Three-dimensional visualization of the renal proximal tubules in developing mice / 解剖学报

Objective Adult proximal tubule ( PT ) is not only the segment most frequently involved in acute renal tubule injury, but also the easiest to repair. It may be consistent with the rapid growth and differentiation mechanism of this segment during the development of the kidney, while the developing information is insufficient. Therefore, we three- dimensional visualized the developing PT to analysis its spatiotemporal morphogenesis. Methods The kidneys were obtained from mice at various developing time point, embryonic day ( E ), postnatal day ( P ). The volume density of Claudin-2 positive PT in the cortex was measured using a stereological method in paraffin sections. After image recording and alignment of the serial sections, the spatial courses of the developing PT were traced and visualized in three dimensions using computer-assisted program. The length of the developing PT was calculated at the same time. Results The volume density of PT in the cortex of PI mice was significantly higher than that in the embryonic stage. Then it experienced a decline ( P3, P5 ), an increase ( start at P7 ) to a stable adult level ( P28 ). The tubular tracing showed that the lengths of developing PT and the number of convolutions of their convoluted part increased with the maturation, but lower than that of adultin E14. 5, E17. 5 and P5 PT in E14. 5 and E17. 5 mice were similar to that of adult with respect to general spatial courses. They were, however, significantly different from adult in the initial direction of PT and the arrangement of the straight part of PT in the medullary rays. While, it was in P5 that the spatial pattern of some PT was gradually approaching to the adult model. Conclusion This study demonstrated that the development of PT was consistent with the kidney development in terms of its volume density in cortex, length and spatial course. It started at the S-shaped body, kept throughout the embryonic period and continued to postnatal, ended at kidney maturation ( P28 ).

Effect of Liraglutide on Toll-like Receptor 4 and Na+/K+-ATPase Activity in Proximal Tubular Epithelial Cells of Rats Induced by High Glucose / 医药导报

Objective To explore the protective effect of liraglutide ( Li) on the proximal tubular cells ( PTECs) of rats induced by model control. Methods The PTECs of rats were obtained by primary culture and divided into normal control group ( NC) , model control group ( HG) , HG+low-dose Li group, HG+medium-dose Li group, and HG+high-dose Li group ( n-6 in each group) . After 72 h of incubation, the activity of Na+/K+-ATPase in PTECs was measured by the liquid scintillation counter. Protein expression of Toll-like receptor 4 ( TLR4 ) was measured by Western blotting. Interleukin-6 ( IL-6 ) , tumor necrosis factor-α (TNF-α) and plasminogen activator inhibitor-1 (PAI-1) in culture supernatants were measured by the enzyme linked immunosorbent assay (ELISA). Results Compared with NC, activity of Na+/K+-ATPase [(2 737. 88±317. 22)μmol·g-1 ·h-1 ] and the protein expression of TLR4 in PTECs, IL-6, TNF-α and PAI-1 in culture supernatants were significantly increased in HG (P<0. 05, or P<0. 01). Compared with HG, activity of Na+/ K+-ATPase and the protein expression of TLR4 in cells, IL-6, TNF-αand PAI-1 in culture supernatants reduced significantly in HG+medium-or high dose Li groups (P<0. 05, or P<0. 01). Conclusion Liraglutide can inhibit the expression of inflammatory cytokines and stabilize the Na+/ K+-ATPase’ s activity in PTECs in high glucose environment. Therefore, it may play a role in kidney protection.

The Role of Proximal Nephron in Cyclophosphamide-Induced Water Retention: Preliminary Data

Cyclophosphamide is clinically useful in treating malignancy and rheumatologic disease, but has limitations in that it induces hyponatremia. The mechanisms by which cyclophosphamide induces water retention in the kidney have yet to be identified. This study was undertaken to test the hypothesis that cyclophosphamide may produce water retention via the proximal nephron, where aquaporin-1 (AQP1) and aquaporin-7 (AQP7) water channels participate in water absorption. To test this hypothesis, we gave a single dose of intraperitoneal cyclophosphamide to male Sprague-Dawley rats and treated rabbit proximal tubule cells (PTCs) with 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide. In the short-term 3-day rat study, AQP1 protein expression was significantly increased in the whole kidney homogenates by cyclophosphamide administration at 48 (614 +/- 194%, P < 0.005), and 96 (460 +/- 46%, P < 0.05) mg/kg BW compared with vehicle-treated controls. Plasma sodium concentration was significantly decreased (143 +/- 1 vs. 146 +/- 1 mEq/L, P < 0.05) by cyclophosphamide 100 mg/kg BW in the long-term 6-day rat study. When primary cultured rabbit PTCs were treated with 4-HC for 24 hours, the protein expressions of AQP1 and AQP7 were increased in a dose-dependent manner. Quantitative polymerase chain reaction revealed no significant changes in the mRNA levels of AQP1 and AQP7 from cyclophosphamide-treated rat renal cortices. From these preliminary data, we conclude that the proximal nephron may be involved in cyclophosphamide-induced water retention via AQP1 and AQP7 water channels. Further studies are required to demonstrate intracellular mechanisms that affect the expression of AQP proteins.

Effect of Insulin on D_5 Dopamine Receptor Expression and Function in Renal Proximal Tubule Cells / 中华高血压杂志

Objective To investigate the effect of insulin on D_5 dopamine receptor expression and function in renal proximal tubule (RPT).Methods Immortalized RPT cells and D_5 receptor transfected HEK293 (HEK-D_5) cells were used in the study to investigate the effect of insulin on D_5 receptor expression and function,and those effects were compared in RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The function of D_5 receptor was determined by measurement of the Na~+-K~+-ATPase activity in HEK-D_5 cells. Results Insulin increased D_5 receptor protein expression in a concentration and time-dependent manner in WKY RPT cells,but not in SHR.The basal level of D_5 receptor expression was higher in WKY cells than that in SHR cells. Stimulation with fenoldopam(D_1-like dopamine receptor agonist) inhibited the Na~+-K~+-ATPase activity;pretreat- ment with insulin increased the inhibitory effect of fenoldopam on Na~+-K~+-ATPase activity in HEK-D_5 cells. Conclusion The abnormal regulation of insulin on D_5 receptor expression and function might be involved in the path- ogenesis of essential hypertension.

Effects of bioartificial kidney on cardiovascular function and cytokine response in endotoxic shock pigs / 中华急诊医学杂志

Objective To test the role of a bioartificial kidney which consisted of a continuous vein-vein hemofiltration (CVVH) system and renal proximal tubule cells (PTCs)device, also called the renal tubule assist device (RAD),in endotoxin shock.Methods Female crossbred pigs 25 to 30 kg received with 3×1011 bacteria / kg body weight of E.coli intraperitoneally followed by treatment with a sham RAD without cells (n=7) or a RAD with cells (n=7). Data on blood pressure, cardiac output, renal blood flow and systemic marker of inflammation (IL-6) were measured.Results Cardiac outputs and renal blood flows were higher in the RAD group with cells than in the sham RAD group after E.coli infusion (P＜0.05). Plasma IL-6 levels were lower in the cell RAD group than in the sham RAD group after bacterial administration (P＜0.05). A significant difference on survival time was also observed between the two groups,with(9.07 ± 0.88) h in the cell RAD group vs. (5.10 ± 0.46)h in the sham RAD group, P＜ 0.01.Conclusion The bioartificial kidney containing PTCs can improve cardiovascular function,reduce cytokine levels,and prolong survival time.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Renal Transport of Urate

All urate transport occurs across the renal epithelial cells of the proximal tubule. Most of the filtered urate is reabsorbed in the S1 segment of the early proximal tubule. This is followed by tubular secretion in the S2 segment of the proximal tubule and approximately 50% of the filtered urate flows back into the tubular lumen. Most of the secreted urate undergoes postsecretory reabsorption that occurs predominantly in the last S3 segment of the proximal tubule. Recently, four proteins that transport urate have been identified at the molecular level. These proteins are an electrogenic urate uniporter, urate transporter/channel (UAT), two members of the organic anion transporter (OAT) family, OAT1 and OAT3, and a protein with some homology to OAT4, designated URAT1.

Adaptive Responses to Dexamethasone Treatment in Rat Kidney Proximal Tubules / 대한해부학회지

Excess accumulation of glucocorticoid increases acid secretion and HCO3- reabsorption in the kidney. Reabsorption of HCO3-, which almost occurs at the proximal tubule, is mediated Na+ / H+ exchanger-3 (NHE-3) and H+ -ATPase on the apical membrane and the Na + /HCO3- cotransporter-1 (NBC-1)on the basolateral membrane. Impact of glucocorticoid was investigated by immunohistochemistry and electron microscopy to correlate the changes with the effect of in vivo dexamethasone treatment for the rat kidney proximal tubule. In a control group, immunoreactivity of NHE-3 was detected in the apical membrane and the brush borders of S1, S2 and 3 segments of the proximal tubule. Immunoreactivity of NBC-1 was detected in the basolateral membrane of S1 and S2 segments of the proximal tubule. Immunoreactivity of NHE-3 and NBC-1 protein was more pronounced in dexamethasone treated groups than the control group. Dexamethasone 1 mg/kg caused most intense immunoreactivity for NHE-3 and NBC-1 protein, however, 0.01 mg/kg and 0.1 mg/kg produced less intense immunoreactivity with no appreciable differences between these lower doses of dexamethasone groups. By electron microscopy, the tubular cells of S1 segment of the control group revealed numerous mitochondria, endocytic apparatuses, lysosomes and many basal cytoplasmic processes. In dexamethasone treated groups, the cells of S1 and S2 segments of the proximal tubule had more mitochodria and more basolateral invaginations and had an increased number of more elongated microvilli, compared with the control group. The cells of the S3 segment of the control group showed scant lateral interdigitations and had a few smaller mitochondria. The cells of the S3 segment of dexamethasone treated groups had many mitochodria and an increased number of microvilli in the brush border, but revealed no difference of basolateral invaginations among the different groups of dexamethasone. These results indicate that prolonged administration of excess glucocorticoid increases NHE-3 and NBC-1 protein, and the up-regulation of these proteins could result in increased HCO3 - reabsorption in the rat renal proximal tubules. It also suggests that these adaptive responses closely correlate to morphological alterations of proximal tubular epithelial cells.

Changes of Na~+,K~+-ATPase activity in proximal tubules of type 2 diabetic rats / 第三军医大学学报

Objective To investigate the changes of Na~+,K~+-ATPase activity in the proximal tubules (PT) of type 2 diabetes mellitus(T2DM) and the correlation of PT Na~+,K~+-ATPase activity and endogenous digitalis-like substance (EDLS). Methods Female Wistar rats were developed a rat model of T2DM and then divided into 3 groups after injected stretozotocin, that is, 5 weeks group, 7 weeks group and 9 weeks group. The PT were microdissection by freehand. Na~+,K~+ -ATPase activity of PT were determined by liquid scintillation counter. Serum EDLS and insulin were determined using radioimmunoassay. Results Compared with control group, T2DM rats were associated with hyperglycemia, hypertriglyceridemia, dyslipidemia, and insulin resistance; Na~+,K~+-ATPase activity of PT in T2DM rats was significantly increased, and there was no significant difference among 3 sub-groups. Serum EDLS level was significantly enhanced in T2DM, however, EDLS levels were no significant difference among 3 sub-groups. Na~+,K~+-ATPase activity of PT had negative relation with EDLS level in serum. Conclusion Na~+,K~+-ATPase activity of PT in T2DM rats is increased and EDLS level in serum decrease may play a important role.

Effects of Dopamine on Intracellular pH in Opossum Kidney Cells

Na+/H+ exchanger (NHE) has a critical role in regulation of intracellular pH (pHi) in the renal proximal tubular cells. It has recently been shown that dopamine inhibits NHE in the renal proximal tubules. Nevertheless, there is a dearth of information on the effects of long-term (chronic) dopamine treatment on NHE activities. This study was performed to elucidate the pHi regulatory mechanisms during the chronic dopamine treatments in renal proximal tubular OK cells. The resting pHi was greatly decreased by chronic dopamine treatments. The initial rate and the amplitude of intracellular acidification by isosmotical Na+ removal from the bath medium in chronically dopamine-treated cells were much smaller than those in control. Although it seemed to be attenuated in Na+-dependent pH regulation system, Na+-dependent pHi recovery by NHE after intracelluar acid loading in the dopamine-treated groups was not significantly different from the control. The result is interpreted to be due to the balance between the stimulation effects of lower pHi on the NHE activity and counterbalance by dopamine. Our data strongly suggested that chronic dopamine treatment increased intrinsic intracellular buffer capacity, since higher buffer capacity was induced by lower resting pHi and this effect could attenuate pHi changes under extracellular Na+-free conditions in chronically dopamine-treated cells. Our study also demonstrated that intracellular acidification induced by chronic dopamine treatments was not mediated by changes in NHE activity.

Effects of breviscapine on Na~+,K~+-ATPase activity of proximal tubule in early diabetic rats / 中国药理学通报

Aim To investigate the effects of breviscapine(Bre)on Na+,K+-ATPase activity of proximal tubule(PT) and its renal protection in early diabetic rats. Methods The rats were divided into three groups: diabetic model (DM group), Bre treatment (Bre group) and normal control (NC group). Rats were administrated with Bre(20 mg?kg-1?d-1, ip) in Bre group, with normal saline(ip) in DM and NC groups for four weeks after diabetes induction with streptozotocin (STZ 65 mg?kg-1, ip) in Bre and DM groups. The urine and blood samples were collected from two intra-ureteral cannulas and the heart, respectively, under anesthesia four weeks after diabetes induction. After one of the renal arteries being perfused, the renal cortex was incubated and PT segments were microdissected freehand under microscope before the Na+,K+-ATPase activity in the segments were assessed by liquid scintillation counter. The blood glucose, levels of creatinine in serum and in urine were assayed. The microalbumin , ?2-microglobulin(?2-MG) of urine and endogenous digitalis-like substance (EDLS) of serum were measured respectively by radioimmunoassay. Results The PT Na+,K+-ATPase activity in NC group was(959.11?117.35) pmolPi?mm-1?h-1, and that in DM group was significant higher for (1893.53?383.90) pmolPi?mm-1?h-1 than it(P

Morphometrical and Histological Changes of Glomeruli and Proximal tubules of Contralateral Kidney in Uninephrectomized Rat / 체질인류학회지

This study was to investigate the histomorphomeric and histological changes of glomeruli and proximal tubules of contralateral kidney in uninephrectomized rat. Rats were Sprague Dawley strain and female 1-year-old. Uninephrectomized rats were sacrificed after 20 days, 30 days, and 40 days. The results were as follows: 1. The mean areas and diameters of the glomeruli were markedly increased at the 30 days. 2. In the glomeruli, the mean nuclear areas of podocytes were markedly increased, but those of cells of the parietal layer were decreased. At the 20 days, those of the endothelial cells were slightly incerased. 3. The lumens of the endothelial cells were irregularly expanded and folded at the 20 days. The basal lamina of the 30 days was markedly thickened in the electron microscopic finding. 4. At the 30 days, the mean areas and mean thicknesses of proximal tubules were markedly increased, but those were not normal. At the 40 days, the mean thicknesses of proximal tubules were restored to the almost normal. 5. Light microscopic changes of the proximal tubules had no difference between the control and the experimental group. In electron microscopical findings, the cytoplasm of the proximal tubules were densely filled with the enlarged mitochondria at the 30 days. At the 40 days, some large vacuoles appeared in the cytoplasm.

Study of K~(+) channels at the apical membrane of mouse renal proximal tubule cell with patch-clamp / 第三军医大学学报

Objective To study the property of the K~(+) channels at the apical membrane of mouse renal proximal tubule cells.Methods Single-channel recording of the K~(+) channel currents in apical membrane of freshly isolated renal proximal tubule cells was performed using the cell-attached mode of the patch-clamp technique.Results This kind of potassium channel appeared to be an inward rectifier,and its limiting inward slope conductance was(11.4?0.6) pS.The channel activity increased with hyperpolarization.Conclusion The potassium channel with spontaneous current was observed in most of the cell-attached patches,which is thought to be involved in volume regulation,potassium recycling and stabilization of the apical potential.

THE CULTURE OF PROXIMAL TUBULE CELLS OF RABBIT KIDNEY / 解剖学报

It is a new approach to investigate the ultrastructure, function and metabolism of each segment of uriniferous tubule using dual environment culture chambers (DECC). In the present study the DECC culture of proximal tubule cells was undertaken with millcell-HA culture insert in the mixed medium of Ham's F12 and Dulbecco's modified Eagle's medium.A monolayer of proximal tubule cells was obtained in the medium mixed by constant bubling of oxygen at 3 weeks after seeding. Transmission electron microscopy revealed the cuboidal cells with microvilli, abundant mitochondria, vacuoles, lysosomes, and irregular plasma membrane infoldings. Also, the monolayer exhibited active metabolism from the release of ~(14)C-I-carbon dioxide and absorption ability of ~(14)C-I-glucose across the cultured monolayer. In addition, the culture procedure has been introduced in detail.