 21-day-old pups and adult Sprague awley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D(28k) .Intercalated cells were identified by immunostaining for H+ -ATPase and by electron microscopy.Calbindin D(28k) immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus.In the connecting tubule,calbindin D(28k) was expressed only in H+ -ATPase negative connecting tubule cells,and there was no labeling of intercalated cells.In the medullary collecting duct,calbindin D(28k) immunostaining was observed in a few cells with apical H+ -ATPase,characteristic of type A intercalated cells.The numbers of calbindin D(28k) -positive type A intercalated cells increased from day 18 of gestation.In contrast,there was little or no calbindin D(28k) immunoreactivity in the type B intercalated cells or principal cells.During the first two weeks after birth,calbindin D(28k) -positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks,calbindin D(28k) immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct.However,the immunoreactivity of calbindin D(28k) in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups.Thus,we propose that the different expression of calbindin D(28k) in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

The Role of HMGB1 and the Effect of Ethyl Pyruvate on Ischemia and Reperfusion Injury of Rat Kidney / 대한이식학회지

PURPOSE: High mobility group box-1 (HMGB1) was identified as a DNA binding protein that functions as a co-factor for proper transcriptional regulation in somatic cells. Extra- cellular HMGB1 acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Ethyl pyruvate (EP), a stable aliphatic ester derived from pyruvic acid is the first described pharmacological inhibitor for HMGB1 secretion. We designed this study to identify the change of HMGB1 expression in rat kidney tissues of ischemia reperfusion injury and the effect of EP on the expression of HMGB1. METHODS: Sprague-Dawley rats (200~300 g) were subjected to 40 minutes of renal warm ischemia. The animals were divided into three groups: sham group without warm ischemia, the EP group (EP given before ischemia), and the ischemic control group. Kidneys were harvested and serum creatinine, IL-1 and IL-6 were measured at 6hours, 1 day, 3 days and 5 days after reperfusion. Immunohistochemical stain of HMGB-1 was done. RESULTS: Serum creatinine and IL-1 level were elevated in ischemic control group and EP injection group. In EP injection group, serum creatinine and IL-1 level were lower than the ischemic control group. In the rat 40minutes ischemia reperfusion model, HMGB1 expression was increased at 6 hours after reperfusion. which was decreased gradually at 1 day, 3 days, and 5 days after reperfusion. HMGB1 expression was more distinct at outer medullary area. intraperitoneal EP injection has no effect on the expression of HMGB1. CONCLUSION: From these results, we deduced a conclusion that the preventive effect of EP on the rat kidney ischemia reperfusion injury is not by the decreased expression of HMGB1 but by the prevention of the release of the HMGB1.

Roles of rat TPP-I in angiotensins I,Ⅱ,Ⅲ hydrolysis / 基础医学与临床

angiotensi I.Conclusion Angiotensin Ⅲ is a good natural substrate for TPP Ⅰ.

Developmental Expression of Epidermal Growth Factor in Rat Kidney / 대한해부학회지

Epidermal growth factor (EGF) is well known to be one of regulating factor in proliferation. The aims of the present study were to elucidate the expression time and localization of expression of EGF in developing rat (Sprague-Dawley) kidney. Kidneys of 16-, 18- and 20-day-old fetuses, 1-, 3-. 7-, 14-, and 21-day-old pups and adult (3months) were preserved for light and electron microscopic immunocytochemistry. In adult rat kidney, EGF immunoreactivity was well localized in thick ascending limb and distal convoluted tubule. In developing rat kidney, EGF-positive cells appeared first in cortical thick ascending limbs and distal convoluted tubules of juxtamedullary nephrons at 3 days and 7 days after birth respectively, in cortical thick ascending limbs and distal convoluted tubules of cortical nephrons at 14 days after birth, and in medullary thick ascending at 21 days after birth. These findings suggest that renal EGF synthesized and secreted from distal tubule may possibly accelerate not proliferation but differentiation of the renal tubular epithelium at least in neonatal rat kidney.

Expression and Distribution of Aquaporin-2, -3 and -4 in Developing Rat Kidney / 대한비뇨기과학회지

PURPOSE: The aquaporins (AQPs) are transmembrane water channel proteins. It is well known that AQP2, -3 and -4 contribute to the urinary concentration in collecting duct (CD) and also reported the presence of these three AQPs in the connecting tubule (CNT). Newborn rats are not capable of producing a concentrated urine. Rats develop the ability to concentrate urine after birth. The purpose of this study was to establish the time of the expression and the distribution of AQP2, -3 and -4 in the developing rat kidney. MATERIALS AND METHODS: Sprague-Dawley rats were used in all experiments. Kidneys were obtained from 16, 18 and 20-day-old fetuses and 1, 4, 7, 14 and 21-day-old pups and preserved and processed for immunohistochemical studies using a preembedding immunoperoxidase procedure. AQP2, -3 and -4 immunoreactivity was detected using rabbit polyclonal antibody and donkey anti-rabbit IgG. RESULTS: AQP2, -3 and -4 appeared first in 16-day-old fetuses in the CD and in 18-day-old fetuses in the CNT. Immunoreactivity for AQP2, -3 and -4 was markedly increased after birth and gradually increased during development. In CNT cells and principal cells, AQP2, -3 and -4 were not distinctly demonstrated on the apical, lateral and basal plasma membrane respectively until 21 days after birth. Distinct polarity of these AQPs both in CNTcells and principal cells were observed at 21 days after birth. CONCLUSIONS: AQP2 -3, and -4 were expressed not only in CD but also in CNT before developing of urine concentrating ability during development and it is concluded that their expression and distribution in CNT may play a role in the development of urine concentration abilities in rat kidney.

The Effects of Externally Administered Insulin-like Growth Factor-I on Glomerulosclerosis during Compensatory Renal Hypertrophy in Rats / 대한비뇨기과학회지

PURPOSE: Among renotropic agents, a growth hormone (GH) independent insulin-like growth factor-I (IGF-I) improves renal function without inducing glomerulosclerosis and its potential for treating renaldisease is increasing. With this in mind, this study was designed to find out the effects of externallyadministered IGF-I toward preventing glomerulosclerosis after renal volume loss in rats. MATERIALS AND METHODS: Sprague-Dawley rats (110-130gm) were divided into four groups in accordance with 3/4 nephrectomyand/or IGF-I administration. The 3/4 nephrectomy was performed at 30 days after birth, and recombinanthuman IGF-I was administered for 60 days after 3/4 nephrectomy. The change of body weight and wet weight of the remnant kidney were determined. The glomerular planar area and percentage of glomerulosclerosiswere measured. RESULTS: The body weight andthe wet weight of remnant kidney were significantly increased after administration of IGF-I in the3/4 nephrectomy group. The glomerular planar area was significantly increased after administration of IGF-I in the 3/4 nephrectomy group, and significant increase in glomerular planar area was observed in the 3/4 nephrectomy group regardless of IGF-I administration. And the percentage of glomerulosclerosis was significantly decreased. CONCLUSIONS: Thus it is concluded that this study proved that the externally administered IGF-I prevents lomerulosclerosis after severe renal volume loss in rats. It may have a potential to become a useful medical agent for suppressing glomerular sclerotic change and facilitating renal function in chronic renal failure patients.

Effect of acute respiratory acidosis and alkalosis on the subtypes of intercalated cells in rat kidney / 대한해부학회지

Intercalated cells play a major role in proton and bicarbonate secretion in the collecting duct of kidney. A third type of intercalated cell (non A-non B cell), besides type A and B intercalated cells, and a bipolar cell are known to exist in the kidneys of the rat or the mouse. The third type cell has H(+)-ATPase in the apical membrane like the type A intercalated cell, but has no Cl(-)-HCO(3)- exchanger (AE1) on the basolateral membrane. The bipolar cell was shown to express H(+)-ATPase on both the apical and basolateral membranes. The functions of these cells, however, are not determined yet. This study was intended to know the immunohistochemical changes of the intercalated cell subtypes in the acute respiratory acidosis and alkalosis. After midline tracheostomy, respiratory acidosis and alkalosis were induced and maintained for 4 hours in the Sprague-Dawley rats (450~500 g) using a Rodent Ventilator. The kidneys were preserved for immunohistochemical studies by in vivo perfusion fixation with periodate-lysine-paraformaldehyde solution through the abdominal aorta. To identify the subtypes of intercalated cells and the tubule segments in which they are located, a triple immunolabeling procedure was used. Distal convoluted tubule cells and principal cells in the collecting duct were identified using antibody to thiazide sensitive Na(+)Cl(-) cotransporter and antibody to aquaporin-2, respectively. Antibodies to H(+)-ATPase and AE1 were used to identify subpopulation of intercalated cells. Type A cells were activated in respiratory acidosis with enhanced AE1 activity on the basolateral membrane and H(+)-ATPase reactivity moved to the apical membrane, whereas inactivated in respiratory alkalosis with decreased AE1 reactivity and H(+)-ATPase reactivity moved to the supranuclear cytoplasm. The change in reactivity of type A cells in respiratory acidosis or alkalosis was shown to differ depending on the tubular segments: most of the intercalated cells were activated in the outer medullary collecting duct while only a portion of the type A cells activated in the distal convoluted tubule, connecting tubule and cortical collecting duct. No changes were observed in type B cells in respiratory acidosis and alkalosis. In non A-non B cell which was increased in size in respiratory acidosis, H(+)-ATPase reactivity was seen on the apical membrane in respiratory acidosis, while seen in the supranuclear cytoplasm in respiratory alkalosis. These findings indicated that the renal compensation for respiratory acid-base imbalance was mediated mainly by type A cells rather than by type B or non A-non B cells. Among type A cells, more of those of outer medullary collec-ting duct were thought to be recruited compared with those of the cortical collecting duct and connecting tubule.

Effects of Vitamin D, PTH and Calcitonin on Osteopontin Expression in Rat Kidney / 대한신장학회잡지

Osteopontin(OPN) is a secreted phosphoprotein that is expressed constitutively in the descending thin limb(DTL) and papillary surface epithelium (PSE). Although the function of OPN is not known with certainty, it has been suggested that OPN may play a role in the regulation of calcium-mediated or calcium-dependent processes. The aim of this study was to compare the effects of 1,25-dihydroxyvitamin D3(VitD), parathyroid hormone(PTH) and calcitonin, the hormones involved in the regulation of calcium homeostasis, on renal OPN expression. Three groups of rats were studied:1)acute(single injection of VitD, 200ng/100g BW s.c., 12h before sacrifice) and chronic VitD(daily injection of VitD 50ng/day/100g BW s.c. for 7d). 2) acute(single injection of PTH 50 microgram/100g BW i.p., 30min before sacrifice) and chronic PTH(infusion of PTH 6 microgram/day/100g BW s.c., for 7d via miniosmotic pump). 3) acute(single injection of calcitonin 25U/100g BW i.p., 1h before sacrifice) and chronic calcitonin(infusion of calcitonin 0.2U/ hr/kg BW s.c., for 7d via miniosmotic pump). Each of the study was compared with vehicle-treated animals. Kidneys were processed for immunohistochemistry and OPN expression was examined using a monoclonal antibody to OPN. In vehicle-treated animals, OPN was expressed primarily in DTL and PSE. In the acute VitD and PTH groups, OPN immunoreactivity appeared strongly in proximal tubule, and increased slightly in DTL and PSE. In the chronic VitD and PTH groups, there was a marked increase in OPN immunoreactivity in DTL, PSE, distal convoluted tubule(DCT) and thick ascending limb(TAL) of Henle's loop. Calcitonin groups showed no apparent change. In summary, this study demonstrates that OPN is constitutively expressed in the cells of the DTL and PSE, and induced in proximal tubule(PT), DCT and TAL by vitD and PTH. From these results we conclude that vitD and PTH play an important role in regulation of OPN expression not only in DTL and PSE but also in PT, DCT and TAL.

Effect of Extracorporeal Shock Waves on Immature Rat Kidney / 대한비뇨기과학회지

Although extracorporeal shock wave lithotripsy(ESWL) has been used to treat renal stones for several years, little is known of its effect on developing tissue. To study the long-term bio-effects of this mode of treatment on the immature animal, we used 32 Sprague-Dawley rats at 4weeks of age and divided 4 groups which consisted of 8 rats respectively. They were weighted and left nephrectomy was then performed. 10 days later, 3 groups received extracorporeal shock waves (16 kV) of 500, 1,000, 1,500 times respectively to the right kidney using Lithoring(3rd generation pendulum-ESWL), but control group didn't received shock waves. They were allowed to mature, and at 16 weeks of age they were evaluated for weight and serum creatinine. The right kidney was then harvested, weighted and stained with hematoxylin and eosin. There were no significant changes in over-all animal growth, renal growth and renal function in the post-treatment groups when compared to the control group. At comparison of histological changes, the grade of interstitial nephritis was proportional to the number of shock wave received In conclusion, shock waves delivered to immature animals do not significantly affect animal growth, renal growth and function, but it can cause significant permanent histological renal changes even at low doses and further studies are needed with an adult control group in an attempt to delineate whether the immature kidney is, indeed, more vulnerable to the shock waves.

Uranyl Nitrate Induced Polyuric Acute Tubular Necrosis in Rats

We investigated the pathobiological course of uranyl nitrate (UN) induced polyuric acute tubular necrosis (ATN) in male Sprague Dawley rats. UN (5mg/kg 15mg/kg and 3Omg/kg) in 5% NaHCO3 induced weight loss, polydipsia, and polyuria 24 hrs after injection when compared to the controls which were treated with 5% NaHCO3 only. Twenty four hours following the injection of UN, serum creatinine and blood urea nitrogen levels had increased. These changes continued for at least 72 hours, although the concentration of uranium had decreased. Light microscopic studies conducted 24 hours after injection, revealed partial degeneration and necrosis of the proximal tubules and many casts m the distal convoluted tubules. These changes progressed for 72 hours. Despite this tubular damage, the glomeruli were relatively intact. 5 days after injection, the epithelial cells lining the proximal tubules displayed regenerative activities; these findings were more prominent after 10 days. Through electron microscopic examination, we observed the destruction of mitochondria in the proximal tubular cells, a possible cause of polyuria. Ten days post injection regenerative activities in the proximal tubular cells showed that the maturation of intracellular organelles followed the proliferation of the premature cells.

Interactions of progesterone with D-amino acid oxidase–Different effects on apo- and holo-enzyme.

A simple method using charcoal treatment was developed for the preparation of apo-D-amino acid oxidase from rat kidney homogenates. This apo-D-amino acid oxidase was used to study the effect of progesterone on the apo- and holo-enzyme. Progesterone inhibited the activity of D-amino acid oxidase, when the apo- enzyme, preincubated with saturating amounts of FAD was used; this effect varied with FAD concentration. Progesterone did not inhibit the activity when added to a mixture of non-preincubated apo-enzyme and FAD; this suggests that progesterone has different effects on apo- and holo D-amino acid oxidase.