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BACKGROUND: Primary membranous nephropathy (MN) is an autoimmune kidney disease in which immune complexes are deposited beneath the epithelium in the glomeruli. The condition introduces a high risk for end-stage kidney disease. Seventy to 80 percent of patients with MN have circulating antibodies against phospholipase A2 receptor 1 (PLA2R1), and levels correlate with treatment response and prognosis. However, experimental evidence that human anti-PLA2R1 antibodies induce MN has been elusive.METHODS: In passive transfer experiments, minipigs received plasma or purified IgG from patients with PLA2R1-associated MN or from healthy controls. Anti-PLA2R1 antibodies and proteinuria were monitored using Western blot, ELISA, and Coomassie staining. Kidney tissues were analyzed using immunohistochemistry, immunofluorescence, electron microscopy, and proteomic analyses.RESULTS: Minipigs, like humans, express PLA2R1 on podocytes. Human anti-PLA2R1 antibodies bound to minipig PLA2R1 in vitro and in vivo. Passive transfer of human anti-PLA2R1 antibodies from patients with PLA2R1-associated MN to minipigs led to histological characteristics of human early-stage MN, activation of components of the complement cascade, and low levels of proteinuria. We observed development of an autologous, later phase of disease.CONCLUSIONS: A translational approach from humans to minipigs showed that human anti-PLA2R1 antibodies are pathogenic in MN, although in the heterologous phase of disease only low-level proteinuria developed.

Calcimimetic agents allosterically increase the calcium ion sensitivity of the calcium-sensing receptor (CaSR), which is expressed in the tubular system and to a lesser extent in podocytes. Activation of this receptor can reduce glomerular proteinuria and structural damage in proteinuric animal models. However, the precise role of the podocyte CaSR remains unclear. Here, a CaSR knockdown in cultured murine podocytes and a podocyte-specific CaSR knockout in BALB/c mice were generated to study its role in proteinuria and kidney function. Podocyte CaSR knockdown abolished the calcimimetic R-568 mediated calcium ion-influx, disrupted the actin cytoskeleton, and reduced cellular attachment and migration velocity. Adriamycin-induced proteinuria enhanced glomerular CaSR expression in wild type mice. Albuminuria, podocyte foot process effacement, podocyte loss and glomerular sclerosis were significantly more pronounced in adriamycin-treated podocyte-specific CaSR knockout mice compared to wild type littermates. Co-treatment of wild type mice with adriamycin and the calcimimetic cinacalcet reduced proteinuria in wild type, but not in podocyte-specific CaSR knockout mice. Additionally, four children with nephrotic syndrome, whose parents objected to glucocorticoid therapy, were treated with cinacalcet for one to 33 days. Proteinuria declined transiently by up to 96%, serum albumin increased, and edema resolved. Thus, activation of podocyte CaSR regulates key podocyte functions in vitro and reduced toxin-induced proteinuria and glomerular damage in mice. Hence, our findings suggest a potential novel role of CaSR signaling in control of glomerular disease.

γδ T cells are involved in the control of

Background Renal denervation (RDN) is an invasive intervention to treat drug-resistant arterial hypertension. Its therapeutic value is contentious. Here we examined the effects of RDN on inflammatory and infectious kidney disease models in mice. Methods Mice were unilaterally or bilaterally denervated, or sham operated, then three disease models were induced: nephrotoxic nephritis (NTN, a model for crescentic GN), pyelonephritis, and acute endotoxemic kidney injury (as a model for septic kidney injury). Analytical methods included measurement of renal glomerular filtration, proteinuria, flow cytometry of renal immune cells, immunofluorescence microscopy, and three-dimensional imaging of optically cleared kidney tissue by light-sheet fluorescence microscopy followed by algorithmic analysis. Results Unilateral RDN increased glomerular filtration in denervated kidneys, but decreased it in the contralateral kidneys. In the NTN model, more nephritogenic antibodies were deposited in glomeruli of denervated kidneys, resulting in stronger inflammation and injury in denervated compared with contralateral nondenervated kidneys. Also, intravenously injected LPS increased neutrophil influx and inflammation in the denervated kidneys, both after unilateral and bilateral RDN. When we induced pyelonephritis in bilaterally denervated mice, both kidneys contained less bacteria and neutrophils. In unilaterally denervated mice, pyelonephritis was attenuated and intrarenal neutrophil numbers were lower in the denervated kidneys. The nondenervated contralateral kidneys harbored more bacteria, even compared with sham-operated mice, and showed the strongest influx of neutrophils. Conclusions Our data suggest that the increased perfusion and filtration in denervated kidneys can profoundly influence concomitant inflammatory diseases. Renal deposition of circulating nephritic material is higher, and hence antibody- and endotoxin-induced kidney injury was aggravated in mice. Pyelonephritis was attenuated in denervated murine kidneys, because the higher glomerular filtration facilitated better flushing of bacteria with the urine, at the expense of contralateral, nondenervated kidneys after unilateral denervation.

Protein homeostasis strongly depends on the targeted and selective removal of unneeded or flawed proteins, of protein aggregates, and of damaged or excess organelles by the two main intracellular degradative systems, namely the ubiquitin proteasomal system (UPS) and the autophagosomal lysosomal system. Despite representing completely distinct mechanisms of degradation, which underlie differing regulatory mechanisms, growing evidence suggests that the UPS and autophagy strongly interact especially in situations of overwhelming and impairment, and that both are involved in podocyte proteostasis and in the pathogenesis of podocyte injury. The differential impact of autophagy and the UPS on podocyte biology and on podocyte disease development and progression is not understood. Recent advances in understanding the role of the UPS and autophagy in podocyte biology are reviewed here.

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. In vivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10–12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer’s disease and cancer treatments.

Immune-mediated glomerular diseases are characterized by infiltration of T cells, which accumulate in the periglomerular space and tubulointerstitium in close contact to proximal and distal tubuli. Recent studies described proximal tubular epithelial cells (PTECs) as renal non-professional antigen-presenting cells that stimulate CD4+ T-cell activation. Whether PTECs have the potential to induce activation of CD8+ T cells is less clear. In this study, we aimed to investigate the capacity of PTECs for antigen cross-presentation thereby modulating CD8+ T-cell responses. We showed that PTECs expressed proteins associated with cross-presentation, internalized soluble antigen via mannose receptor-mediated endocytosis, and generated antigenic peptides by proteasomal degradation. PTECs induced an antigen-dependent CD8+ T-cell activation in the presence of soluble antigen in vitro. PTEC-activated CD8+ T cells expressed granzyme B, and exerted a cytotoxic function by killing target cells. In murine lupus nephritis, CD8+ T cells localized in close contact to proximal tubuli. We determined enhanced apoptosis in tubular cells and particularly PTECs up-regulated expression of cleaved caspase-3. Interestingly, induction of apoptosis in the inflamed kidney was reduced in the absence of CD8+ T cells. Thus, PTECs have the capacity for antigen cross-presentation thereby inducing cytotoxic CD8+ T cells in vitro, which may contribute to the pathology of immune-mediated glomerulonephritis.

A wide spectrum of immunological functions has been attributed to Interleukin 9 (IL-9), including effects on the survival and proliferation of immune and parenchymal cells. In recent years, emerging evidence suggests that IL-9 expression can promote tissue repair in inflammatory conditions. However, data about the involvement of IL-9 in kidney tissue protection is very limited. Here, we investigated the role of IL-9 in Adriamycin-induced nephropathy (AN), a mouse model for proteinuric chronic kidney disease. Compared to wild type mice, IL-9 knockout (Il9−/−) mice with AN displayed accelerated development of proteinuria, aggravated glomerulosclerosis and deterioration of kidney function. At an early stage of disease, the Il9−/− mice already displayed a higher extent of glomerular podocyte injury and loss of podocyte number compared to wild type mice. In the kidney, T cells and innate lymphoid cells produced IL-9. However, selective deficiency of IL-9 in the innate immune system in Il9−/−Rag2−/− mice that lack T and B cells did not alter the outcome of AN, indicating that IL-9 derived from the adaptive immune system was the major driver of tissue protection in this model. Mechanistically, we could show that podocytes expressed the IL-9 receptor in vivo and that IL-9 signaling protects podocytes from Adriamycin-induced apoptosis in vitro. Finally, in vivo treatment with IL-9 effectively protected wild type mice from glomerulosclerosis and kidney failure in the AN model. The detection of increased serum IL-9 levels in patients with primary focal and segmental glomerulosclerosis further suggests that IL-9 production is induced by glomerular injury in humans. Thus, IL-9 confers protection against experimental glomerulosclerosis, identifying the IL-9 pathway as a potential therapeutic target in proteinuric chronic kidney disease.

The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments in vivo.

Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation in vitro. However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions.

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. ispartof: HUMAN MOLECULAR GENETICS vol:29 issue:5 pages:803-816 ispartof: location:England status: published

Background The glomerulus comprises podocytes, mesangial cells, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods We separated glomerular cell types from wild-type or mT/mG mice via a novel FACS approach, and validated their purity. Cell type proteomes were compared between strains, ages, and sex. We applied timMEP to the podocyte-targeting, immunologic, THSD7A-associated, model of membranous nephropathy. Results timMEP enabled protein-biochemical analyses of podocytes, mesangial cells, and endothelial cells derived from reporter-free mice, and allowed for the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. We identified marker proteins for mesangial and endothelial proteins, and outlined protein-based, potential communication networks and phosphorylation patterns. The analysis detected cell type-specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response, chiefly in podocytes, that could not be detected by bulk glomerular analyses. The combination of proteomics with super-resolution imaging revealed a specific loss of slit diaphragm, but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. Conclusion timMEP enables glomerular cell type-resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way toward the comprehensive and systematic characterization of glomerular cell biology.

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow–derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1–deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.

Background About 3%–5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain–containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease. Methods Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays. Results We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV–coated plates but also, with decreased ability to migrate. Conclusions Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm’s permeability to protein.

In recent years, the cytokine interleukin (IL)-22 attracted considerable attention due to its important immunoregulatory function in barrier tissues, such as the gut, lung, and skin. Although a regenerative role of IL-22 in renal tubular damage has been demonstrated, the role of IL-22 in the immunopathogenesis of glomerular injury is still unknown. Here, we demonstrate that the IL-22 receptor is expressed in the glomerular compartment of the kidney and that IL-22 expression increases in the renal cortex after induction of glomerular injury in a mouse model for crescentic glomerulonephritis (cGN, nephrotoxic nephritis). We identified γδ T cells and TH17 cells as major sources for IL-22 in the nephritic kidney. However, neither genetic or antibody-mediated deletion of IL-22 nor genetic deficiency in its endogenous inhibitor IL-22Rα2 (IL-22 binding protein) resulted in substantial phenotypic differences in mice with cGN with respect to crescent formation, tubulointerstitial damage, and kidney function impairment. Similarly, we did not observe significant differences between wild-type or IL-22-deficient mice in a mouse model of secondary focal and segmental glomerulosclerosis (adriamycin-induced nephropathy). As shown previously, we detected concomitant upregulation of IL-17A and IFN-γ production by T cells during the course of cGN, providing alternative cytokine pathways that mediate glomerular injury in this model. In conclusion, we show here that endogenous IL-22 expression is redundant in different forms of glomerular injury, indicating that the IL-22-directed therapies that are being tested in various human diseases might not affect the kidney in patients with glomerular disease.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1–deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1–deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1–deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1–deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1–dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.

Background Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. Methods Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. Results ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. Conclusions ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.

Besides its involvement in blood and bone physiology, the kidney's main function is to filter substances and thereby regulate the electrolyte composition of body fluids, acid-base balance and toxin removal. Depending on underlying conditions, the nephron must undergo remodeling and cellular adaptations. The proteolytic removal of cell surface proteins via ectodomain shedding by A Disintegrin and Metalloproteases (ADAMs) is of importance for the regulation of cell-cell and cell-matrix adhesion of renal cells. ADAM10 controls glomerular and tubule development in a Notch1 signaling-dependent manner and regulates brush border composition. ADAM17 regulates the renin angiotensin system and is together with ADAM10 involved in calcium phosphate homeostasis. In kidney disease ADAMs, especially ADAM17 contribute to inflammation through their involvement in IL-6 trans-signaling, Notch-, epithelial growth factor receptor-, and tumor necrosis factor α signaling. ADAMs are interesting drug targets to reduce the inflammatory burden, defective cell adhesion and impaired signaling pathways in kidney diseases.

Immune cells at sites of inflammation are continuously activated by local antigens and cytokines, and regulatory mechanisms must be enacted to control inflammation. The stepwise hydrolysis of extracellular ATP by ectonucleotidases CD39 and CD73 generates adenosine, a potent immune suppressor. Here we report that human effector CD8 T cells contribute to adenosine production by releasing CD73-containing extracellular vesicles upon activation. These extracellular vesicles have AMPase activity, and the resulting adenosine mediates immune suppression independently of regulatory T cells. In addition, we show that extracellular vesicles isolated from the synovial fluid of patients with juvenile idiopathic arthritis contribute to T cell suppression in a CD73-dependent manner. Our results suggest that the generation of adenosine upon T cell activation is an intrinsic mechanism of human effector T cells that complements regulatory T cell-mediated suppression in the inflamed tissue. Finally, our data underscore the role of immune cell-derived extracellular vesicles in the control of immune responses., Ectonucleotidases associated to regulatory T cells are known modulators in the inflammatory environment. Here the authors describe CD8 T cell-derived extracellular vesicles bearing CD73 and suggest they function as an additional intrinsic modulator of immune responses.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.

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.

A key finding supporting a causal role of the immune system in the pathogenesis of hypertension is the observation that RAG1 knockout mice on a C57Bl/6J background (B6.Rag1 −/ − ), which lack functional B and T cells, develop a much milder hypertensive response to Ang II (angiotensin II) than control C57Bl/6J mice. Here, we report that we never observed any Ang II resistance of B6.Rag1 −/− mice purchased directly from the Jackson Laboratory as early as 2009. B6.Rag1 −/− mice displayed nearly identical blood pressure increases monitored via radiotelemetry and hypertensive end-organ damage in response to different doses of Ang II and different levels of salt intake (0.02%, 0.3%, and 3% NaCl diet). Similarly, restoration of T-cell immunity by adoptive cell transfer did not affect the blood pressure response to Ang II in B6.Rag1 −/− mice. Full development of the hypertension-resistant phenotype in B6.Rag1 −/− mice appears to depend on the action of yet unidentified nongenetic modifiers in addition to the absence of functional T cells.

²²⁵Ac-PSMA-617 targeted-therapy has demonstrated efficacy in 75-85% of patients; however, responses are not durable. We aimed to establish translatable mouse models of disseminated prostate cancer (PCa) to evaluate effectiveness of ²²⁵Ac-PSMA-617 at various disease stages. Methods: C4-2, C4-2B, or 22Rv1 cells were injected into the left ventricle of male NSG mice. Disease progression was monitored using bioluminescence imaging (BLI). For treatment, mice were injected with 40 kBq ²²⁵Ac-PSMA-617 at one (early treatment cohort) or three weeks (late treatment cohort) post-inoculation. Treatment efficacy was monitored by BLI of whole-body tumor burden. Mice were sacrificed based on body conditioning score. Results: C4-2 cells yielded metastases in liver, lungs, spleen, stomach, bones, and brain - achieving a clinically relevant model of widespread metastatic disease. The disease burden in the early treatment cohort was stable over 27 weeks in 5/9 mice and progressive in 4/9 mice. These mice were sacrificed due to brain metastases. Median survival of the late treatment cohort was superior to controls (13 vs. 7 weeks; p

Purpose: Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor. Experimental Design: RM1 cells expressing different levels of PSMA (PSMA−, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA− RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1–3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified. Results: 177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups. Conclusions: In the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT. See related commentary by Ravi Kumar and Hofman, p. 2774

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Background The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. Methods MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. Results Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. Conclusions Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

In immune-mediated glomerular diseases like crescentic glomerulonephritis (cGN), inflammatory CD4

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist–, miR193a inhibitor–, and interferon-γ–induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA–silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

Adaptive and innate immune responses contribute to hypertension and hypertensive end-organ damage. Here, we determined the role of anaphylatoxin C5a, a major inflammatory effector of the innate immune system that is generated in response to complement activation, in hypertensive end-organ damage. For this purpose, we assessed the phenotype of C5a receptor 1 (C5aR1)-deficient mice in ANG II-induced renal and cardiac injury. Expression of C5aR1 on infiltrating and resident renal as well as cardiac cells was determined using a green fluorescent protein (GFP)-C5aR1 reporter knockin mouse. Flow cytometric analysis of leukocytes isolated from the kidney of GFP-C5aR1 reporter mice showed that 28% of CD45-positive cells expressed C5aR1. Dendritic cells were identified as the major C5aR1-expressing population (88.5%) followed by macrophages and neutrophils. Using confocal microscopy, we detected C5aR1 in the kidney mainly on infiltrating cells. In the heart, only infiltrating cells stained C5aR1 positive. To evaluate the role of C5aR1 deficiency in hypertensive injury, an aggravated model of hypertension was used. Unilateral nephrectomy was performed followed by infusion of ANG II (1.5 ng·g−1·min−1) and salt in wild-type ( n = 34) and C5aR1-deficient mice ( n = 32). C5aR1-deficient mice exhibited less renal injury, as evidenced by significantly reduced albuminuria. In contrast, cardiac injury was accelerated with significantly increased cardiac fibrosis and heart weight in C5aR1-deficient mice after ANG II infusion. No effect was found on blood pressure. In summary, the C5a:C5aR1 axis drives end-organ damage in the kidney but protects from the development of cardiac fibrosis and hypertrophy in experimental ANG II-induced hypertension.

Background Antibody-mediated rejection is a leading cause for renal transplant loss. Rodent models are useful to dissect pathomechanisms and to develop treatment strategies. Although used for decades as a model, glomerular histopathological findings of Fischer-344 kidneys transplanted into Lewis rats have never been comprehensively described. Methods Kidneys from Fischer-344 rats were transplanted into Lewis rats as life-sustaining allografts without immunosuppression. Lewis isografts and normal Fischer-344 kidneys served as controls. Grafts were harvested at 9 days, 6 and 26 weeks. Histopathological examination included light microscopy, immunohistochemistry, and morphometry. Findings were compared with 51 human biopsies with transplant glomerulopathy. Results Most glomerular findings in rat allografts resembled human acute and chronic antibody-mediated rejection with glomerulitis, microthrombosis, microaneurysms, glomerular hypertrophy, podocyte loss, glomerular basement membrane splitting, and secondary focal and segmental glomerulosclerosis. In line with previous reports on nonendothelial antigens, glomerular immunoglobulin and C4d deposition was mostly nonendothelial. Only in 26-week allografts, we found mesangial and subendothelial immune complex-type electron-dense deposits. Similar deposits were found in 8 of 51 human biopsies with transplant glomerulopathy after rigorous exclusion of immune complexes of other cause, particularly recurrent glomerulonephritis and hepatitis C. Conclusions Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.

The adult kidney has a remarkable ability to survive injury and restore function despite a limited turnover of cells under physiologic conditions. This accounts both for the tubular and to a lesser extent for the glomerular compartment. It is an ongoing debate whether renal repair is carried out by self-duplication/de-differentiation of mature resident renal cells, or by specialized renal progenitors residing in specific niches or by circulating bone marrow-derived stem cells. In this review, the existence of renal progenitor cells and their contribution for regeneration of the tubular and the glomerular compartment are discussed, highlighting landmark publications of recent years.

Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFNγ treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.

The role of CX

The principal function of glomeruli is to filter blood through a highly-specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial ADAM10 (a disintegrin and metalloprotease 10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR, and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.

Summary: Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons. : Schmiesing et al. show that the lack of GCDH results in glutarylation of mitochondrial proteins in glial cells affecting amino acid metabolism and the tricarboxylic acid cycle. They identify glutamate dehydrogenase as a target suppressed by glutarylation that is linked to glial glutamate metabolism and anaplerosis in GCDH-deficient neuronal cells. Keywords: glutaryl-CoA-dehydrogenase, glutaryl-proteome, glutamate dehydrogenase, carboanhydrase 5B, protein-protein interaction, immunogold electron microscopy, glutaric aciduria type 1/brain

Thrombospondin type 1 domain-containing 7A (THSD7A) is a target for autoimmunity in patients with membranous nephropathy (MN). Circulating autoantibodies from patients with THSD7A-associated MN have been demonstrated to cause MN in mice. However, THSD7A-associated MN is a rare disease, preventing the use of patient antibodies for larger experimental procedures. Therefore, we generated antibodies against the human and mouse orthologs of THSD7A in rabbits by coimmunization with the respective cDNAs. Injection of these anti-THSD7A antibodies into mice induced a severe nephrotic syndrome with proteinuria, weight gain, and hyperlipidemia. Immunofluorescence analyses revealed granular antigen-antibody complexes in a subepithelial location along the glomerular filtration barrier 14 days after antibody injection, and immunohistochemistry for rabbit IgG and THSD7A as well as ultrastructural analyses showed the typical characteristics of human MN. Mice injected with purified IgG from rabbit serum that was taken before immunization failed to develop any of these changes. Notably, MN developed in the absence of detectable complement activation, and disease was strain dependent. In vitro, anti-THSD7A antibodies caused cytoskeletal rearrangement and activation of focal adhesion signaling. Knockdown of the THSD7A ortholog, thsd7aa, in zebrafish larvae resulted in altered podocyte differentiation and impaired glomerular filtration barrier function, with development of pericardial edema, suggesting an important role of THSD7A in glomerular filtration barrier integrity. In summary, our study introduces a heterologous mouse model that allows further investigation of the molecular events that underlie MN.

Myeloperoxidase (MPO) is an enzyme expressed in neutrophils and monocytes/macrophages. Beside its well-defined role in innate immune defence, it may also be responsible for tissue damage. To identify the role of MPO in the progression of chronic kidney disease (CKD), we investigated CKD in a model of renal ablation in MPO knockout and wild-type mice. CKD was induced by 5/6 nephrectomy. Mice were followed for 10 wk to evaluate the impact of MPO deficiency on renal morbidity. Renal ablation induced CKD in wild-type mice with increased plasma levels of MPO compared with controls. No difference was found between MPO-deficient and wild-type mice regarding albuminuria 1 wk after renal ablation, indicating similar acute responses to renal ablation. Over the next 10 wk, however, MPO-deficient mice developed significantly less albuminuria and glomerular injury than wild-type mice. This was accompanied by a significantly lower renal mRNA expression of the fibrosis marker genes plasminogen activator inhibitor-I, collagen type III, and collagen type IV as well as matrix metalloproteinase-2 and matrix metalloproteinase-9. MPO-deficient mice also developed less renal inflammation after renal ablation, as indicated by a lower infiltration of CD3-positive T cells and F4/80-positive monocytes/macrophages compared with wild-type mice. In vitro chemotaxis of monocyte/macrophages isolated from MPO-deficient mice was impaired compared with wild-type mice. No significant differences were observed for mortality and blood pressure after renal ablation. In conclusion, these results demonstrate that MPO deficiency ameliorates renal injury in the renal ablation model of CKD in mice.

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokine-dependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5(-/-) mice and analyzed their immune response against M. tuberculosis. Both Cxcr2(-/-) mice and Cxcl5(-/-) mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5(-/-) mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

The metabolic disorder glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of the pathologic metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in blood, urine and tissues. Affected patients are prone to metabolic crises developing during catabolic conditions, with an irreversible destruction of striatal neurons and a subsequent dystonic–dyskinetic movement disorder. The pathogenetic mechanisms mediated by GA and 3OHGA have not been fully characterized. Recently, we have shown that GA and 3OHGA are translocated through membranes via sodium-dependent dicarboxylate cotransporter (NaC) 3, and organic anion transporters (OATs) 1 and 4. Here, we show that induced metabolic crises in Gcdh−/− mice lead to an altered renal expression pattern of NaC3 and OATs, and the subsequent intracellular GA and 3OHGA accumulation. Furthermore, OAT1 transporters are mislocalized to the apical membrane during metabolic crises accompanied by a pronounced thinning of proximal tubule brush border membranes. Moreover, mitochondrial swelling and increased excretion of low molecular weight proteins indicate functional tubulopathy. As the data clearly demonstrate renal proximal tubule alterations in this GA1 mouse model during induced metabolic crises, we propose careful evaluation of renal function in GA1 patients, particularly during acute crises. Further studies are needed to investigate if these findings can be confirmed in humans, especially in the long-term outcome of affected patients.

Summary Th17 cells are most abundant in the gut, where their presence depends on the intestinal microbiota. Here, we examined whether intestinal Th17 cells contribute to extra-intestinal Th17 responses in autoimmune kidney disease. We found high frequencies of Th17 cells in the kidneys of patients with antineutrophil cytoplasmatic antibody (ANCA)-associated glomerulonephritis. We utilized photoconversion of intestinal cells in Kaede mice to track intestinal T cell mobilization upon glomerulonephritis induction, and we found that Th17 cells egress from the gut in a S1P-receptor-1-dependent fashion and subsequently migrate to the kidney via the CCL20/CCR6 axis. Depletion of intestinal Th17 cells in germ-free and antibiotic-treated mice ameliorated renal disease, whereas expansion of these cells upon Citrobacter rodentium infection exacerbated pathology. Thus, in some autoimmune settings, intestinal Th17 cells migrate into target organs, where they contribute to pathology. Targeting the intestinal Th17 cell “reservoir” may present a therapeutic strategy for these autoimmune disorders., Graphical Abstract, Highlights • Pathogenic TH17 cells migrate from the gut to the kidney in autoimmunity • TH17 cells egress the intestine in a S1PR1-dependent manner in glomerulonephritis • Targeting microbiota-induced TH17 cells ameliorates extraintestinal TH17 responses, By photolabelling intestinal cells, Krebs and colleagues provide direct evidence that microbiota-induced TH17 cells egress from the gut S1PR1-dependently and infiltrate the kidney via CCL20/CCR6 in immune-mediated diseases. This finding will build the basis for therapies targeting the intestinal TH17 cell “reservoir” to treat extraintestinal TH17 autoimmunity.

Ubiquitin C-terminal hydrolase L1 (UCH-L1), a key protease of the ubiquitin-proteasome system (UPS), is associated with neurodegenerative diseases and cancer. Recently, de novo expression of UCH-L1 was described in podocytes in patients with membranous nephropathy (MN), in which UCH-L1 expression correlated with increased ubiquitin content. The objective of the present study was to investigate the role of UCH-L1 in ubiquitin homeostasis and proteasomal degradation in a rat model of MN. After disease induction, UCH-L1 expression increased in podocytes and coincided with decreased glomerular monoubiquitin content. After an initial increase in proteasomal activity, the UPS was impaired. In addition to an increase of ubiquitin in podocytes, aggregates were observed 1 year after disease induction, as in MN in human beings. Inhibition of UCH-L1 hydrolase function in MN reduced UPS impairment and ameliorated proteinuria. In contrast, inhibition of proteasomal activity enhanced UPS impairment, resulting in increased proteinuria. Stable UCH-L1 overexpression in cultured podocytes resulted in accumulation of monoubiquitin and polyubiquitin proteins. In contrast, stable knock-down of UCH-L1 reduced monoubiquitin and polyubiquitin proteins and significantly increased proteasomal activity, indicating that the observed effects in rat MN also occurred in cultured podocytes. These data demonstrate that UCH-L1 activity results in polyubiquitin accumulation, proteasome inhibition, and disease aggravation in experimental models of MN.

A key event in the progression of glomerular disease is podocyte loss that leads to focal and segmental glomerulosclerosis (FSGS). Because adult podocytes are postmitotic cells, podocyte replacement by bone marrow–derived progenitors could prevent podocytopenia and FSGS. This study uses double immunofluorescence for Wilms' tumor-1 and enhanced green fluorescent protein (eGFP) to examine whether an eGFP-positive bone marrow transplant can replace podocytes under normal circumstances and in 3 different rat models of FSGS: puromycin aminoglycoside nephropathy, subtotal nephrectomy, and uninephrectomy. Bone marrow engraftment was successful, with more than 70% eGFP-positive cells and virtually normal histologic findings. No bone marrow transplant–derived podocytes were found in four control rats after transplantation, in nine rats at up to 10 weeks after puromycin aminoglycoside nephropathy induction, in three rats 23 days after subtotal nephrectomy, and in six rats up to 21 days after uninephrectomy. A total of 2200 glomeruli with 14,474 podocytes were evaluated in all groups. Thus, podocyte replacement by bone marrow–derived cells does not contribute to podocyte turnover in rats, even in models of podocyte damage. This is in contrast to previous studies in mice, in which bone marrow–derived podocytes were found. Further studies will address this discrepancy, which could be explained by species differences or by predominant podocyte regeneration from a parietal epithelial cell niche.

Podocyte damage is the basis of many glomerular diseases with ultrastructural changes and decreased expression of components of the slit diaphragm such as nephrin and podocin. Under physiological conditions it is likely that the slit diaphragm underlies permanent renewal processes to indemnify its stability in response to changes in filtration pressure. This would require constant reorganization of the podocyte foot process and the renewal of slit diaphragm components. Thus far, the mechanisms underlying the turnover of slit diaphragm proteins are largely unknown. In this manuscript we examined a mechanism of nephrin endocytosis via CIN85/Ruk(L)-mediated ubiquitination. We can demonstrate that the loss of nephrin expression and onset of the proteinuria in CD2AP(-/-) mice correlates with an increased accumulation of ubiquitinated proteins and expression of CIN85/Ruk(L) in podocytes. In cultured murine podocytes CD2AP deficiency leads to an early ubiquitination of nephrin and podocin after stimulation with fibroblast growth factor-4. Binding assays with different CIN85/Ruk isoforms and mutants showed that nephrin and podocin are binding to the coiled-coil domain of CIN85/Ruk(L). We found that in the presence of CIN85/Ruk(L), which is involved in down-regulation of receptor-tyrosine kinases, nephrin is internalized after stimulation with fibroblast growth factor-4. Interestingly, coexpression of CIN85/Ruk(L) with CD2AP led to a decreased binding of CIN85/Ruk(L) to nephrin and podocin, which indicates a functional competition between CD2AP and CIN85/Ruk(L). Our results support a novel role for CIN85/Ruk(L) in slit diaphragm turnover and proteinuria.

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 ( E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-Pitype 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a “branchless” UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFRα1 (a receptor for GDNF) to tips. Such “quality control” by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

Infiltration of T cells into the kidney is a typical feature of human and experimental lupus nephritis that contributes to renal tissue injury. The chemokine receptor CXCR3 is highly expressed on Th1 cells and is supposed to be crucial for their trafficking into inflamed tissues. In this study, we explored the functional role of CXCR3 using the MRL/MpJ-Faslpr (MRL/lpr) mouse model of systemic lupus erythematosus that closely resembles the human disease. CXCR3−/− mice were generated and backcrossed into the MRL/lpr background. Analysis of 20-wk-old CXCR3−/− MRL/lpr mice showed amelioration of nephritis with reduced glomerular tissue damage and decreased albuminuria and T cell recruitment. Most importantly, not only the numbers of renal IFN-γ-producing Th1 cells, but also of IL-17-producing Th17 cells were significantly reduced. Unlike in inflamed kidneys, there was no reduction in the numbers of IFN-γ- or IL-17-producing T cells in spleens, lymph nodes, or the small intestine of MRL/lpr CXCR3−/− mice. This observation suggests impaired trafficking of effector T cells to injured target organs, rather than the inability of CXCR3−/− mice to mount efficient Th1 and Th17 immune responses. These findings show a crucial role for CXCR3 in the development of experimental lupus nephritis by directing pathogenic effector T cells into the kidney. For the first time, we demonstrate a beneficial effect of CXCR3 deficiency through attenuation of both the Th1 and the newly defined Th17 immune response. Our data therefore identify the chemokine receptor CXCR3 as a promising therapeutic target in lupus nephritis.

Angiotensin II (Ang II) activates at least two receptors, AT1 and AT2, with the majority of its effects-such as vasoconstriction, inflammation, and matrix deposition-mediated by the AT1 receptor. It is thought that the AT2 receptor counteracts these processes; however, recent studies have found proinflammatory and hypertrophic effects of this receptor subtype. To identify the physiological roles of the AT2 receptor in chronic kidney disease, we performed renal ablation in AT2 receptor knockout and wild-type mice. Renal injury caused a greater impairment of renal function, glomerular injury, albuminuria, and mortality in the knockout mice than in the wild-type mice. There was increased fibronectin expression and inflammation in the knockout mice, as shown by augmented monocyte/macrophage infiltration and higher chemokine monocyte chemotactic protein-1 (MCP-1) and RANTES expression in the remnant kidney. The higher mortality and renal morbidity of the knockout mice was not due to differences in systemic blood pressure, glomerular volume, AT1 receptor, renin, or endothelial nitric oxide synthase expression. Whether activation of the AT2 receptor will have therapeutic benefit in chronic kidney disease will require further study.

Rho kinase signaling regulates inflammatory cell migration and chemokine production. We therefore investigated the mechanisms of Rho-kinase-dependent inflammation in lipopolysaccharide (LPS)-induced renal failure. C57/BL6 mice received intraperitoneal LPS with or without daily treatment with specific Rho kinase inhibitors (Y-27632 or HA-1077; 5 mg/kg). Rho kinase inhibitors were applied in a preventive (12 or 1 h before LPS) or a therapeutic (6 h after LPS) scheme. Both protected renal function and decreased tubular injury in LPS-treated mice. Enhanced Rho kinase activity was inhibited by HA-1077 in capillary endothelial cells, inflammatory cells, and tubuli by analysis of Rho kinase substrate phosphorylation. Early neutrophil influx was reduced by HA-1077 without reduction of the proinflammatory cytokine TNFalpha. In contrast, HA-1077 decreased the influx of monocytes/macrophages coinciding with reduced expression of the NF-kappaB-regulated chemokines CCL5 and CCL2. We therefore examined NF-kappaB signal transduction and found that NF-kappaB p65 phosphorylation and nuclear translocation were reduced by Rho kinase inhibition. IkappaBalpha degradation was not altered during the first 6 h but was reduced by HA-1077 at later time points. NF-kappaB p50-deficient mice were similarly protected from renal injury by Rho kinase inhibition further supporting the prominent role for p65 in Rho kinase inhibition. Together, these data suggest that Rho kinase inhibition by preventive or therapeutic treatment effectively reduced endotoxic kidney injury in part by attenuation of NF-kappaB p65 activation.