Epithelial Cells Of Distal Tubules Research Articles

The effect of ginger essential oil (Zingiber officinale) on catalase in rat kidney tissue

We were aimed to investigate the effect of ginger essential oil (Zingiber officinale) on catalase release in rat kidney tissue by histopathological and immunohistochemically method. This study, 21 male Wistar albino rats were used. Rats were divided into three groups: control, 100 mg/kg ginger essential oil (G100), and 500 mg/kg ginger essential oil (G500). Hematoxylin-eosin staining method was used for histopathological evaluations. Immunohistochemically localization of catalase in kidney tissue was determined by streptavidin-biotin peroxidase method. As a result of histopathological evaluations, an increase in glomerulus diameter was observed in kidney tissues of G100 and G500 groups. In addition, vacuolar degeneration was observed in the proximal and distal tubule epithelial cells in the renal cortex of the G100 group. The immunoreactivity of catalase in the renal cortex region; In the control group, it is strong in the proximal tubules and weak in the mesangial cells. While moderate severity in the proximal tubules and weak in the mesangial cells in the G100 group, very weak catalase immunoreactivity was observed in the G500 group. Strong catalase immunoreactivity was detected in the proximal and collecting ducts of the kidney medulla regions of the rats in all groups. We think that ginger essential oil can be used in appropriate doses and times to reduce kidney damage caused by oxidative stress in the kidney.

Continuous genetic monitoring of transient mesenchymal gene activities in distal tubule and collecting duct epithelial cells during renal fibrosis.

Epithelial cells (ECs) have been proposed to contribute to myofibroblasts or fibroblasts through epithelial-mesenchymal transition (EMT) during renal fibrosis. However, since EMT may occur dynamically, transiently, and reversibly during kidney fibrosis, conventional lineage tracing based on Cre-loxP recombination in renal ECs could hardly capture the transient EMT activity, yielding inconsistent results. Moreover, previous EMT research has primarily focused on renal proximal tubule ECs, with few reports of distal tubules and collecting ducts. Here, we generated dual recombinases-mediated genetic lineage tracing systems for continuous monitoring of transient mesenchymal gene expression in E-cadherin+ and EpCAM+ ECs of distal tubules and collecting ducts during renal fibrosis. Activation of key EMT-inducing transcription factor (EMT-TF) Zeb1 and mesenchymal markers αSMA, vimentin, and N-cadherin, were investigated following unilateral ureteral obstruction (UUO). Our data revealed that E-cadherin+ and EpCAM+ ECs did not transdifferentiate into myofibroblasts, nor transiently expressed these mesenchymal genes during renal fibrosis. In contrast, in vitro a large amount of cultured renal ECs upregulated mesenchymal genes in response to TGF-β, a major inducer of EMT.

Effects of preoperative high-oral protein loading on short- and long-term renal outcomes following cardiac surgery: a cohort study

BackgroundPost-cardiac surgery acute kidney injury (AKI) is associated with increased mortality. A high-protein meal enhances the renal blood flow and glomerular filtration rate (GFR) and might protect the kidneys from acute ischemic insults. Hence, we assessed the effect of a preoperative high-oral protein load on post-cardiac surgery renal function and used experimental models to elucidate mechanisms by which protein might stimulate kidney-protective effects.MethodsThe prospective “Preoperative Renal Functional Reserve Predicts Risk of AKI after Cardiac Operation” study follow-up was extended to postoperative 12 months for 109 patients. A 1:2 ratio propensity score matching method was used to identify a control group (n = 214) to comparatively evaluate the effects of a preoperative protein load and standard care. The primary endpoints were AKI development and postoperative estimated GFR (eGFR) loss at 3 and 12 months. We also assessed the secretion of tissue inhibitor of metalloproteases-2 (TIMP-2) and insulin-like growth factor–binding protein 7 (IGFBP7), biomarkers implicated in mediating kidney-protective mechanisms in human kidney tubular cells that we exposed to varying protein concentrations.ResultsThe AKI rate did not differ between the protein loading and control groups (13.6 vs. 12.3%; p = 0.5). However, the mean eGFR loss was lower in the former after 3 months (0.1 [95% CI − 1.4, − 1.7] vs. − 3.3 [95% CI − 4.4, − 2.2] ml/min/1.73 m2) and 12 months (− 2.7 [95% CI − 4.2, − 1.2] vs − 10.2 [95% CI − 11.3, − 9.1] ml/min/1.73 m2; p < 0.001 for both). On stratification based on AKI development, the eGFR loss after 12 months was also found to be lower in the former (− 8.0 [95% CI − 14.1, − 1.9] vs. − 18.6 [95% CI − 23.3, − 14.0] ml/min/1.73 m2; p = 0.008). A dose–response analysis of the protein treatment of the primary human proximal and distal tubule epithelial cells in culture showed significantly increased IGFBP7 and TIMP-2 expression.ConclusionsA preoperative high-oral protein load did not reduce AKI development but was associated with greater renal function preservation in patients with and without AKI at 12 months post-cardiac surgery. The potential mechanisms of action by which protein loading may induce a kidney-protective response might include cell cycle inhibition of renal tubular epithelial cells.Clinical trial registration ClinicalTrials.gov: NCT03102541 (retrospectively registered on April 5, 2017) and ClinicalTrials.gov: NCT03092947 (retrospectively registered on March 28, 2017).

Renal adaptation in relation to insectivorous feeding habit in the greater mouse-eared bat, Myotis myotis (Chiroptera: Vespertilionidae).

We examined the histological structure of the kidneys of Myotis myotis to better understand their structural adaptations to dietary habits. M. myotis is an insectivorous bat species that belongs to the family Vespertilionidae. The kidneys of M. myotis are unilobular, bean-shaped, and surrounded by a renal capsule. The two parts are distinguished by a thin cortex and a thicker medulla. Renal corpuscles consist of the glomerulus and Bowman's capsule. The proximal tubule consists of cubic cells with a well-developed brush border, whereas the distal tubule is lined with a simple cubic epithelium without a brush border. The Henle's loop, located in the medullary region, was composed of flat cells. The microvilli of proximal tubule epithelial cells and basal lamina of proximal and distal tubule epithelial cells were periodic acid Schiff (PAS)-positive. The PAS-positive reaction of the microvilli of proximal tubular epithelial cells and basal lamina of proximal and distal tubule epithelial cells is due to the presence of glycogen, which may be used as an energy substrate during absorption. The presence of acidic glycoconjugates in the papilla was demonstrated by Alcian blue (pH2.5)-PAS staining. According to the result of silver impregnation staining, it was determined that reticular fibers form a dense fibrillary network in the kidney parenchymal tissue. Reticular fibers are responsible for demarcating and supporting the borders of cells by forming a thin network of fibrils beneath the basal lamina of the cells. Structural features in the kidney, such as a thin cortex and thicker medulla, long conical papilla, and division of the thick medulla into inner and outer regions, of M. myotis may be an adaptation to produce concentrated urine, thereby reducing water loss associated with insectivorous feeding habits.

The protective effects of red ginseng and amifostine against renal damage caused by ionizing radiation.

This study aimed to elucidate the effects of amifostine (ethyol) (AM), a synthetic radioprotector, and red ginseng (RG), a natural radioprotective agent, against the toxic effect of ionizing radiation (IR) on kidney tissues through changes in biochemical and histopathological parameters in addition to contributions to the use of amifostine and RG in clinical studies. Five groups were established: Group I (control, receiving only saline by gavage), Group II (IR only), and Group III (IR+AM, 200mg/kg intraperitoneally (i.p.). Group IV (IR + RG, 200mg/kg orally once a day for 4weeks), and Group V (IR+RG+AM, 200mg/kg orally once/day for 4weeks before IR and 200mg/kg AM administered (i.p.) 30min before IR). All groups, except for the control group, were subject to 6-Gy whole-body IR in a single fraction. 24h after irradiation, all animals were sacrificed under anesthesia. IR enhanced MDA, 8-OHdG, and caspase-3 expression while decreasing renal tissue GSH levels (p < .05). Significant numbers of necrotic tubules together with diffuse vacuolization in proximal and distal tubule epithelial cells were also observed. The examination also revealed substantial brush boundary loss in proximal tubules as well as relatively unusual glomerular structures. While GSH levels significantly increased in the AM, RG, and AM+RG groups, a decrease in KHDS, MDA, 8-OHdG, and caspase-3 expression was observed, compared to the group subject to IR only (p < .05). Therefore, reactive oxygen species-scavenging antioxidants may represent a promising treatment for avoiding kidney damage in patients receiving radiation.

Two transition metal coordination polymers: protection activity on renal ischemia-reperfusion injury combined with chitosan chuanqizine nanoparticles

Two new transition metal coordination polymers (CPs), namely, [Co2(OH)(TZI)(DMA)] (1) and [Mn2(OH)(TZI)(H2O)2] (2) have been synthesized under solvo-/hydrothermal conditions via using a bifunctional organic ligand 5-(1H-tetrazol-5-yl)isophthalic acid (H3TZI). Their treatment activity on the renal ischemia-reperfusion injury was evaluated and the related mechanism was explored at the same time. The ELISA detection kit was conducted to measure the levels of the inflammatory cytokines IL-1β and TNF-α released by the epithelial cells in distal tubules. In addition to this, the HE staining was further conducted to confirm the pathological damage levels in the kidney tissue.

An application of histological technique for monitoring health status of fish species, Leuciscus aspius (Linnaeus, 1758) inhabiting Aras River, Iran

Aras River and its reservoir play an important role in fisheries and water supplies in the northwest of Iran. It is considered that the river water is polluted by various anthropogenic activities. This study was performed to determine the histopathologic effects of water pollution on a fish species, Leuciscus aspius inhabiting the river. In July 2015, the fish were caught in two different stations of Aras River in Poldasht, Iran. The histopathological abnormalities were detected microscopically and evaluated with quantitative analyses in three organs of the fish. Hypertrophy, filament dilatation, lamellar epithelial liftings, thickening of filament, especially curve, a decrease of the mean length, necrosis, fusion, and lifting in lamellae were observed in gills. Main histopathological abnormalities in the liver were non-homogenous parenchyma, the proliferation of hepatopancreas, congestion, and degeneration of central vein, increasing melanomacrophage aggregates, and sinusoidal dilations. The observed changes in kidney included the degenerations of renal tubules, increasing melanomacrophage aggregates, pyknotic nuclei and vacuolization in proximal and distal tubule epithelial cells, and lymphocyte infiltration in renal parenchyma. It was found that the frequencies of the histological lesions were higher in the liver in comparison with other organs. It was concluded that the increased histopathological alteration in the fish tissues may be related to the presence and elevated toxic contaminants in the river.

Renal impairment assessment on adults living nearby a landfill: Early kidney dysfunction biomarkers linked to the environmental exposure to heavy metals.

The aim of this study was to assess the integrity and kidney overall functional capacity of subjects exposed to landfill emissions. Urine and blood levels of Pb and Cd, and several of the newly biomarkers of nephrotoxicity (Kim Injury Molecule 1 (KIM-1), alpha-1 Microglobulin (α1 M), beta-2 Microglobulin (β2 M), Cystatin-C (Cyst C), Clusterin, alpha-glutathione S-transferase (GSTα), pi-glutathione S-transferase (GSTπ), Tissue Inhibitor of Metalloproteinase-1 (TIMP1), Calbindin, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Osteopontin (OPN), (Retinol Binding Protein(RBP), Liver-type Fatty Acid-Binding Protein (FABP-1), Trefoil Factor 3 (TFF3), Collagen VI) were measured in order to assess glomerular and tubule damage in adults living near a landfill. Our results indicate glomerular dysfunction in exposed subjects, and supported evidence of necrosis of proximal and distal tubule epithelial cells as specific biomarkers began to appear in the urine. Positive correlation by Pearson test were obtained between : blood Pb and B-OPN, B-Cyst C, Calbindin, U-KIM-1, TIMP1, U-OPN, and U-Clusterin; and also, between urinary Cd and TIMP1, B-Clusterin, U-OPN, FABP-1, Albumin, and U-Clusterin. The relation between biomarkers of Cd/Pb exposure and early effect biomarkers in this study clearly predicts the future risk of severe kidney injury in subjects living close to the landfill.

Expression and distribution of HIF-1α, HIF-2α, VEGF, VEGFR-2 and HIMF in the kidneys of Tibetan sheep, plain sheep and goat.

The objective of this study was to detect the expression and distribution characteristics of five proteins (the hypoxia-inducible factor 1alpha [HIF-1a], HIF-2a, vascular endothelial growth factor [VEGF], VEGF-2 receptor [VEGFR-2] and hypoxia-induced mitogenic factor [HIMF]) in kidney of Tibetan sheep, plain sheep and goat. The results will provide the basic information for the comparative study of sheep breeds living at different altitudes. The kidney tissues were collected from healthy adult Tibetan sheep, plain sheep and goats and made into paraffin sections. Histological characteristics were assessed by haematoxylin and eosin staining. Expressions of HIF-1a, HIF-2a, VEGF, VEGFR-2 and HIMF proteins were measured by immunohistochemical staining. Immunohistochemistry results showed that the positive expression signals of HIF-1a, HIF-2a, VEGF and VEGFR-2 were detected in epithelial cells of renal tubules and collecting tubules, renal corpuscles in the kidneys of the three sheep breeds. Positive expression signals of HIMF were detected in epithelial cells of proximal tubules and distal tubules in Tibetan sheep and epithelial cells of distal tubules in goat. Immunostaining intensity of HIF-1a, HIF-2a, VEGF and VEGFR-2 proteins in Tibetan sheep was significantly higher than that in both plain sheep and goat (p < 0.05). Immunostaining intensity of HIMF in Tibetan sheep was higher than goat (p < 0.05). Positive expression signals of HIMF were not detected in plain sheep. The expression and distribution characteristics of HIF-1a, HIF-2a, VEGF, VEGFR-2 and HIMF in the studied kidney tissues suggested that these proteins may be related to the physiological regulation of Tibetan sheep kidney in hypoxia environment, and therefore might be important regulating proteins for Tibetan sheep to adapt to high altitude hypoxia environment.

In vitro cyst formation of ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the relentless growth of numerous fluid-filled cysts in the kidneys. Mutations in PKD1 and PKD2, genes that encode polycystin 1 and 2, respectively, are responsible for most cases of ADPKD. Currently, the cellular mechanisms responsible for cyst formation remain poorly understood. In vitro models have been used by researchers to investigate cellular processes for cyst formation in carefully controlled experimental conditions. Madin-Darby canine kidney (MDCK) cells, a distal tubule epithelial cell line, were first used to form 3-dimensional (3-D) cysts within a hydrated collagen gel. This method was applied to epithelial cells cultured from cysts of human ADPKD kidneys, allowing investigators to study cellular mechanisms for cyst growth using cells that harbor the genetic mutations responsible for ADPKD in humans. Studies using ADPKD in vitro cysts have provided insight into cellular processes regulating cell proliferation, fluid secretion, and cell polarity. These assays were used to demonstrate the central role of cAMP agonists, such as arginine vasopressin, on cyst growth; and to test the effectiveness of potential therapeutic agents, including tolvaptan. Results obtained from in vitro cyst experiments demonstrate the translational value of cell model systems for investigating the mechanisms for cyst formation in human ADPKD. In this chapter, we describe protocols for growing ADPKD cells in a 3-D in vitro cyst assay and measuring total cyst volume by microscopy and image analysis.

A novel mechanism of gland formation in zebrafish involving transdifferentiation of renal epithelial cells and live cell extrusion.

Transdifferentiation is the poorly understood phenomenon whereby a terminally differentiated cell acquires a completely new identity. Here, we describe a rare example of a naturally occurring transdifferentiation event in zebrafish in which kidney distal tubule epithelial cells are converted into an endocrine gland known as the Corpuscles of Stannius (CS). We find that this process requires Notch signalling and is associated with the cytoplasmic sequestration of the Hnf1b transcription factor, a master-regulator of renal tubule fate. A deficiency in the Irx3b transcription factor results in ectopic transdifferentiation of distal tubule cells to a CS identity but in a Notch-dependent fashion. Using live-cell imaging we show that CS cells undergo apical constriction en masse and are then extruded from the tubule to form a distinct organ. This system provides a valuable new model to understand the molecular and morphological basis of transdifferentiation and will advance efforts to exploit this rare phenomenon therapeutically.

Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032–1043. DOI: 10.1089/ten.tec.2014.0665

[This corrects the article DOI: 10.1089/ten.tec.2014.0665.].

Abnormal Morphology of Distal Tubular Epithelial Cells Is Regulated by Genetic Factors Derived from Mouse Chromosome 12

The distal tubule (DT) helps regulate blood pressure and electrolytes. We describe a novel, autosomal recessive, morphofunctional DT abnormality in inbred mice evident as columnar alternations and age-related cystic changes. This abnormality developed in both sexes of DBA/2Cr. Similar phenotypes were observed in A/J, C3H/He, DBA/1J, and FVB/N strains, but not in AKR/N, BALB/c, or C57BL/6N strains. In DBA/2Cr, abnormal DT localized to straight and convoluted segments and showed IL-36α DT injury marker expression. However, DT epithelial proliferation, examined by bromodeoxyuridine incorporation, was not remarkably altered with the progression of abnormality. Abnormal DT epithelial cells in DBA/2Cr displayed elongated primary cilia, loose intercellular adhesions, and numerous vesicles with altered localization of CD9, Na+/K+ATPase, and E-cadherin, indicating altered cell function, adhesion, and polarity. DBA/2Cr-type D12Mit182-D12Mit83 was identified as a candidate locus designated DBA/2 renal cyst (drecy). Within drecy, the gene regulated by estrogen in breast cancer protein (Greb1) transcript variant 2 was significantly up-regulated in DBA/2Cr kidney versus C57BL/6N. Greb1 localized to DT cytoplasm in C57BL/6 and to cytoplasm and nucleus in DBA/2Cr. Greb1-overexpressing M-1 kidney cells showed an altered epithelial-mesenchyme phenotype. B6.D2-(D12Mit182-D12Mit83) congenic mice carrying drecy did not show DT abnormalities, whereas DBA/2Cr × B6.D2-(D12Mit182-D12Mit83) mice did. Identification of this novel DT abnormality regulated by a DBA/2Cr mouse chromosome 12-derived locus and additional genetic factors improve the understanding of DT pathogenesis.

Structural basis of TRPV5 channel inhibition by econazole revealed by cryo-EM.

The transient receptor potential vanilloid 5 (TRPV5) channel is a member of the transient receptor potential (TRP) channel family, which is highly selective for Ca2+, that is present primarily at the apical membrane of distal tubule epithelial cells in the kidney and plays a key role in Ca2+ reabsorption. Here we present the structure of the full-length rabbit TRPV5 channel as determined using cryo-EM in complex with its inhibitor econazole. This structure reveals that econazole resides in a hydrophobic pocket analogous to that occupied by phosphatidylinositides and vanilloids in TRPV1, thus suggesting conserved mechanisms for ligand recognition and lipid binding among TRPV channels. The econazole-bound TRPV5 structure adopts a closed conformation with a distinct lower gate that occludes Ca2+ permeation through the channel. Structural comparisons between TRPV5 and other TRPV channels, complemented with molecular dynamics (MD) simulations of the econazole-bound TRPV5 structure, allowed us to gain mechanistic insight into TRPV5 channel inhibition by small molecules.

Urinary semaphorin 3A correlates with diabetic proteinuria and mediates diabetic nephropathy and associated inflammation in mice.

Semaphorin 3A (sema3A) was recently identified as an early diagnostic biomarker of acute kidney injury. However, its role as a biomarker and/or mediator of chronic kidney disease (CKD) related to diabetic nephropathy is unknown. We examined the expression of sema3A in diabetic animal models and in humans and tested whether sema3A plays a pathogenic role in the development of diabetic nephropathy. The expression of sema3A was localized to podocytes and epithelial cells in distal tubules and collecting ducts in control animals, and its expression was increased following the induction of diabetes. Quantification of sema3A urinary excretion in three different diabetic mouse models showed that excretion was increased as early as 2weeks after the induction of diabetes and increased over time, in conjunction with the development of nephropathy. Consistent with the mouse data, increased sema3A urinary excretion was detected in diabetic patients with albuminuria, particularly in those with macroalbuminuria. Genetic ablation of sema3A or pharmacological inhibition with a novel sema3A inhibitory peptide was protected against diabetes-induced albuminuria, kidney fibrosis, inflammation, oxidative stress, and renal dysfunction. We conclude that sema3A is both a biomarker and a mediator of diabetic kidney disease and could be a promising therapeutic target in diabetic nephropathy. Key messages Diabetes induced sema3A excretion in urine. Increased semaphorin 3A was associated with severity of albuminuria. Seme3A-mediated diabetes induced glomerulosclerosis. Peptide-based inhibition of semaphorin3A suppressed diabetic nephropathy.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

Post-Translational Loss of Renal TRPV5 Calcium Channel Expression, Ca2+ Wasting, and Bone Loss in Experimental Colitis

Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1β reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation andcytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors

Gene therapy has been proposed as a novel alternative to treat kidney disease. This goal has been hindered by the inability to reliably deliver transgenes to target cells throughout the kidney, while minimizing injury. Since hydrodynamic forces have previously shown promising results, we optimized this approach and designed a method that utilizes retrograde renal vein injections to facilitate transgene expression in rat kidneys. We show, using intravital fluorescence two-photon microscopy, that fluorescent albumin and dextrans injected into the renal vein under defined conditions of hydrodynamic pressure distribute broadly throughout the kidney in live animals. We found injection parameters that result in no kidney injury as determined by intravital microscopy, histology, and serum creatinine measurements. Plasmids, baculovirus, and adenovirus vectors, designed to express EGFP, EGFP-actin, EGFP-occludin, EGFP-tubulin, tdTomato-H2B, or RFP-actin fusion proteins, were introduced into live kidneys in a similar fashion. Gene expression was then observed in live and ex vivo kidneys using two-photon imaging and confocal laser scanning microscopy. We recorded widespread fluorescent protein expression lasting more than 1 mo after introduction of transgenes. Plasmid and adenovirus vectors provided gene transfer efficiencies ranging from 50 to 90%, compared with 10-50% using baculovirus. Using plasmids and adenovirus, fluorescent protein expression was observed 1) in proximal and distal tubule epithelial cells; 2) within glomeruli; and 3) within the peritubular interstitium. In isolated kidneys, fluorescent protein expression was observed from the cortex to the papilla. These results provide a robust approach for gene delivery and the study of protein function in live mammal kidneys.

Bone loss and renal Ca2+ wasting in experimental colitis is accompanied by downregulation of TRPV5 in renal distal convoluted tubules

Dysregulated Ca2+homeostasis likely contributes to the etiology of IBD‐associated bone loss. Experimental colitis leads to decreased expression of Klotho, a protein which supports renal Ca2+ reabsorption by stabilizing TRPV5 channel on the apical membrane of distal tubule epithelial cells. We investigated whether colitis leads to changes in renal Ca2+ handling and expression of TRPV5 utilizing TNBS, and two adoptive T‐cell transfer models (CD4+CD45RBHi or CD4+IL10−/−). Colitic mice had normal serum levels of Ca2+ and PTH. μCT analysis demonstrated changes in bone structure along with changes in serum sRANKL, IL‐6, osteocalcin, TNF, and total serum tDPD crosslinks. Increased fractional Ca2+excretion was accompanied by reduction of TRPV5 protein expression, with concomitant increase in its sialylation status. In mIMCD3 cells transduced with Ad‐TRPV5, proinflammatory cytokines reduced TRPV5 surface expression followed by its degradation. TNF induced interaction of TRPV5 with UBR4 ubiquitin ligase and siRNA‐mediated UBR4 knockdown prevented TNF induced TRPV5 degradation. The cytokine effect on TRPV5 was not observed in cells stably transfected with membrane‐bound Klotho. We propose that TNF and IFNγ act to decrease Klotho expression and activity, increased sialylation of TRPV5, impaired interaction of TRPV5 and galectin‐1, and ultimately to UBR4‐dependent endocytosis and degradation of TRPV5.

Comparison of expression pattern of monoamine oxidase A with histopathologic subtypes and tumour grade of renal cell carcinoma

SummaryBackgroundStudies of the biochemical properties of MAO-A (monoamine oxidase) are numerous, but the information about determination of MAO-A in human normal and tumour renal tissue is limited. Our objectives in the present study were to determine the localization of MAO-A in normal kidney and level of expression of this protein in tumour kidney.Material/MethodsEnzyme immunohistochemical method was chosen for detection of MAO-A in 63 clinical samples of all histopathological types of RCC (renal cell carcinoma). Our results were compared to basic clinical and histopathological parameters such as histopathological type and tumour grade. We also compared MAO-A expression between normal and tumour tissue samples.ResultsWe confirmed the elevated expression of MAO-A in high-grade tumours of renal cell carcinoma specimens. The percentage of MAO-positive samples progressively increased from 9% in grade 2 to 45% in grade 3. We also noted high levels of MAO-A immunoreactivity in epithelial cells of proximal tubules in normal renal tissue. MAO-A was absent or very low in epithelial cells of distal tubules and glomerular capsule, as well as in endothelial cells of renal vessels.ConclusionsTaken together, our results and findings of other studies show that MAO-A expression in high-grade tumours may have a direct role in maintaining a dedifferentiated phenotype and promoting aggressive behaviour. The ability of clorgyline (an MAO-A inhibitor) to counteract oncogenic pathways and promote differentiation suggests that MAO-A inhibitors, which have been used for many years in clinical practise for treating neurological disorders, could be therapeutic options for advanced stages of tumours.