Soluble Klotho protects against glomerular injury through regulation of ER stress response.

αKlotho (Klotho) has well established renoprotective effects; however, the molecular pathways mediating its glomerular protection remain incompletely understood. Recent studies have reported that Klotho is expressed in podocytes and protects glomeruli through auto- and paracrine effects. Here, we examined renal expression of Klotho in detail and explored its protective effects in podocyte-specific Klotho knockout mice, and by overexpressing human Klotho in podocytes and hepatocytes. We demonstrate that Klotho is not significantly expressed in podocytes, and transgenic mice with either a targeted deletion or overexpression of Klotho in podocytes lack a glomerular phenotype and have no altered susceptibility to glomerular injury. In contrast, mice with hepatocyte-specific overexpression of Klotho have high circulating levels of soluble Klotho, and when challenged with nephrotoxic serum have less albuminuria and less severe kidney injury compared to wildtype mice. RNA-seq analysis suggests an adaptive response to increased endoplasmic reticulum stress as a putative mechanism of action. To evaluate the clinical relevance of our findings, the results were validated in patients with diabetic nephropathy, and in precision cut kidney slices from human nephrectomies. Together, our data reveal that the glomeruloprotective effects of Klotho is mediated via endocrine actions, which increases its therapeutic potential for patients with glomerular diseases.

Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway.

Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.

Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes.

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.

Inactivation of mediator complex protein 22 in podocytes results in intracellular vacuole formation, podocyte loss and premature death.

Podocytes are critical for the maintenance of kidney ultrafiltration barrier and play a key role in the progression of glomerular diseases. Although mediator complex proteins have been shown to be important for many physiological and pathological processes, their role in kidney tissue has not been studied. In this study, we identified a mediator complex protein 22 (Med22) as a renal podocyte cell-enriched molecule. Podocyte-specific Med22 knockout mouse showed that Med22 was not needed for normal podocyte maturation. However, it was critical for the maintenance of podocyte health as the mice developed progressive glomerular disease and died due to renal failure. Detailed morphological analyses showed that Med22-deficiency in podocytes resulted in intracellular vacuole formation followed by podocyte loss. Moreover, Med22-deficiency in younger mice promoted the progression of glomerular disease, suggesting Med22-mediated processes may have a role in the development of glomerulopathies. This study shows for the first time that mediator complex has a critical role in kidney physiology.

GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway.

BACKGROUND: Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. METHODS: We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. RESULTS: We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-κB signaling. CONCLUSIONS: GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-κB signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.

Coro2b, a podocyte protein downregulated in human diabetic nephropathy, is involved in the development of protamine sulphate-induced foot process effacement.

Podocytes have an important role in the pathogenesis of diabetic nephropathy (DN). Podocyte foot process effacement, mediated largely by the actin-based cytoskeleton of foot processes, is commonly detected in DN and is believed to be a key pathogenic event in the development of proteinuria. In this study, we identified coronin 2b (Coro2b), a member of known actin-regulating proteins, the coronins, as a highly podocyte-enriched molecule located at the cytoplasmic side of the apical plasma membrane. Studies in human renal biopsies show that glomerular Coro2b expression is significantly down-regulated in patients with DN. Studies in knockout mice indicate that Coro2b is not required for the development or maintenance of the glomerular filtration barrier. Moreover, inactivation of Coro2b specifically in podocytes does not affect the outcome of nephropathy in a streptozotocin-induced diabetes model. However, Coro2b seems to modulate the reorganization of foot processes under pathological conditions as Coro2b knockout podocytes are partially protected from protamine sulfate perfusion-induced foot process effacement. Taken together, our study suggests a role for Coro2b in the pathogenesis of glomerulopathies. Further studies regarding the involvement of Coro2b in podocyte health and diseases are warranted.

FYVE domain-containing protein ZFYVE28 regulates EGFR-signaling in podocytes but is not critical for the function of filtration barrier in mice.

The kidney ultrafiltration barrier is formed of endothelial cells, the glomerular basement membrane and podocytes. Podocytes have a central role in normal physiology and disease pathogenesis of the glomerulus. Signaling through epidermal growth factor receptor (EGFR) in podocytes mediates development of many glomerular disease processes. In this work, we have identified zinc finger FYVE-type containing 28 (ZFYVE28) as a novel highly podocyte-enriched gene. We localize ZFYVE28 in podocyte foot processes in adult kidney. During glomerulogenesis, Zfyve28 is first expressed at the early capillary loop glomerulus. In cultured podocytes, we show that overexpression of ZFYVE28 promotes EGF-signaling, possibly by up-regulating EGFR expression and by modulating its localization. To study the role of ZFYVE28 in vivo, we generated both conventional and podocyte-specific knockout mouse lines. Kidneys developed normally in ZFYVE28-deficient mice. In adult mice, the absence of ZFYVE28 did not affect the maintenance of the filtration barrier. Moreover, ZFYVE28-deficiency did not affect the outcome of glomerular damage induced by injection of nephrotoxic serum. Taken together, we have identified Zfyve28 as a new molecular component of podocyte foot processes and show that it mediates EGF-signaling in podocytes. However, ZFYVE28 is not essential for the development or maintenance of the glomerulus filtration barrier.

Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic TH17 cells.

The cellular sources of interleukin 6 (IL-6) that are relevant for differentiation of the TH17 subset of helper T cells remain unclear. Here we used a novel strategy for the conditional deletion of distinct IL-6-producing cell types to show that dendritic cells (DCs) positive for the signaling regulator Sirpα were essential for the generation of pathogenic TH17 cells. Using their IL-6 receptor α-chain (IL-6Rα), Sirpα+ DCs trans-presented IL-6 to T cells during the process of cognate interaction. While ambient IL-6 was sufficient to suppress the induction of expression of the transcription factor Foxp3 in T cells, trans-presentation of IL-6 by DC-bound IL-6Rα (called 'IL-6 cluster signaling' here) was needed to prevent premature induction of interferon-γ (IFN-γ) expression in T cells and to generate pathogenic TH17 cells in vivo. Our findings should guide therapeutic approaches for the treatment of TH17-cell-mediated autoimmune diseases.

Proteolytic Cleavage Governs Interleukin-11 Trans-signaling.

Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11.

T-cell immunoglobulin and mucin domain 2 (TIM-2) is a target of ADAM10-mediated ectodomain shedding.

T-cell immunoglobulin and mucin domain (TIM)-2 is expressed on activated B cells. Here, we provide evidence that murine TIM-2 is a target of ADAM10-mediated ectodomain shedding, resulting in the generation of a soluble form of TIM-2. We identified ADAM10 but not ADAM17 as the major sheddase of TIM-2, as shown by pharmacological ADAM10 inhibition and with ADAM10-deficient and ADAM17-deficient murine embryonic fibroblasts. Ionomycin-induced or 2'(3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt-induced shedding of TIM-2 was abrogated by deletion of 10 juxtamembrane amino acids from the stalk region but not by deletion of two further N-terminally located blocks of 10 amino acids, indicating a membrane-proximal cleavage site. TIM-2 lacking the intracellular domain was cleaved after ionomycin or 2' (3')-O-(4-benzoylbenzoyl) ATP triethylammonium salt treatment, indicating that this domain was not involved in the regulation of ectodomain shedding. Moreover, TIM-2 shedding was negatively controlled by calmodulin. Shed and soluble TIM-2 interacted with H-ferritin. In summary, we describe TIM-2 as a novel target for ADAM10-mediated ectodomain shedding, and reveal the involvement of ADAM proteases in cellular iron homeostasis.

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine.

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.

A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are major sheddases of T cell immunoglobulin and mucin domain 3 (Tim-3).

T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4(+) and CD8(+) effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu(181)-Asp(190) of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14(+) monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14(+) monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14(+) monocytes.

Effects of blockade of peripheral interleukin-6 trans-signaling on hippocampus-dependent and independent memory in mice.

Besides functions of the interleukin-6 (IL-6)/gp130 cytokine family in immunology, IL-6 signaling has influence on memory processes. IL-6 acts on target cells via a membrane-bound IL-6 receptor (IL-6R) and subsequent association with the signal-transducing protein gp130. While gp130 is expressed on all cells in the body, IL-6R is expressed in only on few cells such as hepatocytes and some leukocytes. Cells lacking IL-6R were shown not to be responsive to the cytokine. Interestingly, a soluble form of the IL-6R in complex with IL-6 can stimulate cells that do not express the membrane-bound IL-6R. This signaling pathway has been called IL-6 trans-signaling. IL-6 trans-signaling can specifically be blocked by a soluble gp130 protein (sgp130Fc) without affecting IL-6 classic signaling via the membrane-bound IL-6R. Transgenic mice expressing sgp130Fc in the blood, but not in the central nervous system, were analyzed for hippocampus-dependent and independent memory, together with exploratory- and anxiety-related behavior. Transgenic animals did not show impaired hippocampus-dependent or independent learning and memory. However, compared to wild-type animals, they showed reduced exploratory behavior and an increased thermal pain threshold, indicating that these effects depend on IL-6 trans-signaling. These results bear important consequences for the therapeutic blockade of IL-6 activity in autoimmune diseases.