The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury (AKI). While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA sequencing (scRNAseq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNAseq and spatial transcriptomics revealed seven distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature suggesting distinct functions. Specific protein markers were identified for two clusters allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation is lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs is not fully restored for at least 28 days post-injury. The change in KRM localization confirms a long hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.
Matthew D. Cheung, Elise N. Erman, Kyle H. Moore, Jeremie M.P. Lever, Zhang Li, Jennifer R. LaFontaine, Gelare Ghajar-Rahimi, Shanrun Liu, Zhengqin Yang, Rafay Karim, Bradley K. Yoder, Anupam Agarwal, James F. George
BACKGROUND. Metabolomic profiling in individuals with chronic kidney disease (CKD) has the potential to identify novel biomarkers and provide insight into disease pathogenesis. Methods: We examined the association between blood metabolites and CKD progression, defined as the subsequent development of end-stage renal disease (ESRD) or estimated glomerular filtrate rate (eGFR) halving, in 1773 participants of the Chronic Renal Insufficiency Cohort (CRIC) study, 962 participants of the African American Study of Kidney Disease and Hypertension (AASK), and 5305 participants of the Atherosclerosis Risk in Communities (ARIC) study. RESULTS. In CRIC, more than half of measured metabolites were associated with CKD progression in minimally adjusted Cox proportional hazards models, but the number and strength of associations were markedly attenuated by serial adjustment for covariates, particularly eGFR. Ten metabolites were significantly associated with CKD progression in fully-adjusted models in CRIC; three of these metabolites were also significant in fully-adjusted models in AASK and ARIC, highlighting potential markers of glomerular filtration (pseudouridine), histamine metabolism (methylimidazoleacetate), and azotemia (homocitrulline). Our findings also nominate N-acetylserine as a potential marker of kidney tubular function, with significant associations with CKD progression observed in CRIC and ARIC. CONCLUSION. Together, our findings demonstrate the application of metabolomics to identify potential biomarkers and causal pathways in CKD progression. TRIAL REGISTRATION. Not applicable FUNDING. This study was supported by the NIH (U01 DK106981, U01 DK106982, U01 DK085689, R01 DK108803, R01 DK124399)
Donghai Wen, Zihe Zheng, Aditya Surapaneni, Bing Yu, Linda Zhou, Wen Zhou, Dawei Xie, Haochang Shou, Julian Avila-Pacheco, Sahir Kalim, Jiang He, Chi-yuan Hsu, Afshin Parsa, Panduranga Rao, James Sondheimer, Raymond Townsend, Sushrut S. Waikar, Casey M. Rebholz, Michelle R. Denburg, Paul L. Kimmel, Ramachandran S. Vasan, Clary B. Clish, Josef Coresh, Harold I. Feldman, Morgan E. Grams, Eugene P. Rhee
Energy metabolism failure in proximal tubule cells (PTC) is a hallmark of chronic kidney injury. We combined transcriptomic, metabolomic and lipidomic approaches in experimental models and patient cohorts to investigate the molecular bases of the progression to chronic kidney allograft injury initiated by ischemia-reperfusion injury (IRI). The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was significantly enriched with long chain fatty acids (FA). We identified a renal FA-related gene signature with low levels of Cpt2 and Acsm5 and high levels of Acsl4 and Acsm5 associated with IRI, transition to chronic injury, and established CKD in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-, Acsl4+, Acsl5+, Acsm5- PTC failing to recover from IRI as identified by snRNAseq. In vitro experiments indicated that endoplasmic reticulum (ER) stress contributes to CPT2 repression, which, in turn, promotes lipids accumulation, drives profibrogenic epithelial phenotypic changes, and activates the unfolded protein response. ER stress through CPT2 inhibition and lipid accumulation, engages an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule sustaining the progression to chronic kidney allograft injury.
Anna Rinaldi, Hélène Lazareth, Virginie Poindessous, Ivan Nemazanyy, Julio L. Sampaio, Daniele Malpetti, Yohan Bignon, Maarten Naesens, Marion Rabant, Dany Anglicheau, Pietro E. Cippà, Nicolas Pallet
Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial cancer. Although the proximal tubule has been defined as the primary target of aristolochic acids I (AAI), the mechanistic underpinning of gross renal deterioration caused by AAI has not been explicitly explained, prohibiting effective therapeutic intervention. To this point, we employed integrated single-cell RNA-sequencing, bulk RNA-sequencing, and mass spectrometry-based proteomics to analyze mouse kidney post-acute AAI exposure. Our results revealed a dramatic reduction of proximal tubule epithelial cells, associated with apoptotic and inflammatory pathways, indicating permanent damage beyond repair. We found the enriched development pathways in other nephron segments, suggesting activation of reparative programs triggered by AAI. The divergent response may be attributed to the segment-specific distribution of organic anions channels along the nephron, including OAT1 and OAT3. Moreover, we observed dramatic activation and recruitment of cytotoxic T and macrophage M1 cells, highlighting inflammation as a principal contributor to permanent renal injury. Ligand-receptor pairing revealed critical intercellular crosstalk underpins damage-induced activation of immune cells. These results provide novel insight into AAI-induced kidney injury and point out potential pathways for future therapeutic intervention.
Jiayun Chen, Piao Luo, Chen Wang, Chuanbin Yang, Yunmeng Bai, Xueling He, Qian Zhang, Junzhe Zhang, Jing Yang, Shuang Wang, Jigang Wang
Acute and chronic kidney injuries induce increased cell cycle progression in renal tubules. While increased cell cycle progression promotes repair after acute injury, the role of ongoing tubular cell cycle progression in chronic kidney disease is unknown. Two weeks after initiation of chronic kidney disease, we blocked cell cycle progression at G1/S phase by using an FDA-approved, selective inhibitor of CDK4/6. Blocking CDK4/6 improved renal function and reduced tubular injury and fibrosis in 2 murine models of chronic kidney disease. However, selective deletion of cyclin D1, which complexes with CDK4/6 to promote cell cycle progression, paradoxically increased tubular injury. Expression quantitative trait loci (eQTLs) for CCND1 (cyclin D1) and the CDK4/6 inhibitor CDKN2B were associated with eGFR in genome-wide association studies. Consistent with the preclinical studies, reduced expression of CDKN2B correlated with lower eGFR values, and higher levels of CCND1 correlated with higher eGFR values. CDK4/6 inhibition promoted tubular cell survival, in part, through a STAT3/IL-1β pathway and was dependent upon on its effects on the cell cycle. Our data challenge the paradigm that tubular cell cycle progression is beneficial in the context of chronic kidney injury. Unlike the reparative role of cell cycle progression following acute kidney injury, these data suggest that blocking cell cycle progression by inhibiting CDK4/6, but not cyclin D1, protects against chronic kidney injury.
Yosuke Osaki, Marika Manolopoulou, Alla V. Ivanova, Nicholas Vartanian, Melanie Phillips Mignemi, Justin Kern, Jianchun Chen, Haichun Yang, Agnes B. Fogo, Mingzhi Zhang, Cassianne Robinson-Cohen, Leslie S. Gewin
Acute kidney injury increases morbidity and mortality and previous studies have shown that remote ischemic preconditioning (RIPC) reduces the risk of acute kidney injury after cardiac surgery. RIPC increases urinary HMGB1 (high mobility group box protein-1) levels in patients which correlates with kidney protection. Here, we show that RIPC reduces renal ischemia-reperfusion injury and improves kidney function in mice. Mechanistically, RIPC increases HMGB1 levels in the plasma and urine and HMGB1 binds to Toll-like receptor 4 (TLR4) on renal tubular epithelial cells, inducing transcriptomic modulation of renal tubular epithelial cells and providing renal protection, whereas TLR4 activation on non-renal cells was shown to contribute to renal injury. This protection is mediated by activation of induction of AMPK⍺ and NF-kB, which induces the upregulation of Sema5b that triggers a transient, protective G1 cell-cycle arrest. In cardiac surgery patients at high risk for postoperative acute kidney injury, increased HMGB1 and Sema5b levels after RIPC were associated with renal protection after surgery. The results may help to develop future clinical treatment options for acute kidney injury.
Jan Rossaint, Melanie Meersch, Katharina Thomas, Sina Mersmann, Martin Lehmann, Jennifer Skupski, Tobias Tekath, Peter Rosenberger, John A. Kellum, Hermann Pavenstädt, Alexander Zarbock
Although macrophages are undoubtedly attractive therapeutic targets for acute kidney injury (AKI) because of their critical roles in renal inflammation and repair, the underlying mechanisms of macrophage phenotype switching and efferocytosis in the regulation of inflammatory responses during AKI are still largely unclear. The present study was to elucidate the role of JAML (junctional adhesion molecule-like protein) in the pathogenesis of AKI. We found that JAML was significantly up-regulated in the kidney from two different murine AKI models including renal ischemia/reperfusion injury (IRI) and cisplatin-induced AKI. By generation of bone marrow chimeric mice, macrophage-specific and tubular-specific Jaml conditional knockout mice, we demonstrated JAML promotes AKI mainly via a macrophage-dependent mechanism and found that JAML-mediated macrophage phenotype polarization and efferocytosis is one of critical signal transduction pathways linking inflammatory responses to AKI. Mechanistically, the effects of JAML on the regulation of macrophages was at least in part, associated with a Mincle-dependent mechanism. Collectively, our studies for the first time explore new biological functions of JAML in macrophages and conclude that JAML is an important mediator and biomarker of AKI. Pharmacologic targeting of JAML mediated signaling pathways at multiple levels may provide a novel therapeutic strategy for patients with AKI.
Wei Huang, Bi-Ou Wang, Yunfeng Hou, Yi Fu, Sijia Cui, Jinghan Zhu, Xinyu Zhan, Rongkun Li, Wei Tang, Jichao Wu, Ziying Wang, Mei Wang, Xiaojie Wang, Yan Zhang, Min Liu, Yusheng Xie, Yu Sun, Fan Yi
Nephrolithiasis is a common and recurrent disease affecting 9% of the US population. Hyperoxaluria is major risk factor for calcium oxalate kidney stones, which constitutes two-thirds of all kidney stones. Gastrointestinal epithelia play an important role in oxalate handling due to the presence of SLC26A family anion exchangers that facilitate oxalate transport. SLC26A3 (originally named DRA, down-regulated in adenoma) is an anion exchanger of chloride, bicarbonate and oxalate thought to facilitate intestinal oxalate absorption, as evidenced by ~70% reduced urine oxalate excretion in knock-out mice. We previously identified, by high-throughput screening and medicinal chemistry, a small molecule SLC26A3 inhibitor (DRAinh-A270) that selectively inhibited SLC26A3-mediated chloride/bicarbonate exchange (IC50 ~ 35 nM), and, as found here, oxalate/chloride exchange (IC50 ~ 60 nM). In colonic closed loops in mice, luminal DRAinh-A270 inhibited oxalate absorption by 70%. In a model of acute hyperoxaluria produced by oral sodium oxalate loading, DRAinh-A270 largely prevented the ~2.5-fold increase in urine oxalate/creatinine ratio. In a mouse model of oxalate nephropathy produced by a high-oxalate low-calcium diet, vehicle-treated mice by day 14 developed marked hyperoxaluria with elevated serum creatinine, renal calcium oxalate crystal deposition and renal injury, which were largely prevented by DRAinh-A270 (10 mg/kg twice daily). DRAinh-A270 administered over 7 days to healthy mice did not show significant toxicity as assessed by CBC, serum chemistries and tissue histology. Our findings support a major role of SLC26A3 in intestinal oxalate absorption and suggest the therapeutic utility of SLC26A3 inhibition for treatment of hyperoxaluria and prevention of calcium oxalate nephrolithiasis.
Onur Cil, Qi Tifany Chu, Sujin Lee, Peter M. Haggie, Alan S. Verkman
COVID-19 infection causes collapse of glomerular capillaries and loss of podocytes, terminating in a severe kidney disease called COVID-19 associated nephropathy (COVAN). The underlying mechanism of COVAN is unknown. We hypothesized that cytokines induced by COVID-19 trigger expression of pathogenic APOL1 via JAK-STAT signaling, resulting in podocyte loss and COVAN phenotype. Here, based on nine biopsy-proven COVAN cases, we demonstrated for the first time that APOL1 protein is abundantly expressed in podocytes and glomerular endothelial cells (GECs) of COVAN kidneys but not in controls. Moreover, a majority (77.8%) of COVAN patients carried two APOL1 risk alleles. We showed that recombinant cytokines induced by SARS-CoV-2 act synergistically to drive APOL1 expression through the JAK-STAT pathway in primary human podocytes, GECs, and kidney micro-organoids derived from a carrier of two APOL1 risk alleles but was blocked by JAK1/2-inhibitor, baricitinib. We demonstrated for the first time that cytokine-induced JAK-STAT-APOL1 signaling reduced the viability of kidney organoid podocytes but was rescued by baricitinib. Together, our results support the conclusion that COVID-19-induced cytokines are sufficient to drive COVAN-associated podocytopathy via JAK-STAT-APOL1 signaling and that JAK-inhibitor could block this pathogenic process. These findings suggest that JAK-inhibitors may have therapeutic benefits for managing cytokine-induced APOL1-mediated podocytopathy.
Sarah E. Nystrom, Guojie Li, Somenath Datta, Karen Soldano, Daniel Silas, Astrid Weins, Gentzon Hall, David B. Thomas, Opeyemi A. Olabisi
Inherited kidney diseases are the fifth most common cause of the end-stage renal disease. Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mt-tRNA variants and their pathogenic contributions to the kidney disease remain largely unclear. In this study, we identified the pathogenic mitochondrial tRNAPhe 616T>C mutation in three families; and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, one proband with homoplamic m.616T>C presented with end-stage renal disease as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m. 616T>C exhibited swollen mitochondria, underwent active mitophagy and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial reactive oxygen species. Pathogenic m. 616T>C abolished a highly conserved base-pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mt-DNA encoded polypeptides. Collectively, these findings provide new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.
Chengxian Xu, Lingxiao Tong, Jia Rao, Qing Ye, Yuxia Chen, Yingying Zhang, Jie Xu, Xiaoting Mao, Feilong Meng, Huijun Shen, Zhihong Lu, Xiaohui Cang, Haidong Fu, Shugang Wang, Weiyue Gu, En Yin Lai, Minxin Guan, Pingping Jiang, Jianhua Mao
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