The Physiology and Pathophysiology of Branched-Chain Amino Acids in the Kidney

˜The œNephron journals/Nephron journals · 2026-03-05

BACKGROUND: Kidneys require large amounts of energy to maintain function and are highly metabolically active. Acute kidney injury (AKI) and diseases including chronic kidney disease (CKD), diabetic kidney disease (DKD), and polycystic kidney disease (PKD) are strongly associated with metabolic disturbances. SUMMARY: While most research to date has focused on glucose and fatty acid metabolism, the catabolism of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine is an emerging area of importance across different kidney pathologies. BCAAs can be used in protein synthesis or catabolized to provide tricarboxylic acid (TCA) cycle intermediates. BCAAs and their metabolites can also act as signaling molecules. Disturbances of BCAA catabolism have recently been described in AKI, CKD, DKD, and PKD, driven by both transcriptional and posttranslational mechanisms. This results in accumulation of BCAAs in the kidneys and the loss of a source of TCA cycle intermediates. In addition, accumulated BCAAs, especially leucine, can activate mechanistic target of rapamycin complex 1 (mTORC1) signaling. In addition to the described disturbances in BCAA catabolism, recent preclinical studies have shown that reactivation of BCAA catabolism could be a potential therapeutic strategy. KEY MESSAGES: This review will describe the process of BCAA catabolism and its disturbances in AKI, CKD, DKD, and PKD.

Evolving Understanding of RNA Biology in Kidney Disease

Journal of the American Society of Nephrology · 2025-12-24

Krüppel-Like Factor 6 Induces RNA Polymerase II Subunit RPB1 to Promote Kidney Injury

Journal of the American Society of Nephrology · 2025-05-06 · 4 citations

Key Points Single nuclear RNA sequencing after DNA damage–induced AKI identified an injured proximal tubule cluster with high Polr2a (RNA polymerase subunit B1), an RNA polymerase II subunit. POLR2A knockdown in injured cells decreased inflammatory and fibrotic gene expression, dedifferentiation, DNA damage, and cell cycle arrest. RNA polymerase subunit B1 was higher in mice overexpressing transcription factor Krüppel-like factor 6 and associated with worse injury after DNA damage. Background Initial proximal tubule cell injury and dedifferentiation contribute to AKI, and persistent dedifferentiation drives fibrosis and CKD. Proximal tubule–specific knockdown of zinc-finger transcription factor Krüppel-like factor 6 ( Klf6 ) attenuates the AKI to CKD transition. Our aim was to study the early transcriptional mechanisms by which KLF6 induction exacerbates proximal tubular injury and eventual fibrosis. Methods Aristolochic acid I–treated wild-type and KLF6 overexpression mice underwent single nucleus (sn)RNA-seq and single nucleus assay for transposase-accessible chromatin sequencing (acute phase) and assessment of kidney function, injury, and fibrosis (remodeling phase). POLR2A was knocked down in human kidney cells and cell number, gene expression, differentiation, DNA damage, and cell cycle assessed. Kidney sections from fibrotic mouse models and human CKD secondary to aristolochic acid and diabetes were assessed for RNA polymerase subunit B1 (RPB1) expression. Results snRNA-seq identified an injured proximal tubule cluster with high expression of Klf6 and RNA polymerase II subunit a ( Polr2a ) encoding RPB1. After injury, RPB1-positive cells accumulated and were associated with dedifferentiated proximal tubules. POLR2A knockdown in injured cells increased cell death, but reduced inflammatory and fibrotic gene expression, dedifferentiation, DNA damage, and G2/M cell cycle arrest, with a transcriptional switch from long genes to short genes. Single nucleus assay for transposase-accessible chromatin sequencing demonstrated an open chromatin region in Polr2a intron 1 in injured proximal tubule cells, containing a KLF6-binding site. Knockdown of KLF6 reduced POLR2A induction, while proximal tubule–specific KLF6 further increased Polr2a levels after injury. Mice with tubule-specific KLF6 induction had more RPB1-positive proximal tubules and more injury post-AKI. Human kidney samples with DNA damage–induced CKD and diabetic kidney disease also had high POLR2A /RPB1 expression in dedifferentiated proximal tubule cells. Conclusions Prolonged high expression of RPB1 is associated with dedifferentiated proximal tubule cells. Mice overexpressing KLF6 had higher expression of RPB1 and worse kidney injury after DNA damage.

Disruption of Branched-Chain Amino Acid (BCAA) Catabolism in Proximal Tubule Cells Impairs Mitochondrial Function and Induces Injury

Journal of the American Society of Nephrology · 2025-10-01

Background: Branched-chain amino acids (BCAAs) catabolism is increasingly acknowledged as a key factor in the metabolic disturbances associated with kidney diseases. Targeting BCAA catabolism through the branched-chain alpha-ketoacid dehydrogenase complex (BCKDH), the enzyme responsible for the rate-limiting step in this pathway, represents a valuable strategy for understanding the BCAAs metabolic dysfunction underlying kidney pathologies, including acute kidney injury (AKI), and addresses an important gap in the field. Methods: A mouse model with floxed Bckdhb (a key subunit of BCKDH) on a C57BL/6J background was generated using CRISPR/Cas9-mediated genome editing. Primary proximal tubule cells were isolated from 3.5- to 4-week-old male pups and cultured until 85-95% confluency. Cells were then transduced with adenovirus-Cre (50 MOI) to induce Bckdhb knockdown, with adenovirus-GFP (50 MOI) serving as a control. Mitochondrial function was evaluated using Seahorse MitoStress test. In parallel, quantitative PCR was performed to assess the expression of injury and inflammation markers associated with Bckdhb knockdown. Results: Quantitative PCR of RNA from adenovirus-Cre–treated cells confirmed successful knockdown of the Bckdhb gene without compensatory changes in Bckdha levels. Functionally, Bckdhb knockdown impaired mitochondrial respiration, with significant reductions observed in ATP-linked respiration, basal respiration, and spare respiratory capacity. In parallel, expression of injury markers, including connective tissue growth factor (Ctgf), vimentin (Vim), and kidney injury molecule-1 (Havcr1), were significantly elevated. Additional markers, such as Cdkn1a (p21), Krt20 (Cytokeratin-20), and Tgfb, also trended upward. Notably, expression of pro-inflammatory cytokines Tnfa and Il6 remained unchanged compared to controls. Conclusion: Proximal tubule-specific knockdown of Bckdhb disrupts mitochondrial bioenergetics and induces injury-associated gene expression without triggering a strong inflammatory response. These findings highlight the critical role of BCAA catabolism in maintaining renal mitochondrial function and epithelial cell health, and suggest that dysregulation of this pathway may contribute to the development or progression of kidney injury. Funding: NIDDK Support - NIDDK Support, NIDDK Support

Fenofibrate Attenuates Kidney Injury by Enhancing Use of Fatty Acids from Lipid Droplets in Proximal Tubules

Journal of the American Society of Nephrology · 2025-10-01

Background: Previous studies demonstrate decreased PPARα- and KLF15-mediated fatty acid beta-oxidation (FAO) and increased lipid droplet formation in proximal tubules (PTs) in AKI. However, it remains unclear whether this increase in lipid droplets is a consequence or a driver of PT injury. The aim of the study is to test the hypothesis that fenofibrate, a PPARα agonist, attenuates PT injury, AKI and eventual interstitial fibrosis by enhancing the utilization of fatty acids from lipid droplets. Methods: 9-week-old FVB/N mice were fed a 0.2% fenofibrate or regular chow diet for 12 days, followed by vehicle or aristolochic acid I (AAI) (2 mg/kg, IP, every 72 hours for 9days). Acute phase is 3 days after last dose of AAI and remodeling phase is 3 weeks after the last dose of AAI. Kidney function was assessed by blood urea nitrogen (BUN), serum creatinine, and FITC sinistrin-based GFR. Histological analyses (PAS, H&E) and immunostaining with RT-PCR were conducted for markers of FAO, lipid droplet formation, PT and fibrotic markers. SnRNA-seq was conducted in WT and PT-specific Klf15 knockout (Klf15PTKO) mice post-AAI. Results: Fenofibrate restored kidney function (BUN, creatinine, and GFR) and lotus lectin expression, and reduced fibrotic markers (vimentin, a-sma, sirus red) as compared to control mice, during the acute and remodeling phase post-AAI treatment. The number of lipid droplets in PT cells, per cross-sectional area, were reduced in fenofibrate- as compared to vehicle-treated-AAI mice (Oil-Red-O, Perilipin 2 (PLIN2) staining). Fenofibrate also increased the expression of FAO and lipid droplet genes (Ppara, Klf15, Pgc1a, Cpt1a, Acaa2, Acox1, Lpin2) and reduced the expression of fibrotic genes (Tgfb, Col1a1) as compared to vehicle-treated mice. Klf15PTKO attenuated these salutary effects of fenofibrate post-AAI treatment. SnRNA-seq also showed a reduction in transcripts involved in utilization of lipid droplets (Lpin2, Gpat4, Gpam, and Dgat1) in PT cells in Klf15PTKO as compared to Klf15fl/fl mice post-AAI. Conclusion: PPARα-KLF15 agonism attenuates AKI to CKD transition by enhancing fatty acid oxidation metabolism through increased utilization of lipid droplets in PT cells post-AAI treatment. Funding: NIDDK Support, Veterans Affairs Support

Differential Alteration of Branched-Chain Amino Acid Catabolism in Mouse Primary Proximal Tubules

Journal of the American Society of Nephrology · 2025-10-01

Background: Acute kidney injury (AKI) disrupts key metabolic pathways in proximal tubule (PT) cells, including fatty acid oxidation (FAO) and branched-chain amino acid (BCAA) catabolism, both essential for energy production. Our previous results showed pharmacological activation of BCAA catabolism could protect against nephrotoxic AKI in male mice. Females are more resistant to AKI than males, and RNA-seq analysis of human AKI patients revealed sex-specific differences in BCAA catabolic enzyme expression. Our aim was to investigate the alteration of BCAA catabolism in nephrotoxin-treated primary PT cells from male and female mice in vitro. Methods: Mouse primary PT cells were isolated from wild-type C57Bl/6 littermates and treated with aristolochic acid I (AAI) to induce injury, with or without the BCAA catabolism activator BT2. Injury markers and BCAA genes and transporters were analyzed by qRT-PCR, mitochondrial respiration by Seahorse assay, intracellular BCAA levels by BCAA-Glo, and mTORC1 signaling by Western blot. Leucine treatment effects on injury markers and mTORC1 activation were also tested. Results: AAI induced injury in both sexes, with slightly higher Krt20 expression in males. Cell cycle arrest markers Cdkn1a and Ccng1 increased in both sexes but were more elevated in females. BCAA catabolic genes were downregulated after AAI, with baseline expression of known sex-dependent genes Oxct1, Mut, Ivd, Auh and Acadm higher in females. BCAA transporter genes were downregulated to similar levels in both sexes after AAI treatments. AAI reduced mitochondrial respiration similarly in both sexes, but BT2 improved respiration in males only. In male cells, AAI resulted in BCAA accumulation and mTORC1 activation. In female cells, BCAA levels were lower at baseline and did not accumulate after AAI, and mTORC1 signaling was not activated. Leucine treatments increased mTORC1 signaling pathway in male cells but not female cells. Conclusion: These findings suggest a sex-specific role of BCAA catabolism in the pathophysiology of AKI. The differential impact of BCAA catabolism on cellular respiration, mTORC1 signaling, BCAA accumulation and response to therapeutic intervention with BT2 highlights the importance of considering sex as a biological variable in the study and treatment of AKI. Funding: NIDDK Support, Private Foundation Support

Sex-Specific Differences in Branched Chain Amino Acid (BCAA) Catabolism and Mitochondrial Respiration in Healthy and Injured Kidneys

Journal of the American Society of Nephrology · 2024-10-01

Background: Proximal tubule (PT) cells are highly susceptible to acute kidney injury (AKI) and undergo significant changes in cellular metabolism, directly contributing to injury. PT cells use fatty acid oxidation (FAO) and branched chain amino acid (BCAA) catabolism to generate ATP, however during AKI, both pathways are downregulated. Women and female mice are protected from AKI versus men and male mice. Our aim was to investigate BCAA catabolism in male and female human subjects and mice PT cells. Methods: Publicly available RNA-sequencing data from kidney cortex of males and female subjects with and without AKI (three per group) were used for differential gene expression and pathway enrichment analysis using Seurat and EnrichR. Primary PT cells from Bckdhbfl/fl male and female mice were infected with adenoviral GFP (control) or CRE to excise Bckdhb, which encodes a crucial subunit of BCKDH, the rate-limiting enzyme in BCAA catabolism, followed by qRT-PCR. Wildtype (WT) primary PT cells were treated with aristolochic acid I to induce injury, and BT2 to activate BCKDH and increase BCAA catabolism. All primary PT cells underwent live cell metabolic assays using a Seahorse bioanalyzer. Results: The BCAA catabolism pathway was significantly downregulated in males and females with AKI compared to healthy controls. However, more genes in the pathway were downregulated in males than females, and the downregulated genes were different between males and females. For example, BCKDHB was significantly downregulated in males but not females. To determine the specific role of Bckdhb, CRE recombination was undertaken in Bckdhbfl/fl primary PT cells, resulting in Bckdhb mRNA knockdown of 60% and 80% in male and female cells, respectively. Seahorse assays showed significant downregulation of basal respiration and mitochondrial ATP production rate in male cells but not in female cells upon Bckdhb knockdown, with no significant differences in glycolytic ATP production rate. BT2 treatment protected mitochondrial function in WT male primary PT cells treated with AAI but not female primary PT cells. Conclusion: These findings suggest that BCKDH may play a more important role in males versus females, both in healthy and injured kidneys, which may have implications for targeted therapeutic strategies for AKI. Funding: NIDDK Support - NIDDK Support, NIDDK Support

RNA Polymerase II Subunit POLR2A/RPB1 Upregulation Is Detrimental in Kidney Injury

Journal of the American Society of Nephrology · 2024-10-01

Background: Proximal tubule (PT) injury is a major factor in the severity of acute kidney injury (AKI) and transition to fibrosis. Our single nuclear RNA-sequencing (snRNA-seq) in a PT-specific nephrotoxin [aristolochic acid I (AAI)]-induced AKI in mice revealed an injured PT cluster with high enrichment of Polr2a, encoding RPB1, the largest subunit of RNA polymerase II (RNAPII). Our aim was to study the role of PT-specific RPB1in AKI. Methods: Immunofluorescence (IF) for RPB1 was undertaken in mice after multiple 2mg/kg AAI injections, 7mg/kg repeated low dose cisplatin (RLDC) for 4 weeks, or unilateral ureteral obstruction (UUO) for 3 or 7 days, or in human AA-exposed chronic kidney disease (CKD) patient samples, with PT brush border counterstaining. HK-2 cells were treated with DMSO or 25µM AAI and scrambled or POLR2A siRNA, with quantification of cell number (CyQuant assay); IL6, CTGF, and CDKN1A mRNA expression; and IF for vimentin (dedifferentiation) or gH2A.X (DNA damage) at 48 hours. Single cell ATAC-seq was undertaken in mice after one dose of AAI. Results: Over a series of 4 AAI injections, despite PT loss, RPB1-positive (RPB1+) tubular nuclei accumulated, and were associated with dedifferentiated PT. This was replicated in mice after RLDC and UUO. Knockdown of POLR2A in HK-2 cells treated with AAI resulted in increased cell death, but the remaining cells expressed less IL6 and CTGF, were less dedifferentiated, had less DNA damage, and more normal cell cycle, with reduced CDKN1A expression. ATAC-seq indicated a new region of open chromatin in Polr2a intron 1 in the injured PT cluster, containing AP-1 and Krüppel-like factor 6 (KLF6) transcription factor binding sites. Trajectory and RNA velocity analysis showed that Klf6 upregulation preceded Polr2a induction, suggesting regulation of Polr2a by KLF6. RPB1 remained high in the fibrotic phase after AAI, and mice overexpressing human KLF6 had more RPB1+ nuclei than control mice. Dedifferentiated PT in human AA-exposed CKD patients also had RPB1+ nuclei. Conclusion: To date, this is first study to showthat sustained high RPB1 expression after AKI may trap PT cells in a dedifferentiated state, and clearance of cells by POLR2A knockdown may be beneficial to attenuate the progression of AKI to CKD. Funding: NIDDK Support

Resistance to Aristolochic Acid-Induced AKI in Female Mice

Journal of the American Society of Nephrology · 2024-10-01

Background: Females are less susceptible to acute kidney injury (AKI) compared to males, in both humans and mice. The resistance of female mice to developing AKI poses a significant barrier to pre-clinical therapeutic studies, and many studies only use male mice. Our aim was to establish a protocol to induce AKI in female mice using the proximal tubule (PT)-specific nephrotoxin, aristolochic acid I (AAI). Methods: Male and female C57Bl/6 mice aged 12 weeks were injected intraperitoneally with 2 doses of 3mg/kg AAI, and female mice were also given 2 doses of 4mg/kg – 6.5mg/kg AAI. All doses were given 72 hours apart, followed by euthanasia 24 hours after the last injection. Body and kidney weights were recorded and serum collected for creatinine and urea nitrogen measurements. Kidneys were analyzed using hematoxylin and eosin (H&E) and periodic acid Schiff staining, and immunofluorescence for cytokeratin-20 (KRT-20; injured PT) and Lotus lectin (LL; uninjured PT). Liver morphology was assessed using H&E. Results: Female mice given the standard male AKI dose of 3mg/kg AAI did not show any histological injury, whereas male mice had extensive PT death. Female mice given 4-6.5mg/kg AAI lost weight, but there was no difference between different doses, and kidney weights did not change. Serum creatinine and urea nitrogen concentrations were not elevated in response to any of the AAI doses. Histologically, kidneys showed minimal injury at 4mg/kg and 5mg/kg, and only small areas of PT cell death and mild loss of brush border at 6mg/kg and 6.5mg/kg AAI. LL and KRT-20 staining showed a linear trend towards reduced LL across increasing AAI doses (one-way ANOVA test for trend), and minimal, non-significant increases in KRT-20. AAI is activated in the liver and H&E showed significant liver vacuolization in mice treated with 6mg/kg AAI, despite only mild kidney injury. Conclusion: Female mice had mild kidney injury and no loss of kidney function at AAI doses double that needed to cause AKI in male mice. Furthermore, high doses caused liver injury, thus limiting their utility. Studies using high doses of AAI should be interpreted with caution due to the likelihood of liver injury. Further studies are needed to identify a protocol to induce robust AKI in female mice without liver injury, to facilitate future pre-clinical therapeutic studies. Funding: NIDDK Support

Sex-Dependent Dynamic Transcriptional Responses in AKI in Mice

Journal of the American Society of Nephrology · 2024-10-01

Background: Previous studies demonstrated the role of sex differences in protection from acute kidney injury (AKI). However, the mechanism(s) that increase the susceptibility to AKI in males vs females remains unclear. We aim to investigate the sex-specific transcriptomic differences with single nucleus multiome sequencing data to identify mediators that increases the susceptibility to AKI in males vs females post-aristolochic acid I (AAI) injury in mice. Methods: Male and female mice were injected with one dose of DMSO or AAI (2mg/kg) and perfused after 72 hours. Using 10X Genomics, single nuclear multiome (scRNA and scATAC) sequencing on the kidney samples was performed. CellRanger-ARC aligned the sequencing data and proceeded through the ArchR pipeline for data mining. Alevin-fry generated splice counts on the original data and scVelo performed RNA velocity analysis. Kidneys were stained for FOSL1 and JUNB by immunofluorescence. Results: Compare to females, 4 unique PT clusters formed in males after AAI. Two of these were similar to uninjured PT clusters, but the other two showed increased expression of injury markers, including AP-1 transcription factors(TF) (Fosl1, Junb; injury cluster), NF-kB signaling pathway (Rela, Relb, Nfkb1, Nfkb2; intermediate cluster). Immunostaining validated expression of these unique proteins in injured PT clusters in male mice vs female mice post-AAI. The injury cluster demonstrated high expression of Junb, as well as motif enrichment and footprinting bias. Consistent with its role as an early response gene, RNA velocity showed that Junb was already fully spliced, suggesting earlier upregulation. By contrast Fosl1, had lower expression, but a higher RNA velocity, suggesting ongoing upregulation. Fosl1 motifs were already open, suggesting that chromatin in these cells was already primed for Fosl1 binding. In the intermediate cluster, NFKB pathway genes showed less well defined dynamics, but enrichment for open motifs and footprinting analysis suggested active transcription. Analysis of downstream target genes showed high expression and dynamics of Plau, Tnfaip3, and Vcam1. Expression, motifs, and footprinting analyses for these TFs in female PT clusters after AAI were all negative. Conclusion: AKI induced dynamic AP-1 and NFKB transcriptional activity in two distinct injured PT clusters in male, but not female mice. Funding: NIDDK Support, Veterans Affairs Support