WT1 Is Necessary for the Proliferation and Migration of Cells of Renin Lineage Following Kidney Podocyte Depletion.

Wilms' tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. METHODS: Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues. RESULTS: Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity. CONCLUSIONS: The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.

TGFß-Induced Actin Cytoskeleton Rearrangement in Podocytes Is Associated with Compensatory Adaptation of Mitochondrial Energy Metabolism.

BACKGROUND/AIMS: In podocytes, the overexpression of TGFß ligands and receptors during glomerulosclerosis could be a causal factor for injury induction and perpetuation in glomerular tufts. Mitochondrial dysfunction and oxidative stress are emerging as potential therapeutic targets in glomerular injury, and TGFß has been shown to modulate mitochondrial metabolism in different cell types. This study aims at investigating the role of TGFß in podocyte energy metabolism and cytoskeleton dynamics. METHODS: Mitochondrial function and cytoskeleton dynamics were analyzed in TGFß-treated WT and Smad2/3 double KO podocytes. RESULTS: TGFß treatment in podocytes induced a significant Smad-dependent increase of mitochondrial oxygen consumption rate (OCR). ATP content was unchanged and increased respiration was not associated with increased mitochondrial mass. Increased cellular reactive oxygen species induced by Smad-mediated TGFß signaling were reverted by NADPH oxidase inhibitor apocynin. TGFß treatment did not induce mitochondrial oxidative stress, and Smad2/3-dependent TGFß signaling and increased mitochondrial OCR were found to be associated with actin cytoskeleton dynamics. The role of motor proteins myosin II and dynamin in TGFß-induced actin polymerization was demonstrated by specific inhibition, resulting in actin stabilization and normalization of mitochondrial OCR. CONCLUSION: TGFß-induced rearrangements of actin cytoskeleton are controlled by Smad2/3 signaling pathways and coupled with the activation of mitochondrial ATP synthesis as bioenergetic adaptation to ATP consumption by ATP- and GTP-dependent motor proteins, myosin II and dynamin.

Krüppel-like factor 6 regulates mitochondrial function in the kidney.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.

Epithelial cell TGFß signaling induces acute tubular injury and interstitial inflammation.

TGFß signaling plays a central role in the development of acute and chronic kidney diseases. Previous in vivo studies involved systemic alteration of TGFß signaling, however, limiting conclusions about the direct role of TGFß in tubular cell injury. Here, we generated a double transgenic mouse that inducibly expresses a ligand-independent constitutively active TGFß receptor type 1 (TßR1) kinase specifically in tubular epithelial cells, with expression restricted by the Pax8 promoter. In this model, activation of TGFß signaling in the tubular epithelium alone was sufficient to cause AKI characterized by marked tubular cell apoptosis and necrosis, oxidative stress, dedifferentiation and regenerative cell proliferation, reduced renal function, and interstitial accumulation of inflammatory cells. This tubular injury was associated with mitochondrial-derived generation of reactive oxygen species (ROS), but cell damage and apoptosis were partially independent of mitochondrial-derived ROS. TßR1 signaling-induced tubular injury also associated with significant leukocyte infiltration consisting of F4/80(+) macrophages, CD11c(+) F4/80(+) dendritic cells, CD11c(+) F4/80(-) Ly6C(high) dendritic cells/monocytes, and T cells. Inhibition of mitochondrial-derived ROS significantly reduced accumulation of CD11c(+) F4/80(+) dendritic cells and T cells, suggesting a role for ROS in the activation and recruitment of the adaptive immune response to tubular injury. Taken together, these results suggest that TGFß signaling in the tubular epithelium alone is sufficient to cause acute tubular injury and inflammation; therefore, TGFß may be a mechanistic link between acute injury and chronic progression of kidney disease.

Transforming growth factor-ß, bioenergetics, and mitochondria in renal disease.

The transforming growth factor-ß (TGF-ß) family comprises more than 30 family members that are structurally related secreted dimeric cytokines, including TGF-ß, activins, and bone morphogenetic proteins/growth and differentiation factors. TGF-ß are pluripotent regulators of cell proliferation, differentiation, apoptosis, migration, and adhesion of many different cell types. TGF-ß pathways are highly evolutionarily conserved and control embryogenesis, tissue repair, and tissue homeostasis in invertebrates and vertebrates. Aberrations in TGF-ß activity and signaling underlie a broad spectrum of developmental disorders and major pathologies in human beings, including cancer, fibrosis, and autoimmune diseases. Recent observations have indicated an emerging role for TGF-ß in the regulation of mitochondrial bioenergetics and oxidative stress responses characteristic of chronic degenerative diseases and aging. Conversely, energy and metabolic sensory pathways cross-regulate mediators of TGF-ß signaling. Here, we review TGF-ß and regulation of bioenergetic and mitochondrial functions, including energy and oxidant metabolism and apoptotic cell death, as well as their emerging relevance in renal biology and disease.

6-Thioguanine damages mitochondrial DNA and causes mitochondrial dysfunction in human cells.

The anticancer and immunosuppressant thiopurines cause 6-thioguanine (6-TG) to accumulate in nuclear DNA. We report that 6-TG is also readily incorporated into mitochondrial DNA (mtDNA) where it is rapidly oxidized. The oxidized forms of mtDNA 6-TG inhibit replication by DNA Pol-γ. Accumulation of oxidized 6-TG is associated with reduced mtDNA transcription, a decline in mitochondrial protein levels, and loss of mitochondrial function. Ultraviolet A radiation (UVA) also oxidizes mtDNA 6-TG. Cells without mtDNA are less sensitive to killing by a combination of 6-TG and UVA than their mtDNA-containing counterparts, indicating that photochemical mtDNA 6-TG oxidation contributes to 6-TG-mediated UVA photosensitization.

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation.

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.

T-lymphocyte-induced, Fas-mediated apoptosis is associated with early keratinocyte differentiation.

The development of eczematous lesions is thought to be due in part to a breakdown in skin barrier function as a result of T lymphocytes (T cells) invading the skin causing epidermal keratinocyte apoptosis. In this study, we investigated the interaction of T cells and keratinocytes on apoptosis and terminal differentiation using an in vitro co-culture system. Experiments were performed using the HaCaT keratinocyte cell line or normal human epidermal keratinocytes. Activated human peripheral blood-derived T cells were found to induce Fas-dependent keratinocyte apoptosis by up to sixfold. Increased Fas was associated with increased IFN-gamma. The T-cell apoptotic signal was found to target preferentially keratinocytes in the very early stages of terminal differentiation, such as those with low levels of alpha 6-integrin expression, and result in subsequent increased caspase 3 activity. This observation was accompanied by a marked increase in keratinocyte ICAM-1 expression and its ligand LFA-1 on T cells. Our data suggest that T cells may initiate the onset of keratinocyte terminal differentiation making them more susceptible to Fas-dependent cell death signals delivered by the T cells.

Immune effector cells produce lethal DNA damage in cells treated with a thiopurine.

Azathioprine, a widely used immunosuppressant, is also used in the control of inflammatory disorders. These are characterized by the local accumulation of immune effector cells that produce reactive oxygen species (ROS). The DNA of azathioprine-treated patients contains 6-thioguanine (6-TG), a base analogue that is particularly susceptible to oxidation. Here, we show that 6-TG is vulnerable to ROS produced by chemical oxidants and that cells containing DNA 6-TG are hypersensitive to these oxidants. We also show that 6-TG incorporated into the DNA of macrophages sensitizes them to killing by endogenously produced ROS. ROS generated by macrophages are also a hazard for cocultured nonmacrophage cells containing DNA 6-TG. This bystander vulnerability of cells containing DNA 6-TG to oxidation by ROS generated by immune effector cells has implications for the long-term use of azathioprine in the management of inflammatory disorders.

Sodium butyrate induced keratinocyte apoptosis.

Apoptosis of keratinocytes is a key mechanism required for epidermal homeostasis and the renewal of damaged cells. Its dysregulation has been implicated in many skin diseases including cancer and hyperproliferative disorders. In the present study, the effect of sodium butyrate, a histone deacetylase inhibitor, on keratinocyte apoptosis was investigated using the HaCaT human keratinocyte cell line. Sodium butyrate induced morphological changes associated with apoptosis and nuclear fragmentation of HaCaTs. Annexin V staining demonstrated that sodium butyrate induced apoptosis in a dose and time-dependent manner with 50% of HaCaTs apoptotic after exposure to 0.8 mg/ml sodium butyrate for 24 h. Apoptosis was associated with upregulation of cell surface expression of the death receptor Fas and activation of the extrinsic caspase pathway, with induction of caspase 8 activity peaking after 8 h. Caspase 3 activity peaked after 24 h and was associated with cleavage of the caspase 3 substrate, poly (ADP-ribose) polymerase (PARP). The intrinsic caspase pathway was not activated as caspase 9 activity was not detected, and there was no change in the expression of terminal differentiation markers keratin 10 and involucrin following sodium butyrate treatment. Together these results indicate that sodium butyrate is a potent inducer of Fas associated apoptosis via caspase activation in HaCaT keratinocytes, an effect that is independent of the induction of terminal differentiation.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin.

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E(17)) in the rat such that embryonic day 19 (E(19)) wounds do not re-epithelialize. Moreover, wounds created in E(17) fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E(17) and E(19) skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E(17) and E(19) skin. c-fos and c-jun induction was transient in E(17) skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E(19) skin, AP-1 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E(17) skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Bcl-xL overexpression protects from apoptosis induced by HMG-CoA reductase inhibitors in murine tubular cells.

BACKGROUND: Hyperplasia is attributed to enhanced tubular cell proliferation with unbalanced cell death. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors induce apoptosis in a variety of cell lines, including proximal tubular cells. However, the mechanisms by which statins induce apoptosis in tubular cells have not been fully addressed. METHODS: Apoptosis induced by simvastatin was measured in murine tubular cells with and without overexpressing Bcl-xL. Expression of genes implicated in cell death was studied by Northern and Western blot. RESULTS: The treatment of proliferating murine tubular cells (MCT) with simvastatin induced apoptosis in a time- and dose-dependent manner (0.1 to 1 micromol/L). Apoptosis was correlated with Bcl-xL mRNA and protein down-regulation. By contrast, the treatment with simvastatin did not modify the expression of the proapoptotic protein Bax. Simvastatin treatment was associated with cytochrome C release from the mitochondria to the cytosol. We also observed the presence of active caspase 9 and 3 during apoptosis induced by simvastatin. These effects were reversed by mevalonate, farnesylpyrophosphate (FPP), and geranylgeranylpyrophosphate (GGPP), suggesting the involvement of protein prenylation. Simvastatin appears to alter the balance between cell-life and death-promoting genes, as reflected by the decreased Bcl-xL/Bax ratio. Supporting this hypothesis, overexpression of Bcl-xL reduced the amount of apoptosis induced by simvastatin by 80% when compared with control vector-expressing cells. The overexpression of Bcl-xL also prevented the activation of caspase 9 and 3. CONCLUSION: Our results indicate that down-regulation of Bcl-xL expression mediates apoptosis induced by statins in tubular cells. These results may be relevant to the treatment of disorders characterized by altered tubular proliferation.