Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

Trabecular architecture of the distal femur in extant hominids.

Extant great apes are characterized by a wide range of locomotor, postural and manipulative behaviours that each require the limbs to be used in different ways. In addition to external bone morphology, comparative investigation of trabecular bone, which (re-)models to reflect loads incurred during life, can provide novel insights into bone functional adaptation. Here, we use canonical holistic morphometric analysis (cHMA) to analyse the trabecular morphology in the distal femoral epiphysis of Homo sapiens (n = 26), Gorilla gorilla (n = 14), Pan troglodytes (n = 15) and Pongo sp. (n = 9). We test two predictions: (1) that differing locomotor behaviours will be reflected in differing trabecular architecture of the distal femur across Homo, Pan, Gorilla and Pongo; (2) that trabecular architecture will significantly differ between male and female Gorilla due to their different levels of arboreality but not between male and female Pan or Homo based on previous studies of locomotor behaviours. Results indicate that trabecular architecture differs among extant great apes based on their locomotor repertoires. The relative bone volume and degree of anisotropy patterns found reflect habitual use of extended knee postures during bipedalism in Homo, and habitual use of flexed knee posture during terrestrial and arboreal locomotion in Pan and Gorilla. Trabecular architecture in Pongo is consistent with a highly mobile knee joint that may vary in posture from extension to full flexion. Within Gorilla, trabecular architecture suggests a different loading of knee in extension/flexion between females and males, but no sex differences were found in Pan or Homo, supporting our predictions. Inter- and intra-specific variation in trabecular architecture of distal femur provides a comparative context to interpret knee postures and, in turn, locomotor behaviours in fossil hominins.

Immunosuppressive calcineurin inhibitor cyclosporine A induces proapoptotic endoplasmic reticulum stress in renal tubular cells.

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA-like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA-like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.

Transgenic angiotensin-converting enzyme 2 overexpression in the rat vasculature protects kidneys from ageing-induced injury.

Chronic kidney disease is one of the leading causes of morbidity and mortality especially among the aged population. A decline in kidney function with ageing comparable to ageing-related processes in human kidneys has also been described in Sprague-Dawley (SD) rats. The renin-angiotensin-system (RAS) plays a pivotal role in the pathophysiology of cardiovascular and kidney disease and is a successful therapeutic target. The discovery of angiotensin-(1-7) (Ang(1-7)), mainly produced by angiotensin-converting enzyme 2 (ACE2), and its receptor MAS offered a new view on the RAS. This ACE2/Ang(1-7)/MAS axis counteracts most deleterious actions of the RAS in the kidney. In order to evaluate if activation of this axis has a protective effect in ageing-induced kidney disease we generated a transgenic rat model (TGR(SM22hACE2)) overexpressing human ACE2 in vascular smooth muscle cells. These animals showed a specific transgene expression pattern and increased ACE2 activity in the kidney. Telemetric recording of cardiovascular parameters and evaluation of kidney function by histology and urine analysis revealed no alterations in blood pressure regulation and basal kidney function in young transgenic rats when compared to young SD rats. However, with ageing, SD rats developed a decline in kidney function characterized by severe albuminuria which was significantly less pronounced in TGR(SM22hACE2) rats. Concomitantly, we detected lower mRNA expression levels of kidney damage markers in aged transgenic animals. Thus, our results indicate that vascular ACE2-overexpression protects the kidney against ageing-induced decline in kidney function, supporting the kidney-protective role of the ACE2/Ang(1-7)/MAS axis.

LRP2 contributes to planar cell polarity-dependent coordination of motile cilia function.

Motile cilia are protruding organelles on specialized epithelia that beat in a synchronous fashion to propel extracellular fluids. Coordination and orientation of cilia beating on individual cells and across tissues is a complex process dependent on planar cell polarity (PCP) signaling. Asymmetric sorting of PCP pathway components, essential to establish planar polarity, involves trafficking along the endocytic path, but the underlying regulatory processes remain incompletely understood. Here, we identified the endocytic receptor LRP2 as regulator of PCP component trafficking in ependyma, a multi-ciliated cell type that is involved in facilitating flow of the cerebrospinal fluid in the brain ventricular system. Lack of receptor expression in gene-targeted mice results in a failure to sort PCP core proteins to the anterior or posterior cell side and, consequently, in the inability to coordinate cilia arrangement and to aligned beating (loss of rotational and translational polarity). LRP2 deficiency coincides with a failure to sort NHERF1, a cytoplasmic LRP2 adaptor to the anterior cell side. As NHERF1 is essential to translocate PCP core protein Vangl2 to the plasma membrane, these data suggest a molecular mechanism whereby LRP2 interacts with PCP components through NHERF1 to control their asymmetric sorting along the endocytic path. Taken together, our findings identified the endocytic receptor LRP2 as a novel regulator of endosomal trafficking of PCP proteins, ensuring their asymmetric partition and establishment of translational and rotational planar cell polarity in the ependyma.

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

BACKGROUND: Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known. METHODS: Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of Lrrc8b-e, or with inducible or cell-specific ablation of Lrrc8a, were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics. RESULTS: The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms. CONCLUSIONS: VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments despite their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.

Connexin43 is differentially distributed within renal vasculature and mediates profibrotic differentiation in medullary fibroblasts.

Connexins (Cxs) form gap junctions for intercellular exchange of inorganic ions and messenger molecules. In the kidney, Cxs play essential roles within its compartments, but data on the precise cellular localization and cell type-related function of their isoforms are scarce. We tested whether Cx43 distribution is restricted to vascular and interstitial cells and whether medullary fibroblasts express Cx43 to coordinate profibrotic signaling. Confocal immunofluorescence techniques, ultrastructural labeling, and functional experiments in cell culture were performed. Cx43 was chiefly expressed in the vasculature but was absent from tubular epithelia. All arterial, arteriolar, and lymphatic endothelia showed continuous Cx43 signal along their borders. In the inner medulla, only the interstitium showed Cx43 signals, which were assigned to fibroblasts and their processes. Cultured Cx43-expressing medullary fibroblasts served to study the role of gap junctions in a profibrotic context. In a dye spreading assay, Cx43-sensitive diffusion of Lucifer yellow was dependent on gap junctional passage. The addition of transforming growth factor-ß1 (5 ng/mL for 48 h) activated Cx43 biosynthesis and caused Cx43-sensitive transformation of the fibroblasts into a myofibroblast phenotype. This suggested that Cx43 gap junctional channels enable the coordination of profibrotic signaling between cells of the medullary interstitium. In summary, we demonstrate the presence of Cx43-expressing gap junctions within the two major renal compartments, the vasculature and interstitium. Endothelial Cx43 likely provides functions of an earlier-defined "electrical syncytium" within the vascular wall. Additionally, Cx43 facilitates profibrotic signaling between medullary interstitial fibroblasts.

Neutrophil Gelatinase-Associated Lipocalin Protects from ANCA-Induced GN by Inhibiting TH17 Immunity.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown. METHODS: We measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of TH17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice. RESULTS: Mice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating TH17 cells. NGAL-expressing neutrophils and CD3+ T cells were in close proximity in kidney and spleen. CD4+ T cells showed no intrinsic difference in proliferation and polarization in vitro, whereas iron siderophore-loaded NGAL suppressed TH17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras. CONCLUSIONS: Our findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating TH17 immunity.

Preparation of Samples for Large-Scale Automated Electron Microscopy of Tissue and Cell Ultrastructure.

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time-consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by the automated acquisition of virtually all structures in a given EM section. Resulting datasets allow the convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We, therefore, established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared with other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.

WNK bodies cluster WNK4 and SPAK/OSR1 to promote NCC activation in hypokalemia.

K+ deficiency stimulates renal salt reuptake via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K+ losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K+ deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed "WNK bodies," the significance of which is undetermined. By feeding diets of varying salt and K+ content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K+ deprivation, and it was promptly suppressed by K+ loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K+ channel-forming protein Kir4.1, which is required for the DCT to sense plasma K+ concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K+ deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K+ concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.

Vasopressin Increases Urinary Acidification via V1a Receptors in Collecting Duct Intercalated Cells.

BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.

Patients with hypokalemia develop WNK bodies in the distal convoluted tubule of the kidney.

Hypokalemia contributes to the progression of chronic kidney disease, although a definitive pathophysiological theory to explain this remains to be established. K+ deficiency results in profound alterations in renal epithelial transport. These include an increase in salt reabsorption via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes electroneutral K+ loss in downstream nephron segments. In experimental conditions of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases have been discovered in the murine DCT and termed "WNK bodies," referring to their component, with no K (lysine) kinases (WNKs). We hypothesized that in humans, WNK bodies occur in hypokalemia as well. Renal needle biopsies of patients with chronic hypokalemic nephropathy and appropriate controls were examined by histological stains and immunofluorescence. Segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely, WNKs and Ste20-related proline-alanine-rich kinase (SPAK). In both patients with hypokalemia, WNKs and SPAK concentrated in non-membrane-bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region close to, but not within, the endoplasmic reticulum. They were closely adjacent to microtubules but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients.

Hyperkalemia and blood pressure regulation.

Hypertension is common in the general population. Management of hypertensive patients at risk of hyperkalemia is challenging due to potential life-threatening complications such as cardiac arrest. Chronic hyperkalemia is often associated with impaired renal ability to excrete excessive potassium ions (K+). This may refer to chronic kidney disease or certain pharmacological interventions, including broadly used renin-angiotensin-aldosterone system and calcineurin inhibitors. Understanding the intrinsic mechanisms permitting kidney adaptations to hyperkalemia is critical for choosing therapeutic strategies. Valuable insights were obtained from the analysis of familial hyperkalemic hypertension (FHHt) syndrome, which became a classic model for coincidence of high blood pressure and hyperkalemia. FHHt can be caused by mutations in several genes, all of them resulting in excessive activity of with-no-lysine kinases (WNKs) in the distal nephron of the kidney. WNKs have been increasingly recognized as key signalling enzymes in the regulation of renal sodium ions (Na+) and K+ handling, enabling adaptive responses to systemic shifts of potassium homoeostasis consequent to variations in dietary potassium intake or disease. The WNK signalling pathway recruits a complex protein network mediating catalytic and non-catalytic effects of distinct WNK isoforms on relevant Na+- or K+-transporting proteins. In this review article, we summarize recent progress in understanding WNK signalling. An update of available models for renal adaptation to hyperkalemic conditions is presented. Consequences for blood pressure regulation are discussed. Pharmacological targeting of WNKs or their substrates offers promising options to manage hypertension while preventing hyperkalemia.

GRHL2 Is Required for Collecting Duct Epithelial Barrier Function and Renal Osmoregulation.

Collecting ducts make up the distal-most tubular segments of the kidney, extending from the cortex, where they connect to the nephron proper, into the medulla, where they release urine into the renal pelvis. During water deprivation, body water preservation is ensured by the selective transepithelial reabsorption of water into the hypertonic medullary interstitium mediated by collecting ducts. The collecting duct epithelium forms tight junctions composed of barrier-enforcing claudins and exhibits a higher transepithelial resistance than other segments of the renal tubule exhibit. However, the functional relevance of this strong collecting duct epithelial barrier is unresolved. Here, we report that collecting duct-specific deletion of an epithelial transcription factor, grainyhead-like 2 (GRHL2), in mice led to reduced expression of tight junction-associated barrier components, reduced collecting duct transepithelial resistance, and defective renal medullary accumulation of sodium and other osmolytes. In vitro, Grhl2-deficient collecting duct cells displayed increased paracellular flux of sodium, chloride, and urea. Consistent with these effects, Grhl2-deficient mice had diabetes insipidus, produced dilute urine, and failed to adequately concentrate their urine after water restriction, resulting in susceptibility to prerenal azotemia. These data indicate a direct functional link between collecting duct epithelial barrier characteristics, which appear to prevent leakage of interstitial osmolytes into urine, and body water homeostasis.

A novel role for Tamm-Horsfall protein (uromodulin) in the renal tubule.

Tamm-Horsfall protein (THP)/uromodulin, the most abundant urinary protein, is produced in the thick ascending limb of the loop of Henle. Besides immunological functions, it has regulatory impact on ion transport and volume regulation. It had been debated whether the distal convoluted tubule is a source for THP as well. Novel results suggest that in its early portions, THP synthesis affects adaptive plasticity of the epithelium, stabilizes calcium homeostasis, and activates the thiazide-sensitive Na+,Cl--cotransporter.

Modern field emission scanning electron microscopy provides new perspectives for imaging kidney ultrastructure.

Recent progress in electron microscopy (EM) techniques has opened new pathways to study renal tissue in research and pathology. Modern field emission scanning EM may be utilized to scan thin sections of resin-embedded tissue mounted on a conductive support. Here we sought to achieve automated imaging without the typical limitations of transmission EM with equivalent or superior quality. Extended areas of tissue were either imaged in two (nanotomy) or in three dimensions (volume EM) by serial-section-based array tomography. Single-beam and fast-recording multi-beam field emission scanning EM instruments were compared using perfusion-fixed rodent kidneys. High-resolution scans produced excellent images of tissue, cells, and organelles down to macromolecular complexes. Digital stitching of image tiles in both modes allowed seamless Google Earth-like zooming from overview to regions of interest at the nanoscale. Large datasets were created that can be rapidly shared between scientists of different disciplines or pathologists using open source software. Three-dimensional array tomography of thin sections was followed by segmentation to visualize selected features in a large volume. Furthermore, correlative light-EM enabled the identification of functional information in a structural context. Thus, limitations in biomedical transmission EM can be overcome by introducing field emission scanning EM-based technology that permits high-quality, large field-of-view nanotomy, volume EM, and correlative light-EM modes. Advantages of virtual microscopy in clinical and experimental nephrology are illustrated.

Inverse correlation between vascular endothelial growth factor back-filtration and capillary filtration pressures.

Background: Vascular endothelial growth factor A (VEGF) is an essential growth factor during glomerular development and postnatal homeostasis. VEGF is secreted in high amounts by podocytes into the primary urine, back-filtered across the glomerular capillary wall to act on endothelial cells. So far it has been assumed that VEGF back-filtration is driven at a constant rate exclusively by diffusion. Methods: In the present work, glomerular VEGF back-filtration was investigated in vivo using a novel extended model based on endothelial fenestrations as surrogate marker for local VEGF concentrations. Single nephron glomerular filtration rate (SNGFR) and/or local filtration flux were manipulated by partial renal mass ablation, tubular ablation, and in transgenic mouse models of systemic or podocytic VEGF overexpression or reduction. Results: Our study shows positive correlations between VEGF back-filtration and SNGFR as well as effective filtration rate under physiological conditions along individual glomerular capillaries in rodents and humans. Conclusion: Our results suggest that an additional force drives VEGF back-filtration, potentially regulated by SNGFR.

The A-kinase Anchoring Protein GSKIP Regulates GSK3ß Activity and Controls Palatal Shelf Fusion in Mice.

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3ß (GSK3ß) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3ß. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3ß at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3ß activity. At embryonic day 18.5, GSK3ß activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3ß signaling in palatal shelf fusion.

Regulation of renal Na-(K)-Cl cotransporters by vasopressin.

Vasopressin (AVP) induces antidiuresis, thus playing an essential role in body water and electrolyte homeostasis. Its antidiuretic effects are mediated chiefly by V2 vasopressin receptors (V2R) expressed along the distal nephron and collecting duct epithelia. NaCl reabsorption in the distal nephron, which includes the thick ascending limb (TAL) and distal convoluted tubule (DCT), largely depends on the activity of two structurally related Na-(K)-Cl cotransporters, NKCC2 in TAL and NCC in DCT. AVP-induced activation of these transporters contributes to urine concentration and renal electrolyte reabsorption. Previous work has specified molecular pathways mediating the effects of V2R activation in TAL and DCT, and protein networks involved in intracellular trafficking and phosphoregulation of the two transporters have been identified. This review summarizes recent progress in understanding AVP signalling mechanisms that are responsible for the activation of NKCC2 and NCC. Implications in the pathophysiology of diseases such as nephrogenic diabetes insipidus, diabetes mellitus and salt-sensitive hypertension are discussed in this context.