The AE4 transporter mediates kidney acid-base sensing

The kidney plays a key role in the correction of systemic acid-base imbalances. Central for this regulation are the intercalated cells in the distal nephron, which secrete acid or base into the urine. How these cells sense acid-base disturbances is a long-standing question. Intercalated cells exclusively express the Na$^{+}$-dependent Cl$^{−}$/HCO$_{3}$$^{−}$ exchanger AE4 (Slc4a9). Here we show that AE4-deficient mice exhibit a major dysregulation of acid-base balance. By combining molecular, imaging, biochemical and integrative approaches, we demonstrate that AE4-deficient mice are unable to sense and appropriately correct metabolic alkalosis and acidosis. Mechanistically, a lack of adaptive base secretion via the Cl$^{−}$/HCO$_{3}$$^{−}$ exchanger pendrin (Slc26a4) is the key cellular cause of this derailment. Our findings identify AE4 as an essential part of the renal sensing mechanism for changes in acid-base status

Enzyme replacement therapy in mice lacking arylsulfatase B targets bone-remodeling cells, but not chondrocytes

Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient

Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H

Podocytes are an important part of the glomerular filtration barrier and the key player in the development of proteinuria, which is an early feature of complement mediated renal diseases. Complement factors are mainly liver-born and present in circulation. Nevertheless, there is a growing body of evidence for additional sites of complement protein synthesis, including various cell types in the kidney. We hypothesized that podocytes are able to produce complement components and contribute to the local balance of complement activation and regulation. To investigate the relevant balance between inhibiting and activating sides, our studies focused on complement factor H (CFH), an important complement regulator, and on C3, the early key component for complement activation. We characterized human cultured podocytes for the expression and secretion of activating and regulating complement factors, and analyzed the secretion pathway and functional activity. We studied glomerular CFH and C3 expression in puromycin aminonucleoside (PAN) -treated rats, a model for proteinuria, and the physiological mRNA-expression of both factors in murine kidneys. We found, that C3 and CFH were expressed in cultured podocytes and expression levels differed from those in cultivated glomerular endothelial cells. The process of secretion in podocytes was stimulated with interferon gamma and located in the Golgi apparatus. Cultured podocytes could initiate the complement cascade by the splitting of C3, which can be shown by the generation of C3a, a functional C3 split product. C3 contributed to external complement activation. Podocyte-secreted CFH, in conjunction with factor I, was able to split C3b. Podocytes derived from a patient with a CFH mutation displayed impaired cell surface complement regulation. CFH and C3 were synthesized in podocytes of healthy C57Bl/6-mice and were upregulated in podocytes of PAN treated rats. These data show that podocytes produce functionally active complement components, and could therefore influence the local glomerular complement activation and regulation. This modulating effect should therefore be considered in all diseases where glomerular complement activation occurs. Furthermore, our data indicate a potential novel role of podocytes in the innate immune system

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation

International audienceIon channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5′-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1b (IL-1b). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1b release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeu-tics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

Non-functional ubiquitin C-terminal hydrolase L1 drives podocyte injury through impairing proteasomes in autoimmune glomerulonephritis

In membranous nephropathy autoantibodies target podocytes of the kidney filter resulting in injury. Here the authors show that the ensuing proteostatic disturbances and proteinuria relate to aberrant interactions of non-functional UCH-L1 enzyme with the proteasome, curtailing its capacity.Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.Bio-organic Synthesi

UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation

Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN