The organic anion transporter OAT1 (SLC22A6, originally identified as NKT) is a multispecific transporter responsible for the elimination by the kidney of small organic anions that derive from the gut microbiome. Many are uremic toxins associated with chronic kidney disease (CKD). OAT1 is among a group of “drug” transporters that act as hubs in a large homeostatic network regulating interorgan and interorganismal communication via small molecules. The Remote Sensing and Signaling Theory predicts that genetic deletion of such a key hub in the network results in compensatory interorganismal communication (e.g., host-gut microbe dynamics). Recent metabolomics data from Oat1-KO mice indicate that some of the most highly affected metabolites derive from bacterial tyrosine, tryptophan, purine, and fatty acid metabolism. Functional metagenomic analysis of fecal 16S amplicon and whole-genome sequencing revealed that loss of OAT1 was impressively associated with microbial pathways regulating production of urate, gut-derived p-cresol, tryptophan derivatives, and fatty acids. Certain changes, such as alterations in gut microbiome urate metabolism, appear compensatory. Thus, Oat1 in the kidney appears to mediate remote interorganismal communication by regulating the gut microbiome composition and metabolic capability. Since OAT1 function in the proximal tubule is substantially affected in CKD, our results may shed light on the associated alterations in gut-microbiome dynamics.
Vladimir S. Ermakov, Jeffry C. Granados, Sanjay K. Nigam
Albuminuria and podocyte injury are the key cellular events in the progression of diabetic nephropathy (DN). Acetyl-CoA synthetase 2 (ACSS2) is a nucleocytosolic enzyme responsible for the regulation of metabolic homeostasis in mammalian cells. This study aimed to investigate the possible roles of ACSS2 in kidney injury in DN. We constructed an ACSS2-deleted mouse model to investigate the role of ACSS2 in podocyte dysfunction and kidney injury in diabetic mouse models. In vitro, podocytes were chosen and transfected with ACSS2 siRNA and ACSS2 inhibitor and treated with high glucose. We found that ACSS2 expression was significantly elevated in the podocytes of patients with DN and diabetic mice. ACSS2 upregulation promoted phenotype transformation and inflammatory cytokine expression while inhibiting podocytes’ autophagy. Conversely, ACSS2 inhibition improved autophagy and alleviated podocyte injury. Furthermore, ACSS2 epigenetically activated raptor expression by histone H3K9 acetylation, promoting activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Pharmacological inhibition or genetic depletion of ACSS2 in the streptozotocin-induced diabetic mouse model greatly ameliorated kidney injury and podocyte dysfunction. To conclude, ACSS2 activation promoted podocyte injury in DN by raptor/mTORC1-mediated autophagy inhibition.
Jian Lu, Xue Qi Li, Pei Pei Chen, Jia Xiu Zhang, Liang Liu, Gui Hua Wang, Xiao Qi Liu, Ting Ting Jiang, Meng Ying Wang, Wen Tao Liu, Xiong Zhong Ruan, Kun Ling Ma
Transglutaminase 2 (TGase2) has been shown to contribute to the mesangial IgA1 deposition in a humanized mouse model of IgA nephropathy (IgAN), but the mechanism is not fully understood. In this study, we found that inhibition of TGase2 activity could dramatically decrease the amount of polymeric IgA1 (pIgA1) isolated from patients with IgAN that interacts with human mesangial cells (HMC). TGase2 was expressed both in the cytosol and on the membrane of HMC. Upon treatment with pIgA1, there were more TGase2 recruited to the membrane. Using a cell model of mesangial deposition of pIgA1, we identified 253 potential TGase2-associated proteins in the cytosolic fraction and observed a higher concentration of cellular vesicles and increased expression of Ras homolog family member A (RhoA) in HMC after pIgA1 stimulation. Both the amount of pIgA1 deposited on HMC and membrane TGase2 level were decreased by inhibition of the vesicle trafficking pathway. Mechanistically, TGase2 was found to be coprecipitated with RhoA in the cellular vesicles. Membrane TGase2 expression was greatly increased by overexpression of RhoA, while it was reduced by knockdown of RhoA. Our in vitro approach demonstrated that TGase2 was transported from the cytosol to the membrane through a RhoA-mediated vesicle-trafficking pathway that can facilitate pIgA1 interaction with mesangium in IgAN.
Zhong Zhong, Zhijian Li, Yanjie Li, Lanping Jiang, Qingyu Kong, Wei Chen, Shaozhen Feng
Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway have few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2 knockdown kidneys. The ketone bodies β-hydroxybutyrate and ATP production were increased in CNN2 knockdown mice. Mechanistically, CNN2 interacts with ESR2 to negatively regulate activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylates Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.
Yuan Gui, Zachary Palanza, Priya R. Gupta, Hanwen Li, Yuchen Pan, Yuanyuan Wang, Geneva R. Hargis, Donald L. Kreutzer, Yanlin Wang, Sheldon I. Bastacky, Yansheng Liu, Silvia Liu, Dong Zhou
Background: Cardiorenal syndrome (CRS)—renal injury during heart failure (HF)—is linked to higher morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear. Methods: We investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip model (KOC). The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.Results: Renal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from HF patients. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. 15 EV-miRNAs were associated with creatinine, targeting 1143 gene targets specifying pathways relevant to renal injury, including TGF beta and AMPK signaling. We observed directionally consistent changes in the expression of TGF beta pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function. Conclusion: Plasma EVs in CRS patients elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.
Emeli Chatterjee, Rodosthenis S. Rodosthenous, Ville J. Kujala, Priyanka Gokulnath, Michail Spanos, H. Immo Lehmann, Getulio P de Oliveira-Jr, Mingjian Shi, Tyne W. Miller-Fleming, Guoping Li, Ionita Ghiran, Katia Karalis, JoAnn Lindenfeld, Jonathan D. Mosley, Emily S. Lau, Jennifer E. Ho, Quanhu Sheng, Ravi Shah, Saumya Das
Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γ ENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR-Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared to wild type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo, but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.
Andrew J. Nickerson, Stephanie M. Mutchler, Shaohu Sheng, Natalie A. Cox, Evan C. Ray, Ossama B. Kashlan, Marcelo D. Carattino, Allison L. Marciszyn, Aaliyah Winfrey, Sebastien Gingras, Annet Kirabo, Rebecca P. Hughey, Thomas R. Kleyman
Retinoic acid receptor (RAR) signaling is essential for mammalian kidney development, but in the adult kidney is restricted to occasional collecting duct epithelial cells. We now show that there is widespread reactivation of RAR signaling in proximal tubular epithelial cells (PTECs) in human sepsis-associated acute kidney injury (AKI), and in mouse models of AKI. Genetic inhibition of RAR signaling in PTECs protected against experimental AKI but was unexpectedly associated with increased expression of the PTEC injury marker, Kim1. However, the protective effects of inhibiting PTEC RAR signaling were associated with increased Kim1 dependent apoptotic cell clearance, or efferocytosis, and this was associated with de-differentiation, proliferation, and metabolic reprogramming of PTECs. These data demonstrate the functional role that reactivation of RAR signaling plays in regulating PTEC differentiation and function in human and experimental AKI.
Min Yang, Lauren N. Lopez, Maya Brewer, Rachel Delgado, Anna Menshikh, Kelly Clouthier, Yuantee Zhu, Thitinee Vanichapol, Haichun Yang, Raymond C. Harris, Leslie Gewin, Craig R. Brooks, Alan J. Davidson, Mark de Caestecker
Sialidosis is an ultrarare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe Type II form of the disease, manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology and visceromegaly. A subset of these patients presents with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in two NEU1-deficient mouse models, a constitutive Neu1 knockout Neu1∆Ex3 and a conditional phagocyte-specific knockout Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal and distal tubules was drastically increased including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.
Ikhui Kho, Ekaterina P. Demina, Xuefang Pan, Irene Londono, Christopher W. Cairo, Luisa Sturiale, Angelo Palmigiano, Angela Messina, Domenico Garozzo, Roth-Visal Ung, Fabrice Mac‐Way, Éric Bonneil, Pierre Thibault, Mathieu Lemaire, Carlos R. Morales, Alexey V. Pshezhetsky
Intact fibroblast growth factor 23 (iFGF23) is a phosphaturic hormone that is cleaved by furin into N-terminal and C-terminal fragments. Several studies have implicated vitamin D in regulating furin in infections. Thus, we investigated the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and the vitamin D receptor (VDR) on furin-mediated iFGF23 cleavage. Mice lacking VDR (Vdr–/–) had a 25-fold increase in iFGF23 cleavage, with increased furin levels and activity compared with wild-type (WT) littermates. Inhibition of furin activity blocked the increase in iFGF23 cleavage in Vdr–/– animals and in a Vdr-knockdown osteocyte OCY454 cell line. Chromatin immunoprecipitation revealed VDR binding to DNA upstream of the Furin gene, with more transcription in the absence of VDR. In WT mice, furin inhibition reduced iFGF23 cleavage, increased iFGF23, and reduced serum phosphate levels. Similarly, 1,25(OH)2D reduced furin activity, decreased iFGF23 cleavage, and increased total FGF23. In a post hoc analysis of a randomized clinical trial, we found that ergocalciferol treatment, which increased serum 1,25(OH)2D, significantly decreased serum furin activity and iFGF23 cleavage, compared with placebo. Thus, 1,25(OH)2D inhibits iFGF23 cleavage via VDR-mediated suppression of Furin expression, thereby providing a mechanism by which vitamin D can augment phosphaturic iFGF23 levels.
Han Xie, Isinsu Bastepe, Wen Zhou, Birol Ay, Zara Ceraj, Ignacio A. Portales-Castillo, Eva S. Liu, Sherri-Ann M. Burnett-Bowie, Harald Jüppner, Eugene P. Rhee, Murat Bastepe, Petra Simic
BACKGROUND. Severe forms of idiopathic nephrotic syndrome require prolonged immune-suppressive therapies and repeated courses of high-dose glucocorticoids. Mesenchymal stromal cells (MSCs) have promising immunomodulatory properties that may be employed therapeutically to reduce patient exposure to medications and their side effects. METHODS. We performed a phase 1 open-label trial assessing safety and feasibility of autologous bone marrow-derived (BM) MESenchymal stromal cells in children and young adults with severe forms of steroid-dependent NEPHrotic syndrome, (MESNEPH). Following autologous BM-MSC preparation and infusion, oral immunosuppression was tapered. Safety, efficacy and immunomodulatory effects in vivo were monitored for 12 months. RESULTS. Sixteen patients (10 children, 6 adults) were treated. Adverse events were limited and not related to BM-MSC infusions. All patients relapsed during follow-up, but in the 10 treated children, time to first relapse was delayed (p=0.02) and number of relapses was reduced (p=0.002) after BM-MSC infusion, compared to the previous 12 months. Cumulative prednisone dose was also reduced at 12 months compared to baseline (p<0.05). No treatment benefit was observed in adults. In children, despite tapering of immunosuppression, clinical benefit was mirrored by a significant reduction of total CD19+, mature and memory B cells and an increase of regulatory T cells in vivo up to 3-6 months following BM-MSC infusion. CONCLUSION. Treatment with autologous BM-MSC is feasible and safely reduces relapses and immunosuppression at 12 months in children with severe steroid-dependent INS. Immunomodulatory studies suggest that repeating MSC infusions at 3-6 months may sustain benefit. TRIAL REGISTRATION. EudraCT 2016-004804-77 FUNDING. AIFA Ricerca Indipendente 2016-02364623
Marina Vivarelli, Manuela Colucci, Mattia Algeri, Federica Zotta, Francesco Emma, Ines L'Erario, Marco Busutti, Stefano Rota, Chiara Capelli, Martino Introna, Marta Todeschini, Federica Casiraghi, Annalisa Perna, Tobia Peracchi, Andrea De Salvo, Nadia Rubis, Franco Locatelli, Giuseppe Remuzzi, Piero Ruggenenti
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