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BACKGROUND: The root of many kidney diseases in humans can be traced to alterations or damage to subcellular organelles. Mitochondrial fragmentation, endoplasmic reticulum (ER) stress, and lysosomal inhibition, among others, ultimately contribute to kidney injury and are the target of therapeutics in development. Although recent technological advancements allow for the understanding of disease states at the cellular level, investigating changes in subcellular organelles from kidney tissue remains challenging. METHODS: Using structured illumination microscopy, we imaged mitochondria and other organelles from paraffin sections of mouse tissue and human kidney biopsy specimens. The resulting images were 3D rendered to quantify mitochondrial size, content, and morphology. Results were compared with those from transmission electron microscopy and segmentation. RESULTS: Super-resolution imaging reveals kidney tubular epithelial cell mitochondria in rodent and human kidney tissue form large, interconnected networks under basal conditions, which are fragmented with injury. This approach can be expanded to other organelles and cellular structures including autophagosomes, ER, brush border, and cell morphology. We find that, during unilateral ischemia, mitochondrial fragmentation occurs in most tubule cells, and they remain fragmented for >96 hours. Promoting mitochondrial fusion with the fusion promotor M1 preserves mitochondrial morphology and interconnectivity and protects against cisplatin-induced kidney injury. CONCLUSIONS: We provide, for the first time, a nonbiased, semiautomated approach for quantification of the 3D morphology of mitochondria in kidney tissue. Maintaining mitochondrial interconnectivity and morphology protects against kidney injury. Super-resolution imaging has the potential to both drive discovery of novel pathobiologic mechanisms in kidney tissue and broaden the diagnoses that can be made on human biopsy specimens.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2–Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

Chronic kidney disease (CKD) is a global health problem. CKD patients are at high risk of developing cardiovascular disease (CVD), including coronary artery disease, heart failure and stroke. Several factors invoke a vicious cycle of CKD and CVD, which is referred as to "cardiorenal syndrome". Among these factors, the compounds retained through loss of renal excretion play a pathological role in causing atherosclerosis and CVD. These compounds have been broadly classified as uremic toxins because of their accumulation with declining renal function and cytotoxicity. The major uremic toxins contributing to CVD are asymmetric dimethylarginine (ADMA), advanced glycation endproducts (AGE), and trimethyl amine N-oxide (TMAO). ADMA is linked to CVD through regulation of nitric oxide, reactive oxygen species, and renal anemia. AGE not only directly accumulates in the heart and kidney, but interacts with the receptor for AGE (RAGE), leading to cell damage in CVD. TMAO correlates with a high prevalence of CVD and promotes organ fibrosis by itself. The levels of these and other uremic toxins rise with worsening CKD, inducing multiplicative damage in the heart and kidney. Therefore, a better understanding of uremic toxins has great clinical importance for preventing cardiorenal syndrome. This review highlights the molecular mechanism by which these uremic toxins are implicated in CVD and suggests the possible mutual relationship between them.

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin β1 subunit in these cells converts them from a "loose" epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a "tight" epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin β1 cytoplasmic tail and does not entail β1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the β1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin β1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.

Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.

Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction (TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-Calpha and PP1alpha accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of occludin and ZO-1 during TJ assembly. Phosphorylation of occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase (GST) pull-down assay using recombinant GST-occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of occludin, and these interactions were also reduced during the assembly of TJ. A pairwise binding assay using GST-occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of occludin. In vitro incubation of phospho-occludin with PP2A or PP1 indicated that PP2A dephosphorylates occludin on phospho-Thr residues, whereas PP1 dephosphorylates it on phospho-Ser. This study shows that PP2A and PP1 directly interact with occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of occludin.

The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation, and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development and maintenance, its exact molecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either lineage results in abnormal tubulogenesis, with profound defects in polarity, lumen formation, and the actin cytoskeleton. Ultimately, these defects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processes by regulating the polarity Par complex (Par3/Par6/aPKC/Cdc42) and the cytoskeletal proteins N-Wasp and ezrin. Thus, we conclude that the principal role of Cdc42 in ureteric bud and metanephric mesenchyme development is to regulate epithelial cell polarity and the actin cytoskeleton.

Integrin receptors cluster on the cell surface and bind to extra cellular matrix (ECM) proteins triggering the formation of focal contacts and the activation of various signal transduction pathways that affect the morphology, motility, gene expression and survival of adherent cells. Polyamine depletion prevents the increase in autophosphorylation of focal adhesion kinase (FAK) and Src during attachment. Rac activity also shows a steady decline, and its upstream guanine nucleotide exchange factor (GEF), Tiam1 also shows a reduction in total protein level when cells are depleted of polyamines. When Tiam1 and Rac1 interaction was inhibited by NSC-23766, there was not only a decrease in Rac1 activity as expected but also a decrease in FAK auto-phosphorylation. Inhibition of Src activity by PP2 also reduced FAK autophosphorylation, which implies that Src modulates FAK autophosphorylation. From the data obtained in this study we conclude that FAK and Src are rapidly activated upon fibronectin mediated signaling leading to Tiam1-mediated Rac1 activation and that intracellular polyamines influence the signaling strength by modulating interaction of Src with Tiam1 using focal adhesion kinase as a scaffolding site.

PKCη is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKCη phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKCη by specific pseudo substrate inhibitor or knockdown of PKCη by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKCη K394R disrupts the TJ and barrier function, whereas wild-type PKCη and constitutively active PKCη A161E enhance the TJ integrity. Inhibition and knockdown of PKCη or expression of PKCη K394R induce dephosphorylation of occludin on threonine residues, whereas active PKCη elevates occludin phosphorylation. PKCη directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKCη inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKCη.

The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pair-wise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Intriguingly, the activities of these proteins are interdependent; MdmX stabilizes Mdm2, enabling its activities towards p53, but it also requires Mdm2 for its nuclear localization. Here we demonstrate that via its phosphorylation by CDK2/Cdc2p34, MdmX regulates nuclear export of Mdm2. Cdc2p34 phosphorylates MdmX on Ser 96 in vitro. Mutation within this site (MdmX(S96A)) impairs, whereas phosphomimic substitution (MdmX(S96D)) increases the cytoplasmic localization of MdmX, suggesting that CDK2/Cdc2p34 phosphorylation is required for export of MdmX from the nucleus. Consequently, cells that express MdmX(S96A) retain Mdm2 in their nuclei, suggesting that export of Mdm2 to the cytoplasm is MdmX-dependent. Similarly, treatment of cells with the pharmacological inhibitor of CDK2/Cdc2p34 or with a dominant-negative Cdc2 results in nuclear localization of MdmX and Mdm2 and decreases the level of Mdm2 expression. Since Cdc2p34 is active in nonstressed conditions, our finding provides a novel insight into the signaling cascade involved in the regulation of MdmX localization and for regulation of Mdm2 localization and stability.

We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli . At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP– N -acetyl muramylpentapeptide and UDP–[ 3 H]- N -acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.