Acute kidney injury strongly upregulates the transcription factor Foxm1 in proximal tubule in vivo and Foxm1 drives epithelial proliferation in vitro. Here we report that deletion of Foxm1 either with a nephron specific Cre driver or by inducible global deletion reduces proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI-to-CKD transition with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report extracellular signal-regulated kinase (ERK) mediates FOXM1 induction downstream of the epidermal growth factor receptor (EGFR) in primary proximal tubule cells. We defined FOXM1 genomic binding sites by Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned datasets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identify two cis regulatory elements that bind FOXM1 and regulate CCNF expression, demonstrate that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK-FOXM1-CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.
Megan L. Noonan, Yoshiharu Muto, Yasuhiro Yoshimura, Aidan Leckie-Harre, Haojia Wu, Vladimir V. Kalinichenko, Benjamin D. Humphreys, Monica Chang-Panesso
Our previous study identified 8 risk and 9 protective plasma miRNAs associated with progression to end-stage kidney disease (ESKD) in diabetes. This study aimed to elucidate preanalytical factors that influence the quantification of circulating miRNAs. Using the EdgeSeq platform, which quantifies 2,002 miRNAs in plasma, including ESKD-associated miRNAs, we compared miRNA profiles in whole plasma versus miRNA profiles in RNA extracted from the same plasma specimens. Less than half of the miRNAs were detected in standard RNA extraction from plasma. Detection of individual and concentrations of miRNAs were much lower when RNA extracted from plasma was quantified by RNA sequencing (RNA-Seq) or quantitative reverse transcription PCR (qRT-PCR) platforms compared with EdgeSeq. Plasma profiles of miRNAs determined by the EdgeSeq platform had excellent reproducibility in assessment and had no variation with age, sex, hemoglobin A1c, BMI, and cryostorage time. The risk ESKD-associated miRNAs were detected and measured accurately only in whole plasma and using the EdgeSeq platform. Protective ESKD-associated miRNAs were detected by all platforms except qRT-PCR; however, correlations among concentrations obtained with different platforms were weak or nonexistent. In conclusion, preanalytical factors have a profound effect on detection and quantification of circulating miRNAs in ESKD in diabetes. Quantification of miRNAs in whole plasma and using the EdgeSeq platform may be the preferable method to study profiles of circulating cell-free miRNAs associated with ESKD and possibly other diseases.
Eiichiro Satake, Bozena Krolewski, Hiroki Kobayashi, Zaipul I. Md Dom, Joseph Ricca, Jonathan M. Wilson, Dave S.B. Hoon, Kevin L. Duffin, Marcus G. Pezzolesi, Andrzej S. Krolewski
Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1–/– C3–/– mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1–/– C3–/– mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1–/– C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.
Sofia Bin, Miran Yoo, Paolo Molinari, Micaela Gentile, Kelly Budge, Chiara Cantarelli, Yaseen Khan, Gaetano La Manna, William M. Baldwin, Nina Dvorina, Paolo Cravedi, G. Luca Gusella
Lactate elevation is a well-characterized biomarker of mitochondrial dysfunction, but its role in diabetic kidney disease (DKD) is not well defined. Urine lactate was measured in patients with type 2 diabetes (T2D) in 3 cohorts (HUNT3, SMART2D, CRIC). Urine and plasma lactate were measured during euglycemic and hyperglycemic clamps in participants with type 1 diabetes (T1D). Patients in the HUNT3 cohort with DKD had elevated urine lactate levels compared with age- and sex-matched controls. In patients in the SMART2D and CRIC cohorts, the third tertile of urine lactate/creatinine was associated with more rapid estimated glomerular filtration rate decline, relative to first tertile. Patients with T1D demonstrated a strong association between glucose and lactate in both plasma and urine. Glucose-stimulated lactate likely derives in part from proximal tubular cells, since lactate production was attenuated with sodium-glucose cotransporter-2 (SGLT2) inhibition in kidney sections and in SGLT2-deficient mice. Several glycolytic genes were elevated in human diabetic proximal tubules. Lactate levels above 2.5 mM potently inhibited mitochondrial oxidative phosphorylation in human proximal tubule (HK2) cells. We conclude that increased lactate production under diabetic conditions can contribute to mitochondrial dysfunction and become a feed-forward component to DKD pathogenesis.
Manjula Darshi, Luxcia Kugathasan, Soumya Maity, Vikas S. Sridhar, Roman Fernandez, Christine P. Limonte, Brian I. Grajeda, Afaf Saliba, Guanshi Zhang, Viktor R. Drel, Jiwan J. Kim, Richard Montellano, Jana Tumova, Daniel Montemayor, Zhu Wang, Jian-Jun Liu, Jiexun Wang, Bruce A. Perkins, Yuliya Lytvyn, Loki Natarajan, Su Chi Lim, Harold Feldman, Robert Toto, John R. Sedor, Jiten Patel, Sushrut S. Waikar, Julia Brown, Yahya Osman, Jiang He, Jing Chen, W. Brian Reeves, Ian H. de Boer, Sourav Roy, Volker Vallon, Stein Hallan, Jonathan A.L. Gelfond, David Z.I. Cherney, Kumar Sharma, for the Kidney Precision Medicine Project, and the CRIC Study Investigators
BACKGROUND. Identifying patients with acute kidney injury (AKI) who are at higher risk of chronic kidney disease (CKD) progression at time of AKI diagnosis remains a major challenge in clinical practice. METHODS. Kidney transcriptome sequencing was applied to identify the top up-regulated genes in mice with AKI. The product of the top-ranked gene was identified in the tubular cells and urine both in mouse and human AKI. Data from two cohorts of patients with a prehospitalization estimated glomerular filtration rate (eGFR) ≥ 45 ml/min/1.73m2 who survived for at least 90 days after AKI were used to derive and validate multivariable prediction models. AKI to CKD progression was defined as a persistent eGFR < 60 ml/min/1.73m2 and with a minimum 25% reduction from baseline eGFR 90 days after AKI in patients with prehospitalization eGFR ≥ 60 ml/min/1.73m2. AKI to advanced CKD was defined by a sustained reduction of eGFR < 30 ml/min/1.73m2 90 days after AKI in those with prehospitalization eGFR 45–60 ml/min/1.73m2. RESULTS. Kidney cytokeratin 20 (CK20) was up-regulated in injured proximal tubular cells and detectable in urine within 7 days after AKI. High concentrations of urinary CK20 (uCK20) were independently associated with the severity of histological AKI and the risk of AKI to CKD or advanced CKD progression. In Test set, the AUC of uCK20 for predicting AKI to CKD or advanced CKD was 0.80, outperformed currently used biomarkers for detecting kidney tubular injury. Addition of uCK20 to an established clinical model improved the ability for predicting AKI-CKD progression with an AUC of 0.90, and largely improved the risk reclassification. CONCLUSION. This finding highlighted uCK20 as a useful predictor for AKI to CKD progression, and may provide a tool to early identify patients at high risk of CKD following AKI. FUNDING. The National Natural Science Foundation of China (Key Program).
Rui Ma, Han Ouyang, Shihong Meng, Jun Liu, Jianwei Tian, Nan Jia, Youhua Liu, Xin Xu, Xiaobing Yang, Fan Fan Hou
Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a key regulator of plasma triglycerides, and it has recently been suggested to play a role in both type 1 diabetes-accelerated atherosclerosis and kidney disease progression. To investigate if APOC3 plays a role in kidney disease in people with type 2 diabetes, we analyzed plasma levels of APOC3 from the Veterans Affairs Diabetes Trial (VADT). Elevated baseline APOC3 levels predicted a greater loss of renal function. To mechanistically test if APOC3 plays a role in diabetic kidney disease and associated atherosclerosis, we treated BTBR wildtype (WT) and leptin-deficient (OB; diabetic) mice, a model of type 2 diabetes, with an antisense oligonucleotide (ASO) to APOC3 or a control ASO (cASO), all in the setting of human-like dyslipidemia. Silencing APOC3 prevented diabetes-augmented albuminuria, renal glomerular hypertrophy, monocyte recruitment, and macrophage accumulation, partly driven by reduced ICAM1 expression. Furthermore, reduced levels of APOC3 suppressed atherosclerosis associated with diabetes. This suggests that targeting APOC3 might benefit both diabetes-accelerated atherosclerosis and kidney disease.
Jocelyn Cervantes, Juraj Koska, Farah Kramer, Shreeram Akilesh, Charles E. Alpers, Adam E. Mullick, Peter Reaven, Jenny E. Kanter
Chronic kidney disease (CKD) causes an accumulation of uremic metabolites that negatively impact skeletal muscle function. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR) which has been shown to be activated in the blood of CKD patients. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared to control participants with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD (P=0.032) and the magnitude of AHR activation was inversely correlated with mitochondrial respiration (P<0.001). In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was significantly impaired and strongly correlated with both the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR significantly improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD+probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite-AHR-mitochondrial axis in skeletal muscle was further confirmed using muscle-specific AHR knockdown in C57BL6J that harbour a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were only present when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significant increases in Pdk4 expression (P<0.05) and phosphorylation of pyruvate dehydrogenase enzyme (P<0.05). These findings establish a uremic metabolite-AHR-Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.
Trace Thome, Nicholas A. Vugman, Lauren E. Stone, Keon Wimberly, Salvatore T. Scali, Terence E. Ryan
Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.
Safaa Hammoud, Alla Ivanova, Yosuke Osaki, Steven Funk, Haichun Yang, Olga Viquez, Rachel Delgado, Dongliang Lu, Melanie Phillips Mignemi, Jane Tonello, Selene Colon, Louise Lantier, David Wasserman, Benjamin D. Humphreys, Jeffrey Koenitzer, Justin Kern, Mark de Caestecker, Toren Finkel, Agnes Fogo, Nidia Messias, Irfan J. Lodhi, Leslie Gewin
Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle–dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.
Geremy Clair, Hasmik Soloyan, Paolo Cravedi, Andrea Angeletti, Fadi Salem, Laith Al-Rabadi, Roger E. De Filippo, Stefano Da Sacco, Kevin V. Lemley, Sargis Sedrakyan, Laura Perin
Sodium-glucose cotransporter 2 (SGLT2) inhibitor, dapagliflozin (Dapa), exhibited nephroprotective effects in patients with chronic kidney disease (CKD). We assessed the efficacy of short-term Dapa administration following acute kidney injury (AKI) in preventing CKD. Male Wistar rats were randomly assigned to Sham surgery, bilateral ischemia for 30 minutes (abbreviated as IR), and IR + Dapa groups. Daily treatment with Dapa was initiated just 24 hours after IR and maintained for only 10 days. Initially, rats were euthanized at this point to study early renal repair. After severe AKI, Dapa promptly restored creatinine clearance (CrCl) and significantly reduced renal vascular resistance compared with the IR group. Furthermore, Dapa effectively reversed the mitochondrial abnormalities, including increased fission, altered mitophagy, metabolic dysfunction, and proapoptotic signaling. To study this earlier, another set of rats was studied just 5 days after AKI. Despite persistent renal dysfunction, our data reveal a degree of mitochondrial protection. Remarkably, a 10-day treatment with Dapa demonstrated effectiveness in preventing CKD transition in an independent cohort monitored for 5 months after AKI. This was evidenced by improvements in proteinuria, CrCl, glomerulosclerosis, and fibrosis. Our findings underscore the potential of Dapa in preventing maladaptive repair following AKI, emphasizing the crucial role of early intervention in mitigating AKI long-term consequences.
Miguel Ángel Martínez-Rojas, Hiram Balcázar, Isaac González-Soria, Jesús Manuel González-Rivera, Mauricio E. Rodríguez-Vergara, Laura A. Velazquez-Villegas, Juan Carlos León-Contreras, Rosalba Pérez-Villalva, Francisco Correa, Florencia Rosetti, Norma A. Bobadilla
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