IL-22 is secreted by proximal tubule cells and regulates DNA damage response and cell death in acute kidney injury.

Acute kidney injury (AKI) affects over 13 million people worldwide annually and is associated with a 4-fold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces cell death and worsens AKI. The factors that trigger DDR to switch from pro-repair to pro-cell death remain to be resolved. Here we investigated the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identified PTCs as a novel source of urinary IL-22. Functionally, IL-22 binding IL-22RA1 on PTCs amplified the DDR. Treating primary PTCs with IL-22 alone induced rapid activation of the DDR. The combination of IL-22 and either cisplatin- or AA-induced cell death in primary PTCs, while the same dose of cisplatin or AA alone did not. Global deletion of IL-22 protected against cisplatin- or AA-induced AKI, reduced expression of DDR components, and inhibited PTC cell death. To confirm PTC IL-22 signaling contributed to AKI, we knocked out IL-22RA1 specifically in kidney tubule cells. IL-22RA1ΔTub mice displayed reduced DDR activation, cell death, and kidney injury compared to controls. Thus, targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with repair of damaged DNA.

IL-22 promotes acute kidney injury through activation of the DNA damage response and cell death in proximal tubule cells.

Acute kidney injury (AKI) affects over 13 million people world-wide annually and is associated with a fourfold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces to cell death and worsens AKI. The factors that trigger the switch from pro-reparative to pro-cell death DDR remain to be resolved. Here we investigate the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identify PTCs as a novel source of urinary IL-22, making PTCs the only epithelial cells known to secret IL-22, to our knowledge. Functionally, IL-22 binding its receptor (IL-22RA1) on PTCs amplifies the DDR. Treating primary PTCs with IL-22 alone induces rapid activation of the DDR in vitro. The combination of IL-22 + cisplatin or AA treatment on primary PTCs induces cell death, while the same dose of cisplatin or AA alone does not. Global deletion of IL-22 protects against cisplatin or AA induced AKI. IL-22 deletion reduces expression of components of the DDR and inhibits PTC cell death. To confirm PTC IL-22 signaling contributes to AKI, we knocked out IL-22RA1 in renal epithelial cells by crossing IL-22RA1floxed mice with Six2-Cre mice. IL-22RA1 KO reduced DDR activation, cell death, and kidney injury. These data demonstrate that IL-22 promotes DDR activation in PTCs, switching pro-recovery DDR responses to a pro-cell death response and worsening AKI. Targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with the processes necessary for repair of damaged DNA.

Cyclin G1 induces maladaptive proximal tubule cell dedifferentiation and renal fibrosis through CDK5 activation.

Acute kidney injury (AKI) occurs in approximately 13% of hospitalized patients and predisposes patients to chronic kidney disease (CKD) through the AKI-to-CKD transition. Studies from our laboratory and others have demonstrated that maladaptive repair of proximal tubule cells (PTCs), including induction of dedifferentiation, G2/M cell cycle arrest, senescence, and profibrotic cytokine secretion, is a key process promoting AKI-to-CKD transition, kidney fibrosis, and CKD progression. The molecular mechanisms governing maladaptive repair and the relative contribution of dedifferentiation, G2/M arrest, and senescence to CKD remain to be resolved. We identified cyclin G1 (CG1) as a factor upregulated in chronically injured and maladaptively repaired PTCs. We demonstrated that global deletion of CG1 inhibits G2/M arrest and fibrosis. Pharmacological induction of G2/M arrest in CG1-knockout mice, however, did not fully reverse the antifibrotic phenotype. Knockout of CG1 did not alter dedifferentiation and proliferation in the adaptive repair response following AKI. Instead, CG1 specifically promoted the prolonged dedifferentiation of kidney tubule epithelial cells observed in CKD. Mechanistically, CG1 promotes dedifferentiation through activation of cyclin-dependent kinase 5 (CDK5). Deletion of CDK5 in kidney tubule cells did not prevent G2/M arrest but did inhibit dedifferentiation and fibrosis. Thus, CG1 and CDK5 represent a unique pathway that regulates maladaptive, but not adaptive, dedifferentiation, suggesting they could be therapeutic targets for CKD.

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection.

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes patients to CKD later in life. Kidney epithelial cells have been identified as key signaling nodes in both AKI and CKD, whereby the cells can determine the course of the disease through the secretion of cytokines and other proteins. In CKD especially, several lines of evidence have demonstrated that maladaptively repaired tubular cells drive disease progression through the secretion of transforming growth factor-beta (TGF-ß), connective tissue growth factor (CTGF), and other profibrotic cytokines. However, identifying the source and the relative number of secreted proteins from different cell types in vivo remains challenging. This paper describes a technique using brefeldin A (BFA) to prevent the secretion of cytokines, enabling the staining of cytokines in kidney tissue using standard immunofluorescent techniques. BFA inhibits endoplasmic reticulum (ER)-to-Golgi apparatus transport, which is necessary for the secretion of cytokines and other proteins. Injection of BFA 6 h before sacrifice leads to a build-up of TGF-ß, PDGF, and CTGF inside the proximal tubule cells (PTCs) in a mouse cisplatin model of AKI and TGF-ß in a mouse aristolochic acid (AA) model of CKD. Analysis revealed that BFA + cisplatin or BFA + AA increased TGF-ß-positive signal significantly compared to BFA + saline, cisplatin, or AA alone. These data suggest that BFA can be used to identify the cell type producing specific cytokines and quantify the relative amounts and/or different types of cytokines produced.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

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.

Dysbiosis-Related Advanced Glycation Endproducts and Trimethylamine N-Oxide in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a public health concern that affects approximately 10% of the global population. CKD is associated with poor outcomes due to high frequencies of comorbidities such as heart failure and cardiovascular disease. Uremic toxins are compounds that are usually filtered and excreted by the kidneys. With the decline of renal function, uremic toxins are accumulated in the systemic circulation and tissues, which hastens the progression of CKD and concomitant comorbidities. Gut microbial dysbiosis, defined as an imbalance of the gut microbial community, is one of the comorbidities of CKD. Meanwhile, gut dysbiosis plays a pathological role in accelerating CKD progression through the production of further uremic toxins in the gastrointestinal tracts. Therefore, the gut-kidney axis has been attracting attention in recent years as a potential therapeutic target for stopping CKD. Trimethylamine N-oxide (TMAO) generated by gut microbiota is linked to the progression of cardiovascular disease and CKD. Also, advanced glycation endproducts (AGEs) not only promote CKD but also cause gut dysbiosis with disruption of the intestinal barrier. This review summarizes the underlying mechanism for how gut microbial dysbiosis promotes kidney injury and highlights the wide-ranging interventions to counter dysbiosis for CKD patients from the view of uremic toxins such as TMAO and AGEs.

Quantitative super-resolution microscopy reveals promoting mitochondrial interconnectivity protects against AKI.

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.

Uremic Toxin-Targeting as a Therapeutic Strategy for Preventing Cardiorenal Syndrome.

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.

Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses.

Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin-positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC-/-) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC-/- mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.

Quantifying autophagic flux in kidney tissue using structured illumination microscopy.

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.

Cdc42 regulates epithelial cell polarity and cytoskeletal function during kidney tubule development.

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.

The integrin ß1 subunit regulates paracellular permeability of kidney proximal tubule cells.

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 ß1 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 ß1 cytoplasmic tail and does not entail ß1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the ß1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin ß1 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.

Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation.

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.

PKC eta regulates occludin phosphorylation and epithelial tight junction integrity.

PKC eta is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC eta 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 PKC eta by specific pseudo substrate inhibitor or knockdown of PKC eta by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC eta(K394R) disrupts the TJ and barrier function, whereas wild-type PKC eta and constitutively active PKC eta(A161E) enhance the TJ integrity. Inhibition and knockdown of PKC eta or expression of PKC eta(K394R) induce dephosphorylation of occludin on threonine residues, whereas active PKC eta elevates occludin phosphorylation. PKC eta 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 PKC eta inhibitor-mediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC eta.

Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its interaction with ZO-1 and destabilizes its assembly at the tight junctions.

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.

Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer.

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.