Cells Of Proximal Convoluted Tubules Research Articles

Internalization and effects on cellular ultrastructure of nickel nanoparticles in rat kidneys.

Purpose: Since nanoparticles (NPs) are beginning to be introduced in medicine and industry, it is mendatory to evaluate their biological side-effects, among other things. The present study aimed to investigate the pathways by which nickel nanoparticles (NiNPs) enter nephrons and to evaluate their localization and effects on cellular ultrastructure.Methods: Rats were injected intraperitoneally with 20 nm NiNPs (20 mg/Kg/b.w./day) for 28 consecutive days. Transmission electron microscope technique was used to detect localization of NiNPs and their effects on cellular ultrastructure in rat kidneys. Additionally, measurements of certain biochemical parameters such as creatinine, urea, uric acid and phosphorus for investigating renal function following NiNPs treatment were taken.Results: The presence of NiNPs in the nephrons in treated rats was confirmed by transmission electron microscopy. NiNPs entered the renal tubules cells via various pathways. The results indicated that NiNPs administration induced ultrastructural changes in the proximal cells of renal tubules and certain glomerular cells (podocytes and mesangial cells). Additionally, NiNPs were found to be localized in the mitochondria, which led to a significant decrease in their density and morphology. Furthermore, cell death was induced in the glomerular cells as found with a Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay and through detection of p35 using immunohistochemical staining.Conclusion: Herein, NiNPs were found to induce various cellular ultrastructural changes in the kidneys of rats. NiNPs used diverse pathways to internalize into the cytoplasm of the proximal convoluted tubules (PT) cells across the basement membrane, and also through the plasma membrane of two adjacent PT cells. NiNPs internalization, accumulation and their alterations of the cellular ultrastructure affected rat renal function.

3‐D Electron Microscopy of Mouse Proximal Convoluted Tubule Endo‐lysosomal System

Blood low molecular weight proteins (LMWPs) are freely filtered in the kidney glomeruli and subsequently reabsorbed via receptor‐mediated endocytosis (RME) by epithelial cells lining the proximal convoluted tubule (PCT). After RME, the destiny of LMWPs that enter the endo‐lysosomal system (ELS) is to either be degraded in lysosomes or recycled to the blood stream via transcytosis. The PCT is classically divided into early and late segments (S1 and S2, respectively), based on differences in cellular ultrastructure observed with electron microscopy (EM). We recently found that RME of LMWPs takes place almost entirely in S1 (JASN 2018). The aim of this study was to investigate how the structure of the ELS in S1 cells is adapted to this purpose.To further investigate the reasons for axial differences in LMWP reabsorption capacity, we first performed transmission‐EM of C57Bl/6J‐Rj mouse PCTs using a fixation strategy optimized to preserve the structure of the ELS. We found electron‐transparent small apical vesicles densely packed beneath the apical brush border, which formed a much thicker layer in S1 than in S2. Moreover, S1 cells contained numerous large apical vacuoles (LAVs) heterogeneous in electron density, shape and dimension. Lysosomes were observed as rounded structures that could be easily distinguished from the irregular LAVs in S1. Lysosomes in S1 had a different appearance to those in S2, being much more electron‐dense in the latter.To gain deeper insights into the micro‐anatomical features responsible for the greater capacity of LMWP reabsorption in S1, we performed high resolution 3‐D imaging using focused ion beam‐scanning electron microscopy (FIB‐SEM). Sections were acquired every 5nm through the entire thickness of a single S1 cell. Afterwards, using a machine‐learning based approach (ilastik‐1.3.2), we segmented the different components of the ELS and reconstructed their 3‐D appearance. Using this approach, we found that the vast majority of the electron‐transparent vesicles found underneath the apical brush border actually have direct openings into the tubular lumen, and thus represent apical membrane invaginations. LAVs, which are about 1.5μm in diameter, exhibited complex and irregular 3‐D structures, suggesting they are highly dynamic, In contrast, lysosomes were spherical in shape and ranged from 500nm to 1μm in diameter. Finally, we observed a few electron‐transparent small vesicles in the basal third of the cell, which may represent organelles responsible for transcytosis of internalized cargoes to the basolateral membrane.In summary, 2‐D electron microscopy in C57Bl/6J‐Rj mice revealed micro‐anatomical differences in ELS structure between S1 and S2 PCT cells, which may help to explain differences in endocytic capacity. Furthermore, using 3‐D EM we were able to reconstruct the complex organization of the endocytic machinery of S1 cells and to visualize the connections between its components. Taken together, these findings have important implications for understanding reabsorption and handling of LMWPs in the PCT in vivo.Support or Funding InformationNCCR Kidney. CH and S NSF grant 31003A166507This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Histological and immunohistochemical evaluation of the dose-dependent effect of gold nanoparticles on the renal cortex of adult female albino rat

Introduction: Applications of gold nanoparticles (GNPs) in modern life lead to the release of GNPs into the environment, thus representing a potential hazard to human health.Aim of the work: To evaluate the effect of GNPs on the renal cortex of adult female rats employing different histological and immunohistochemical techniques.Materials and Methods: Thirty adult female albino rats were divided into three equal groups; Group I (Control), in groups II and group III, GNPs were administered daily as 40μg/kg and 400μg/kg for 14 days, respectively. Specimens were processed for light and electron microscopy, in addition to immunohistochemical staining for tumor necrosis factor-alpha (TNF-α).Results: GNPs-treated animals showed many hypercellular glomeruli. Most proximal convoluted tubules (PCTs) were severely distorted, their cells showed vacuolated cytoplasm and pyknotic nuclei. Many distal convoluted tubules (DCTs) showed exfoliated cells and some karyolytic nuclei. Some tubules were totally distorted. Dilated congested blood vessels together with extensive mononuclear cellular aggregations were observed. Ultrastructural examination revealed podocytes with small heterochromatic nuclei and interrupted processes with distortion and effacement. Many GNPs resided in the podocytic terminal processes. Some cells of PCTs showed irregular nuclei, variably sized and shaped mitochondria with loss of their basal orientation. Focal areas of rarefied cytoplasm and apical vacuoles were observed. Cells of DCTs showed irregular nuclei and numerous abnormally shaped mitochondria. Irregularly thickened basal lamina and GNPs were clearly observed in their cytoplasm. The immunohistochemical study showed a highly significant increase in TNF-α immunoreaction.Conclusion: GNPs cause dose-dependent structural changes in renal cortex with various degrees of inflammation.

MICROSCOPICAL STUDIES OF THE EFFECT OF THE ORGANOPHOSPHOROUS INSECTICIDE MONOCROTOPHOS ON THE KIDNEYS OF MALE ALBINO MICE

The present study aimed to throw more light on the effect of the insecticide monocrotophos on the kidneys of mice treated orally with the insecticide. The applied doses were 1/20, 1/6 and 1/3 LD50 (0.38, 1.25 and 2.5 mg/kg body weight), respectively. Each dose was given for seven successive days. The observed histopathological and ultrastructural alterations were found to be dose-dependent. The low dose (1/20 LD50) caused shrinkage of some glomeruli; some were congested and the cellularity in some others were increased. Both of the proximal and distal convoluted tubules cells showed many cytoplasmic vacuoles and pyknotic nuclei and disappearance of microvilli of the proximal convoluted tubule cells. The degenerative alterations became more severe in case of the median (1/6 LD50) and the high (1/3 LD50) doses of monocrotophos. In such cases, the glomeruli revealed cellular abnormalities, necrosis and some of them were deeply clefted. Dissociated and degenerated renal tubule cells with pyknotic nuclei, and cell debris in the tubules lumina were observed in the kidney cortex. In case of the cumulative treatment, monocrotophos in a dose of 0.19 mg/kg given for twenty successive day, caused similar degenerative changes to the afore-mentioned alterations. These histopathological alterations showed partial recovery when inspected on the seventh day after the last dose of treatment, and the recovery became more pronounced on the fourteenth days after the last treatment. This may indicate withdrawal of the insecticide from the experimental animal after stopping treatment. The ultrastructural alterations showed increased mesangial matrix and atrophied podocytes with fusion of their secondary foot processes. The cells of the convoluted renal tubules displayed degenerated mitochondria, short profiles of RER, pyknotic and chromatolytic nuclei and presence of cytosegrosomes (sequestration of parts of their cytoplasm).

Calcifying nanoparticles induce cytotoxicity mediated by ROS-JNK signaling pathways.

Calcifying nanoparticles (CNPs) play an important role in kidney stone formation, but the mechanism(s) are unclear. CNPs were isolated and cultured from midstream urine of patients with kidney stones. CNP morphology and characteristics were examined by electron microscopy and electrophoresis analysis. Chemical composition was analyzed using energy-dispersive X-ray microanalysis and Western blotting. Human renal proximal convoluted tubule cell (HK-2) cultures were exposed to CNPs for 0, 12 and 72h, and production of reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis levels were evaluated. CNPs isolated from patients showed classical morphology, the size range of CNPs were 15-500nm and negative charge; they were found to contain fetuin-A. Exposure of HK-2 cells to CNPs induced ROS production, decreased mitochondrial membrane potential and decreased cell viability. Transmission electron microscopy showed that CNPs can enter the cell by phagocytosis, and micrographs revealed signs of apoptosis and autophagy. CNPs increased the proportion of apoptotic cells, down-regulated Bcl-2 expression and up-regulated Bax expression. CNPs also up-regulated expression of LC3-B, Beclin-1and p-JNK.CNPs are phagocytosed by HK-2 cells, leading to autophagy, apoptosis and ROS production, in part through activation of JNK signaling pathways. ROS and JNK pathways may contribute to CNP-induced cell injury and kidney stone formation.

The mechanism of action of sodium–glucose co‐transporter 2 inhibitors is similar to carbonic anhydrase inhibitors

Butler et al.,1 in their review about the potential role of sodium–glucose co-transporter-2 inhibitors (SGLT2i) in the treatment of heart failure (HF), do not mention the carbonic anhydrase (CA) inhibitors, whose mechanism of action has remarkable similarities with SGLT2i. Although there are no studies in humans,2 it is established that around 30% of the filtered Na+ is reabsorbed through the Na+/H+ isoform 3 (NHe3) exchanger, which is located in the proximal convoluted tubule (PCT), whereas up to 15% of tubular Na+ is reabsorbed through SGLT2 in situations of hyperglycaemia. The SGLT2i inhibit both the SGLT2 and the NHe3 exchangers that share the microdomain with SGLT2,2 and they have a natriuretic effect independent of the glycaemic level.3 On the other hand, NHe3 is activated in HF and is involved in the states of resistance to diuretics.4 Carbonic anhydrase is an enzyme located on the luminal membrane of PCT cells that accelerates the formation of H2CO3 and its subsequent conversion into H2O and CO2 using H+ and HCO3−, which favours the mobilization of NHe3 substrates and increases the tubular reabsorption of Na+. CA inhibitors, such as acetazolamide, act to inhibit the CA, slowing down the H2CO3 circuit and decreasing NHe3 activity, leading to natriuresis.5 The SGLT2i and CA inhibitors indirectly inhibit NHe3, cause mild to moderate natriuresis and bicarbonaturia, and activate the juxtaglomerular system by decreasing hyperfiltration and proteinuria, secondary to increased Na+ and Cl− reabsorption by macula densa (MD). This reabsorption would activate the juxtaglomerular system allowing the local secretion of adenosine that in turn stimulates the afferent arteriole vasoconstriction, which leads to a decrease in hyperfiltration. This haemodynamic fact does not occur with loop diuretics, thiazides, or K+-sparing diuretics. The loop diuretics block the carrier Na+/K+/2Cl− in the MD, and the others act beyond the ascending loop of Henle and MD, so these drugs would have no proteinuric or nephroprotective effect.5 There are differences in the natriuretic effect between SGLT2i and CA inhibitors. CA inhibitors lose their diuretic effect in the long term due to compensatory mechanisms of distal reabsorption.5 However, the SGLT2i maintain their long-term diuretic effect. Thus, currently, the clinical use of acetazolamide in HF is restricted to patients with diuretic resistance. In contrast, the diuretic effect of SGLT2i is maintained over time, probably due to the glucosuric effect6 that modifies renin levels.1 Despite these differences in their long-term temporal effect considering the similarities in the mechanism of action of SGLT2i and CA inhibitors, and considering the benefit demonstrated by SGLT2i, it would be interesting to develop specific clinical trials with CA inhibitors in patients with diabetes and HF (https://clinicaltrials.gov/ct2/show/NCT01973335).

Dietary fat composition influences glomerular and proximal convoluted tubule cell structure and autophagic processes in kidneys from calorie-restricted mice.

SummaryCalorie restriction (CR) has been repeatedly shown to prevent cancer, diabetes, hypertension, and other age‐related diseases in a wide range of animals, including non‐human primates and humans. In rodents, CR also increases lifespan and is a powerful tool for studying the aging process. Recently, it has been reported in mice that dietary fat plays an important role in determining lifespan extension with 40% CR. In these conditions, animals fed lard as dietary fat showed an increased longevity compared with mice fed soybean or fish oils. In this paper, we study the effect of these dietary fats on structural and physiological parameters of kidney from mice maintained on 40% CR for 6 and 18 months. Analyses were performed using quantitative electron microcopy techniques and protein expression in Western blots. CR mitigated most of the analyzed age‐related parameters in kidney, such as glomerular basement membrane thickness, mitochondrial mass in convoluted proximal tubules and autophagic markers in renal homogenates. The lard group showed improved preservation of several renal structures with aging when compared to the other CR diet groups. These results indicate that dietary fat modulates renal structure and function in CR mice and plays an essential role in the determination of health span in rodents.

MP19-02 HUMAN RENAL PROXIMAL TUBULE CELLS EXPRESSING FUNCTIONAL CHARACTERISTICS WITH MEMBRANOUS AND 3D TUBULAR CHITOSAN SCAFFOLD

You have accessJournal of UrologyStem Cell Research1 Apr 2015MP19-02 HUMAN RENAL PROXIMAL TUBULE CELLS EXPRESSING FUNCTIONAL CHARACTERISTICS WITH MEMBRANOUS AND 3D TUBULAR CHITOSAN SCAFFOLD Ini Chiang Ini ChiangIni Chiang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.994AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Human renal proximal tubule cells (HRPTCs) are responsible for reabsorption of glomerular filtrations and maintenance of water/electrolytes internal balance. Primary culture of HRPTCs is a promising target for applications in bioengineering, reconstructive medicine, nephrotoxicity assay, physiological studies, and even bioartificial kidney. In this study, we chose chitosan to fabricate membranous and tubular scaffolds for HRPTCs cultivation due to biocompatibility, biodegradability, antibacterial and wound-healing activity. METHODS First, primary HRPTCs digested and retrieved from nephrectomy/nephroureterectomy specimen were seeded on chitosan membrane, collagen-coated well, and blank petri dish (TCPS). Cell viability, dome quantitation, immunofluorescence characterization, western blot assay and transepithelial electrical resistance (TEER) were compared. RESULTS HRPTCs could be maintained on chitosan membrane with good viability and differentiation characteristics for at least six months in serum-free condition. Dome formation is a specific marker of renal proximal tubule cell differentiation. HRPTCs on chitosan membrane showed higher dome numbers than cells on collagen and TCPS. HRPTCs also showed highest expression of Na+-K+ ATPase £∖-1 subunit and lowest expression of Vimentin on chitosan membrane, which indicated that HRPTCs would be functional and of specific differentiation characteristics on this biomaterial. Then, HRPTCs were further cultivated on a 3D chitosan tubular scaffold successfully with cell full confluence and dome formation. Graph 1 (A) Cross-sectional view of the tubular chitosan scaffold (B) Longitudinal incision of the tube was done and the inner side of the tube was noted with confluent PTCs. (C) Dome formation was noted under x50 SEM field. Arrow: Dome (D) Dome formation under x500 SEM Graph 2 Human renal proximal convoluted tubule cell dome formation on chitosan membrane demonstrated by confocal microscopy with horizontal and vertical view CONCLUSIONS Human renal proximal tubule cells could be maintained with good viability and functional characteristics on chitosan membranous and tubular scaffold © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e215-e216 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ini Chiang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The vitamin D receptor in the proximal renal tubule is a key regulator of serum 1α,25-dihydroxyvitamin D₃.

It is well established that the mitochondria of proximal convoluted tubule cells of the kidney are the site of production of circulating 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The production of 1,25(OH)2D3 at this site is tightly regulated. Parathyroid hormone markedly stimulates 1,25(OH)2D3 production, whereas 1,25(OH)2D3 itself suppresses production. The mechanism of suppression by 1,25(OH)2D3 has not yet been elucidated. We have now found that in the absence of vitamin D (vitamin D deficiency), the vitamin D receptor (VDR) is found in the interior of the apical brush border of the proximal tubule cells. This is unique for the proximal tubule cells, since this has not been observed in the distal tubule cells or in other epithelial cells, such as intestinal mucosa. Administration of 1,25(OH)2D3 to vitamin D-deficient rats results in the movement of VDR from the brush border to the cytoplasm and nucleus presumably bound to reabsorbed 1,25(OH)2D3. The VDR bound to 1,25(OH)2D3 suppresses expression of 25-hydroxyvitamin D3 1α-hydroxylase and stimulates the 25-hydroxyvitamin D3 24-hydroxylase. Thus, VDR in the apical brush border of the proximal convoluted tubule cells serves to "sense" the level of circulating 1,25(OH)2D3 and modulates the activity of the 1α-hydroxylase and the 24-hydroxylase accordingly.

Role of losartan on the renal cortex damage induced by cisplatin in adult male albino rats

Introduction Cisplatin is a potent anticancer agent for the treatment of solid tumors, but its use is limited by its adverse effects. Aim This research aimed to evaluate the effect of losartan on cisplatin nephrotoxicity in adult male albino rats. Materials and methods Twenty-five male albino rats were divided into four groups. The first was the control group. Group II received a single injection of cisplatin. Group III received losartan for 6 days. Group IV received a single injection of cisplatin in addition to losartan ingestion for 6 days. Renal tissues were prepared for light and transmission electron microscopic examinations. Glomerular diameter and cell number were measured by means of an image analyzer. Also, glomerular capillary basement membrane (GBM) thickness, filtration slit width, and pedicel length were recorded and statistically analyzed. Results The renal cortical sections of group II rats showed loss of normal appearance of renal corpuscles with enlarged glomeruli and increased glomerular cellularity. Degenerative changes and necrosis of the lining cells of proximal convoluted tubule were noted. Intertubular hemorrhage and cellular infiltration were detected. Electron microscopic examination revealed fusion of the foot processes with significant increase in the thickness of GBM and filtration slit width. Damage of proximal convoluted tubule cells was seen more than damage of distal convoluted tubules. The renal cortical sections of group III rats showed vacuolated cytoplasm of some tubules lining cells. Electron microscopic examination revealed some ultrastructural changes in the distal convoluted tubule lining cells. Most of the renal corpuscles of rats treated with losartan and cisplatin appeared normal, with presence of some enlarged corpuscles as well as cytoplasmic vacuolation of the tubular cells. Electron microscopic examination revealed a significant decrease in GBM thickness and filtration slit width in comparison with cisplatin-received rats. Conclusion From this study, it was obvious that losartan ameliorates the histological changes induced by cisplatin given at a dose of 3 mg/kg in adult male rats.

Role of caveolin 1 in AT1a receptor-mediated uptake of angiotensin II in the proximal tubule of the kidney.

Caveolin 1 (CAV-1) functions not only as a constitutive scaffolding protein of caveolae but also as a vesicular transporter and signaling regulator. In the present study, we tested the hypothesis that CAV-1 knockout (CAV-1 KO) inhibits ANG II type 1 [AT1 (AT1a)] receptor-mediated uptake of ANG II in the proximal tubule and attenuates blood pressure responses in ANG II-induced hypertension. To determine the role of CAV-1 in mediating the uptake of FITC-labeled ANG II, wild-type (WT) mouse proximal convoluted tubule cells were transfected with CAV-1 small interfering (si)RNA for 48 h before AT1 receptor-mediated uptake of FITC-labeled ANG II was studied. CAV-1 siRNA knocked down CAV-1 expression by >90% (P < 0.01) and inhibited FITC-labeled ANG II uptake by >50% (P < 0.01). Moreover, CAV-1 siRNA attenuated ANG II-induced activation of MAPK ERK1/2 and Na(+)/H(+) exchanger 3 expression, respectively (P < 0.01). To determine whether CAV-1 regulates ANG II uptake in the proximal tubule, Alexa 488-labeled ANG II was infused into anesthetized WT and CAV-1 KO mice for 60 min (20 ng/min iv). Imaging analysis revealed that Alexa 488-labeled ANG II uptake was decreased by >50% in CAV-1 KO mice (P < 0.01). Furthermore, Val(5)-ANG II was infused into WT and CAV-1 KO mice for 2 wk (1.5 mg·kg(-1)·day(-1) ip). Basal systolic pressure was higher, whereas blood pressure and renal excretory and signaling responses to ANG II were attenuated, in CAV-1 KO mice (P < 0.01). We concluded that CAV-1 plays an important role in AT1 receptor-mediated uptake of ANG II in the proximal tubule and modulates blood pressure and renal responses to ANG II.

Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1–239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240–361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin−/− mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1UF. Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1UF were compared with full-length Sepp1, Sepp1Δ240–361, and Sepp1U40S as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1U40S, which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1UF from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1UF to function by itself as a peroxidase. Ultimately, plasma Sepp1UF and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3.

Histological Effects of Different Levels of Sorghum Grain on the Liver and Kidney of Ghezel×Arkhar-Merino Crossbred Lambs

Sorghum grains with variable concentrations of tannin (antinutritional factor) are becoming an increasingly important source of energy in animal nutrition. A large intake of tannins may cause gastrointestinal irritation, kidney and liver damage. Our experiment was conducted to study the effects of replacement different levels of barley grain with sorghum grain on the kidney and liver tissue. In this study sixteen male Ghezel×Arkhar-merino crossbred lambs were used. Dietary treatment were contain alfalfa hay (20% total DM), as roughage part of the diets and grain part (80% total DM) had different levels of the barley grain substituted with sorghum grain during 90 days. Lambs were randomly assigned to to receive one of the four dietary treatments in a completely randomized design assignment, in which sorghum grain was used in the levels of 60, 70 and 80 percent of total ration. The gross examination of kidney and liver did not reveal any significant histological changes. The microscopic results showed that, increases of the sorghum grain levels resulting histological changes in kidney and liver parenchyma. Histological examination of kidney of group A showed a little change as degeneration of brush borders in some of the proximal convoluted tubule (PCT) cells. In B group, results appeared which brush borders of PCT were destroyed therefore the high cuboidal cells of PCT were observed as low cuboidal epithelium and diameter of PCT increased. Hemorrhage and congestion is not observed in cortex but it was observed in medulla. In C group, degeneration of cells of PCT and DCT, hemorrhage in cortex, congestion in medulla which they are more in area near apex of renal pyramids were observed. Decrease length of cuboidal cells in medullary collecting ducts. Amyloidal sedimentation in PCT and DCT. In the liver of A and B group animals, histological structure of liver was normal, but sinusoidal abnormality was observed in A group and only a little congestion observed in some central venules in B group animals. Liver of C group animals had many histological changes as shrinkage of hepatocyte, degeneration and necrosis of hepatocytes around central venules, degeneration of some hepatic sinusoids, enlargement hepatic sinusoid in order to congestion and reduce length of hepatocytes, hemorrhage around central venuls, a little distribution of cirrhosis. In generally high level of sorghum grains in diet can induced hisological changs in kidney and liver of sheep.

Molecular evidence for a role for K+-Cl− cotransporters in the kidney

K(+)-Cl(-) cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K(+) diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K(+) diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.

PROLACTIN BINDING SITES IN XENOPUS LAEVIS (DAUDIN) (ANURA PIPIDAE) KIDNEY: AN AUTORADIOGRAPHIC AND IMMUNOCYTOCHEMICAL STUDY

SUMMARY We have studied prolactin binding sites in Xenopus laevis (Daudin) (Anura Pipidae) kidney by autoradiography and immunocytochemistry. By the autoradiographic technique after intracardiac injection of 125I- oPRL labelling is appreciable in Xenopus laevis kidney at the level of the large vessels and capillaries, the renal corpuscle and, to a variable extent, within the lumina and epithelial cells of the proximal convoluted tubules. Compared to the autoradiographic reaction, the immunocytochemical method failed to reveal the hormone when extremely low doses of it were injected but allowed a much more exact localization of prolactin uptake by the epithelial cells of the proximal convoluted tubules. On the basis of the autoradiographs we could not ascertain wheter, or to what extent, the hormone was bound to specific renal cell PRL receptors: in fact, not only insulin and luteinizing hormone, but also unlabelled prolactin, injected as a specific competitor, failed to bring about the recognizable displa...

Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis.

Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry (MS/MS). Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by Western blotting. Data from the cytosolic fractions were then combined with previous proteomic data, and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.

Ultrastructural evaluation of the effect of N-acetylcysteine on methotrexate nephrotoxicity in rats.

The aim of this study is to investigate the possible protective effect of N-Acetylcysteine (NAC) against the likely methotrexate (MTX) toxicity on the kidney using ultrastructural together with biochemical data. Moreover, the immunohistochemical detection of Ki67 nuclear antigen is to be evaluated. Fifteen male Wistar albino rats, weighing 240-290 g, were divided into three equal groups: Rats receiving MTX alone, rats receiving MTX plus NAC treatment, and rats comprising the control group. MTX (18 mg/kg/day, body weight) in dissolved physiologic saline was administered intraperitoneally to rats during 3 days. For the MTX plus NAC group, N-Acetylcysteine (300 mg/kg/day, body weight) was administered together with MTX. At the end of the third day, all the rats were killed with cervical dislocation to obtain blood and tissue samples. Application of MTX principally induced prominent large vacuolization in the proximal convoluted tubule cells, and focal thickening in the glomerular basal lamina of some glomeruli. A decrease in tissue SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase), and an increase in serum urea nitrogen and creatinine and in tissue MDA (malondialdehyde) levels were also seen in the MTX group. These changes were significantly reversed in the MTX-plus-NAC-treated group. Most of the vacuoles in the proximal convoluted tubule cells disappeared. Furthermore, an increase in antioxidant enzyme activities, a decrease in serum urea nitrogen and creatinine, and tissue MDA levels were all significant. Additionally, an increase in the number of Ki67 positive-stained cells in proximal tubules was also noted. In conclusion, NAC may be a promising substance against MTX-induced renal damage. It might be useful to use NAC supplementally to minimize MTX-induced nephrotoxicity.

Study the Anatomical Descriptions and Histological Observations of the Kidney in Golden Eagles (Aquila Chrysaetos)

The present study is preliminary investigate of the kidney gross morphology and some histological observations was studied in golden eagle (Aquila Chrysaetus). The anatomical descriptions of the kidneys had revealed a paired kidney and ureters were protrude, flattened and extra-peritoneal organs. The kidney tissue is transverse by major nerve trunks and blood vessels, securing the kidneys tightly in place. Each kidney consists of three lobes: cranial, middle and caudal lobes. The cranial lobe was the largest and wider than the other two lobes and the caudal lobe was appeared similar to middle lobe but little smaller than it. There is no line of demarcation between cortex and medulla as in mammals. The glomerulus consisted of a tightly packed central core of mesangial cells, surrounded by capillary loops. The cytoplasm of the proximal convoluted tubules cells contains condensing vesicles and a large apically situated nucleus. The distal convoluted tubules and collecting ducts were distinguished on a topographical basis revealed that varied slightly in their reactions with PAS stain and appeared as vacuoles or look like vesicles contains secretion from lining epithelial cells of distal convoluted and collecting tubules which appeared diffused in cross section. The cells of distal convoluted tubules and collecting tubules possessed vesicles with a clearly defined coated outer membrane and some had small blebs invaginated membrane. However in this study found vesicles or vacuoles in the lumen of collecting tubules revealed the secreting cells had margins that were often incompletely membrane –bound and continuous with the apical cell margin as if discharging their contents to the tubular lumen.