Outer retinal function depends on two supporting tissues: the retinal pigment epithelium (RPE) and the choroid. Limited molecular information is available on the intercellular networks that sustain RPE/choroid tissue in both healthy and pathological states. Galectin-1 (Gal1), a β-galactoside-binding lectin, has recently emerged as a key regulator of angiogenesis and a potential therapeutic target in vascular pathologies, including age-related macular degeneration. Here, we studied the expression of Gal1 in the outer retina and its regulatory role in the RPE/choroid under physiological and pathological conditions. Our findings indicate that Gal1 is predominantly associated with stromal cells in the RPE/choroid. In Gal1-deficient (Lgals1-/-) mice, the RPE/choroid ultrastructure and gene expression profiles were altered, and choroidal explants exhibited reduced sprouting compared to those of wild-type mice. Consistently, recombinant Gal1 promoted choroidal sprouting under hypoxic conditions, and stromal-like cells modulated pro-angiogenic and antiangiogenic gene expression in vitro under pathological conditions. Interestingly, Gal1 was also expressed by the RPE, with apical secretion under normoxia that shifted toward a basolateral phenotype under hypoxia. These findings identify stromal-like cells and RPE as key sources of Gal1 in the choroid, highlighting its distinct roles in maintaining RPE/choroid homeostasis in healthy or pathological microenvironments.

The Na/K/2Cl cotransporter NKCC2 mediates the NaCl reabsorption in the thick ascending limb (TAL). Phosphorylation at Thr-96,101 and traffcking to the surface are increased in the Dahl salt-sensitive (DSS) rat together with SPAK phosphorylation, an upstream kinase for Thr-96,101 in NKCC2. We previously found that deletion of SPAK in the DSS, lowers NKCC2 phosphorylation and blunts salt-sensitive hypertension, but doesn’t completely abolish NKCC2 phosphorylation. Therefore, other kinases may be involved in NKCC2 phosphorylation. We recently found the Traf2 and NCK interacting kinase (TNIK) phosphorylates NKCC2 and is overexpressed in the TALs of DSS rats. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 hyperphosphorylation in DSS rats. We approached TNIK inhibition in two ways, using a pharmacological inhibitor and its genomic deletion with CRISPR/Cas9. First, we studied the small molecule TNIK inhibitor NCB-0846, and whether it could decrease baseline NKCC2 phosphorylation in TALs from DSS rats. Treating TALs from DSS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr-96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.001, n=3). To study in vivo effcacy of NCB-0846, we directly infused NCB (or vehicle) into the left kidney via implanted renal medullary catheters connected to minipumps in DSS rats fed 4% NaCl diet. We found that NCB-0846 decreased SBP by 30 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs control). To decrease TNIK gene expression we used Cas9 mediated gene editing. For this, we designed gRNA sequences targeting TNIK and tested their effcacy in C6 cells. One of the gRNAs designed decreased TNIK expression by 48 ± 22% compared to control (p=0.101, n=3). To decrease TNIK in TALs in vivo, we injected the renal medulla of the left kidney with AAV-gRNA-TNIK together with AAV-NKCC2 promoter-Cas9. After 2 weeks, we isolated medullary TALs and measured TNIK expression and NKCC2 phosphorylation. TNIK expression was decreased by 50 ± 5.5% (n=4, p<0.0001), and pThr-96,101-NKCC2 was decreased by 38.1 ± 10.6% in transduced TALs compared to kidneys transduced with AV-control (n=4, p<0.05). The expression of related kinases SPAK and OSR1 was not modified (SPAK 79.2 ± 18.3%, OSR1 81.3 ± 13.3%, n=4, ns). We conclude that NKCC2 phosphorylation in the DSS rat is in part caused by TNIK and that this new kinase could be a target for salt sensitive hypertension. Future experiments will test the effect of gene editing TNIK in salt sensitive hypertension. 23POST1019882 AHA15GRNT25710369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microvasculature-secreted factors create a local niche for tissue specialization and homeostasis. Diseases like diabetes are considered a microvascular disease. However, the consequences of microvascular alteration in surrounding tissues like epithelial barriers have not been suffciently explored. The objective of this study is to determine whether a cell-cell communication protein, Semaphorin-3E (Sema3E), which we have previously found increased in diabetic mice and in endothelial cells cultured on high glucose, impairs the barrier properties of epithelial cells. We determined by ELISA that HUVEC endothelial cells secreted Sema3E (7.8 ±1.8 pg/sq.cm/day) and while mannitol osmolality control 15mM had no effect, high glucose (25mM) increased Sema3E secretion 6-fold (ANOVA, p<0.01). To test the effect of Sema3E in the tight junction barrier properties of epithelial cells, we measured trans-epithelial electrical resistance (TEER) in Calu-3 epithelial cells cultured in trans-well permeable support. We found that 200ng/ml of recombinant Sema3E inhibited the acquisition of TEER over the course of 2 weeks (vehicle: 803 ± 31 Ω/cm^2 vs. Sema3E: 465 ± 15 Ω/cm^2; t-test p<0.01) The weakening of tight junctions was due to decreased presence of Claudin-2 at the cell surface as measured by surface biotinylation (Surface/intracellular ratio= vehicle: 0.356 ± 0.043 vs. Sema3E: 0.020 ± 0.003; t-test p<0.01). Consequently, the polarity of Calu-3 cells was also impaired as we determined by measuring the polarized distribution of the basolateral protein E-cadherin. We observed a reduction of basolateral E-cadherin from 88 ± 3% (vehicle) to 65 ± 6% (Sema3E), t-test p<0.05. Finally, to determine whether surface Claudin-2 expression was decreased in epithelial cells of diabetic mice, we performed surface biotinylation in freshly isolated kidney renal tubules. We observed that the surface/intracellular ratio of Claudin-2 was decreased by 76% in kidney tubules from type-1 diabetic mice (Akita strain), compared to healthy control mice (control: 4.23 ± 1.44 ratio vs. Akita: 1.03 ± 0.16 ratio, t-test p<0.05). We conclude that glucose stimulates Sema3E secretion from endothelial cells, and this signal protein impairs the epithelial barrier properties of epithelial cells by reducing Claudin-2 presence at tight junctions. Reduced surface Claudin-2 may be a feature of diabetic epithelial cells. American Heart Association NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p<0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p<0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p<0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hyperglycemia in the diabetic kidney causes tubular and microvascular abnormalities that eventually lead to kidney damage. Given the close association between the proximal tubule and peritubular capillaries, a thorough understanding of their interaction could help identify mechanisms of diabetic kidney disease. Microvascular abnormalities are known to occur in diabetic patients and animal models. Despite known growth factors produced by the proximal tubule that could potentially target neighboring endothelial cells, it is not well understood whether the proximal tubule regulates angiogenesis. Here we propose that the proximal tubule secretes Semaphorin-3G (Sema3G), an anti-angiogenic protein. We hypothesize, that high glucose stimulates Sema3G secretion by proximal tubule cells and inhibits angiogenesis in peritubular endothelial cells.To study the role of secreted Sema3G, we collected the conditioned media from cultured polarized proximal tubule cells (RPTEC cell line) where we silenced Sema3G via lentivirus-delivered shRNAs. To study angiogenesis, we developed primary cultures of mouse kidney peritubular endothelial cells and analyzed angiogenesis via tubulogenesis assays in matrigel.We observed that the basolateral conditioned medium from RPTEC cells stimulated angiogenesis of peritubular endothelial cells in matrigel over 8 hours (p<0.01). However, RPTEC cells pre-grown on high glucose (25 mM) lost this ability. The apical media did not have any effect. To identify a potential proximal tubule anti-angiogenic factor, we measured release of Sema3G from polarized RPTEC cells. We found that RPTEC cells secreted Sema3G basolaterally and high glucose stimulated this by 5.5-fold. (p<0.01). Next, we tested the anti-angiogenic potential of Sema3G in peritubular endothelial cells. While adding recombinant Sema3G (200ng/ml) produced a modest inhibition (28%, p<0.05) of baseline peritubular endothelial cell angiogenesis, instead it completely blocked the stimulatory effect of a dose-response treatment (50–100ng/ml) with the potent angiogenic factor VEGF (p<0.01). Finally, to test the role of endogenous proximal tubule Sema3G, we silenced Sema3G via lentivirus-delivered shRNA in RPTEC cells. We then collected the conditioned medium from cells on normal or high glucose and performed angiogenesis assays. We found that silencing Sema3G in RPTEC cells on high glucose restored the ability of the conditioned medium to stimulate peritubular endothelial angiogenesis.We conclude that RPTEC proximal tubule cells secrete Sema3G, which is stimulated by high glucose, and this anti-angiogenic factor inhibits angiogenesis in mouse peritubular endothelial cells. Henry Ford Hospital, American Heart Association This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated fructose consumption is associated with hypertension in humans. In normal Sprague Dawley rats, a high fructose intake (20% fructose drinking water) induced salt sensitive hypertension. We found that fructose directly stimulates the apical Na/K/2Cl cotransporter NKCC2 in the thick ascending limb (TAL), and when consumed for more than 7 days, increases NKCC2 phosphorylation at Thre 96,101. SPAK and OSR1 kinases are known to phosphorylate NKCC2, but it is not known how fructose activates SPAK or OSR1. We hypothesized that fructose intake activates several signaling cascades in TALs that selectively stimulate SPAK. Using an antibody that recognizes activated SPAK and OSR1 (P-Ser 373 SPAK; Ser325 in OSR1), we found that 20% fructose (14 d) enhanced SPAK/OSR1 phosphorylation by 179±32% (p<0.05). This effect was specific for SPAK because phosphorylation of OSR1, immunoprecipitated from TALs, was not increased by fructose. To study the mechanism of SPAK activation, we isolated TALs from rats on control or 20% fructose diet for (14 d, n=6) and performed RNAseq. Over 12,000 genes were statistically compared and 500 were significantly different (p<0.05). Expression of some of the most highly upregulated genes (PRKRIP1 and SOCS3) was confirmed by Western blot in TALs. However, expression of upstream SPAK activators was unchanged by fructose (WNK1/3/4). We developed a targeted bioinformatics approach that included angiotensin signaling, nitric oxide and lipid metabolism, plus protein-protein interaction networks for SPAK. We identified PRKCQ (PKC Theta), a kinase upstream of SPAK, that was upregulated in TALs by fructose. In fructose fed rats, PRKCQ expression was 31±11% higher than control, as was Phospho-PRKCQ (223±45% increase, p<0.05). We then measured systolic BP (SBP) by telemetry in Dahl SS rats with genetic deletion of SPAK (SPAK KO). In WT Dahl SS rats, 4 weeks of fructose intake increased SBP by 25±4 mmHg (p<0.05), whereas in SPAK KO the increase in SBP was reduced (11±4 mmHg, p<0.05). We concluded that high fructose intake induces several prohypertensive signaling pathways in the TAL that lead to SPAK and NKCC2 activation. We identified PRKCQ as a potential activator of SPAK. Fructose induced hypertension in Dahl SS rats was in part mediated by SPAK.

Endothelial cells secrete trophic factors that contribute to the maturation of surrounding tissues. The notion of the microvasculature as niche for local differentiation and homeostasis has implication in diseases like diabetic kidney disease, in which the microvasculature is lost at early stages. Endothelial cells are known to produce semaphorins (Sema) as signal molecules that target cells in proximity. The isoform Sema3F is secreted by endothelial cells throughout the body, and it is known to regulate angiogenesis in an autocrine fashion. However, Sema3F can potentially reach other neighboring cell types, in particular kidney proximal tubule, since this epithelium is closely associated with peritubular capillaries. It is not known whether Sema3F influences proximal tubule homeostasis. We hypothesize that endothelial cells secrete Sema3F, which in turn promotes proximal tubule epithelial maturation and polarization. We utilized the human proximal tubule cell line RPTEC/hTERT since these cells polarize in trans‐well permeable support by 2 weeks, and they retain expression and apical‐basolateral polarization of proximal tubule proteins. To study the role of endothelial cells in RPTEC/hTERT maturation, we co‐culture them with the human endothelial cell line HUVEC. First, we measured Sema3F release from HUVEC endothelial cells by ELISA and observed that they secrete 206 ± 3 pg Sema3F/cm2/day. Next, to determine whether HUVEC endothelial cells promote proximal tubule maturation, we monitored the progressive acquisition of trans‐epithelial resistance as a measure of tight junction maturation. We observed that in co‐culture with HUVEC endothelial cells, RPTEC/hTERT cells developed a trans‐epithelial resistance that was 11±3% higher at 1 week, and 27±5% higher at 2 weeks compared to monoculture (p<0.05). To test whether Sema3F stimulates RPTEC/hTERT trans‐epithelial resistance, we added 200ng/mL of recombinant human Sema3F to cell cultures. We observed that Sema3F increased RPTEC/hTERT trans‐epithelial resistance at 1 week (vehicle= 152±5 Ω/cm2 vs. Sema3F= 171±3 Ω/cm2; p<0.05) and 2 weeks (vehicle= 150±2 Ω/cm2 vs. Sema3F= 182±4 Ω/cm2; p<0.05). Also, Sema3F accelerated polarization of the basolateral protein E‐cadherin measured by surface biotinylation over 2 weeks. Finally, to determine whether Sema3F produced by endothelial cells stimulate RPTEC/hTERT maturation, we silenced Sema3F in HUVEC endothelial cells via lentivirus‐transduction of silencing shRNAs. We observed that silencing Sema3F in HUVEC cells prevented the stimulation of RPTEC/hTERT trans‐epithelial resistance, while control shRNA transduction showed the expected stimulation at 7 days (monoculture= 222±4 Ω/cm2 vs. coculture‐shSema3F= 218±7 Ω/cm2; p=NS vs. coculture‐shControl= 231±8 Ω/cm2; p<0.05). We conclude that HUVEC endothelial cells secrete Sema3F, which reaches RPTEC/hTERT proximal tubule cells in co‐culture and stimulates tight junction maturation and polarization. These findings have relevance as a possible mechanism of disease in pathologies like diabetic kidney disease where the microvasculature is compromised.

Background AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. Methods The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. Results A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. Conclusion Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

In most cells, intracellular cytoplasmic Cl− is maintained between 10 and 30 mM by a combination of entry and exit transport pathways. However, in endocrine cells intra‐granule Clin secretory granules is higher than cytoplasmic Cl−. This, is in part due to an intragranular positive H+ gradient driven by V‐ATPases, which generates low intra‐granular pH (4–5), and provides an electrochemical gradient for Cl− entry via CLC‐6/7. The role of V‐ATPases in exocytosis is well known, but less is known about how changes in Cl− contribute. There are very few methods available to measure intra‐granule Cl− concentration and monitor how it changes. ClopHensor is a genetically encoded protein sensor based on a Cl ‐sensitive GFP mutant (E2‐GFP) and DsRed ( Nature Methods, v7, pages 516–518, 2010). When excited by a 457 nm laser, E2‐GFP emission is sensitive to Cl− whereas at 488 nm it is sensitive to pH, whereas DsRed (insensitive to Cl− or pH) serves to control for volume changes. Renal endocrine juxtaglomerular (JG) cells, store renin, the rate‐limiting step enzyme in the production of angiotensin II, which play an essential role in blood pressure control. Renin is stored in large secretory granules and its exocytosis is stimulated primarily by increases in cAMP. We hypothesized that targeting ClopHensor to secretory granules would allow measurement of intra‐granular Cl− in JG cells and changes in its concentration during stimulation. We first tested the ability of ClopHensor to measure intracellular Cl− in M1 cells, clamped at pH 5.5 (reported for secretory granules). M1 cells transduced with adenovirus coding for ClopHensor were permeabilized to H+ with nygericin and bathed at pH 5.5, and permeabilized to Cl−. Cells were imaged by confocal microscopy under 457, 491 and 568 nm laser excitation while simultaneously acquiring emissions at 525 (E2‐GFP) and 590nm (Dsred). Under these conditions increasing Cl− from 0, to 20, 80 and 120 mM caused a logarithmic reduction in the 457/568 emission ratio (Cl‐sensitive) with a half‐max inhibition at 10.7±1.4 mM (n =6). To monitor intra‐granule Cl− we generated adenoviruses expressing NeuroPeptide Y targeting motif (NPY)‐ClopHensor. When primary mouse JG cells were transduced with NPY‐ClopHensor fluorescence was restricted to large granules. We could not permeabilize granules to Cl‐ in live JG cells Thus we measured baseline intra‐granule Cl− immediately after a daily calibration of ClopHensor in M1 cells. These results yielded an intra‐granule Cl− concentration of 77±15 mM (n=8). After measuring baseline Cl− in granules JG cells were treated with forskolin/IBMX to increase cAMP, and then 0 mM extracellular Cl− and bafilomycin. cAMP decreased intra‐granule Cl−, evidenced by a 22±5% increase (p<0.05, n= 11) in 457/568 Cl‐sensitive ratio, which was further increased by inhibiting V‐ATPse with bafilomycin by 40±6% (p<0.05, n=13). Our data show for the first time, measurements of intra‐granule Cl− in renal JG cells and suggest that NPY‐ClopHensor can be used to monitor the effect of agonists, or V‐ATPase regulators on intra‐granule Cl−. The role of intra‐granule Cl− in renin exocytosis and renin‐angiotensin system physiology remains to be studied.Support or Funding InformationHFH Research fund: Pablo A Ortiz

BackgroundPolarized epithelial cells express distinct sets of surface proteins at the apical and basolateral membranes. This surface proteome is crucial for epithelial physiology as it includes channels and transporters, surface receptors and adhesion molecules among other proteins. We recently developed a quantitative approach to obtain polarized surface proteomes of epithelial cells in culture. This information could benefit efforts to understand epithelial cell/extracellular medium interactions as mechanisms to maintain higher order multi‐tissue structures. Such interactions could represent organ‐specific pathways of homeostasis and disease. In order to exploit the potential of our polarized epithelial surface proteomics approach, we pursued a detailed analysis of members of the Plexin/Semaphorin/Integrin (PSI) superfamily we found in the proteome of retinal pigment epithelial cells. In other cells, plexins are known receptors for semaphorins and mediate cell‐cell communication, while integrins bind components of the extracellular matrix. In epithelial cells, some PSI isoforms have well known roles in cell adhesion, cytoskeletal remodeling and homotypic cell‐cell junctions. However, the wide range of isoforms identified by surface proteomics provides a unique opportunity to analyze less‐known isoforms as well as cell‐type specific pathways.ObjectiveTo identify semaphorins and integrin isoforms in retinal pigment epithelium that mediate interactions with the extracellular matrix and other cell types.MethodsWe identified possible targets of interests based on their surface expression in retinal pigment epithelium and absence in the generic epithelial cell line MDCK. Next, we utilized gene knockdown via CRISPR or shRNA to analyze their role in cell‐adhesion, migration and communication with endothelial cells. To study interaction with endothelial cells in vivo, we measured angiogenesis in a mouse model of matrigel‐induced neovascularization.ResultsWe determined that retinal pigment epithelial cells expressed semaphorin‐4A (Sema4A) and Beta‐8 integrin (ItgB8) at the surface, but they were absent in MDCK epithelial cells. Knockdown of ItgB8 significantly decreased retinal pigment epithelial cell migration in wound healing assays and cell adhesion to uncoated and collagen IV‐coated substrate. Knockdown of Sema4A in retinal pigment epithelial cells enhanced angiogenesis in co‐cultured choroidal explants (contains native endothelial cells that supply the retinal pigment epithelium in vivo). This enhanced angiogenesis suggests a baseline inhibitory role of secreted Sema4A. To test this possibility in vivo, we injected recombinant Sema4A intraocularly in mice, and we observed that this reduced matrigel‐induced neovascularization.ConclusionAnalysis of the surface proteome of epithelial cells led to identification of ItgB8 as mediator of retinal pigment epithelial cell adhesion and migration, and Sema4A as an anti‐angiogenic cell‐cell communication signal.Support or Funding InformationNIH EY008538 and EY008538‐28S1

The polarized phenotype of the retinal pigment epithelium is crucial for the outer retina-blood barrier and support of photoreceptors and underlying choroid, and its disruption plays a central role in degenerative retinopathies. Although the mechanisms of polarization remain mostly unknown, they are fundamental for homeostasis of the outer retina. Recent research is revealing a growing picture of interconnected tissues in the outer retina, with the retinal pigment epithelium at the center. This review discusses how elements of epithelial polarity relate to emerging apical interactions with the neural retina, basolateral cross-talk with the underlying Bruch's membrane and choriocapillaris, and tight junction biology. An integrated view of outer retina physiology is likely to provide insights into the pathogenesis of blinding diseases.

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity w as observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

Purpose of review The apical Na+/K+/2Cl(-) cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb, contributing to maintenance of blood pressure (BP). Despite effective NKCC2 inhibition by loop diuretics, these agents are not viable for long-term management of BP due to side effects. Novel molecular mechanisms that control NKCC2 activity reveal an increasingly complex picture with interacting layers of NKCC2 regulation. Here, we review the latest developments that shine new light on NKCC2-mediated control of BP and potential new long-term therapies to treat hypertension. Recent findings Emerging molecular NKCC2 regulators, often binding partners, reveal a complex overlay of interacting mechanisms aimed at fine tuning NKCC2 activity. Different factors achieve this by shifting the balance between trafficking steps like exocytosis, endocytosis, recycling and protein turnover, or by balancing phosphorylation vs. dephosphorylation. Further molecular details are also emerging on previously known pathways of NKCC2 regulation, and recent in-vivo data continues to place NKCC2 regulation at the center of BP control. Several layers of emerging molecular mechanisms that control NKCC2 activity may operate simultaneously, but they can also be controlled independently. This provides an opportunity to identify new pharmacological targets to fine-tune NKCC2 activity for BP management.

The ability to detect and track single molecules presents the advantage of visualizing the complex behavior of transmembrane proteins with a time and space resolution that would otherwise be lost with traditional labeling and biochemical techniques. Development of new imaging probes has provided a robust method to study their trafficking and surface dynamics. This mini-review focuses on the current technology available for single-molecule labeling of transmembrane proteins, their advantages, and limitations. We also discuss the application of these techniques to the study of renal transporter trafficking in light of recent research.

Elevated blood pressure (BP) and renal dysfunction are complex traits representing major global health problems. Single nucleotide polymorphisms identified by genome-wide association studies have identified the Alström syndrome 1 (ALMS1) gene locus to render susceptibility for renal dysfunction, hypertension, and chronic kidney disease (CKD). Mutations in the ALMS1 gene in humans causes Alström syndrome, characterized by progressive metabolic alterations including hypertension and CKD. Despite compelling genetic evidence, the underlying biological mechanism by which mutations in the ALMS1 gene lead to the above-mentioned pathophysiology is not understood. We modeled this effect in a KO rat model and showed that ALMS1 genetic deletion leads to hypertension. We demonstrate that the link between ALMS1 and hypertension involves the activation of the renal Na+/K+/2Cl- cotransporter NKCC2, mediated by regulation of its endocytosis. Our findings establish a link between the genetic susceptibility to hypertension, CKD, and the expression of ALMS1 through its role in a salt-reabsorbing tubular segment of the kidney. These data point to ALMS1 as a potentially novel gene involved in BP and renal function regulation.