VEGF-C overexpression in kidney progenitor cells is a model of renal lymphangiectasia.

Background: Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease.As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined. Methods: We generated a new mouse model expressing Vegf-C under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegf-C). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging. Results: Six2Vegf-C mice had reduced body weight and kidney function compared to littermate controls. Six2Vegf-C kidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density. Conclusions: Kidney lymphangiogenesis was robustly induced in the Six2Vegf-C mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.

Activation of Angiopoietin-Tie2 Signaling Protects the Kidney from Ischemic Injury by Modulation of Endothelial-Specific Pathways.

SIGNIFICANCE STATEMENT: Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. BACKGROUND: Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). METHODS: Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. RESULTS: The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. CONCLUSIONS: Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.

Formation of the glomerular microvasculature is regulated by VEGFR-3.

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.

N6-Adenosine Methylation of Socs1 mRNA Is Required to Sustain the Negative Feedback Control of Macrophage Activation.

Bacterial infection triggers a cytokine storm that needs to be resolved to maintain the host's wellbeing. Here, we report that ablation of m6A methyltransferase subunit METTL14 in myeloid cells exacerbates macrophage responses to acute bacterial infection in mice, leading to high mortality due to sustained production of pro-inflammatory cytokines. METTL14 depletion blunts Socs1 m6A methylation and reduces YTHDF1 binding to the m6A sites, which diminishes SOCS1 induction leading to the overactivation of TLR4/NF-κB signaling. Forced expression of SOCS1 in macrophages depleted of METTL14 or YTHDF1 rescues the hyper-responsive phenotype of these macrophages in vitro and in vivo. We further show that LPS treatment induces Socs1 m6A methylation and sustains SOCS1 induction by promoting Fto mRNA degradation, and forced FTO expression in macrophages mimics the phenotype of METTL14-depleted macrophages. We conclude that m6A methylation-mediated SOCS1 induction is required to maintain the negative feedback control of macrophage activation in response to bacterial infection.

ATP-citrate lyase is an epigenetic regulator to promote obesity-related kidney injury.

Obesity is a leading cause of chronic kidney disease (CKD), but how obesity promotes renal injury remains poorly understood. Here we showed that ATP-citrate lyase (ACL), an enzyme converting citrate to acetyl-CoA, is highly induced in the kidney of overweight or obese patients with CKD and ob/ob BTBR mice. ACL induction is associated with increased ectopic lipid accumulation (ELA), glomerulosclerosis, and albuminuria. Acetyl-CoA is the substrate for de novo lipogenesis as well as for histone acetylation. By raising acetyl-CoA concentration ACL promotes H3K9/14 and H3K27 hyperacetylation leading to up-regulation of several rate-limiting lipogenic enzymes and fibrogenic factors. On the other hand, the excess acetyl-CoA generated as a result of ACL induction provides the substrate for these lipogenic enzymes to drive de novo lipogenesis leading to ELA, a detrimental event toward renal injury. In mesangial cells, ACL is synergistically induced by high glucose, palmitate, and TNF-α via NF-κB and PKA pathways. Under these conditions, H3K9/14 and H3K27 hyperacetylation, as well as the induction of the lipogenic and fibrogenic proteins, are completely blocked in the presence of an ACL inhibitor. Collectively, these data suggest that ACL is an epigenetic regulator that promotes renal ELA and fibrogenesis leading to renal injury in obesity.-Chen, Y., Deb, D. K., Fu, X., Yi, B., Liang, Y., Du, J., He, L., Li, Y. C. ATP-citrate lyase is an epigenetic regulator to promote obesity-related kidney injury.

Gut Epithelial Vitamin D Receptor Regulates Microbiota-Dependent Mucosal Inflammation by Suppressing Intestinal Epithelial Cell Apoptosis.

Recent studies show that colonic vitamin D receptor (VDR) signaling protects the mucosal epithelial barrier and suppresses colonic inflammation, but the underlying molecular mechanism remains to be fully understood. To investigate the implication of colonic VDR downregulation seen in patients with inflammatory bowel disease, we assessed the effect of gut epithelial VDR deletion on colonic inflammatory responses in an experimental colitis model. In a 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, mice carrying VDR deletion in gut epithelial cells [VDRflox/flox (VDRf/f);Villin-Cre or VDRΔIEC] or in colonic epithelial cells (VDRf/f;CDX2-Cre or VDRΔCEC) developed more severe clinical colitis than VDRf/f control mice, characterized by more robust T-helper (TH)1 and TH17 responses, with greater increases in mucosal interferon (IFN)-γ+, interleukin (IL)-17+, and IFN-γ+IL-17+ T cells. Accompanying the severe mucosal inflammation was more profound colonic epithelial cell apoptosis in the mutant mice. Treatment with caspase inhibitor Q-VD-OPh dramatically reduced colitis severity and attenuated TH1 and TH17 responses in VDRΔCEC mice. The blockade of cell apoptosis also prevented the increase in mucosal CD11b+CD103+ dendritic cells (DCs), known to be critical for TH17-cell activation. Moreover, depletion of gut commensal bacteria with antibiotics eliminated the robust TH1 and TH17 responses and CD11b+CD103+ DC induction. Taken together, these observations demonstrate that gut epithelial VDR deletion aggravates epithelial cell apoptosis, resulting in increases in mucosal barrier permeability. Consequently, invading luminal bacteria activate CD11b+CD103+ DCs, which promote mucosal TH1 and TH17 responses. Therefore, gut epithelial VDR signaling controls mucosal inflammation by suppressing epithelial cell apoptosis.

ATP-citrate lyase is essential for high glucose-induced histone hyperacetylation and fibrogenic gene upregulation in mesangial cells.

The goal of this study was to address the role of ATP-citrate lyase (ACL), an enzyme that converts citrate to acetyl-CoA, in high glucose (HG)-induced histone acetylation and profibrotic gene expression. Our recent ChIP-Seq studies have demonstrated that HG induces genome-wide histone hyperacetylation in mesangial cells (MCs). Here, we showed that exposure of MCs to HG markedly increased histone acetylation at the H3K9/14 and H3K18 marks and induced the expression of potent profibrotic factors TGF-ß1, TGF-ß3, and connective tissue growth factor (CTGF). The induction of these profibrotic factors was further enhanced by histone deacetylase inhibitor but suppressed by histone acetyl-transferase inhibitor, confirming the importance of histone acetylation in this regulation. Interestingly, HG not only upregulated ACL expression but also promoted ACL nuclear translocation, evidenced by increased ACL concentration and activity in the nuclear extracts. Consistent with this observation, transfection of MCs with a plasmid-carrying green fluorescent protein (GFP)-ACL fusion protein led to GFP nuclear accumulation when cultured in HG condition. Silencing ACL with siRNAs alleviated HG-induced histone hyperacetylation, as well as upregulation of TGF-ß1, TGF-ß3, CTGF, and extracellular matrix (ECM) proteins fibronectin and collagen type IV, whereas ACL overexpression further enhanced HG induction of histone acetylation, as well as these profibrotic factors and ECM proteins. Collectively, these observations demonstrate that HG promotes ACL expression and translocation into the nucleus, where ACL converts citrate to acetyl-CoA to provide the substrate for histone acetylation, leading to upregulation of fibrogenic genes. Therefore, ACL plays a critical role in epigenetic regulation of diabetic renal fibrosis.

Critical role of the cAMP-PKA pathway in hyperglycemia-induced epigenetic activation of fibrogenic program in the kidney.

Hyperglycemia is a major pathogenic factor that promotes diabetic nephropathy, but the underlying mechanism remains incompletely understood. Here, we show that high glucose induced cAMP response element-binding protein (CREB)-binding protein (CBP)-mediated H3K9/14 hyperacetylation in approximately 5000 gene promoters in glomerular mesangial cells, including those of Tgfb1, Tgfb3, and Ctgf, the major profibrotic factors that are known to drive diabetic renal fibrogenesis. In these promoters, H3K9/14 hyperacetylation was closely associated with NF-κB or CREB motifs. Chromatin immunoprecipitation assays confirmed that hyperglycemia promoted phospho-p65 or phospho-CREB and CBP bindings and RNA polymerase II recruitment to these promoters in mesangial cells as well as in glomeruli that were purified from type I and type II diabetic mice. Under hyperglycemia, cAMP production and PKA activity were markedly increased as a result of glucose transporter 1-mediated glucose influx that drives glucose metabolism and ATP production, which led to increased phosphorylation of p65 and CREB. Inhibition of adenylyl cyclase or PKA activity blocked p65 and CREB phosphorylation, CBP recruitment, and histone acetylation in these promoters. Collectively, these data demonstrate that the cAMP-PKA pathway plays a key role in epigenetic regulation of key profibrotic factors in diabetes.-Deb, D. K., Bao, R., Li, Y. C. Critical role of the cAMP-PKA pathway in hyperglycemia-induced epigenetic activation of fibrogenic program in the kidney.

Activation of the Renin-Angiotensin System Promotes Colitis Development.

The renin-angiotensin system (RAS) plays pathogenic roles in renal and cardiovascular disorders, but whether it is involved in colitis is unclear. Here we show that RenTgMK mice that overexpress active renin from the liver developed more severe colitis than wild-type controls. More than 50% RenTgMK mice died whereas all wild-type mice recovered. RenTgMK mice exhibited more robust mucosal TH17 and TH1/TH17 responses and more profound colonic epithelial cell apoptosis compared to wild-type controls. Treatment with aliskiren (a renin inhibitor), but not hydralazine (a smooth muscle relaxant), ameliorated colitis in RenTgMK mice, although both drugs normalized blood pressure. Chronic infusion of angiotensin II into wild-type mice mimicked the severe colitic phenotype of RenTgMK mice, and treatment with losartan [an angiotensin type 1 receptor blocker (ARB)] ameliorated colitis in wild-type mice, confirming a colitogenic role for the endogenous RAS. In human biopsies, pro-inflammatory cytokines were suppressed in patients with inflammatory bowel disease who were on ARB therapy compared to patients not receiving ARB therapy. These observations demonstrate that activation of the RAS promotes colitis in a blood pressure independent manner. Angiotensin II appears to drive colonic mucosal inflammation by promoting intestinal epithelial cell apoptosis and mucosal TH17 responses in colitis development.

Chronic Activation of the Renin-Angiotensin System Induces Lung Fibrosis.

Pulmonary fibrosis is a serious lung disorder that can lead to respiratory failure. Here we show that transgenic mice expressing active renin from the liver (RenTgMK) developed progressive pulmonary fibrosis leading to impaired pulmonary function. Histological analyses revealed a marked increase in extracellular matrix (ECM) deposition and decrease in alveolar size in the lungs of RenTgMK mice compared to wild-type (WT) littermates, accompanied with increased expression of ECM proteins and fibrogenic factors. The increase in lung fibrosis led to a substantial decrease in respiratory system compliance. Two-week treatment with aliskiren (renin inhibitor) or losartan (AT1 antagonist) ameliorated pulmonary ECM deposition, blocked the induction of ECM proteins and fibrogenic factors and improved respiratory compliance in RenTgMK mice, confirming a critical role of the renin-Ang II-AT1 cascade in promoting pulmonary fibrogenesis. However, when RenTgMK mice were treated with hydralazine (a smooth muscle relaxant), the blood pressure was normalized but the lung fibrotic abnormalities, fibrogenic gene induction and pulmonary elasticity were not corrected. Moreover, intratracheal instillation of lipopolysaccharide induced more severe lung injury in RenTgMK mice compared to WT littermates. These observations demonstrate that the renin-angiotensin system is a key mediator of lung fibrosis, and its pro-fibrotic effect is independent of blood pressure.

GLUT1 regulation of the pro-sclerotic mediators of diabetic nephropathy.

Diabetic glomerulosclerosis is characterized by accumulation of extracellular matrix proteins, mesangial expansion, and tubulointerstitial fibrosis. Hyperglycemia accelerates development of the disease, a direct result of increased intracellular glucose availability. The facilitative glucose transporter GLUT1 mediates mesangial cell glucose flux which leads to activation of signaling cascades favoring glomerulosclerosis, including pathways mediated by angiotensin II (Ang II), transforming growth factor ß (TGF-ß), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). Ang II has both hemodynamic and metabolic effects directly inducing GLUT1 and/or matrix protein synthesis through diacyl glycerol (DAG) or protein kinase C (PKC) induction, mesangial cell stretch, and/or through transactivation of the epidermal growth factor receptor, the platelet-derived growth factor receptor, and the insulin-like growth factor-1 receptor, all of which may stimulate GLUT1 synthesis via an ERK-mediated pathway. Conversely, inhibition of Ang II effects suppresses GLUT1 and cellular glucose uptake. GLUT1-mediated glucose flux leads to metabolism of glucose via glycolysis, with induction of DAG, PKC, TGF-ß1, CTGF and VEGF. VEGF in turn triggers both GLUT1 and matrix synthesis. New roles for GLUT1-mTOR and GLUT1-mechano-growth factor interactions in diabetic glomerulosclerosis have also recently been suggested. Recent mouse models confirmed roles for GLUT1 in vivo in stimulating glomerular growth factor expression, growth factor receptors and development of glomerulosclerosis. GLUT1 may therefore act in concert with cytokines and growth factors to induce diabetic glomerulosclerosis. Further clarification of the pathways involved may prove useful for the therapy of diabetic nephropathy. New directions for investigation are discussed.

Vitamin D receptor inhibits nuclear factor κB activation by interacting with IκB kinase ß protein.

1,25-Dihydroxyvitamin D (1,25(OH)2D3) is known to suppress NF-κB activity, but the underlying mechanism remains poorly understood. Here we show that the vitamin D receptor (VDR) physically interacts with IκB kinase ß (IKKß) to block NF-κB activation. 1,25(OH)2D3 rapidly attenuates TNFα-induced p65 nuclear translocation and NF-κB activity in a VDR-dependent manner. VDR overexpression inhibits IKKß-induced NF-κB activity. GST pull-down assays and coimmunoprecipitation experiments demonstrated that VDR physically interacts with IKKß and that this interaction is enhanced by 1,25(OH)2D3. Protein mapping reveals that VDR-IKKß interaction occurs between the C-terminal portions of the VDR and IKKß proteins. Reconstitution of VDR(-/-) cells with the VDR C terminus restores the ability to block TNFα-induced NF-κB activation and IL-6 up-regulation. VDR-IKKß interaction disrupts the formation of the IKK complex and, thus, abrogates IKKß phosphorylation at Ser-177 and abolishes IKK activity to phosphorylate IκBα. Consequently, stabilization of IκBα arrests p65/p50 nuclear translocation. Together, these data define a novel mechanism whereby 1,25(OH)2D3-VDR inhibits NF-κB activation.

1,25-Dihydroxyvitamin D promotes negative feedback regulation of TLR signaling via targeting microRNA-155-SOCS1 in macrophages.

The negative feedback mechanism is essential to maintain effective immunity and tissue homeostasis. 1,25-dihydroxyvitamin D (1,25[OH]2D3) modulates innate immune response, but the mechanism remains poorly understood. In this article, we report that vitamin D receptor signaling attenuates TLR-mediated inflammation by enhancing the negative feedback inhibition. Vitamin D receptor inactivation leads to hyperinflammatory response in mice and macrophage cultures when challenged with LPS, because of microRNA-155 (miR-155) overproduction that excessively suppresses suppressor of cytokine signaling 1, a key regulator that enhances the negative feedback loop. Deletion of miR-155 attenuates vitamin D suppression of LPS-induced inflammation, confirming that 1,25(OH)2D3 stimulates suppressor of cytokine signaling 1 by downregulating miR-155. 1,25(OH)2D3 downregulates bic transcription by inhibiting NF-κB activation, which is mediated by a κB cis-DNA element located within the first intron of the bic gene. Together, these data identify a novel regulatory mechanism for vitamin D to control innate immunity.

Vitamin D receptor signaling in podocytes protects against diabetic nephropathy.

Vitamin D and its analogs have antiproteinuric activity and podocytes express the vitamin D receptor, but whether vitamin D signaling in podocytes accounts for this renoprotection is unknown. To investigate this question, we used the 2.5 kb podocin promoter to target Flag-tagged human vitamin D receptor (hVDR) to podocytes in DBA/2J mice. After the induction of diabetes with streptozotocin, transgenic mice had less albuminuria than wild-type controls. In transgenic mice, a low dose of the vitamin D analog doxercalciferol prevented albuminuria, markedly attenuated podocyte loss and apoptosis, and reduced glomerular fibrosis, but it had little effect on the progression of diabetic nephropathy in wild-type mice. Moreover, reconstitution of VDR-null mice with the hVDR transgene in podocytes rescued VDR-null mice from severe diabetes-related renal damage. In culture, 1,25-dihydroxyvitamin D suppressed high-glucose-induced apoptosis of podocytes by blocking p38- and ERK-mediated proapoptotic pathways. Taken together, these data provide strong evidence that vitamin D/VDR signaling in podocytes plays a critical role in the protection of the kidney from diabetic injury.

Inactivation of the vitamin D receptor in APC(min/+) mice reveals a critical role for the vitamin D receptor in intestinal tumor growth.

Emerging evidence supports an inhibitory role for vitamin D in colorectal carcinogenesis; however, the mechanism remains unclear. The adenomatous polyposis coli (APC)/ß-catenin pathway plays a critical role in colorectal carcinogenesis. The purpose of our study is to explore the interactions of vitamin D and APC/ß-catenin pathways in intestinal tumor development. APC(min/+) mice with genetic inactivation of the vitamin D receptor (VDR) were generated through breeding. Intestinal tumorigenesis was compared between APC(min/+) and APC(min/+) VDR(-/-) mice at different ages. No differences were seen in the number of small intestinal and colonic tumors between APC(min/+) and APC(min/+) VDR(-/-) mice aged 3, 4, 6 and 7 months. The size of the tumors, however, was significantly increased in APC(min/+) VDR(-/-) mice in all age groups. Immunostaining showed significant increases in ß-catenin, cyclin D1, phosphorylated Stat-3 and MSH-2 levels and decreases in Stat-1 in APC(min/+) VDR(-/-) tumors compared to APC(min/+) tumors. These observations suggest that VDR signaling inhibits tumor growth rather than tumor initiation in the intestine. Thus, the increased tumor burden in APC(min/+) VDR(-/-) mice is likely due to the loss of the growth-inhibiting effect of VDR. This study provides strong evidence for the in vivo relevance of the interaction demonstrated in vitro between the vitamin D and ß-catenin signaling pathways in intestinal tumorigenesis.

Targeted expression of human vitamin D receptor in adipocytes decreases energy expenditure and induces obesity in mice.

Our previous studies demonstrated a high fat diet-resistant lean phenotype of vitamin D receptor (VDR)-null mutant mice mainly due to increased energy expenditure, suggesting an involvement of the VDR in energy metabolism. Here, we took a transgenic approach to further define the role of VDR in adipocyte biology. We used the aP2 gene promoter to target the expression of the human (h) VDR in adipocytes in mice. In contrast to the VDR-null mice, the aP2-hVDR Tg mice developed obesity compared with the wild-type counterparts without changes in food intake. The increase in fat mass was mainly due to markedly reduced energy expenditure, which was correlated with decreased locomotive activity and reduced fatty acid ß-oxidation and lipolysis in the adipose tissue in the transgenic mice. Consistently, the expression of genes involved in the regulation of fatty acid transport, thermogenesis, and lipolysis were suppressed in the transgenic mice. Taken together, these data confirm an important role of the VDR in the regulation of energy metabolism.

Molecular mechanism underlying 1,25-dihydroxyvitamin D regulation of nephrin gene expression.

Nephrin plays a key role in maintaining the structure of the slit diaphragm in the glomerular filtration barrier. Our previous studies have demonstrated potent renoprotective activity for 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)). Here we showed that in podocytes 1,25(OH)(2)D(3) markedly stimulated nephrin mRNA and protein expression. ChIP scan of the 6-kb 5' upstream region of the mouse nephrin gene identified several putative vitamin D response elements (VDREs), and EMSA confirmed that the VDRE at -312 (a DR4-type VDRE) could be bound by vitamin D receptor (VDR)/retinoid X receptor. Luciferase reporter assays of the proximal nephrin promoter fragment (-427 to +173) showed strong induction of luciferase activity upon 1,25(OH)(2)D(3) treatment, and the induction was abolished by mutations within -312VDRE. ChIP assays showed that, upon 1,25(OH)(2)D(3) activation, VDR bound to this VDRE leading to recruitment of DRIP205 and RNA polymerase II and histone 4 acetylation. Treatment of mice with a vitamin D analog induced nephrin mRNA and protein in the kidney, accompanied by increased VDR binding to the -312VDRE and histone 4 acetylation. 1,25(OH)(2)D(3) reversed high glucose-induced nephrin reduction in podocytes, and vitamin D analogs prevented nephrin decline in both type 1 and 2 diabetic mice. Together these data demonstrate that 1,25(OH)(2)D(3) stimulates nephrin expression in podocytes by acting on a VDRE in the proximal nephrin promoter. Nephrin up-regulation likely accounts for part of the renoprotective activity of vitamin D.

1,25-Dihydroxyvitamin D3 suppresses inflammation-induced expression of plasminogen activator inhibitor-1 by blocking nuclear factor-κB activation.

Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at -299. EMSA and ChIP assays showed that TNF-α increased p50/p65 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D.

Vitamin D receptor attenuates renal fibrosis by suppressing the renin-angiotensin system.

Analogs of vitamin D attenuate renal injury in several models of kidney disease, but the mechanism underlying this renoprotective effect is unknown. To address the role of the vitamin D receptor (VDR) in renal fibrogenesis, we subjected VDR-null mice to unilateral ureteral obstruction for 7 days. Compared with wild-type mice, VDR-null mice developed more severe renal damage in the obstructed kidney, with marked tubular atrophy and interstitial fibrosis. Significant induction of extracellular matrix proteins (fibronectin and collagen I), profibrogenic and proinflammatory factors (TGF-beta, connective tissue growth factor, and monocyte chemoattractant protein 1), and epithelial-to-mesenchymal transition accompanied this histologic damage. Because VDR ablation activates the renin-angiotensin system and leads to accumulation of angiotensin II (AngII) in the kidney, we assessed whether elevated AngII in the VDR-null kidney promotes injury. Treatment with the angiotensin type 1 antagonist losartan eliminated the difference in obstruction-induced interstitial fibrosis between wild-type and VDR-null mice, suggesting that AngII contributes to the enhanced renal fibrosis observed in obstructed VDR-null kidneys. Taken together, these results suggest that the VDR attenuates obstructive renal injury at least in part by suppressing the renin-angiotensin system.

Transgenic overexpression of GLUT1 in mouse glomeruli produces renal disease resembling diabetic glomerulosclerosis.

Previous work identified an important role for hyperglycemia in diabetic nephropathy (The Diabetes Control and Complications Trial Research Group. N Engl J Med 329: 977-986, 1993; UK Prospective Diabetes Study Group. Lancet 352: 837-853, 1998), and increased glomerular GLUT1 has been implicated. However, the roles of GLUT1 and intracellular glucose have not been determined. Here, we developed transgenic GLUT1-overexpressing mice (GT1S) to characterize the roles of GLUT1 and intracellular glucose in the development of glomerular disease without diabetes. GLUT1 was overexpressed in glomerular mesangial cells (MC) of C57BL6 mice, a line relatively resistant to diabetic nephropathy. Blood pressure, blood glucose, glomerular morphometry, matrix proteins, cell signaling, transcription factors, and selected growth factors were examined. Kidneys of GT1S mice overexpressed GLUT1 in glomerular MCs and small vessels, rather than renal tubules. GT1S mice were neither diabetic nor hypertensive. Glomerular GLUT1, glucose uptake, mean capillary diameter, and mean glomerular volume were all increased in the GT1S mice. Moderately severe glomerulosclerosis (GS) was established by 26 wk of age in GT1S mice, with increased glomerular type IV collagen and fibronectin. Modest increases in glomerular basement membrane thickness and albuminuria were detected with podocyte foot processes largely preserved, in the absence of podocyte GLUT1 overexpression. Activation of glomerular PKC, along with increased transforming growth factor-beta1, VEGFR1, VEGFR2, and VEGF were all detected in glomeruli of GT1S mice, likely contributing to GS. The transcription factor NF-kappaB was also activated. Overexpression of glomerular GLUT1, mimicking the diabetic GLUT1 response, produced numerous features typical of diabetic glomerular disease, without diabetes or hypertension. This suggested GLUT1 may play an important role in the development of diabetic GS.