Biology subjects

Pabla, N. S.

Publications and source records attributed to Pabla, N. S..

10 recordsLinked to original sources

bioRxiv2025

Androgens Drive SOX9 Upregulation in Injured Proximal Tubular Cells

IntroductionSex influences susceptibility, recovery, and long-term outcomes after acute kidney injury (AKI) in humans. Rodent models have been invaluable for elucidating AKI mechanisms; however, most studies have focused on males, assuming direct applicability to females, an assumption that remains largely untested. In males, the transcription factor SOX9 regulates injury-associated proximal tubule cell states, supporting survival during injury but also contributing to maladaptive repair. Given the well-described role of SOX9 in sex determination during development and its emerging importance in AKI, we investigated whether it mediates sex-specific tubular responses in the adult kidney. MethodsWe first optimized differential ischemic and cisplatin conditions in males and females to generate injury-matched models. We then used these models to ask whether proximal tubular cell responses, particularly Sox9 upregulation, are sex dependent. Tubule-specific knockout models of Sox9, Sox4, Sox11, Vgf, Zfp24, and the androgen receptor (Ar), along with gonadectomy and hormone replacement studies, combined with gene and protein analyses, were used to define regulatory networks. ResultsIn both ischemic and nephrotoxic AKI, SOX9 expression was markedly blunted in females, with levels more than fivefold lower than in males. While injury biomarkers such as NGAL and KIM1 were equally induced in both sexes, Sox9 and its downstream target Vgf showed markedly reduced induction in females, whereas the upstream regulator Zfp24 was functionally relevant only in males. Deletion of Sox9, Zfp24, or Vgf worsened injury in males but not in females. In contrast, Sox4 and Sox11 were equally upregulated and protective in both sexes. Castration or proximal tubule-specific deletion of Ar in males abolished Sox9 induction, establishing a testosterone-dependent regulatory axis. ConclusionsThese findings define a hormone-driven, male-specific tubular repair program and demonstrate that injury and recovery pathways differ fundamentally between sexes, underscoring the need for sex-inclusive therapeutic strategies for AKI. Translational StatementSex influences susceptibility and outcomes after AKI, but the molecular basis remains unclear. SOX9, a transcription factor recently identified as one of the most highly upregulated genes in proximal tubular cells during AKI in both mice and humans, has been presumed to mediate a universal protective program. Using injury-matched murine models of AKI, we now demonstrate that this response is restricted to males, driven by testosterone and androgen receptor signaling, while SOX9 induction is markedly blunted and functionally dispensable in females. These findings reveal that tubular protective pathways differ fundamentally between sexes despite equivalent injury severity. Because SOX9 activation confers epithelial protection in males, therapeutic targeting of this pathway may have clinical relevance primarily in male patients. These data underscore the importance of incorporating biological sex into mechanistic, translational, and clinical studies of kidney injury and recovery.

cell biology↗

bioRxiv2025

Myeloid-specific TFAM deficiency drives mitochondrial DNA stress and exacerbates allergic airway inflammation

Asthma is the most prevalent chronic inflammatory lung disease in adolescents and young adults, characterized by persistent airway inflammation and remodeling. Increasing evidence indicates that activated lung macrophages play a significant role in the initiation, intensity, progression, and resolution of allergic airway inflammation. However, the underlying mechanisms regulating macrophage-mediated inflammation in asthma remain incompletely understood. Our previous work revealed increased mitochondrial DNA (mtDNA) depletion and mitochondrial damages in the lungs of asthmatic mice, implicating mitochondrial dysfunction in disease pathogenesis. Given that mitochondrial transcription factor A (TFAM) is essential for mtDNA maintenance and integrity, we hypothesized that TFAM has a fundamental role in regulating mtDNA stress and downstream inflammtroy response in asthma. Using myeloid-specific TFAM knockout (TFAMfl/flLysMcre, TFAM KO) mice subjected to allergens sensitization and challenge, we observed pronounced mitochondrial dysfunction and accentuated asthmatic inflammation. This was accompanied by elevated expression of asthma-associated mediators, including il-13, muc5a/c, muc5b, and ccl17. In addition, TFAM deficiency was associated with increased eosinophilia and and cytosolic mtDNA release, contributing to exacerbated airway pathology. Together, we have identified a critical role of TFAM in myeloid cells that contributes to asthmatic airway inflammation. These results suggest that therapeutic restoration of TFAM function may offer a novel strategy to mitigate mitochondrial stress, reduce airway inflammation, and improve outcomes in patients with moderate to severe asthma.

immunology↗

bioRxiv2024

Circadian clock disruption and growth of kidney cysts in autosomal dominant polycystic kidney disease

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 and PKD2 genes, and often progresses to kidney failure. ADPKD progression is not uniform among patients, suggesting that factors secondary to the PKD1/2 gene mutation could regulate the rate of disease progression. Here we tested the effect of circadian clock disruption on ADPKD progression. Circadian rhythms are regulated by cell-autonomous circadian clocks composed of clock proteins. BMAL1 is a core constituent of the circadian clock. MethodsTo disrupt the circadian clock, we deleted Bmal1 gene in the renal collecting ducts of the Pkd1RC/RC (RC/RC) mouse model of ADPKD (RC/RC;Bmal1f/f;Pkhd1cre, called DKO mice), and in Pkd1 knockout mouse inner medullary collecting duct cells (Pkd1Bmal1KO mIMCD3 cells). Only male mice were used. ResultsHuman nephrectomy ADPKD kidneys and Pkd1KO mIMCD3 cells showed reduced Bmal1 gene expression compared to normal controls. When compared to RC/RC kidneys, DKO kidneys showed significantly altered clock gene expression, increased cyst growth, cell proliferation, apoptosis and fibrosis. DKO kidneys also showed increased lipogenesis and cholesterol synthesis-related gene expression, and increased tissue triglyceride levels compared to RC/RC kidneys. Similarly, in vitro, Pkd1Bmal1KO cells showed altered clock genes, increased lipogenesis and cholesterol synthesis-related genes, and reduced fatty-acid oxidation-related gene expression compared to Pkd1KO cells. The Pkd1Bmal1KO cells showed increased cell proliferation compared to Pkd1KO cells, which was rescued by pharmacological inhibition of lipogenesis. ConclusionRenal collecting duct specific Bmal1 gene deletion disrupts the circadian clock and triggers accelerated ADPKD progression by altering lipid metabolism-related gene expression. Key pointsO_LILack of BMAL1, a circadian clock protein in renal collecting ducts disrupted the clock and increased cyst growth and fibrosis in an ADPKD mouse model. C_LIO_LIBMAL1 gene deletion increased cell proliferation by increasing lipogenesis in kidney cells. C_LIO_LIThus, circadian clock disruption could be a risk factor for accelerated disease progression in patients with ADPKD. C_LI

molecular biology↗

bioRxiv2024

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.

cell biology↗

bioRxiv2023

IL-22 promotes acute kidney injury through activation of the DNA damage response and cell death in proximal tubule cells

Acute kidney injury (AKI) affects over 13 million people world-wide annually and is associated with a fourfold increase in mortality. Our lab and others have shown that DNA damage response (DDR) governs the outcome of AKI in a bimodal manner. Activation of DDR sensor kinases protects against AKI, while hyperactivation of DDR effector proteins, such as p53, induces to cell death and worsens AKI. The factors that trigger the switch from pro-reparative to pro-cell death DDR remain to be resolved. Here we investigate the role of interleukin 22 (IL-22), an IL-10 family member whose receptor (IL-22RA1) is expressed on proximal tubule cells (PTCs), in DDR activation and AKI. Using cisplatin and aristolochic acid (AA) induced nephropathy as models of DNA damage, we identify PTCs as a novel source of urinary IL-22, making PTCs the only epithelial cells known to secret IL-22, to our knowledge. Functionally, IL-22 binding its receptor (IL-22RA1) on PTCs amplifies the DDR. Treating primary PTCs with IL-22 alone induces rapid activation of the DDR in vitro. The combination of IL-22 + cisplatin or AA treatment on primary PTCs induces cell death, while the same dose of cisplatin or AA alone does not. Global deletion of IL-22 protects against cisplatin or AA induced AKI. IL-22 deletion reduces expression of components of the DDR and inhibits PTC cell death. To confirm PTC IL-22 signaling contributes to AKI, we knocked out IL-22RA1 in renal epithelial cells by crossing IL-22RA1floxed mice with Six2-Cre mice. IL-22RA1 KO reduced DDR activation, cell death, and kidney injury. These data demonstrate that IL-22 promotes DDR activation in PTCs, switching pro-recovery DDR responses to a pro-cell death response and worsening AKI. Targeting IL-22 represents a novel therapeutic approach to prevent the negative consequences of the DDR activation while not interfering with the processes necessary for repair of damaged DNA. Translational statementAcute kidney injury, which affects 10-20% of hospitalized patients, is associated with a fourfold increase in mortality and predisposes patients to chronic kidney disease. In the present study, we identify interleukin 22 as a cofactor which worsens acute kidney injury. Interleukin 22 activates the DNA damage response, which in combination with nephrotoxic drugs amplifies the injury response in kidney epithelial cells and increases cell death. Deletion of interleukin 22 from mice or its receptor from mouse kidneys ameliorates cisplatin induced nephropathy. These findings may help clarify the molecular mechanisms of DNA damage induced kidney injury and identify interventions that can help treat acute kidney injury.

cell biology↗

bioRxiv2023

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3{beta}, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3{beta} activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3{beta} activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3{beta}. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces post-synaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated, key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity and human neuropathology.

neuroscience↗

bioRxiv2023

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3/{beta} affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3/{beta} signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3/{beta}. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3/{beta} promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

cell biology↗

bioRxiv2023

Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

biochemistry↗

bioRxiv2021

Nephrotoxicity of the BRAF-kinase inhibitor Vemurafenib isdriven by off-target Ferrochelatase inhibition

A multitude of disease and therapy related factors drive the frequent development of renal disorders in cancer patients. Along with chemotherapy, the newer targeted therapeutics can also cause renal dysfunction through on and off-target mechanisms. Interestingly, among the small-molecule inhibitors approved for the treatment of cancers that harbor BRAF-kinase activating mutations, vemurafenib can trigger tubular damage and acute kidney injury (AKI). To investigate the underlying mechanisms, here, we have developed cell culture and mouse models of vemurafenib nephrotoxicity. Our studies show that at clinically relevant concentrations vemurafenib induces cell-death in transformed and primary murine and human renal tubular epithelial cells (RTEC). In mice, two weeks of daily vemurafenib treatment causes moderate AKI with histopathological characteristics of RTEC injury. Importantly, RTEC-specific BRAF gene deletion did not influence renal function under normal conditions or alter the severity of vemurafenib-associated renal impairment. Instead, we found that inhibition of ferrochelatase (FECH), an enzyme involved in heme biosynthesis contributes to vemurafenib nephrotoxicity. FECH overexpression protected RTECs and conversely FECH knockdown increased the sensitivity to vemurafenib nephrotoxicity. Collectively, these studies suggest that vemurafenib-associated RTEC dysfunction and nephrotoxicity is BRAF-independent and caused in part by off-target FECH inhibition. Translational StatementBRAF is the most frequently mutated protein kinase and a critical oncogenic driver in human cancers. In melanoma and other cancers with BRAF activating mutations, BRAF targeted small-molecule therapeutics such as vemurafenib, and dabrafenib have shown remarkable clinical benefits. However, recent clinical studies have shown that a significant number of patients that receive vemurafenib develop AKI through mechanisms that remain unknown. The present study describes the development of novel experimental models of vemurafenib nephrotoxicity and reveals the underlying off-target mechanisms that contribute to renal injury.

pharmacology and toxicology↗

bioRxiv2020

SOX9 promotes stress-responsive transcription of VGF nerve growth factor inducible gene in kidney epithelial cells

Acute kidney injury (AKI) is a common clinical condition associated with diverse etiologies and abrupt loss of renal function. In patients with sepsis, rhabdomyolysis, cancer, as well as cardiovascular disorders, the underlying disease or associated therapeutic interventions can cause hypoxic, cytotoxic, and inflammatory insults to renal tubular epithelial cells (RTECs) resulting in the onset of AKI. To uncover stress-responsive disease-modifying genes, here we have carried out renal transcriptome profiling in three distinct murine models of AKI. We find that Vgf nerve growth factor inducible gene upregulation is a common transcriptional stress response in RTECs to ischemia, cisplatin, and rhabdomyolysis-associated renal injury. The Vgf gene encodes a secretory peptide precursor protein that has critical neuro-endocrine functions; however, its role in the kidneys remains unknown. Our functional studies show that RTEC-specific Vgf gene ablation exacerbates ischemia, cisplatin, and rhabdomyolysis-associated AKI in vivo and cisplatin-induced RTEC cell death in vitro. Importantly, addback experiments showed that aggravation of cisplatin-induced renal injury caused by Vgf gene ablation is partly reversed by TLQP-21, a Vgf-derived peptide. Finally, in vitro and in vivo mechanistic studies showed that injury-induced Vgf upregulation in RTECs is driven by the transcriptional regulator Sox9. These findings reveal a crucial downstream target of the Sox9-directed transcriptional program and identify Vgf as a stress-responsive protective gene in kidney epithelial cells.AbbreviationsAKIAcute kidney injuryRTECrenal tubular epithelial cellsBUNblood urea nitrogenChIPchromatin immunoprecipitationSox9SRY-Box transcription factor 9.View Full Text

cell biology↗