Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.

Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability to Inhibit Cyst Enlargement of ADPKD Cells Compared with Nonsecreted Form.

Polycystic kidney disease (PKD) is one of the most common inherited diseases and is characterized by the development of fluid-filled cysts along multiple segments of the nephron. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of PKD, which is caused by mutations in either PKD1 or PKD2 genes that encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. As ADPKD progresses, cysts enlarge and disrupt normal kidney architecture, eventually leading to kidney failure. Our previous study showed that overexpression of exogenous kidney-specific neutrophil gelatinase-associated lipocalin (NGAL) reduced cyst progression and prolonged the lifespan of ADPKD mice (Pkd1L3/L3, 2L3 for short). In this study, we attempted to explore the underlying mechanism of reduced cyst progression in the presence of NGAL using immortalized 2L3 cells. The results of MTT and BrdU incorporation assays showed that recombinant mouse NGAL (mNGAL) protein significantly decreased the viability and proliferation of 2L3 cells. Flow cytometry and western blot analyses showed that mNGAL inhibited activation of the ERK and AKT pathways and induced apoptosis and autophagy in 2L3 cells. In addition, a 3D cell culture platform was established to identify cyst progression in 2L3 cells and showed that mNGAL significantly inhibited cyst enlargement in 2L3 cells. Overexpression of secreted mNGAL (pN + LS) and nonsecreted mNGAL (pN - LS) repressed cell proliferation and cyst enlargement in 2L3 cells and had effects on markers involved in proliferation, apoptosis, and autophagy. However, secreted mNGAL had a more pronounced and consistent effect than that of nonsecreted form. These results reveal that secreted mNGAL has stronger ability to inhibit cyst enlargement of ADPKD cells than that of nonsecreted form. These findings could help to identify strategies for the future clinical treatment of ADPKD.

Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis, leading to polycystic kidney disease in mice.

WD repeat domain 19 (Wdr19) is a major component of the intraflagellar transport (IFT) machinery, which is involved in the function of primary cilia. However, the effects of Wdr19 on primary cilia formation, cystogenesis, and polycystic kidney disease (PKD) progression remain unclear. To study these effects, we generated three lines of kidney-specific conditional knockout mice: Wdr19-knockout (Wdr19-KO, Wdr19f/- ::Cdh16-CreTg/0 ), Pkd1-knockout (Pkd1-KO, Pkd1f/- ::Cdh16-CreTg/0 ), and Wdr19/Pkd1-double knockout (Wdr19&Pkd1-dKO, Wdr19f/- ;Pkd1f/- ::Cdh16-CreTg/0 ) mice. Ultrastructural analysis using transmission electron microscopy (TEM) indicated that the primary cilia were almost absent at postnatal day 10 in Wdr19-KO mice compared with Pkd1-KO and wild-type (WT) mice. However, the primary cilia appeared structurally normal even if malfunctional in Pkd1-deficient cysts. The Pkd1-KO mice had the most severe PKD progression, including the shortest lifespan (14 days) and the largest renal cysts, among the three knockout lines. Thus, the molecular mechanism of renal cystogenesis in Wdr19-KO mice (primary cilia abrogation) was different from that in Pkd1-KO mice (primary cilia malfunction). In summary, Wdr19 deficiency leads to primary cilia abrogation and renal cyst formation. Wdr19 is primarily proposed to participate in retrograde IFT and to be crucial for the construction of primary cilia, which are critical organelles for tubulogenesis in the developing kidneys. © 2022 The Pathological Society of Great Britain and Ireland.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2- CD34- Lin- Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor ß subunit (CSF2RB). As a result, Flk2- CD34- LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2- CD34- LSK-HSCs.

Kidney-based in vivo model for drug-induced nephrotoxicity testing.

The need is critical and urgent for a real-time, highly specific, and sensitive acute kidney injury biomarker. This study sought to establish a sensitive and specific Miox-NanoLuc transgenic mouse for early detection of drug-induced nephrotoxicity. We generated Miox-NanoLuc transgenic mice with kidney-specific NanoLuc overexpression. Our data showed that Miox-NanoLuc-produced luminescence was kidney-specific and had good stability at room temperature, 4 °C, - 20 °C, and repeated freeze-thaw cycles. Serum levels of BUN and creatinine were significantly increased at day 2 or 3 in cisplatin-treated mice and at day 5 in aristolochic acid (AAI)-treated mice. Particularly, the serum and urine Miox-NanoLuc luminescence levels were significantly increased at day 1 in cisplatin-treated mice and at day 3 in AAI-treated mice. Renal pathological analysis showed that the kidney sections of cisplatin-treated mice at day 5 and AAI-treated mice at day 13 showed cytolysis and marked vacuolization of tubular cells. In conclusion, we developed a new platform to early quantify drug-induced nephrotoxicity before serum BUN and creatinine levels increased and pathological tubular cell injury occurred. This model may serve as an early detection for drug- and food-induced nephrotoxicity and as an animal model to investigate tubular cell injury.

A reporter mouse for non-invasive detection of toll-like receptor ligands induced acute phase responses.

The acute phase response (APR) is a systemic first-line defense against challenges including infection, trauma, stress, and neoplasia. Alteration of acute phase protein (APP) levels in plasma is the most important change during acute phase response. C-reactive protein (CRP), which increases dramatically during inflammation onset, is an indicator of inflammation. To monitor the process of APR, we generated human CRP promoter-driven luciferase transgenic (hCRP-Luc) mice to quantify the hCRP promoter activation in vivo. The naïve female hCRP-Luc mice express low basal levels of liver bioluminescence, but the naïve male hCRP-Luc mice do not. Thus, female hCRP-Luc mice are suitable for monitoring the process of APR. The liver bioluminescence of female hCRP-Luc mice can be induced by several toll-like receptor (TLR) ligands. The expression of liver bioluminescence was highly sensitive to endotoxin stimulation in a dose-dependent manner. On-off-on bioluminescence response was noted in female hCRP-Luc mice upon two endotoxin stimulations one month apart. The LPS-induced bioluminescence of the female hCRP-Luc mice was IL-6-mediated and associated with APP alpha-1-acid glycoprotein expression. In conclusion, the female hCRP-Luc mouse is a non-invasive, sensitive and reusable reporter tool for APR.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Targeted Delivery of Curcumin Rescues Endoplasmic Reticulum-Retained Mutant NOX2 Protein and Avoids Leukocyte Apoptosis.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitors could be used to rescue mutant H338Y-gp91phox protein of a particular type of CGD with a CybbC1024T mutation, leading to endoplasmic reticulum (ER) retention of the mutant protein. In this study, we developed a novel mouse model with the CybbC1024T mutation on a Cybb knockout background and investigated the therapeutic effects of ER-targeted delivery of the SERCA inhibitor, curcumin, with poly(lactic-coglycolic acid) (PLGA) nanoparticles (NPs). We found that PLGA encapsulation improved the efficacy of curcumin as a SERCA inhibitor to induce ER calcium release. ER-targeting curcumin-loaded PLGA NPs reduced and delayed extracellular calcium entry and protected the cells from mitochondrial damage and apoptosis. In vivo studies showed that ER-targeting curcumin-loaded PLGA NPs treatment enhanced neutrophil gp91phox expression, ROS production and peritoneal bacterial clearance ability of the CybbC1024T transgenic Cybb -/- mice. Our findings indicate that ER-targeted delivery of curcumin not only rescues ER-retained H338Y-gp91phox protein, and hence leukocyte function, but also enhances the bioavailability and reduces cytotoxicity. Modulation of ER function by using organelle-targeted NPs may be a promising strategy to improve the therapeutic potential of curcumin as a treatment for CGD.

Safe Nanocomposite-Mediated Efficient Delivery of MicroRNA Plasmids for Autosomal Dominant Polycystic Kidney Disease (ADPKD) Therapy.

There is currently no cure for gene mutation-caused autosomal dominant polycystic kidney disease (ADPKD). Over half of patients with ADPKD eventually develop kidney failure, requiring dialysis or kidney transplantation. Current treatment modalities for ADPKD focus on reducing morbidity and mortality from renal and extrarenal complications of the disease. MicroRNA has been shown to be useful in treating ADPKD. This study combines anti-miRNA plasmids and iron oxide/alginate nanoparticles for conjugation with antikidney antibodies. These nanocomposites can specifically target renal tubular cells, providing a potential treatment for ADPKD. Magnetic resonance imaging and in vivo imaging system results show effective targeting of renal cells. Anti-miRNA plasmids released from the nanocomposites inhibit cell proliferation and cyst formation in the PKD cellular and animal models. The results suggest the novel combination of the anti-miRNA plasmids and nanomaterials provides potential clinical implications for ADPKD treatment.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

Casein kinase 1α (CK1α), a component of the ß-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by ß-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

Overexpression of exogenous kidney-specific Ngal attenuates progressive cyst development and prolongs lifespan in a murine model of polycystic kidney disease.

Neutrophil gelatinase-associated lipocalin (Ngal) is a biomarker for acute and chronic renal injuries, including polycystic kidney disease (PKD). However, the effect of Ngal on PKD progression remains unexplored. To study this, we generated 3 strains of mice with different expression levels of Ngal within an established PKD model (Pkd1L3/L3): Pkd1L3/L3 (with endogenous Ngal), Pkd1L3/L3; NgalTg/Tg (with endogenous and overexpression of exogenous kidney-specific Ngal) and Pkd1L3/L3; Ngal-/- mice (with Ngal deficiency). Knockout of endogenous Ngal had no effect on phenotypes, cystic progression, or survival of the PKD mice. However, the transgenic mice had a significantly longer lifespan, smaller (but not fewer) renal cysts, and less interstitial fibrosis than the mice without or with endogenous Ngal. Western-blot analyses showed significant increases in Ngal and cleaved caspase-3 and decreases in α-smooth muscle actin, hypoxia-inducible factor 1-α, pro-caspase 3, proliferating cell nuclear antigen, Akt, mammalian target of rapamycin, and S6 Kinase in the transgenic mice as compared with the other 2 strains of PKD mice. Thus, overexpression of exogenous kidney-specific Ngal reduced cystic progression and prolonged the lifespan in PKD mice, was associated with reductions in interstitial fibrosis and proliferation, and augmented apoptosis.

Insulin Receptor Substrate-1 Activation Mediated p53 Downregulation Protects Against Hypoxic-Ischemia in the Neonatal Brain.

This study determined if dietary restriction (DR) protects against hypoxic-ischemia (HI) in the neonatal brain via insulin receptor substrate-1 (IRS-1)/Akt pathway-mediated downregulation of p53 in the neurovascular unit. On postnatal (P) day 7, HI was induced in rat pups grouped from P1 into normal litter size (NL, 12 pups/dam) and increased litter size (DR, 18 pups/dam). In vivo IRS-1 anti-sense oligonucleotide and IRS-1 overexpressed recombinant adenovirus were given, and neurovascular damage was assessed. In vitro models of oxygen-glucose deprivation (OGD) examined the inhibition and overexpression of IRS-1 on p53 and cell death in neurons and endothelial cells. Compared to NL pups, DR pups had significantly higher IRS-1, p-IRS-1, and pAkt levels, decreased p53, more tight junction proteins, reduced blood-brain barrier (BBB) damage after HI, and less infarct volumes at P21. Immunofluorescence revealed that IRS-1 was upregulated in the endothelial cells and neurons of DR pups. IRS-1 downregulation in DR pups reduced p-Akt, increased p53, worsened BBB damage, and increased brain injury, whereas IRS-1 overexpression in NL pups upregulated p-Akt, decreased p53, attenuated BBB damage, and decreased brain injury. In vitro, IRS-1 downregulation aggravated cell death in neurons and endothelial cells and is associated with decreased p-Akt and increased p53. In contrast, IRS-1 overexpression reduced cell death in endothelial cells with increased p-Akt and decreased p53. In conclusion, DR reduces neurovascular damage after HI in the neonatal brain through an IRS-1/Akt-mediated p53 downregulation, suggesting that IRS-1 signaling is a therapeutic target for hypoxic brain injury in neonates.

Adipocyte IL-15 regulates local and systemic NK cell development.

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

Selective inhibition of the NLRP3 inflammasome by targeting to promyelocytic leukemia protein in mouse and human.

The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1ß was strongly impaired. The expression of pro-IL-1ß, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1ß production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.

Different NK cell developmental events require different levels of IL-15 trans-presentation.

NK cell development requires IL-15, which is "trans-presented" to IL-15Rßγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.

Progressive renal distortion by multiple cysts in transgenic mice expressing artificial microRNAs against Pkd1.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, and the PKD1 gene is responsible for most cases of this disease. Previous efforts to establish a mouse model that recapitulates the phenotypic characteristics of ADPKD, which have used conventional or conditional knockout of the mouse orthologue Pkd1, have been unsuccessful or unreliable. In a previous study, we described the generation of a novel Pkd1 hypomorphic allele, in which Pkd1 expression was significantly reduced but not totally blocked. These Pkd1 homozygous mutant mice rapidly developed renal cystic disease, supporting the hypothesis that 'haploinsufficiency' explains development of the ADPKD phenotype. In the present study, we further investigated the Pkd1 haploinsufficiency effect by generating Pkd1 knockdown transgenic mice with co-cistronic expression of two miRNA hairpins specific to Pkd1 transcript and an Emerald GFP reporter driven by a human ubiquitin B promoter. Two transgenic lines which had ∼60-70% reduction of Pkd1 expression developed severe renal cystic disease at a rate similar to that of human ADPKD. These results further support the haploinsufficiency hypothesis, and suggest that the onset and progression of the renal cystic diseases are correlated with the level of Pkd1 expression. The two novel mutant lines of mice appear to be ideal models for the study of ADPKD.

Impaired water reabsorption in mice deficient in the type VI adenylyl cyclase (AC6).

Adenylyl cyclase (AC) type VI (AC6) is a calcium-inhibitable enzyme which produces cAMP upon stimulation. Herein, we characterized the specific role of AC6 in the kidneys using two AC6-knockout mouse lines. Immunohistochemical staining revealed that AC6 exists in the tubular parts of the nephron and collecting duct. Activities of AC evoked by forskolin or a selective agonist of the V2 vasopressin receptor were lower in the kidneys of AC6-null mice compared to those of wildtype mice. Results of a metabolic cage assay and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) showed for the first time that AC6 plays a critical role in regulating water homeostasis.

Mouse kidney progenitor cells accelerate renal regeneration and prolong survival after ischemic injury.

Acute tubular necrosis is followed by regeneration of damaged renal tubular epithelial cells, and renal stem cells are supposed to contribute to this process. The purpose of our study is to test the hypothesis that renal stem cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal stem cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include (1) spindle-shaped morphology, (2) self-renewal of more than 100 passages without evidence of senescence, and (3) expression of Oct-4, Pax-2, Wnt-4, WT-1, vimentin, alpha-smooth muscle actin, CD29, and S100A4 but no SSEA-1, c-kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intrarenal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone, and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult stem cell population, which may contribute to the renal repair and prolong survival after ischemic injury.

Deltex1 is a target of the transcription factor NFAT that promotes T cell anergy.

The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.