Straight Tubules Research Articles

Combinatorial expression of claudins in the proximal renal tubule and its functional consequences

The proximal renal tubule (PT) is characterized by a highly conductive paracellular pathway, which contributes to a significant amount of solute and water reabsorption by the kidney. Claudins are tight junction proteins that, in part, determine the paracellular permeability of epithelia. In the present study, we determined the expression pattern of the major PT claudins. We found that claudin-2 and claudin-10 are coexpressed throughout the PT, whereas claudin-3 is coexpressed with claudin-2 predominantly in the proximal straight tubule. Additionally, claudin-2 and claudin-3 are expressed separately within mutually exclusive populations of descending thin limbs. We developed a novel double-inducible Madin-Darby canine kidney I cell model to characterize in vitro the functional effect of coexpression of PT claudins. In keeping with previous studies, we found that claudin-2 alone primarily increased cation (Na+ and Ca2+) permeability, whereas claudin-10a alone increased anion (Cl-) permeability. Coexpression of claudin-2 and claudin-10a together led to a weak physical interaction between the isoforms and the formation of a monolayer with high conductance but neutral charge selectivity. Claudin-3 expression had a negligible effect on all measures of cell permeability, whether expressed alone or together with claudin-2. In cells coexpressing a claudin-2 mutant, S68C, together with claudin-10a, inhibition of cation permeability through the claudin-2 pore with a thiol-reactive pore blocker did not block anion permeation through claudin-10a. We conclude that claudin-2 and claudin-10a form independent paracellular cation- and anion-selective channels that function in parallel.

Anhydramnios, but prenatally normal kidneys: renal tubular dysgenesis – patient with mutations in the renin-angiotensin system gene AGTR1

Abstract Background Prenatally detected oligo- or even anhydramnios may – beside other reasons – be indicative for a diminished or absent urine production. The resulting clinical picture is a “Potter sequence” with arthrogrypotic joint contractions, a flat face and most importantly pulmonary hypoplasia. In severe cases this pulmonary hypoplasia can be life-limiting irrespective of the underlying lesion. Case presentation Our patient initially presented with anhydramnios and normal kidneys at external ultrasonography after 31 weeks of pregnancy. Following spontaneous birth after 37 weeks of gestation, the baby boy was born with all the clinical signs of a “Potter-sequence” along with a severe pulmonary hypoplasia leading to insufficient oxygenation and ventilation. Despite all measures taken, the child died after 8 h of life. Beside life-limiting pulmonary hypoplasia postmortem examination again confirmed macroscopically normal kidneys, but microscopy showed compact and variable sized glomeruli, numerically reduced and immature tubules, a structurally altered renal vascular bed and an expanded medullar interstitium. Immunohistochemical studies revealed the absence of proximal convoluted tubules, shortened proximal straight tubules beside an immaturity and dysmorphogenesis of the other segments of the nephron, and thus proved renal tubular dysgenesis (RTD) as the underlying disease. A homozygous mutation c.377G>C (p.Arg126Pro) in exon 2 of the AGTR1 gene was found, leading to the exchange of a highly conserved arginine to proline. This mutation has not been reported in public databases so far. As expected, both consanguineous parents were heterozygous for this mutation. Conclusion RTD has to be considered in an anuric fetus with apparently normal renal sonography in order to allow adequate prenatal counseling and – if indicated – palliative postnatal care.

Single Cell RNA Sequencing Analysis of Human Kidney Reveals the Presence of ACE2 Receptor: A Potential Pathway of COVID-19 Infection

Background: A novel coronavirus called COVID-19, which shared 82% similarity of genome sequence with SARS-CoV, was found in Wuhan in late December of 2019, causing an epidemic outbreak of novel coronavirus-induced pneumonia with dramatically increasing number of cases. Several organs are vulnerable to COVID-19 infection. Acute kidney injury (AKI) was reported in parts of case-studies reporting characteristics of COVID-19 patients. This study aimed at analyzing the potential route of COVID-19 entry and mechanism at cellular level. Methods: cRNA-seq technology was used to obtain evidence of potential route and ACE2 expressing cell in renal system for underlying pathogenesis of kidney injury caused by COVID-19. The whole process was performed under R with Seurat packages. Canonical marker genes were used to annotate different types of cells. Results: Ten different clusters were identified and ACE2 was mainly expressed in proximal tubule and glomerular parietal epithelial cells. From GO & KEGG enrichment analysis, imbalance of ACE2 expression, renin-angiotensin system (RAS) activation, and neutrophil-related processes were the main issue of COVID-19 leading kidney injury. Conclusion: Our study provided the cellular evidence that COVID-19 invaded human kidney tissue via proximal convoluted tubule, proximal tubule, proximal straight tubule cells and glomerular parietal cells by means of ACE2-related pathway and used their cellular protease TMPRSS2 for priming. Funding Statement: The authors stated: Not available. Declaration of Interests: All authors have no conflicts of interest to disclose.

The Rat (Rattus norvegicus) as a Model Object for Acute Organophosphate Poisoning. 5. Morphofunctional Alterations in Kidneys

In two rat models of acute paraoxon (POX) poisoning against the background of specific and nonspecific inhibition of blood plasma carboxyl esterase (CE) activity, the POX effect on morphology and functions of the rat kidneys was assessed within a period from 1 day to 6 weeks after the poisoning. During the first 3 days, rats developed transient glycosuria and increased urinary excretion of calbindin and sulfated glycosaminoglycans. One day after the poisoning, a positive reaction for protein deposits (AFOG+) was observed in the descending and ascending limbs of the loop of Henle and collecting ducts against the background of nonspecific CE inhibition. After 2 weeks, the AFOG+ reaction was detected in the proximal straight tubules along with desquamation of epithelial cells. Both 3 days and 2 weeks after the poisoning, regardless of the mode of CE inhibition, a morphometric analysis of individual components of the rat nephron revealed an increase in the size of the renal corpuscle and the glomerular capillary tuft within the Bowman’s capsule. Three days after the poisoning and at every subsequent observations, intoxicated rats exhibited an alteration in the luminal diameter of the proximal tubules and the luminal surface of the collecting ducts. Most pronounced alterations in the luminal diameter of the distal tubules were observed 3 days, 4 and 6 weeks after the poisoning.

Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Increased sodium transporters in distal nephron segments are associated with clozapineinduced hypertension

Dopamine receptor subtype 4 (D4R) antagonists, such as clozapine, are used in the treatment of schizophrenia. A common side effect of clozapine is hypertension but the mechanism of the clozapine‐induced hypertension is unknown. D4R plays an important role in blood pressure (BP) regulation; deletion of the D4R gene in mice causes hypertension. To determine if renal mechanisms are involved in clozapine‐induced hypertension, we characterized the distribution of D4R in mouse kidney and measured BP, water and sodium balance, and renal protein expression of sodium transporters in clozapine‐ and vehicle‐treated mice. D4R (immunocytochemistry) was mainly located in thick ascending limb, distal convoluted tubule and cortical and medullary collecting ducts; there is slight staining of D4R in the proximal convoluted but not straight tubule. Clozapine (20mg/kg/day) or vehicle was delivered subcutaneously to adult male C57BL/6J mice for seven days via osmotic mini‐pump (n=5/group). In clozapine‐treated mice, systolic (110±0.5, mm Hg, under anesthesia) and diastolic BPs (76.6±1),) were elevated, relative to vehicle‐treated mice (SBP=97.6±1.6, DBP=69.5±2.3). Food and water intake, body weight, serum electrolytes and creatinine, urine volume, and creatinine clearance were similar in the two groups. The sodium excretion tended to decrease while sodium excretion and blood pressure plot in clozapine‐treated mice was shifted to the right of the vehicle‐treated mice. Renal protein expressions (immunoblotting) of sodium hydrogen exchanger 3 and sodium phosphate cotransporter 2 were similar in clozapine‐ and vehicle‐treated mice. However, the renal protein expressions of sodium potassium 2 chloride cotransporter (180±21, % of control) and sodium chloride cotransporter (171±22% ), and the three subunits of epithelial sodium channel, α (140±7%), β (166±21%) and γ (55 kDa=130±7%, 85kDa=114±10% ), were greater in clozapine‐than vehicle‐treated mice. Renal protein expression of a‐sodium potassium ATPase was also slightly increased (118±4%) in clozapine‐treated mice. These findings suggest that inhibition of D4R by clozapine increases sodium transporters in distal nephron segments. Furthermore, a similar increase in the protein expression of sodium chloride cotransporter was also found in the immortalized mouse renal distal convoluted tubule cells in vitro with inhibition of D4R with its siRNA and antagonists. Thus, inhibition of renal D4R may cause the impaired sodium excretion that may be responsible for the increased blood pressure in clozap‐inetreated mice.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fructose reabsorption by rat proximal tubules: role of Na+-linked cotransporters and the effect of dietary fructose.

Fructose consumption has increased because of widespread use of high-fructose corn syrup by the food industry. Renal proximal tubules are thought to reabsorb fructose. However, fructose reabsorption (Jfructose) by proximal tubules has not yet been directly demonstrated, nor the effects of dietary fructose on Jfructose. This segment expresses Na+- and glucose-linked transporters (SGLTs) 1, 2, 4, and 5 and glucose transporters (GLUTs) 2 and 5. SGLT4 and -5 transport fructose, but SGLT1 and -2 do not. Knocking out SGLT5 increases urinary fructose excretion. We hypothesize that Jfructose in the S2 portion of the proximal tubule is mediated by luminal entry via SGLT4/5 and basolateral exit by GLUT2 and that it is enhanced by a fructose-enriched diet. We measured Jfructose by proximal straight tubules from rats consuming either tap water (Controls) or 20% fructose (FRU). Basal Jfructose in Controls was 14.1 ± 1.5 pmol·mm-1·min-1. SGLT inhibition with phlorizin reduced Jfructose to 4.9 ± 1.4 pmol·mm-1·min-1 ( P < 0.008), whereas removal of Na+ diminished Jfructose by 86 ± 5% ( P < 0.0001). A fructose-enriched diet increased Jfructose from 12.8 ± 2.5 to 19.3 ± 0.5 pmol·mm-1·min-1, a 51% increase ( P < 0.03). Using immunofluorescence, we detected luminal SGLT4 and SGLT5 and basolateral GLUT2; GLUT5 was undetectable. The expression of apical transporters SGLT4 and SGLT5 was higher in FRU than in Controls [137 ± 10% ( P < 0.01) and 38 ± 14% ( P < 0.04), respectively]. GLUT2 was also elevated by 88 ± 27% ( P < 0.02) in FRU. We conclude that Jfructose by proximal tubules occurs primarily via Na+-linked cotransport processes, and a fructose-enriched diet enhances reabsorption. Transport across luminal and basolateral membranes is likely mediated by SGLT4/5 and GLUT2, respectively.

Ppargc1a controls nephron segmentation during zebrafish embryonic kidney ontogeny

Nephron segmentation involves a concert of genetic and molecular signals that are not fully understood. Through a chemical screen, we discovered that alteration of peroxisome proliferator-activated receptor (PPAR) signaling disrupts nephron segmentation in the zebrafish embryonic kidney (Poureetezadi et al., 2016). Here, we show that the PPAR co-activator ppargc1a directs renal progenitor fate. ppargc1a mutants form a small distal late (DL) segment and an expanded proximal straight tubule (PST) segment. ppargc1a promotes DL fate by regulating the transcription factor tbx2b, and restricts expression of the transcription factor sim1a to inhibit PST fate. Interestingly, sim1a restricts ppargc1a expression to promote the PST, and PST development is fully restored in ppargc1a/sim1a-deficient embryos, suggesting Ppargc1a and Sim1a counterbalance each other in an antagonistic fashion to delineate the PST segment boundary during nephrogenesis. Taken together, our data reveal new roles for Ppargc1a during development, which have implications for understanding renal birth defects.

Immunoexpression of aquaporins 1, 2, 3 and 4 in kidney of yak (Bos grunniens) on the Qinghai-Tibetan Plateau

ABSTRACTAquaporins (AQP) 1, 2, 3 and 4 belong to the aquaporin water channel family and play an important role in urine concentration by reabsorption of water from renal tubule fluid. Renal AQPs have not been reported in the yak (Bos grunniens), which resides in the Qinghai Tibetan Plateau. We investigated AQPs 1−4 expressions in the kidneys of Yak using immunohistochemical staining. AQP1 was expressed mainly in the basolateral and apical membranes of the proximal tubules and descending thin limb of the loop of Henle. AQP2 was detected in the apical plasma membranes of collecting ducts and distal convoluted tubules. AQP3 was located in the proximal tubule, distal tubule and collecting ducts. AQP4 was located in the collecting ducts, distal straight tubule, glomerular capillaries and peritubular capillaries. The expression pattern of AQPs 1−4 in kidney of yak was different from other species, which possibly is related to kidney function in a high altitude environment.

Light and Electron microscopic studies on the sperm host glands in Punjab white quail

The present work was conducted on oviduct of 42 Punjab white quails to observe the histomorphochemical and ultrastructural details of sperm host glands. The tissue samples were collected from the uterovaginal and infundiomagnal junctions of the oviduct in 10% NBF and 2.5% glutaralehyde solutions and processed for histological and electronmicroscopic studies. Fresh tissue samples were collected for cryostat sectioning to demonstrate lipids. It was observed that the UVJ was lined by pseudostratified columnar epithelium with ciliated, non ciliated cells, goblet cells and basal cells. The sperm host glands extended into lamina propria of UVJ as oval, rounded and straight tubules. The proximal part of glandular neck was lined by pseudostratified columnar ciliated epithelium and distal part with non ciliated columnar cells. The ciliated cells showed cilia and microvilli on the apical surface in the neck region, whereas the non ciliated cells present in the distal part of SHG had oval or elongated nuclei in their basal part and were studded with some of the microvilli in their luminal surface. These glands were surrounded by some of the smooth muscle cells and nerve fibers. Histochemical studies revealed a moderate to strong reaction of acid and neutral mucopolysaccharides and lipids whereas reaction for basic proteins was weak in the supranuclear part of SHG.

Microscopic changes in the liver, heart, and kidneys in alloxan diabetes rats

To make future researches of the results of treatment of diabetes possible, it is necessary to create a model of this pathology in experimental animals. It is crucial to trace the correlation between changes in organs when this pathology occurs in clinical cases and when it occurs in experimental cases. Today we have a number of methods used to form alloxan diabetes in animals, but alloxan monohydrate is the most frequently used. Thus, the purpose of our study was to: study microscopic changes in the liver, heart, and kidneys of alloxan diabetes rats. The experimental model of diabetes was reproduced by a 150 mg/kg single-dose subcutaneous administration of alloxan monohydrate in the form of a 5% solution per citrate buffer (pH 4.5). To prove the development of diabetes, blood glucose levels were measured by a Rightest GM500 blood glucose meter on the 20th day after administration of alloxan. There were used animals with medium severity diabetes (fasting glucose test showings 10 to 20 mmol/l). The experimental animals were euthanized by ethereal anesthesia decapitation. Samples of the liver, heart, and kidneys for histological examination were taken on the 20th day after administration of alloxan. Histologic sections were prepared according to the standard method, coloring was made by hematoxylin and eosin and based on Van Gizon method. We have found that on the 20th day after the simulation of alloxan diabetes, the liver showed microscopic signs of hepatitis with dystrophic changes in hepatocytes and intracellular cholestasis. The kidneys showed destruction of all structural components of the glomeruli with subsequent necrosis and dystrophic changes and destruction of the epithelium of the curvy and straight tubules. The heart showed granular degeneration of cardiocytes, in some areas, and distinct swelling between the muscle fibers bundles and inside the muscle fibers bundles, infiltration of the connective tissue stroma by erythrocytes in others. We believe that it is possible to obtain the most optimal spectrum of signs peculiar for type 1 diabetes by administration of alloxan as evidenced by microscopic changes in organs. Further, this model of diabetes will be used to study the influence of cell transplantation upon the development of the disease.

Compartments with predominant ephrin-B1 and EphB2/B4 expression are present alternately along the excurrent duct system in the adult mouse testis and epididymis.

Ephrin receptors (Eph) and ligands are membrane-bound cell-cell communication molecules that regulate the spatial organization of various tissues and organs by repulsive or adhesive signals arising from contact between Eph- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the testis and epididymis are virtually unknown. We aimed to investigate the expression of several EphB receptors and ephrin-B ligands in the testis and epididymis of adult mice. mRNA and protein expression was detected via reverse transcription-polymerase chain reaction amplification and immunostaining, respectively. Complementary expression patterns were observed in the epithelia along the excurrent duct system in the testis and epididymis; ephrin-B1 was strongly expressed in the epithelia of the rete testis and segment I in the ductus epididymis, whereas EphB2 and/or EphB4 were strongly expressed in the epithelia of the straight tubules and efferent ductules. Moreover, ephrin-B1 was expressed in the spermatogonia, Leydig cells, and peritubular myoid cells in the testis, whereas EphB2 was expressed in elongated spermatids and EphB4 was expressed in the spermatogonia and Leydig cells. Furthermore, these receptors were found to be tyrosine-phosphorylated in the testis and/or epididymis. Receptor localization and phosphorylation patterns suggested that EphB/ephrin-B signaling might occur in the seminiferous tubules and epithelial junctions among the straight tubules, rete testis, efferent ductules, and ductus epididymis. Therefore, we propose that EphB/ephrin-B signaling may regulate epithelial boundary formation in the excurrent tubule/ductule/duct system as well as modulate spermatogenesis and spermiation. Overall, this study represents the first analysis of EphB receptor and ephrin-B ligand expression in the normal adult testis and epididymis.

Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney.

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study's purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

Three‐dimensional reconstruction for study of the functional design of the avian lung

Among the air‐breathing vertebrates, the avian respiratory system is anatomically the most complex and physiologically the most efficient. Considerably, these attributes explain how and why birds achieved powered flight, a highly energetically costly form of locomotion. The avian lung which is small and rigid and firmly attached to the vertebral column and the ribs is ventilated continuously and unidirectionally by synchronized activity of capacious air sacs. Although the respiratory system has been studied for nearly five centuries, i.e. since Coiter (1573), certain aspects of its functional design remained unclear. The size, the shape and the arrangement of the blood capillaries and the air capillaries, the minuscule terminal respiratory units, particularly remained uncertain. Various investigators considered the air capillaries to be ‘tiny tubules', ‘intercapillary air passages', ‘straight tubules', ‘terminal air canals' and ‘intercellular passages'. Furthermore, the air capillaries were deemed to terminate in blind expansions, the blood capillaries were not mirror images of the air capillaries and the air capillaries and the blood capillaries run parallel to and in contact with each other across the exchange tissue. Because of the intimate entwining between the air capillaries and the blood capillaries, the exchange tissue was reported to resemble ‘a sponge in structure’, ‘a sinusoidal three‐dimensional network’ and ‘a dexterously crafted blood capillary network that interfaces with the equally complex air capillary labyrinth’. The descriptive (imprecise) terms used on the morphologies of the air capillaries and the blood capillaries and their topographical relationships were based on personal perceptions of microscopic sectional profiles rather than on hard empirical data. Unfortunately, the imprecise details were used in the formulation of a functional model for gas exchange in the avian lung where the air‐ and the blood capillaries were tubular in shape, the air capillaries run outwards from the parabronchial lumen and contacted the blood capillaries which run inwards from the peripherally located interparabronchial arteries and each of the respiratory units terminated blindly. Taking advantage of the recent increase in computing power and availability of appropriate robust software, three‐dimensional reconstruction was performed on serial sections of the avian lung to elucidate the morphologies and the spatial assemblage of the air capillaries and the blood capillaries. Unlike the classical morphology of the blood capillaries, e.g., in skeletal muscle, where the capillaries are longer than they are wide, those of the avian lung comprise distinctive segments that are rather as long as they are wide and are connected in three‐dimensions. The air capillaries are rotund structures which are joined by very narrow passageways. Completely isolated air capillaries were identified: the functional role of such units remains unclear. The air capillaries and the blood capillaries are not mirror images and the structural interdependence between the air capillaries and the blood capillaries may explain the strength of the tiny respiratory units. Because in a exchange tissue the air diffuses outwards from the parabronchial lumen and the blood flows inwards from the peripherally located interparabronchial arteries, existence of an auxiliary countercurrent arrangement was confirmed between the air capillaries and the blood capillaries.Support or Funding InformationSupported by the National Research Foundation (NRF) of South AfricaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The von Hippel-Lindau Gene Is Required to Maintain Renal Proximal Tubule and Glomerulus Integrity in Zebrafish Larvae

Background: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. Methods: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. Results: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl^–/– tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1–43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. Conclusions: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.

Can Randall\u2019s plug composed of calcium oxalate form via the free particle mechanism?

BackgroundThe likelihood of a Randall’s plug composed of calcium oxalate monohydrate (COM) forming by the free particle mechanism in a model of kidney with a structure recently described by Robertson was examined at the most favourable conditions for the considered mechanism.MethodsThe Robertson model of the kidney is used in the following development. The classical theory of crystallization was used for calculations.ResultsInitial COM nuclei were assumed to form at the beginning of the ascending loop of Henle where the supersaturation with respect to COM has been shown to reach the threshold level for spontaneous nucleation. Nucleation proceeds by a heterogeneous mechanism. The formed particles are transported in the nephron by a laminar flow of liquid with a parabolic velocity profile. Particles travel with a velocity dependent on their position in the cross-section of the nephron assumed to be straight tubule with smooth walls and without any sharp bends and kinks. These particles move faster with time as they grow as a result of being surrounded by the supersaturated liquid. Individual COM particles (crystals) can reach maximum diameter of 5.2 × 10−6 m, i.e. 5.2 μm, at the opening of the CD and would thus always be washed out of the CD into the calyx regardless of the orientation of the CD. Agglomeration of COM crystals forms a fractal object with an apparent density lower than the density of solid COM. The agglomerate that can block the beginning of the CD is composed of more crystals than are available even during crystaluria. Moreover the settling velocity of agglomerate blocking the opening of the CD is lower than the liquid flow and thus such agglomerate would be washed out even from upward-draining CD.ConclusionsThe free particle mechanism may be responsible for the formation of a Randall’s plug composed by COM only in specific infrequent cases such as an abnormal structure of kidney. Majority of incidences of Randall’s plug development by COM are caused by mechanism different from the free particle mechanism.

External Morphology of Antennae and Mouthpart Sensillae of the Granary Weevil (Coleoptera: Curculionidae)

Abstract The ultrastructure of the antennal and mouthpart sensillae for the granary weevil, Sitophilus granarius L. (Coleoptera: Curculionidae), adult was examined with scanning electron microscopy. The cephalic capsule bears two types of sensillae—a multibranched sensillum and a multiporous peg sensillum. The multibranched sensillum was a structure not previously reported for the granary weevil or any other insect species, and is distinguished by its torpedo-shaped appearance. The multiporous peg sensillae, formed from multiple straight tubules, are distributed on the distal one-third of the rostrum. The mouthparts bear two types of sensillae—the multiporous peg sensillae on the labrum and mandibles, and short basiconic sensillae on the labial palps. The antenna consists of eight segments arranged on the scape, pedicel, and flagellum. Each antennal segment bears sqaumiform sensillae, and five types of sensillae were identified on the club segment. These sensillae are a trichodea Type I, trichodea Type I...

N-Glycoform-dependent interactions of megalin with its ligands

BackgroundMegalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner. MethodsDistribution of megalin and glycans was histochemically analyzed in mouse kidneys. Kidney absorption of Cy5-labeled ligands was examined in vivo. Megalin–ligand interactions were analyzed using ligand blotting and ELISA. ResultsMegalins expressed on renal proximal convoluted tubules (PCTs) and proximal straight tubules (PSTs) have different N-glycans. PCT megalin stained with Lens culinaris agglutinin (LCA), which recognizes core-fucosyl N-glycans catalyzed by α1,6-fucosyltransferase (Fut8). In contrast, PST megalin stained with wheat germ agglutinin (WGA), which recognizes hybrid-type N-glycans. Retinol-binding protein-Cy5 (RBP-Cy5) was endocytosed by megalin on PCTs but minimally endocytosed by PSTs. BSA-Cy5 was endocytosed nearly equally by both tubules. The purified LCA-positive glycoform megalin had higher binding activity for RBP and vitamin D-binding protein than did WGA-positive glycoform megalin. Both glycoforms had nearly the same BSA- and kanamycin-binding activities. RBP-binding analysis of megalin lacking core fucose, in Fut8–/– mouse kidneys, had significantly decreased binding activity. ConclusionsN-Glycosylation of megalin can modulate its ligand-binding activity. Core fucosylation, in particular, is a modification crucial for megalin–RBP interactions. General significanceCell type-specific glycoforms of megalin exist in the proximal tubular cells and modulate ligand absorption capacity.

Region-specific localization of NOS isoforms and NADPH-diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus).

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage-dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)-NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH-d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH-d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell-specific localization in the efferent ductules and region- and cell-specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation.

Morfologia do escroto, do testículo e das vias espermáticas de Metachirus nudicaudatus (Geoffroy, 1803), Didelphidae-Marsupialia

Resumo: Foram utilizadas as gônadas e vias espermáticas de cinco animais machos, adultos em fase reprodutiva, da espécie Metachirus nudicaudatus Geoffroy 1803, única espécie do gênero, para descrever a morfologia do escroto, do testículo e das vias espermáticas. O Metachirus possui escroto pré-peniano e que contém os testículos permanentemente. A pele escrotal é não pigmentada e com poucos pelos e glândulas. A lâmina parietal da túnica vaginal apresenta-se pouco pigmentada. Os testículos são ovais e ligados ao epidídimo através do pedículo testículo-epididimário. Eles são envolvidos, externamente, pela cápsula testicular e sustentados por um estroma de natureza conjuntiva. As células intersticiais são os elementos predominantes no abundante tecido intertubular. Os túbulos seminíferos são largos, enovelados e envolvidos por uma túnica própria fibroelástica, contendo células mióides. O epitélio seminífero é formado pelas células espermatogênicas e de Sertoli intercaladas. Os túbulos seminíferos convergem em direção à extremidade capitata do testículo, ficando revestidos por apenas células de sustentação, caracterizando uma região de transição entre túbulos seminíferos e túbulos retos, ocupada por uma estrutura tipo "válvula" que obstrui parcialmente o lume tubular. Os túbulos retos reúnem-se para formar um único dúctulo eferente, que percorre uma pequena extensão intratesticular, atravessa a albugínea e penetra no pedículo testículo-epididimário. A parte flexuosa do dúctulo eferente forma um lóbulo separado na parte medial do corpo do epidídimo. O epidídimo é envolvido pela cápsula epididimária e constituído pelo ducto epididimário, que se encontra bastante enovelado. O ducto epididimário é revestido por epitélio simples colunar pseudoestratificado apresentando células principais, basais, apicais e de "halo claro". As células principais são predominantes e apresentam características morfológicas e histoquímicas que diferem ao longo do ducto, possibilitando a caracterização de nove diferentes zonas epididimárias. É no lume da zona sete (início da cauda) que começa o pareamento de espermatozoides. Esse fenômeno coincide com alterações morfológicas bem evidentes e uma maior quantidade de mucossubstâncias neutras é secretada nessa zona.O ducto deferente apresenta-se dividido em três partes: justa-epididimária, funicular e abdominal, baseando nas variações histológicas e histoquímicas de seu epitélio e componentes envolventes. O ducto deferente não apresenta ampola e nem cruza o ureter antes de desembocar na uretra. O funículo espermático contém o ducto deferente, artéria e veias testiculares, vasos linfáticos, nervos e um desenvolvido músculo cremáster. Seus componentes apresentam modificações estruturais nas regiões proximal, média e distal, sendo notável a peculiar rede admirável.