Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease.

Antiretroviral therapy (ART) has profoundly decreased HIV-1 associated morbidity. However, despite ART, immune cells remain latently infected and slowly release viral proteins, leading to chronic inflammation and HIV associated comorbidities. Thus, new strategies are needed to reduce the inflammatory effects of HIV-1. In previous studies we found that gamma secretase inhibitor (GSIXX) ameliorated renal lesions of HIV-Tg26 mice carrying replication defective HIV-1 PNL4-3 by inhibiting Notch activation. Since gamma secretase inhibition is not a safe strategy in humans, here we examined the specific role of the Notch3 pathway in the pathogenesis of the renal lesions and outcome of HIV-Tg26 mice. We found that Notch3 is activated in podocytes and other renal cells in HIV-Tg26 mice and human biopsies with HIV-1 associated Nephropathy (HIVAN). Knockdown of Notch3 in HIV-Tg26 mice revealed a marked reduction in the mortality rate, improvement in renal injury and function. RNA sequencing and immunolabeling data revealed that Notch3 deletion drastically reduced infiltrating renal macrophages in HIV-Tg-N3KO mice in association with renal reduction of HIV-nef mRNA expression levels. In fact, bone marrow derived macrophages from HIV-Tg26 mice showed a significant activation of Notch3 signaling. Further, systemic levels of TNF-alpha and MCP-1 and other inflammatory chemokines and cytokines were reduced in Tg-N3KO mice as compared to HIV-Tg26 mice and this translated to a marked reduction of HIV-induced skin lesions. Taken together, these studies strongly point to a dual inhibitory/therapeutic effect of Notch3 inhibition on HIV-induced systemic, skin and renal lesions independently of ART.

Polycystin-1 Interacting Protein-1 (CU062) Interacts with the Ectodomain of Polycystin-1 (PC1).

The PKD1 gene, encoding protein polycystin-1 (PC1), is responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 has been shown to be present in urinary exosome-like vesicles (PKD-ELVs) and lowered in individuals with germline PKD1 mutations. A label-free mass spectrometry comparison of urinary PKD-ELVs from normal individuals and those with PKD1 mutations showed that several proteins were reduced to a degree that matched the decrease observed in PC1 levels. Some of these proteins, such as polycystin-2 (PC2), may be present in a higher-order multi-protein assembly with PC1-the polycystin complex (PCC). CU062 (Q9NYP8) is decreased in ADPKD PKD-ELVs and, thus, is a candidate PCC component. CU062 is a small glycoprotein with a signal peptide but no transmembrane domain and can oligomerize with itself and interact with PC1. We investigated the localization of CU062 together with PC1 and PC2 using immunofluorescence (IF). In nonconfluent cells, all three proteins were localized in close proximity to focal adhesions (FAs), retraction fibers (RFs), and RF-associated extracellular vesicles (migrasomes). In confluent cells, primary cilia had PC1/PC2/CU062 + extracellular vesicles adherent to their plasma membrane. In cells exposed to mitochondrion-decoupling agents, we detected the development of novel PC1/CU062 + ring-like structures that entrained swollen mitochondria. In contact-inhibited cells under mitochondrial stress, PC1, PC2, and CU062 were observed on large, apically budding extracellular vesicles, where the proteins formed a reticular network on the membrane. CU062 interacts with PC1 and may have a role in the identification of senescent mitochondria and their extrusion in extracellular vesicles.

Myofibroblast depletion reduces kidney cyst growth and fibrosis in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) involves the development and persistent growth of fluid filled kidney cysts. In a recent study, we showed that ADPKD kidney cyst epithelial cells can stimulate the proliferation and differentiation of peri-cystic myofibroblasts. Although dense myofibroblast populations are often found surrounding kidney cysts, their role in cyst enlargement or fibrosis in ADPKD is unclear. To clarify this, we examined the effect of myofibroblast depletion in the Pkd1RC/RC (RC/RC) mouse model of ADPKD. RC/RC;αSMAtk mice that use the ganciclovir-thymidine kinase system to selectively deplete α-smooth muscle actin expressing myofibroblasts were generated. Ganciclovir treatment for four weeks depleted myofibroblasts, reduced kidney fibrosis and preserved kidney function in these mice. Importantly, myofibroblast depletion significantly reduced cyst growth and cyst epithelial cell proliferation in RC/RC;αSMAtk mouse kidneys. Similar ganciclovir treatment did not alter cyst growth or fibrosis in wild-type or RC/RC littermates. In vitro, co-culture with myofibroblasts from the kidneys of patients with ADPKD increased 3D microcyst growth of human ADPKD cyst epithelial cells. Treatment with conditioned culture media from ADPKD kidney myofibroblasts increased microcyst growth and cell proliferation of ADPKD cyst epithelial cells. Further examination of ADPKD myofibroblast conditioned media showed high levels of protease inhibitors including PAI1, TIMP1 and 2, NGAL and TFPI-2, and treatment with recombinant PAI1 and TIMP1 increased ADPKD cyst epithelial cell proliferation in vitro. Thus, our findings show that myofibroblasts directly promote cyst epithelial cell proliferation, cyst growth and fibrosis in ADPKD kidneys, and their targeting could be a novel therapeutic strategy to treat PKD.

Caspase-1 and the inflammasome promote polycystic kidney disease progression.

We and others have previously shown that the presence of renal innate immune cells can promote polycystic kidney disease (PKD) progression. In this study, we examined the influence of the inflammasome, a key part of the innate immune system, on PKD. The inflammasome is a system of molecular sensors, receptors, and scaffolds that responds to stimuli like cellular damage or microbes by activating Caspase-1, and generating critical mediators of the inflammatory milieu, including IL-1ß and IL-18. We provide evidence that the inflammasome is primed in PKD, as multiple inflammasome sensors were upregulated in cystic kidneys from human ADPKD patients, as well as in kidneys from both orthologous (PKD1 RC/RC or RC/RC) and non-orthologous (jck) mouse models of PKD. Further, we demonstrate that the inflammasome is activated in female RC/RC mice kidneys, and this activation occurs in renal leukocytes, primarily in CD11c+ cells. Knock-out of Casp1, the gene encoding Caspase-1, in the RC/RC mice significantly restrained cystic disease progression in female mice, implying sex-specific differences in the renal immune environment. RNAseq analysis implicated the promotion of MYC/YAP pathways as a mechanism underlying the pro-cystic effects of the Caspase-1/inflammasome in females. Finally, treatment of RC/RC mice with hydroxychloroquine, a widely used immunomodulatory drug that has been shown to inhibit the inflammasome, protected renal function specifically in females and restrained cyst enlargement in both male and female RC/RC mice. Collectively, these results provide evidence for the first time that the activated Caspase-1/inflammasome promotes cyst expansion and disease progression in PKD, particularly in females. Moreover, the data suggest that this innate immune pathway may be a relevant target for therapy in PKD.

Critical Role of Osteopontin in Maintaining Urinary Phosphate Solubility in CKD.

Background: Nephron loss dramatically increases tubular phosphate to concentrations that exceed supersaturation. Osteopontin (OPN) is a matricellular protein that enhances mineral solubility in solution; however, the role of OPN in maintaining urinary phosphate solubility in CKD remains undefined. Methods: Here, we examined (1) the expression patterns and timing of kidney/urine OPN changes in CKD mice, (2) if tubular injury is necessary for kidney OPN expression in CKD, (3) how OPN deletion alters kidney mineral deposition in CKD mice, (4) how neutralization of the mineral-binding (ASARM) motif of OPN alters kidney mineral deposition in phosphaturic mice, and (5) the in vitro effect of phosphate-based nanocrystals on tubular epithelial cell OPN expression. Results: Tubular OPN expression was dramatically increased in all studied CKD murine models. Kidney OPN gene expression and urinary OPN/Cr ratios increased before changes in traditional biochemical markers of kidney function. Moreover, a reduction of nephron numbers alone (by unilateral nephrectomy) was sufficient to induce OPN expression in residual nephrons and induction of CKD in OPN-null mice fed excess phosphate resulted in severe nephrocalcinosis. Neutralization of the ASARM motif of OPN in phosphaturic mice resulted in severe nephrocalcinosis that mimicked OPN-null CKD mice. Lastly, in vitro experiments revealed calcium-phosphate nanocrystals to induce OPN expression by tubular epithelial cells directly. Conclusions: Kidney OPN expression increases in early CKD and serves a critical role in maintaining tubular mineral solubility when tubular phosphate concentrations are exceedingly high, as in late-stage CKD. Calcium-phosphate nanocrystals may be a proximal stimulus for tubular OPN production.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked ß-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

Expression of active B-Raf proto-oncogene in kidney collecting ducts induces cyst formation in normal mice and accelerates cyst growth in mice with polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by the formation and progressive enlargement of fluid-filled cysts due to abnormal cell proliferation. Cyclic AMP agonists, including arginine vasopressin, stimulate ERK-dependent proliferation of cystic cells, but not normal kidney cells. Previously, B-Raf proto-oncogene (BRAF), a MAPK kinase kinase that activates MEK-ERK signaling, was shown to be a central intermediate in the cAMP mitogenic response. However, the role of BRAF on cyst formation and enlargement in vivo had not been demonstrated. To determine if active BRAF induces kidney cyst formation, we generated transgenic mice that conditionally express BRAFV600E, a common activating mutation, and bred them with Pkhd1-Cre mice to express active BRAF in the collecting ducts, a predominant site for cyst formation. Collecting duct expression of BRAFV600E (BRafCD) caused kidney cyst formation as early as three weeks of age. There were increased levels of phosphorylated ERK (p-ERK) and proliferating cell nuclear antigen, a marker for cell proliferation. BRafCD mice developed extensive kidney fibrosis and elevated blood urea nitrogen, indicating a decline in kidney function, by ten weeks of age. BRAFV600E transgenic mice were also bred to Pkd1RC/RC and pcy/pcy mice, well-characterized slowly progressive PKD models. Collecting duct expression of active BRAF markedly increased kidney weight/body weight, cyst number and size, and total cystic area. There were increased p-ERK levels and proliferating cells, immune cell infiltration, interstitial fibrosis, and a decline in kidney function in both these models. Thus, our findings demonstrate that active BRAF is sufficient to induce kidney cyst formation in normal mice and accelerate cystic disease in PKD mice.

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin.

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts.

DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.

The tyrosine-kinase inhibitor Nintedanib ameliorates autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1f/fPkhd1Cre and Pkd1RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1f/fPkhd1Cre and Pkd1RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1RC/RC mice, but did not affect renal fibrosis in Pkd1f/fPkhd1Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.

Posttransplant proteinuria due to Apolipoprotein E2 deposition in a kidney allograft.

Lipoprotein deposition disorders limited to the kidney and causing proteinuria are rare. We present a case of nephrotic range proteinuria presenting within 4 months after deceased donor renal transplantation in a patient with end-stage kidney disease presumed secondary to hypertension. Two transplant kidney biopsies were performed sixteen weeks after transplantation, and one year after the first biopsy, both showing lipoprotein deposits in the glomeruli, progressive focal segmental glomerulosclerosis, and effacement of visceral foot processes. The patient had a normal lipid profile. Based on previous case reports of Apolipoprotein E variants causing proteinuria in native kidneys, Apolipoprotein E genotyping was performed. Genotyping showed Apolipoprotein E2 homozygosity. This Apolipoprotein E variant has been associated with lipoprotein deposition, proteinuria, and progressive kidney disease in the native kidneys. However, this is the first case of Apolipoprotein E2 homozygosity-related kidney disease in a transplant recipient. The patient was treated with fenofibrate, angiotensin enzyme inhibition, and angiotensin receptor blockade with reduction in proteinuria, and he kept good stable kidney function.

Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

Glycogen synthase kinase-3ß inhibits tubular regeneration in acute kidney injury by a FoxM1-dependent mechanism.

Acute kidney injury (AKI) is characterized by injury to the tubular epithelium that leads to the sudden loss of renal function. Proper tubular regeneration is essential to prevent progression to chronic kidney disease. In this study, we examined the role of FoxM1, a forkhead box family member transcription factor in tubular repair after AKI. Renal FoxM1 expression increased after renal ischemia/reperfusion (I/R)-induced AKI in mouse kidneys. Treatment with thiostrepton, a FoxM1 inhibitor, reduced FoxM1 regulated pro-proliferative factors and cell proliferation in vitro, and tubular regeneration in mouse kidneys after AKI. Glycogen synthase kinase-3 (GSK3) was found to be an upstream regulator of FoxM1 because GSK3 inhibition or renal tubular GSK3ß gene deletion significantly increased FoxM1 expression, and improved tubular repair and renal function. GSK3 inactivation increased ß-catenin, Cyclin D1, and c-Myc, and reduced cell cycle inhibitors p21 and p27. Importantly, thiostrepton treatment abolished the improved tubular repair in GSK3ß knockout mice following AKI. These results demonstrate that FoxM1 is important for renal tubular regeneration following AKI and that GSK3ß suppresses tubular repair by inhibiting FoxM1.

Epithelial Vasopressin Type-2 Receptors Regulate Myofibroblasts by a YAP-CCN2-Dependent Mechanism in Polycystic Kidney Disease.

BACKGROUND: Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys. METHODS: We treated Pkd1 gene knockout (Pkd1KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated Pkd1KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP). RESULTS: V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation in vitro, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of CCN2 significantly reduced cyst growth and myofibroblasts in Pkd1KO mouse kidneys. We found that YAP regulates CCN2, and YAP inhibition or gene deletion reduces renal fibrosis in Pkd1KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in Pkd1KO kidneys. Further in vitro studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells. CONCLUSIONS: Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.

BCL9/STAT3 regulation of transcriptional enhancer networks promote DCIS progression.

The molecular processes by which some human ductal carcinoma in situ (DCIS) lesions advance to the more aggressive form, while others remain indolent, are largely unknown. Experiments utilizing a patient-derived (PDX) DCIS Mouse INtraDuctal (MIND) animal model combined with ChIP-exo and RNA sequencing revealed that the formation of protein complexes between B Cell Lymphoma-9 (BCL9), phosphoserine 727 STAT3 (PS-727-STAT3) and non-STAT3 transcription factors on chromatin enhancers lead to subsequent transcription of key drivers of DCIS malignancy. Downregulation of two such targets, integrin ß3 and its associated metalloproteinase, MMP16, resulted in a significant inhibition of DCIS invasive progression. Finally, in vivo targeting of BCL9, using rosemary extract, resulted in significant inhibition of DCIS malignancy in both cell line and PDX DCIS MIND animal models. As such, our studies provide compelling evidence for future testing of rosemary extract as a chemopreventive agent in breast cancer.

Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Inhibition of Endothelial PHD2 Suppresses Post-Ischemic Kidney Inflammation through Hypoxia-Inducible Factor-1.

BACKGROUND: Prolyl-4-hydroxylase domain-containing proteins 1-3 (PHD1 to PHD3) regulate the activity of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2, transcription factors that are key regulators of hypoxic vascular responses. We previously reported that deficiency of endothelial HIF-2 exacerbated renal ischemia-reperfusion injury, whereas inactivation of endothelial PHD2, the main oxygen sensor, provided renoprotection. Nevertheless, the molecular mechanisms by which endothelial PHD2 dictates AKI outcomes remain undefined. METHODS: To investigate the function of the endothelial PHD2/HIF axis in ischemic AKI, we examined the effects of endothelial-specific ablation of PHD2 in a mouse model of renal ischemia-reperfusion injury. We also interrogated the contribution of each HIF isoform by concurrent endothelial deletion of both PHD2 and HIF-1 or both PHD2 and HIF-2. RESULTS: Endothelial deletion of Phd2 preserved kidney function and limited transition to CKD. Mechanistically, we found that endothelial Phd2 ablation protected against renal ischemia-reperfusion injury by suppressing the expression of proinflammatory genes and recruitment of inflammatory cells in a manner that was dependent on HIF-1 but not HIF-2. Persistence of renoprotective responses after acute inducible endothelial-specific loss of Phd2 in adult mice ruled out a requirement for PHD2 signaling in hematopoietic cells. Although Phd2 inhibition was not sufficient to induce detectable HIF activity in the kidney endothelium, in vitro experiments implicated a humoral factor in the anti-inflammatory effects generated by endothelial PHD2/HIF-1 signaling. CONCLUSIONS: Our findings suggest that activation of endothelial HIF-1 signaling through PHD2 inhibition may offer a novel therapeutic approach against ischemic AKI.