Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%–40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.
Andrea Fava, Jill Buyon, Laurence Magder, Jeff Hodgin, Avi Rosenberg, Dawit S. Demeke, Deepak A. Rao, Arnon Arazi, Alessandra Ida Celia, Chaim Putterman, Jennifer H. Anolik, Jennifer Barnas, Maria Dall’Era, David Wofsy, Richard Furie, Diane Kamen, Kenneth Kalunian, Judith A. James, Joel Guthridge, Mohamed G. Atta, Jose Monroy Trujillo, Derek Fine, Robert Clancy, H. Michael Belmont, Peter Izmirly, William Apruzzese, Daniel Goldman, Celine C. Berthier, Paul Hoover, Nir Hacohen, Soumya Raychaudhuri, Anne Davidson, Betty Diamond, the Accelerating Medicines Partnership in RA/SLE network, Michelle Petri
Ischemia-reperfusion injury–induced (IRI-induced) acute kidney injury is accompanied by mononuclear phagocyte (MP) invasion and inflammation. However, systematic analysis of extracellular vesicle–carried (EV-carried) proteins mediating intercellular crosstalk in the IRI microenvironment is still lacking. Multiomics analysis combining single-cell RNA-Seq data of kidney and protein profiling of kidney-EV was used to elucidate the intercellular communication between proximal tubular cells (PTs) and MP. Targeted adhesion and migration of various MPs were caused by the secretion of multiple chemokines as well as integrin β1–rich EV by ischemic-damaged PTs after IRI. These recruited MPs, especially Fn1+ macrophagocyte, amplified the surviving PT’s inflammatory response by secreting the inflammatory factors TNF-α, MCP-1, and thrombospondin 1 (THBS-1), which could interact with integrin β1 to promote more MP adhesion and interact with surviving PT to further promote the secretion of IL-1β. However, GW4869 reduced MP infiltration and maintained a moderate inflammatory level likely by blocking EV secretion. Our findings establish the molecular bases by which chemokines and kidney-EV mediate PT-MP crosstalk in early IRI and provide insights into systematic intercellular communication.
Wenjuan Wang, Xuejing Ren, Xiangmei Chen, Quan Hong, Guangyan Cai
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via “centrosome clustering,” a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.
Tao Cheng, Aruljothi Mariappan, Ewa Langner, Kyuhwan Shim, Jay Gopalakrishnan, Moe R. Mahjoub
The deposition of anti-podocyte auto-antibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.
Qi Zhang, Sofia Bin, Kelly L. Budge, Astgik Petrosyan, Valentina Villani, Paola Aguiari, Coralien H. Vink, Jack Wetzels, Hasmik Soloyan, Gaetano La Manna, Manuel Alfredo Podestà, Paolo Molinari, Sargis Sedrakyan, Kevin V. Lemley, Roger E. De Filippo, Laura Perin, Paolo Cravedi, Stefano Da Sacco
Despite clinical use of immunosuppressive agents, the immunopathogenesis of Minimal Change Disease (MCD) and Focal Segmental Glomerulosclerosis (FSGS) remains unclear. SH3BP2, a scaffold protein, forms an immune signaling complex (signalosome) with seventeen other proteins including PLCγ2 and VAV2. Bioinformatic analysis of human glomerular transcriptome (NEPTUNE cohort) revealed upregulated SH3BP2 in MCD (p=0.001) and FSGS (p<0.001). The SH3BP2-signalosome score and downstream MYD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS (p=0.004-0.001). Immune pathway activation scores for Toll-like receptors (p=0.042), Cytokine-Cytokine receptor (p=0.001) and NOD-like receptors (p=0.042) were increased in FSGS. Lower SH3BP2-signalosome score was associated with MCD, higher eGFR and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6 and ~25-fold increase in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin (p=0.002) and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes underscoring the significance of SH3BP2 in immune activation. SH3BP2 and its binding partners likely determine the immune activation pathways resulting in podocyte injury leading to loss of glomerular filtration barrier.
Tarak Srivastava, Robert E. Garola, Jianping Zhou, Varun Chandra Boinpelly, Mohammad H. Rezaiekhaligh, Trupti Joshi, Yuexu Jiang, Diba Ebadi, Siddarth Sharma, Christine Sethna, Vincent S. Staggs, Ram Sharma, Debbie S. Gipson, Wei Hao, Yujie Wang, Laura H. Mariani, Jeffrey B. Hodgin, Robert Rottapel, Teruhito Yoshitaka, Yasuyoshi Ueki, Mukut Sharma
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
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