Claudia A. Bertuccio

Overexpression of VEGF-A in podocytes of adult mice causes glomerular disease

We sought to examine the pathogenic role of excessive VEGF-A expression in podocytes, since it has been reported that diabetic nephropathy and other glomerular diseases are associated with increased VEGF-A expression. The induction of podocyte-specific VEGF164 overexpression in adult transgenic mice led to proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, loss of slit diaphragms, and podocyte effacement. When doxycycline-mediated VEGF164 was stopped, these abnormalities reversed. These findings were associated with reversible downregulation of metalloproteinase 9 and nephrin expression. Using transmission electron microscopy, we established that VEGF-A receptor-2 (VEGFR2) was expressed in podocytes and glomerular endothelial cells. We also found that VEGF164 induced VEGFR2 phosphorylation in podocytes. Further, we were able to co-immunoprecipitate VEGFR2 and nephrin using whole kidney lysates, confirming interaction in vivo. This implies that autocrine and paracrine VEGF-A signaling through VEGFR2 occurs in podocytes and may mediate the glomerular phenotype caused by VEGF164 overexpression. Thus, we suggest that podocyte VEGF164 overexpression in adult mice is sufficient to induce glomerular filtration barrier structural and functional abnormalities similar to those present in murine diabetic nephropathy.

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes

Background: Excess VEGF-A down-regulates nephrin causing glomerular disease. Nephrin interacts with VEGFR2 in vivo. Results: Nephrin-VEGFR2 interaction is direct, modulated by tyrosine phosphorylation, the VEGR2-nephrin complex involves Nck and actin, and VEGF-A signaling via this complex decreases cell size. Conclusion: This interaction links extracellular VEGF-A to slit diaphragms and the podocyte actin cytoskeleton. Significance: Our findings provide a molecular mechanism for VEGF-induced glomerular disease. The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

Acute Podocyte Vascular Endothelial Growth Factor (VEGF-A) Knockdown Disrupts alphaVbeta3 Integrin Signaling in the Glomerulus

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGFKD) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ∼20% of non-induced controls and urine VEGF-A to ∼30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alphaVbeta3 integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in VEGFKD podocytes. Podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGFKD podocytes downregulates fibronectin and disrupts alphaVbeta3 integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alphaVbeta3 integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alphaVbeta3 integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.

Induction of Podocyte VEGF164 Overexpression at Different Stages of Development Causes Congenital Nephrosis or Steroid-Resistant Nephrotic Syndrome

The tight regulation of vascular endothelial growth factor-A (VEGF-A) signaling is required for both the development and maintenance of the glomerular filtration barrier, but the pathogenic role of excessive amounts of VEGF-A detected in multiple renal diseases remains poorly defined. We generated inducible transgenic mice that overexpress podocyte VEGF164 at any chosen stage of development. In this study, we report the phenotypes that result from podocyte VEGF164 excess during organogenesis and after birth. On doxycycline induction, podocin-rtTA:tet-O-VEGF164 mice express twofold higher kidney VEGF164 levels than single transgenic mice, localized to podocytes. Podocyte VEGF164 overexpression during organogenesis resulted in albuminuria at birth and was associated with glomerulomegaly, uniform podocyte effacement, very few and wide foot processes joined by occluding junctions, almost complete absence of slit diaphragms, and swollen endothelial cells with few fenestrae as revealed by transmission electron microscopy. Podocyte VEGF164 overexpression after birth caused massive albuminuria in 70% of 2-week-old mice, glomerulomegaly, and minimal changes on light microscopy. Transmission electron microscopy showed podocyte effacement and fusion and morphologically normal endothelial cells. Podocyte VEGF164 overexpression induced nephrin down-regulation without podocyte loss. VEGF164-induced abnormalities were reversible on removal of doxycycline and were unresponsive to methylprednisolone. Collectively, the data suggest that moderate podocyte VEGF164 overexpression during organogenesis results in congenital nephrotic syndrome, whereas VEGF164 overexpression after birth induces a steroid-resistant minimal change like-disease in mice.

Polycystin-1 C-terminal cleavage is modulated by polycystin-2 expression.

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC-1) and polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway.

Drug Authorization for Sofosbuvir/Ledipasvir (Harvoni) for Chronic HCV Infection in a Real-World Cohort: A New Barrier in the HCV Care Cascade

Background New treatments for hepatitis C (HCV) infection hold great promise for cure, but numerous challenges to diagnosing, establishing care, and receiving therapy exist. There are limited data on insurance authorization for these medications. Materials and Methods We performed a retrospective chart review of patients receiving sofosbuvir/ledipasvir (SOF/LED) from October 11-December 31, 2014 to determine rates and timing of drug authorization. We also determined predictors of approval, and those factors associated with faster decision and approval times. Results Of 174 patients prescribed HCV therapy during this period, 129 requests were made for SOF/LED, of whom 100 (77.5%) received initial approval, and an additional 17 patients (13.9%) ultimately received approval through the appeals process. Faster approval times were seen in patients with Child-Pugh Class B disease (14.4 vs. 24.7 days, p = 0.048). A higher proportion of patients were initially approved in those with Medicare/Medicaid coverage (92.2% vs. 71.4%, p = 0.002) and those with baseline viral load ≥6 million IU/mL (84.1% vs. 62.5%, p = 0.040). Linear regression modeling identified advanced fibrosis, high Model of End Stage Liver Disease (MELD) score, and female gender as significant predictors of shorter decision and approval times. On logistic regression, Medicare/Medicaid coverage (OR 5.96, 95% CI 1.66–21.48) and high viral load (OR 4.52, 95% CI 1.08–19.08) were significant predictors for initial approval. Conclusions Early analysis of real-world drug authorization outcomes between October-December 2014 reveals that nearly one in four patients are initially denied access to SOF/LED upon initial prescription, although most patients are eventually approved through appeal, which delays treatment initiation. Having Medicare/Medicaid and advanced liver disease resulted in a higher likelihood of approval as well as earlier decision and approval times. More studies are needed to determine factors resulting in higher likelihood of denial and to evaluate approval rates and times after implementation of restrictive prior authorization guidelines.

Anterograde Trafficking of KCa3.1 in Polarized Epithelia Is Rab1- and Rab8-Dependent and Recycling Endosome-Independent

The intermediate conductance, Ca2+-activated K+ channel (KCa3.1) targets to the basolateral (BL) membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP)-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia cells.

Polycystin-1 cleavage and the regulation of transcriptional pathways

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease, affecting approximately 1 in 1,000 people. The disease is characterized by the development of numerous large fluid-filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments which manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein.

Relevance of VEGF and nephrin expression in glomerular diseases.

The glomerular filtration barrier is affected in a large number of acquired and inherited diseases resulting in extensive leakage of plasma albumin and larger proteins, leading to nephrotic syndrome and end-stage renal disease. Unfortunately, the molecular mechanisms governing the development of the nephrotic syndrome remain poorly understood. Here, I give an overview of recent investigations that have focused on characterizing the interrelationships between the slit diaphragm components and podocytes-secreted VEGF, which have a significant role for maintaining the normal podocyte structure and the integrity of the filtering barrier.

Dynamin- and Rab5-Dependent Endocytosis of a Ca2+-Activated K+ Channel, KCa2.3

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+-activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.