Klotho and Clinical Outcomes in CKD: Findings From the Chronic Renal Insufficiency Cohort (CRIC) Study.

RATIONALE & OBJECTIVE: Klotho deficiency may affect clinical outcomes in chronic kidney disease (CKD) through fibroblast growth factor-23 (FGF23)-dependent and -independent pathways. However, the association between circulating Klotho and clinical outcomes in CKD remains unresolved and was the focus of this study. STUDY DESIGN: Prospective observational study. SETTING & PARTICIPANTS: 1,088 participants in the Chronic Renal Insufficiency Cohort (CRIC) Study with an estimated glomerular filtration rate (eGFR) of 20-70mL/min/1.73m2. EXPOSURE: Plasma Klotho level at the year-1 study visit. OUTCOMES: 5-year risks of all-cause mortality, heart failure hospitalization, atherosclerotic cardiovascular events, and a composite kidney end point that comprised a sustained 50% decrease in eGFR, dialysis, kidney transplant, or eGFR<15mL/min/1.73m2. ANALYTICAL APPROACH: We divided Klotho into 6 groups to account for its nonnormal distribution. We used Cox proportional hazards regression and subdistribution hazards models to compare survival and clinical outcomes, respectively, between Klotho groups. We sequentially adjusted for demographic characteristics, kidney function, cardiovascular risk factors, sample age, and FGF23. RESULTS: Mean eGFR was 42mL/min/1.73m2, and median Klotho concentration was 0.31ng/mL (IQR, 0.10-3.27ng/mL). When compared with the lowest Klotho group, survival (HR, 0.77; 95% CI, 0.32-1.89), heart failure hospitalization (HR, 1.10; 95% CI, 0.38-3.17), atherosclerotic cardiovascular events (HR, 1.19; 95% CI, 0.57-2.52), and CKD progression (HR, 1.05; 95% CI, 0.58-1.91) did not differ in the high Klotho group. In contrast, FGF23 was significantly associated with mortality and heart failure hospitalization independent of Klotho levels. LIMITATIONS: Despite adjustments, we cannot exclude the potential influence of residual confounding or sample storage on the results. A single measurement of plasma Klotho concentration may not capture Klotho patterns over time. CONCLUSIONS: In a large, diverse, well-characterized CKD cohort, Klotho was not associated with clinical outcomes, and Klotho deficiency did not confound the association of FGF23 with mortality or heart failure hospitalization. PLAIN-LANGUAGE SUMMARY: Klotho is a protein that is vital to mineral metabolism and aging and may protect against cardiovascular disease. Klotho levels decrease in chronic kidney disease (CKD), but the association between Klotho and clinical outcomes in CKD remains uncertain. In a prospective cohort study of more than 1,000 people with CKD, circulating Klotho levels were not associated with kidney disease progression, cardiovascular outcomes, or mortality. These results suggest that the decrease in circulating Klotho levels in CKD does not play a prominent role in the development of poor clinical outcomes.

Why do workers leave geriatric care, and do they come back? A timing of events study.

BACKGROUND: In many industrialized countries, demographic change is accompanied by increasing demand for geriatric care workers, which in turn intensifies the need to retain them or to attract them back after leaving. Although much is known about the intention of workers to leave, little is known about how many of them actually do leave and return to geriatric care at a later stage in a comprehensive framework. OBJECTIVE: To examine and explain how many workers withdraw from geriatric care, take up other occupations, and return to geriatric care at a later stage in their careers, and the reasons behind this. DESIGN: This study used administrative daily data that cover all workers in Germany who started working in geriatric care in the period 1996 to 2018 and observed their employment biographies for a maximum of 26 years. METHODS: Kaplan-Meier survival analysis and parametric regression survival-time analysis were used to estimate the survival rates and to estimate individual, workplace and regional determinants of leaving and returning to geriatric care. RESULTS: Workers in geriatric care were more likely to leave the profession than other workers, two thirds withdrew within five years of first taking up work in geriatric care. If they remained in employment, they generally chose an occupation related to geriatric care. Half of the leavers returned to geriatric care again within eight years. Individual and workplace-related factors, such as age and remuneration, are statistically significant predictors of leaving and returning. CONCLUSIONS: The findings emphasize how important it is for organizations to keep in touch with geriatric care workers leaving their job, since a substantial share of them returns again.

Renal proximal tubule cells: power and finesse.

The proximal tubule is the high-capacity reabsorptive powerhouse of the kidney. Two papers in recent issues of the JCI highlight mechanisms of more delicate effects of the proximal tubule. Yoon et al. demonstrated the intracellular mechanism by which parathyroid hormone (PTH) increases production of 1,25-vitamin D. Activation of PTH receptor 1/cAMP/PKA signaling inhibited salt-inducible kinase 2 (SIK2) and SIK3, which increased CYB27B1 transcription and 1,25-vitamin D production. Replication of these effects with small-molecule SIK inhibitors suggests possible therapeutic applications for patients with disorders characterized by 1,25-vitamin D deficiency. Zhou et al. discovered that proximal tubule glycolysis acts as a phosphate sensor that regulates fibroblast growth factor 23 production in bone. They described several kidney-specific metabolic modifications that enabled glycolysis to be deployed as a phosphate sensor. The provocative results raise intriguing questions with implications for patients with disorders of phosphate homeostasis, including chronic kidney disease.

Localization and characterization of proenkephalin-A as a potential biomarker for kidney disease in murine and human kidneys.

INTRODUCTION: Exact measurement of renal function is essential for the treatment of patients. Elevated serum-creatinine levels, while established, are influenced by other parameters and show a significant time-lag. This drives the search for novel biomarkers of renal function and injury. Beside Lipocalin-2 and kidney-injury-molecule-1 (KIM-1), the endogenous opioid precursor proenkephalin-A (Penk) has recently emerged as a promising marker for renal function. But the cellular origin and regulation of Penk outside the brain has not yet been investigated in depth. MATERIALS AND METHODS: This study characterizes the cellular origin of Penk expression with high-resolution in situ hybridization in two models of renal fibrosis in mice and human tissue. RESULTS: Interstitial cells are the main expression site for renal Penk. This classifies Penk as biomarker for interstitial damage as opposed to tubular damage markers like Lipocalin-2 and KIM-1. Furthermore, our data indicate that renal Penk expression is not regulated by classical profibrotic pathways. DISCUSSION: This study characterizes changing Penk expression in the kidneys. The similarity of Penk expression across species gives rise to further investigations into the function of Penk in healthy and injured kidneys. CONCLUSION: Penk is a promising biomarker for interstitial renal damage that warrants further studies to utilize its predictive potential.Clinical significanceKnowledge of real-time renal function is essential for proper treatment of critically ill patients and in early diagnosis of acute kidney injury (AKI). Proenkephalin-A has been measured in a number of patient cohorts as a highly accurate and predictive biomarker of renal damage.The present study identifies Penk as a biomarker for interstitial damage in contrast to the tubular biomarkers such as Lipocalin-2 or KIM-1.Our data show that Penk is regulated independently of classical profibrotic or proinflammatory pathways, indicating it might be more robust against extra-renal influences.Data presented in this study provide fundamental information about cell type-specific localization and regulation of the potential new biomarker Penk across species as foundation for further research.

Fibroblast Growth Factor (FGF) 23 and FGF Receptor 4 promote cardiac metabolic remodeling in chronic kidney disease.

Chronic kidney disease (CKD) is a global health epidemic that significantly increases mortality due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiac injury in CKD. High serum levels of fibroblast growth factor (FGF) 23 in patients with CKD may contribute mechanistically to the pathogenesis of LVH by activating FGF receptor (FGFR) 4 signaling in cardiac myocytes. Mitochondrial dysfunction and cardiac metabolic remodeling are early features of cardiac injury that predate development of hypertrophy, but these mechanisms of disease have been insufficiently studied in models of CKD. Wild-type mice with CKD induced by adenine diet developed LVH that was preceded by morphological changes in mitochondrial structure and evidence of cardiac mitochondrial and metabolic dysfunction. In bioengineered cardio-bundles and neonatal rat ventricular myocytes grown in vitro, FGF23-mediated activation of FGFR4 caused a mitochondrial pathology, characterized by increased bioenergetic stress and increased glycolysis, that preceded the development of cellular hypertrophy. The cardiac metabolic changes and associated mitochondrial alterations in mice with CKD were prevented by global or cardiac-specific deletion of FGFR4. These findings indicate that metabolic remodeling and eventually mitochondrial dysfunction are early cardiac complications of CKD that precede structural remodeling of the heart. Mechanistically, FGF23-mediated activation of FGFR4 causes mitochondrial dysfunction, suggesting that early pharmacologic inhibition of FGFR4 might serve as novel therapeutic intervention to prevent development of LVH and heart failure in patients with CKD.

The fate of erythropoietin-producing cells: another piece of the puzzle.

In this issue, Kaneko et al. reported the generation of a mouse line that allows for the labeling of cells under control of the erythropoietin (Epo) gene promotor. The authors show that Epo-producing cells become proliferating, profibrotic cells after kidney injury and lose their ability to produce Epo. Furthermore, they show that the fluorescent-labeled cells can recover their Epo synthesis capability subsequently to a recovery period.

Flexible and multifaceted: the plasticity of renin-expressing cells.

The protease renin, the key enzyme of the renin-angiotensin-aldosterone system, is mainly produced and secreted by juxtaglomerular cells in the kidney, which are located in the walls of the afferent arterioles at their entrance into the glomeruli. When the body's demand for renin rises, the renin production capacity of the kidneys commonly increases by induction of renin expression in vascular smooth muscle cells and in extraglomerular mesangial cells. These cells undergo a reversible metaplastic cellular transformation in order to produce renin. Juxtaglomerular cells of the renin lineage have also been described to migrate into the glomerulus and differentiate into podocytes, epithelial cells or mesangial cells to restore damaged cells in states of glomerular disease. More recently, it could be shown that renin cells can also undergo an endocrine and metaplastic switch to erythropoietin-producing cells. This review aims to describe the high degree of plasticity of renin-producing cells of the kidneys and to analyze the underlying mechanisms.

Intact prostaglandin signaling through EP2 and EP4 receptors in stromal progenitor cells is required for normal development of the renal cortex in mice.

Cyclooxygenase (Cox) inhibitors are known to have severe side effects during renal development. These consist of reduced renal function, underdeveloped subcapsular glomeruli, interstitial fibrosis, and thinner cortical tissue. Global genetic deletion of Cox-2 mimics the phenotype observed after application of Cox inhibitors. This study aimed to investigate which cell types express Cox-2 and prostaglandin E2 receptors and what functions are mediated through this pathway during renal development. Expression of EP2 and EP4 mRNA was detected by RNAscope mainly in descendants of FoxD1+ stromal progenitors; EP1 and EP3, on the other hand, were expressed in tubules. Cox-2 mRNA was detected in medullary interstitial cells and macula densa cells. Functional investigations were performed with a cell-specific approach to delete Cox-2, EP2, and EP4 in FoxD1+ stromal progenitor cells. Our data show that Cox-2 expression in macula densa cells is sufficient to drive renal development. Deletion of EP2 or EP4 in FoxD1+ cells had no functional effect on renal development. Codeletion of EP2 and EP4 in FoxD1+ stromal cells, however, led to severe glomerular defects and a strong decline of glomerular filtration rate (1.316 ± 69.7 µL/min/100 g body wt in controls vs. 644.1 ± 64.58 µL/min/100 g body wt in FoxD1+/Cre EP2-/- EP4ff mice), similar to global deletion of Cox-2. Furthermore, EP2/EP4-deficient mice showed a significant increase in collagen production with a strong downregulation of renal renin expression. This study shows the distinct localization of EP receptors in mice. Functionally, we could identify EP2 and EP4 receptors in stromal FoxD1+ progenitor cells as essential receptor subtypes for normal renal development.NEW & NOTEWORTHY Cyclooxygenase-2 (Cox-2) produces prostaglandins that are essential for normal renal development. It is unclear in which cells Cox-2 and the receptors for prostaglandin E2 (EP receptors) are expressed during late nephrogenesis. This study identified the expression sites for EP subtypes and Cox-2 in neonatal mouse kidneys. Furthermore, it shows that stromal progenitor cells may require intact prostaglandin E2 signaling through EP2 and EP4 receptors for normal renal development.

Prolyl-4-hydroxylases 2 and 3 control erythropoietin production in renin-expressing cells of mouse kidneys.

Activation of the hypoxia-signalling pathway induced by deletion of the ubiquitin-ligase von Hippel-Lindau protein causes an endocrine shift of renin-producing cells to erythropoietin (EPO)-expressing cells. However, the underlying mechanisms have not yet been investigated. Since oxygen-regulated stability of hypoxia-inducible transcription factors relevant for EPO expression is dependent on the activity of prolyl-4-hydroxylases (PHD) 2 and 3, this study aimed to determine the relevance of different PHD isoforms for the EPO expression in renin-producing cells in vivo. For this purpose, mice with inducible renin cell-specific deletions of different PHD isoforms were analysed. Our study shows that there are two subgroups of renal renin-expressing cells, juxtaglomerular renin+ cells and platelet-derived growth factor receptor-ß+ interstitial renin+ cells. These interstitial renin+ cells belong to the cell pool of native EPO-producing cells and are able to express EPO and renin in parallel. In contrast, co-deletion of PHD2 and PHD3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular and hyperplastic renin+ cells and downregulates renin expression. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent the hypoxia-inducible transcription factor-2-dependent phenotype shift into EPO cells. In summary, PHDs seem important for the stabilization of the juxtaglomerular renin cell phenotype. Moreover, these findings reveal tubulointerstitial cells as a novel site of renal renin expression and suggest a high endocrine plasticity of these cells. Our data concerning the distinct expression patterns and functions of PHD2 and PHD3 provide new insights into the regulation of renin-producing cells and highlight the need for selective PHD inhibitors. KEY POINTS: Renal renin-expressing cells can be clearly distinguished into two subgroups, the typical juxtaglomerular renin-producing cells and interstitial renin+ cells. Interstitial renin+ cells belong to the cell pool of native erythropoietin (EPO)-producing cells, show a fast EPO response to acute hypoxia-inducible factor-2 (HIF-2) stabilization and are able to express EPO and renin in parallel. Only co-deletion of the prolyl-4-hydroxylases (PHD) 2 and 3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular renin+ cells. Chronic HIF-2 stabilization in juxtaglomerular renin-expressing cells leads to their phenotypic shift into EPO-producing cells. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent a HIF-2-dependent phenotype shift into EPO cells suggesting PHD3 fulfils a stabilizer function for the juxtaglomerular renin cell phenotype.

Endothelin receptors in renal interstitial cells do not contribute to the development of fibrosis during experimental kidney disease.

Renal interstitial fibrosis is characterized by the development of myofibroblasts, originating from resident renal and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various factors. Among these, endothelins have been discussed as potential modulators of renal fibrosis. Utilizing mouse models of adenine nephropathy (AN) and unilateral ureter occlusion (UUO), this study aimed to investigate the contribution of endothelin signaling in stromal mesenchymal resident renal interstitial cells. We found in controls that adenine feeding and UUO caused marked upregulations of endothelin-1 (ET-1) gene expression in endothelial and in tubular cells and a strong upregulation of ETA-receptor (ETA-R) gene expression in interstitial and mesangial cells, while the gene expression of ETB-receptor (ETB-R) did not change. Conditional deletion of ETA-R and ETB-R gene expression in the FoxD1 stromal cell compartment which includes interstitial cells significantly reduced renal ETA-R gene expression and moderately lowered renal ETB-R gene expression. ET receptor (ET-R) deletion exerted no apparent effects on kidney development nor on kidney function. Adenine feeding and UUO led to similar increases in profibrotic and proinflammatory gene expression in control as well as in ETAflflETBflfl FoxD1Cre+ mice (ET-Ko). In summary, our findings suggest that adenine feeding and UUO activate endothelin signaling in interstitial cells which is due to upregulated ETA-R expression and enhanced renal ET-1 production Our data also suggest that the activation of endothelin signaling in interstitial cells has less impact for the development of experimentally induced fibrosis.

Inhibition of transforming growth factor ß1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice.

Kidney fibrosis is characterized by the development of myofibroblasts originating from resident kidney and immigrating cells. Myofibroblast formation and extracellular matrix production during kidney damage are triggered by various cytokines. Among these, transforming growth factor ß1 (TGFß1) is considered a central trigger for kidney fibrosis. We found a highly upregulated expression of TGFß1 and TGFß receptor 2 (TGFß-R2) mRNAs in kidney interstitial cells in experimental fibrosis. Here, we investigated the contribution of TGFß1 signaling in resident kidney interstitial cells to organ fibrosis using the models of adenine induced nephropathy and unilateral ureteral occlusion in mice. For this purpose TGFß1 signaling was interrupted by inducible deletion of the TGFß-R2 gene in interstitial cells expressing the fibroblast marker platelet derived growth factor receptor-ß. Expression of profibrotic genes was attenuated up to 50% in kidneys lacking TGFß-R2 in cells positive for platelet derived growth factor receptor-ß. Additionally, deletion of TGFß-R2 prevented the decline of erythropoietin production in ureter ligated kidneys. Notably, fibrosis associated expression of α-smooth muscle actin as a myofibroblast marker and deposits of extracellular collagens were not altered in mice with targeted deletion of TGFß-R2. Thus, our findings suggest an enhancing effect of TGFß1 signaling in resident interstitial cells that contributes to profibrotic gene expression and the downregulation of erythropoietin production, but not to the development of myofibroblasts during kidney fibrosis.

Localization of angiotensin II type 1 receptor gene expression in rodent and human kidneys.

The kidneys are an important target for angiotensin II (ANG II). In adult kidneys, the effects of ANG II are mediated mainly by ANG II type 1 (AT1) receptors. AT1 receptor expression has been reported for a variety of different cell types within the kidneys, suggesting a broad spectrum of actions for ANG II. Since there have been heterogeneous results in the literature regarding the intrarenal distribution of AT1 receptors, this study aimed to obtain a comprehensive overview about the localization of AT1 receptor expression in mouse, rat, and human kidneys. Using the cell-specific and high-resolution RNAscope technique, we performed colocalization experiments with various cell markers to specifically discriminate between different segments of the tubular and vascular system. Overall, we found a similar pattern of AT1 mRNA expression in mouse, rat, and human kidneys. AT1 receptors were detected in mesangial cells and renin-producing cells. In addition, AT1 mRNA was found in interstitial cells of the cortex and outer medulla. In rodents, late afferent and early efferent arterioles expressed AT1 receptor mRNA, but larger vessels of the investigated species showed no AT1 expression. Tubular expression of AT1 mRNA was species dependent with a strong expression in proximal tubules of mice, whereas expression was undetectable in human tubular cells. These findings suggest that the (juxta)glomerular area and tubulointerstitium are conserved expression sites for AT1 receptors across species and might present the main target sites for ANG II in adult human and rodent kidneys.NEW & NOTEWORTHY Angiotensin II (ANG II) type 1 (AT1) receptors are essential for mediating the effects of ANG II in the kidneys. This study aimed to obtain a comprehensive overview about the cell-specific localization of AT1 receptor expression in rodent and human kidneys using the novel RNAscope technique. We found that the conserved AT1 receptor mRNA expression sites across species are the (juxta)glomerular areas and tubulointerstitium, which might present main target sites for ANG II in adult human and rodent kidneys.

Different subpopulations of kidney interstitial cells produce erythropoietin and factors supporting tissue oxygenation in response to hypoxia in vivo.

Genetic induction of hypoxia signaling by deletion of the von Hippel-Lindau (Vhl) protein in mesenchymal PDGFR-ß+ cells leads to abundant HIF-2 dependent erythropoietin (EPO) expression in the cortex and outer medulla of the kidney. This rather unique feature of kidney PDGFR-ß+ cells promote questions about their special characteristics and general functional response to hypoxia. To address these issues, we characterized kidney PDGFR-ß+ EPO expressing cells based on additional cell markers and their gene expression profile in response to hypoxia signaling induced by targeted deletion of Vhl or exposure to low oxygen and carbon monoxide respectively, and after unilateral ureteral obstruction. CD73+, Gli1+, tenascin C+ and interstitial SMMHC+ cells were identified as zonally distributed subpopulations of PDGFR-ß+ cells. EPO expression could be induced by Vhl deletion in all PDGFR-ß+ subpopulations. Under hypoxemic conditions, recruited EPO+ cells were mostly part of the CD73+ subpopulation. Besides EPO production, expression of adrenomedullin and regulator of G-protein signaling 4 was upregulated in PDGFR-ß+ subpopulations in response to the different hypoxic stimuli. Thus, different kidney interstitial PDGFR-ß+ subpopulations exist, capable of producing EPO in response to different stimuli. Activation of hypoxia signaling in these cells also induces factors likely contributing to improved kidney interstitial tissue oxygenation.

Apparently normal kidney development in mice with conditional disruption of ANG II-AT1 receptor genes in FoxD1-positive stroma cell precursors.

An intact renin-angiotensin system involving ANG II type 1 (AT1) receptors is crucial for normal kidney development. It is still unclear in which cell types AT1 receptor signaling is required for normal kidney development, maturation, and function. Because all kidney cells deriving from stroma progenitor cells express AT1 receptors and because stromal cells fundamentally influence nephrogenesis and tubular maturation, we investigated the relevance of AT1 receptors in stromal progenitors and their descendants for renal development and function. For this aim, we generated and analyzed mice with conditional deletion of AT1A receptor in the FoxD1 cell lineage in combination with global disruption of the AT1B receptor gene. These FoxD1-AT1ko mice developed normally. Their kidneys showed neither structural nor functional abnormalities compared with wild-type mice, whereas in isolated perfused FoxD1-AT1ko kidneys, the vasoconstrictor and renin inhibitory effects of ANG II were absent. In vivo, however, plasma renin concentration and renal renin expression were normal in FoxD1-AT1ko mice, as were blood pressure and glomerular filtration rate. These findings suggest that a strong reduction of AT1 receptors in renal stromal progenitors and their descendants does not disturb normal kidney development.

Activation of Hypoxia Signaling in Stromal Progenitors Impairs Kidney Development.

Intrauterine hypoxia is a reason for impaired kidney development. The cellular and molecular pathways along which hypoxia exerts effects on nephrogenesis are not well understood. They are likely triggered by hypoxia-inducible transcription factors (HIFs), and their effects appear to be dependent on the cell compartment contributing to kidney formation. In this study, we investigated the effects of HIF activation in the developing renal stroma, which also essentially modulates nephron development from the metanephric mesenchyme. HIF activation was achieved by conditional deletion of the von Hippel-Lindau tumor suppressor (VHL) protein in the forkhead box FOXD1 cell lineage, from which stromal progenitors arise. The resulting kidneys showed maturation defects associated with early postnatal death. In particular, nephron formation, tubular maturation, and the differentiation of smooth muscle, renin, and mesangial cells were impaired. Erythropoietin expression was strongly enhanced. Codeletion of VHL together with HIF2A but not with HIF1A led to apparently normal kidneys, and the animals reached normal age but were anemic because of low erythropoietin levels. Stromal deletion of HIF2A or HIF1A alone did not affect kidney development. These findings emphasize the relevance of sufficient intrauterine oxygenation for normal renal stroma differentiation, suggesting that chronic activity of HIF2 in stromal progenitors impairs kidney development. Finally, these data confirm the concept that normal stroma function is essential for normal tubular differentiation.

Functional Roles of Complexin 3 and Complexin 4 at Mouse Photoreceptor Ribbon Synapses.

UNLABELLED: Complexins (Cplxs) are SNARE complex regulators controlling the speed and Ca(2+) sensitivity of SNARE-mediated synaptic vesicle fusion. We have shown previously that photoreceptor ribbon synapses in mouse retina are equipped with Cplx3 and Cplx4 and that lack of both Cplxs perturbs photoreceptor ribbon synaptic function; however, Cplx3/4 function in photoreceptor synaptic transmission remained elusive. To investigate Cplx3/4 function in photoreceptor ribbon synapses, voltage-clamp recordings from postsynaptic horizontal cells were performed in horizontal slice preparations of Cplx3/4 wild-type (WT) and Cplx3/4 double knock-out (DKO) mice. We measured tonic activity in light and dark, current responses to changes in luminous intensity, and electrically evoked postsynaptic responses. Cplx3/4 decreased the frequency of tonic events and shifted their amplitude distribution to smaller values. Light responses were sustained in the presence of Cplx3/4, but transient in their absence. Finally, Cplx3/4 increased synaptic vesicle release evoked by electrical stimulation. Using electron microscopy, we quantified the number of synaptic vesicles at presynaptic ribbons after light or dark adaptation. In Cplx3/4 WT photoreceptors, the number of synaptic vesicles associated with the ribbon base close to the release site was significantly lower in light than in dark. This is in contrast to Cplx3/4 DKO photoreceptors, in which the number of ribbon-associated synaptic vesicles remained unchanged regardless of the adaptational state. Our results indicate a suppressing and a facilitating action of Cplx3/4 on Ca(2+)-dependent tonic and evoked neurotransmitter release, respectively, and a regulatory role in the adaptation-dependent availability of synaptic vesicles for release at photoreceptor ribbon synapses. SIGNIFICANCE STATEMENT: Synaptic vesicle fusion at active zones of chemical synapses is executed by SNARE complexes. Complexins (Cplxs) are SNARE complex regulators and photoreceptor ribbon synapses are equipped with Cplx3 and Cplx4. The absence of both Cplxs perturbs ribbon synaptic function. Because we lack information on Cplx function in photoreceptor synaptic transmission, we investigated Cplx function using voltage-clamp recordings from postsynaptic horizontal cells of Cplx3/4 wild-type and Cplx3/4 double knock-out mice and quantified synaptic vesicle number at the ribbon after light and dark adaptation using electron microscopy. The findings reveal a suppressing action of Cplx3/4 on tonic neurotransmitter release, a facilitating action on evoked release, and a regulatory role of Cplx3/4 in the adaptation-dependent availability of synaptic vesicles at mouse photoreceptor ribbon synapses.

Erythropoietin production by PDGFR-ß(+) cells.

PDGFR-ß-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-ß-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-ß(+) cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-ß. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-ß(+) cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-ß(+) cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-ß(+) cells but exerted no effect in mice lacking HIF-2α in PDGFR-ß(+) cells. These findings suggest that PDGFR-ß(+) cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-ß(+) cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest.

In vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses.

Piccolo is the largest known cytomatrix protein at active zones of chemical synapses. A growing number of studies on conventional chemical synapses assign Piccolo a role in the recruitment and integration of molecules relevant for both endo- and exocytosis of synaptic vesicles, the dynamic assembly of presynaptic F-actin, as well as the proteostasis of presynaptic proteins, yet a direct function in the structural organization of the active zone has not been uncovered in part due to the expression of multiple alternatively spliced isoforms. We recently identified Piccolino, a Piccolo splice variant specifically expressed in sensory ribbon synapses of the eye and ear. Here we down regulated Piccolino in vivo via an adeno-associated virus-based RNA interference approach and explored the impact on the presynaptic structure of mouse photoreceptor ribbon synapses. Detailed immunocytochemical light and electron microscopical analysis of Piccolino knockdown in photoreceptors revealed a hitherto undescribed photoreceptor ribbon synaptic phenotype with striking morphological changes of synaptic ribbon ultrastructure.

Evidence for a Clathrin-independent mode of endocytosis at a continuously active sensory synapse.

Synaptic vesicle exocytosis at chemical synapses is followed by compensatory endocytosis. Multiple pathways including Clathrin-mediated retrieval of single vesicles, bulk retrieval of large cisternae, and kiss-and-run retrieval have been reported to contribute to vesicle recycling. Particularly at the continuously active ribbon synapses of retinal photoreceptor and bipolar cells, compensatory endocytosis plays an essential role to provide ongoing vesicle supply. Yet, little is known about the mechanisms that contribute to endocytosis at these highly complex synapses. To identify possible specializations in ribbon synaptic endocytosis during different states of activity, we exposed mice to controlled lighting conditions and compared the distribution of endocytotic proteins at rod and cone photoreceptor, and ON bipolar cell ribbon synapses with light and electron microscopy. In mouse ON bipolar cell terminals, Clathrin-mediated endocytosis seemed to be the dominant mode of endocytosis at all adaptation states analyzed. In contrast, in mouse photoreceptor terminals in addition to Clathrin-coated pits, clusters of membranously connected electron-dense vesicles appeared during prolonged darkness. These clusters labeled for Dynamin3, Endophilin1, and Synaptojanin1, but not for AP180, Clathrin LC, and hsc70. We hypothesize that rod and cone photoreceptors possess an additional Clathrin-independent mode of vesicle retrieval supporting the continuous synaptic vesicle supply during prolonged high activity.

Identification and immunocytochemical characterization of Piccolino, a novel Piccolo splice variant selectively expressed at sensory ribbon synapses of the eye and ear.

Piccolo is one of the largest cytomatrix proteins present at active zones of chemical synapses, where it is suggested to play a role in recruiting and integrating molecules relevant for both synaptic vesicle exo- and endocytosis. Here we examined the retina of a Piccolo-mutant mouse with a targeted deletion of exon 14 in the Pclo gene. Piccolo deficiency resulted in its profound loss at conventional chemical amacrine cell synapses but retinal ribbon synapses were structurally and functionally unaffected. This led to the identification of a shorter, ribbon-specific Piccolo variant, Piccolino, present in retinal photoreceptor cells, bipolar cells, as well as in inner hair cells of the inner ear. By RT-PCR analysis and the generation of a Piccolino-specific antibody we show that non-splicing of intron 5/6 leads to premature translation termination and generation of the C-terminally truncated protein specifically expressed at active zones of ribbon synapse containing cell types. With in situ proximity ligation assays we provide evidence that this truncation leads to the absence of interaction sites for Bassoon, Munc13, and presumably also ELKS/CAST, RIM2, and the L-type Ca(2) (+) channel which exist in the full-length Piccolo at active zones of conventional chemical synapses. The putative lack of interactions with proteins of the active zone suggests a function of Piccolino at ribbon synapses of sensory neurons different from Piccolo's function at conventional chemical synapses.