American Journal of Physiology-Renal Physiology

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.

Background The relative contributions of molecular size, electrostatic charge, and filtration rate to glomerular transport remain controversial. We hypothesized that glomerular sieving data contain a limited number of underlying transport modes that can be identified directly from experimental measurements. Methods Glomerular sieving coefficients were measured in anesthetized rats using neutral and anionic polysucrose during baseline conditions and glucagon-induced hyperfiltration. Data were analyzed using aligned-rank two-factor ANOVA, nonlinear mixed-effects regression of an electrostatic distributed two-pore model, pairwise correlation analysis, and principal component analysis. Results Hyperfiltration reduced the sieving of small and intermediate polysucrose molecules, whereas anionic polysucrose exhibited lower sieving coefficients than neutral polysucrose over a broad range of molecular sizes. An electrostatic distributed two-pore model accurately reproduced the observed effects of filtration rate and molecular charge and yielded an effective pore-wall charge density of 5.4 mC/m2 (95% confidence interval, 4.5 to 6.6). Pairwise correlation analysis revealed strong coupling between neighboring molecular sizes throughout the entire measured size range. Principal component analysis of the 2.5-8.0 nm size-selective region showed that the first principal component explained 96.3% of the variance and the first two principal components explained 99.9% of the variance. Separate analyses of the 2.5-5.0 nm and 5.0-8.0 nm transport regions showed that the first principal component explained 99.4% and 89.5% of the variance, respectively. Conclusions Glomerular sieving curves exhibited a highly constrained low-dimensional structure despite differences in molecular charge, filtration rate, and individual animals. The observed transport structure was consistent with distinct small-pore and large-pore transport domains and enabled highly effective principal component-based denoising of experimental sieving data.

Spinal cord injury (SCI) disrupts supraspinal autonomic pathways that regulate cardiovascular function, producing marked blood pressure instability and contributing to secondary injury in peripheral organs. The kidney is particularly vulnerable to these disturbances because renal blood flow (RBF) depends on tightly regulated interactions between neural, myogenic, and vascular control mechanisms. However, how SCI level and chronicity alter dynamic renal autoregulation remains poorly defined. Here, we investigated the effects of high- and low-thoracic SCI on renal hemodynamic control using in vivo blood pressure and RBF recordings in female mice. Hemodynamics were assessed at baseline and during acute sympathetic stimulation induced by norepinephrine (NE; 10 g/kg, i.v.) at 24 h and 4 wk following spinal cord transection at thoracic level 3 (T3) or thoracic level 10 (T10). Time-domain analyses quantified systolic blood pressure recovery, while frequency-domain analyses were used to resolve myogenic and sympathetic contributions to RBF regulation. High-thoracic SCI caused marked disruption of renal vascular responses to acute hypertension, producing paradoxical increases in RBF during NE-induced pressure elevations and sustained reductions in baseline and evoked RBF activity within frequency ranges associated with myogenic and sympathetic vasomotion. These impairments were most pronounced during the chronic phase of injury, consistent with loss of dynamic autoregulatory control and vascular remodeling. In contrast, low-thoracic SCI preserved baseline renal vasomotor activity and demonstrated recovery of dynamic autoregulatory responses over time. These findings identify SCI level and chronicity as critical determinants of renal microvascular regulation and demonstrate that high-thoracic SCI produces persistent autonomic-vascular uncoupling. This disruption of dynamic renal autoregulation represents a previously underappreciated mechanism of secondary organ vulnerability following neurotrauma.

Circulating biomarkers are increasingly used for patient risk stratification in chronic kidney disease (CKD) and heart failure with preserved ejection fraction (HFpEF). However, clinically relevant circulating biomarkers remain insufficiently characterized in rodent models recapitulating diabetic cardiorenal disease with HFpEF. To address this gap, we evaluated 20 translationally relevant inflammation-associated biomarkers in the diabetic db/db uninephrectomized (UNx)-ReninAAV mouse model of CKD and HFpEF. db/db UNx-ReninAAV mice exhibited marked increases in circulating soluble urokinase-type plasminogen activator receptor (suPAR) and monocyte chemoattractant protein-1 (MCP-1), and in interleukin 10 (IL-10) at late stages of disease. Histological analyses confirmed increased tissue expression of suPAR in the heart and kidney and of MCP-1 in the heart. Notably, circulating suPAR levels correlated with disease severity, including systolic and diastolic cardiac dysfunction and albuminuria. Together, these results provide a systematic analysis of biomarkers in a rodent model of diabetes, CKD and HFpEF and identify suPAR as the biomarker most closely associated with disease severity.

Blockade by sedimentary particles, such as mineral crystals, is a continuous risk the kidney tubule faces. To prevent that, kidney resident macrophages form transepithelial protrusions and remove intratubular sedimentary particles, a behavior particularly prevailing in the medulla over the cortex. However, the molecular mechanisms underlying this characteristic behavior of medulla macrophages are incompletely understood. In this study, we identified that the medulla had higher mechanical stiffness than the cortex in steady state, which was further elevated when kidney stone formed. Increased tissue rigidity was sensed by medulla macrophages via mechanoreceptor Piezo1, which promoted macrophage protrusion formation and their ability to clean the tubules. Loss of Piezo1 expression in kidney macrophages predisposed mice to intratubular accumulation of mineral crystal in steady state and accelerated kidney stone formation during oxalate intake challenge. Signaling via Piezo1 mobilized molecules involved in cell adhesion and protrusion assembly, including Talin2 and focal adhesion kinase (FAK). Finally, we developed a first-of-its-kind cell-based therapy for the treatment of experimental nephrolithiasis by exploiting macrophage Piezo1 activity, and this strategy shows great promise for future translational research.

Key PointsO_LIPerfused human kidney MPS revealed CsA-associated sublethal tubular stress that was not detected by conventional 2D viability assays or by KIM-1 release in 3D MPS. C_LIO_LIAt matched exposure, VCS preserved mitochondria and activated ER chaperones and iron detoxification, with no p21 arrest compared to CsA. C_LIO_LIMechanistic separation supports VCSs nephroprotection potential and early mechanism-based biomarkers to guide CNI choice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/737071v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@e5e01dorg.highwire.dtl.DTLVardef@1dc9167org.highwire.dtl.DTLVardef@1ce22f8org.highwire.dtl.DTLVardef@5a053a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG BackgroundCalcineurin inhibitors (CNIs) are indispensable for transplantation immunosuppression, yet cyclosporine A (CsA) produces renal toxicity. Voclosporin (VCS), a CsA analog, is proposed to be less nephrotoxic, but mechanisms remain unclear. MethodsPrimary human proximal tubule epithelial cells (PTECs) were exposed to CsA or VCS in 2D monolayers and perfused 3D kidney microphysiological system (MPS). Viability was assessed in 2D cultures by MTS, mitochondrial membrane potential ({Delta}{Psi}m) by TMRM flow cytometry, and soluble injury and inflammatory biomarkers in MPS effluents by ELISA and MSD multiplex assays. RNA sequencing of 3D-cultured PTECs was used to identify differentially expressed genes and pathways. ResultsIn 2D PTECs, neither drug reduced viability. In 3D MPS effluents, KIM-1 did not distinguish CsA from VCS, whereas the MSD biomarker panel showed larger aggregate deviation with CsA. Confocal tomography showed CsA-associated mitochondrial fragmentation, whereas VCS preserved reticular mitochondrial architecture. TMRM flow cytometry showed a treatment-dependent difference in TMRM-positive cells, with VCS yielding the highest TMRM-positive fraction and exceeding CsA, supporting preservation of {Delta}{Psi}m relative to CsA. RNA-seq identified 1188 CsA-specific and 185 VCS-specific differentially expressed genes, with 304 shared. Pathway analysis indicated CsA enrichment of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, p21-associated G2/M checkpoint arrest, and transcriptional signatures consistent with ferroptosis priming, while VCS mainly induced ER chaperone and ER-associated degradation gene programs without activating canonical UPR sensors and showed limited cell-cycle suppression. ConclusionsA physiologically relevant 3D kidney MPS revealed sublethal tubular stress from CsA that is masked in 2D culture, including mitochondrial depolarization, proteostatic stress, and ferroptosis priming. At matched exposure, VCS preserved mitochondrial function and proteostasis while eliciting a narrower, adaptive ER quality control response. These data support VCS as a nephron-sparing immunosuppressant and 3D MPS as a mechanism-based platform for evaluating renal safety of drugs and nominating early sub-lethal tubular injury biomarkers.

Abstract/SummaryHuman variants in renal polycystins (PKD1, PKD2) are responsible for most forms of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder without curative drug treatment. Renal polycystins form ion channels in primary cilia, but our understanding of their molecular dysregulation caused by disease-associated variants is limited. Using cryo-electron microscopy (cryo-EM), primary cilia electrophysiology and super-resolution analysis, we investigated the mechanistic impact and pathogenic potential of the disease-associated PKD2 missense variant (D511V) located within the channels voltage sensor domain (VSD). Our findings define how this mutation neutralizes critical transmembrane charge interactions, which attenuates PKD2 protein stability resulting in abolished ciliary channel trafficking and function in membranes. To assess the pathogenic effect of this variant in vivo, we generated novel mouse strains carrying the analogous PKD2 mutation in combination with a conditional floxed allele (Pkd2D509V/fl) that exhibit renal tubule primary cilia degeneration and develop rapid renal cysts. Our results establish a clear direct correlation between the in vitro molecular dysfunction and phenotypic in vivo consequences while providing a valuable tool to evaluate ADPKD therapeutic interventions. Translational StatementADPKD is a genetic kidney disorder affecting millions of patients globally and is primarily caused by variants in renal polycystin genes (PKD1, PKD2). Polycystins function as ion channel subunits in primary cilia but the mechanistic impact and cystogenic propensity of disease-associated variants remain poorly defined. The authors employ advanced methodologies including cryo-EM to uncover distinct structurally destabilizing effects of a human PKD2 mutation, while generating a new mouse model which genetically expresses the same variant and recapitulates the human disease. The findings define primary cilia degeneration results from PKD2 hypostasis and establish new tools to assess ADPKD therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/734313v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1128cb0org.highwire.dtl.DTLVardef@d2aec1org.highwire.dtl.DTLVardef@1cf3083org.highwire.dtl.DTLVardef@179ee83_HPS_FORMAT_FIGEXP M_FIG C_FIG

Recent initiatives by the U.S. Food and Drug Administration and the National Institutes of Health to reduce animal testing in drug development have highlighted the need for in vitro platforms that better recapitulate human biology for preclinical safety assessment. Drug-induced nephrotoxicity remains a major cause of drug attrition, underscoring the need for human-relevant kidney models. To address this, a pump-free human patient-derived proximal tubule microphysiological system was developed by integrating human renal proximal tubular epithelial cells (hRPTECs), isolated from non-tumorous nephrectomy cortex, with a porous membrane-based microfluidic device. Expanded hRPTECs were cultured for 10 days under static conditions or rocker-driven shear stress approximating physiological proximal tubular flow. Shear stress increased epithelial density, enhanced proximal tubule marker expression (Na+/K+-ATPase and aquaporin-1), and improved Zonula occludens-1 and occludin localization. Bulk RNA sequencing demonstrated transcriptomic changes associated with enhanced apical maturation and epithelial signature. In cisplatin-induced injury assays, shear-conditioned epithelia exhibited reduced cell density and increased {gamma}H2AX staining, indicating greater sensitivity to nephrotoxicity. These findings demonstrate that rocker-driven shear stress promotes epithelial maturation in patient-derived hRPTECs. The pump-free human patient-derived proximal tubule microphysiological system offers a practical, scalable, and physiologically relevant platform for modeling flow-dependent proximal tubule biology and assessing human-relevant nephrotoxicity.

Diabetic nephropathy (DN) is the leading cause of kidney failure in the developed world, but the genetic architecture of DN susceptibility is not well characterised. Here we apply a systems genetics approach in a mouse model of DN to discover novel QTLs for clinically relevant phenotypes including albuminuria, glomerulosclerosis, and macrophage infiltration. For context and prioritisation, we combined single-cell-transcriptomics-guided pQTL and eQTL mapping with cell-type-specific co-expression networks, identifying 192 candidate pGenes for albuminuria. While many were novel, 27% had prior genetic associations, and 40% were validated in a human diabetic cohort. Twelve genes belong to a podocyte network enriched for human GWAS signals. Among those, functional significance of the E3 ubiquitin ligases DCAF6 and ZNRF2 was confirmed by knockdown in Drosophila nephrocytes. Our systems genetics approach identified DN susceptibility genes previously validated in human GWAS while uncovering potential new genes and pathways that could be exploited for risk stratification and therapeutics development.

Polycystic kidney disease (PKD) is the most common cause of end stage renal disease with a known genetic etiology. This disease is characterized by the progressive development and expansion of kidney cysts. While recent studies have shed light on cell types and states contributing to PKD progression following cyst formation, the biological processes at work prior to cyst formation are relatively unexplored. To better understand mechanisms contributing to cystogenesis, we analyze pre-cystic kidneys from Pkd1R3277C/R3277Cmice across multiple early timepoints, generating a transcriptomic atlas of nearly 1 million single nucleus transcriptomes. Activation of a small subset of genes in a precystic signaling pathway drives changes in both the distal convoluted tubule and proximal tubule cells. This pathway overlaps with a recently described "failed repair" transcriptomic signature despite the lack of clear changes in tissue morphology at these early stages of nascent cystogenesis. We identify Creb5 as a critical driver for cystogenesis. This single cell transcriptomic analysis of nascent cystogenesis reveals previously unrecognized cellular signaling at the earliest assessed points in precystic kidneys and provides a foundation for the development of high definition early diagnostic and therapeutic approaches prior to observable cysts in PKD.

Background: Urinary tract infection (UTI) is the most common secondary condition among people with spinal cord injury/disease (SCI/D). Intravesical Lacticaseibacillus rhamnosus GG (LGG) is an antibiotic-sparing approach to managing urinary symptoms. Objective: Determine the optimal number of doses of intravesical LGG for urinary symptom reduction. Design: Prospective, randomized, two-arm dosing trial. Setting: National recruitment with a local subsample providing urine samples in Washington, DC, USA. Participants: Adults with SCI/D and neurogenic lower urinary tract dysfunction (NLUTD) who use intermittent catheterization (IC); 177 enrolled and randomized (intention-to-treat), with 76 compliant instillers (39 low-dose, 37 high-dose) in the per-protocol analytic sample. Interventions: Two (2 doses/24 hours) or four (4 doses/36 hours) intravesical LGG regimens, self-initiated in response to cloudier or malodorous urine per the Self-Management Protocol using Probiotics (SMP-Pro). Main Outcome Measures: Primary: proportion achieving [≥]20% reduction on the Urinary Symptom Questionnaire for Neurogenic Bladder-Intermittent Catheter version (USQNB-IC). Secondary: urinary biomarkers (leukocyte esterase, nitrite, white blood cells, urinary neutrophil gelatinase-associated lipocalin [uNGAL]) and standard urine culture (SUC) in a local subsample. Results: By Day 2, 57.9% (63.8% low-dose; 51.2% high-dose) achieved [≥]20% total symptom reduction; high-dose success rose to 70.0% by Day 4. Thirty percent of high-dose participants did not respond at either time point and could not be distinguished from responders by demographics or urine biomarkers. Urinary biomarkers and SUC were unchanged pre- to post-instillation. No serious adverse events were adjudicated as attributable to intravesical LGG by an independent Data Safety Monitoring Board (DSMB). Conclusions: A two-dose course of intravesical LGG yields clinically meaningful symptom improvement in the majority of people with SCI/D and NLUTD who use IC; four doses benefits a meaningful subgroup of two-day non-responders, while a small cohort remains nonresponsive. These results provide preliminary dosing guidance and support progression to a definitive trial.

The selective autophagy receptor p62/SQSTM1 dynamically shuttles between the nucleus and cytoplasm through distinct nuclear localization and export signals, yet the physiological significance of this trafficking has remained unknown. Here, we generated mice carrying a deletion of the p62 nuclear export signal (dNES) to determine the in vivo role of p62 nuclear export. Homozygous dNES mice developed progressive podocyte injury, glomerulosclerosis, and fatal renal failure by 6-7 weeks of age, whereas heterozygous and dNES/- mice did not develop renal dysfunction. Loss of nuclear export caused constitutive nuclear accumulation of p62, accompanied by the formation of insoluble ubiquitin-positive aggregates and widespread alterations in the renal proteome, including activation of energy metabolism-related pathways and suppression of developmental programs. We previously demonstrated that the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) inhibits the nuclear export receptor XPO1, resulting in nuclear retention of p62 in cultured cells. The present findings provide in vivo evidence that continuous nuclear export of p62 is indispensable for maintaining kidney homeostasis and reveal that excessive nuclear accumulation, rather than cytoplasmic depletion, underlies p62-mediated toxicity. Collectively, these findings establish continuous nuclear export of p62 as an essential mechanism for maintaining kidney homeostasis. Significance StatementThe adaptor protein p62/SQSTM1 continuously shuttles between the nucleus and cytoplasm, but the physiological significance of this trafficking has remained unknown. Here, we show that disrupting the nuclear export signal of p62 causes progressive podocyte injury, glomerulosclerosis, and fatal kidney failure through excessive nuclear accumulation and aggregate formation. In contrast, dNES/+ and dNES/- mice remain healthy, demonstrating that excessive nuclear accumulation, rather than cytoplasmic depletion, drives disease. These findings identify continuous nuclear export as a fundamental mechanism that prevents toxic nuclear accumulation of p62 and preserves kidney homeostasis.

Introduction Acute interstitial nephritis is an important differential diagnosis in patients with deteriorating kidney function. Diagnosis currently requires kidney biopsy, an invasive procedure associated with risks. We hypothesized that urinary T cells may serve as a non-invasive biomarker for acute interstitial nephritis. Methods A total of 320 patients undergoing clinically indicated kidney biopsy were enrolled in a discovery cohort at Charite Berlin (n = 80), an internal validation cohort at Charite (n = 100), and an external validation cohort at The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou (n = 140). Urinary immune cells were assessed by flow cytometry. Renal T cell infiltration was evaluated by immunofluorescence in kidney biopsy specimens from the discovery and internal validation cohorts, including 16 patients with acute interstitial nephritis and 9 patients without acute interstitial nephritis. Additionally, CXCL9 was measured by ELISA in 102 urine samples from these cohorts. Results Across all cohorts, 27 patients (8.4%) were diagnosed with acute interstitial nephritis. In the discovery cohort, multiple urinary T cell subsets were increased in acute interstitial nephritis, with activated CD4+ effector memory T cells expressing CD38 and HLA-DR showing the strongest diagnostic performance. This marker outperformed urinary monocytes, eosinophils, and CXCL9 and was validated in both independent cohorts. Across all cohorts, the area under the receiver operating characteristic curve was 0.84 and increased to 0.91 after exclusion of 8 patients receiving corticosteroids. A cutoff of 211 activated CD4+ effector memory T cells per 100 mL urine yielded a sensitivity of 78% and a specificity of 81%. Urinary activated CD4+ effector memory T cell counts correlated with renal CD4+ and CD4+ CD38+ T cell infiltration in acute interstitial nephritis. Conclusions Urinary activated CD4+ effector memory T cells expressing CD38 and HLA-DR represent a promising non-invasive biomarker for the diagnosis of acute interstitial nephritis.

Background. The 2024 international reference standard for urinary tract infection (UTI) research scores four domains - symptoms and signs, systemic criteria, pyuria, and culture - to classify samples as No UTI, Possible UTI, Probable UTI, or Definite UTI. It explicitly identifies spinal cord injury (SCI) as a condition of impaired symptom perception, and states that catheter-associated UTI requires a separate standard. We applied it to verified-asymptomatic samples from adults with neurogenic lower urinary tract dysfunction (NLUTD) due to spinal cord injury or disease (SCI/D) who use intermittent catheterization (IC), to test whether it can identify UTI likelihood in this population. Methods. The reference standard was applied to 224 samples from 99 adults with NLUTD due to SCI/D using IC, all verified asymptomatic by the Urinary Symptom Questionnaire for Neurogenic Bladder-Intermittent Catheter (USQNB-IC) at sampling and for 72 hours prior. Because no participant was febrile and no blood markers are drawn in this population, the systemic-criteria domain scored zero for every sample; the reported classifications are therefore a floor. Pyuria was scored under conservative and inclusive interpretations of categorical urinary white blood cell (WBC) bins. Results. Under conservative interpretation, 40.2% of samples were classified No UTI, 21.9% Possible UTI, and 37.9% Probable UTI; under inclusive interpretation, 11.6% No UTI, 38.4% Possible UTI, and 50.0% Probable UTI. No sample reached Definite UTI - a structural consequence of the empty systemic domain. Conclusions. The consensus reference standard classifies 38-50% of fully asymptomatic NLUTD-IC samples as Probable UTI, a floor estimate that could only rise if blood markers were available. This confirms the 2024 framework's own prediction that a separate standard is needed for populations with altered symptom expression and baseline-positive urinary markers.

Type 2 diabetes is characterized by progressive aggregation of islet amyloid polypeptide (IAPP) within the islets of Langerhans, a process strongly implicated in beta-cell dysfunction and loss. Although oligomeric IAPP intermediates are widely considered the principal cytotoxic species, the relative contributions of the many biological and kinetic processes governing their formation, clearance, and conversion into fibrils remain poorly quantified. Here, a mathematical model of IAPP aggregation is developed that incorporates the physiology of beta-cell secretion and the microanatomy of the islet, including capillary-mediated clearance, enzymatic degradation, and the kinetics of oligomer and fibril formation within a well-mixed control volume. Building on the hypothesis that oligomers are the major cytotoxic species, the concept of accumulated cytotoxicity is introduced, defined as the time integral of the oligomer concentration, and a systematic sensitivity analysis of this quantity with respect to all model parameters is performed. The results reveal a striking hierarchy: only two parameters, the basal rate of IAPP monomer secretion and the rate constant for spontaneous oligomer dissociation, exert a first-order influence on long-term accumulated cytotoxicity, with dimensionless sensitivities approaching +1 and -1, respectively, while the effect of all other parameters remains subordinate and decays at long times. The model further shows that capillary clearance, owing to the physical exclusion of oligomers from fenestrated capillaries, selectively reduces fibril accumulation and amyloid deposition without affecting oligomer-mediated cytotoxicity, indicating that amyloid area fraction, the standard histological metric of disease severity, may not be a reliable surrogate for cytotoxic burden. The model predicts that approximately 48% of the islet area is replaced by amyloid after 30 years, broadly consistent with histological observations of advanced disease. These findings identify monomer secretion and oligomer dissociation as the most promising therapeutic targets to limit cytotoxic damage in type 2 diabetes and provide a quantitative framework for evaluating candidate intervention strategies.

The mature kidney contains approximately one million nephrons, and defects arising during nephron development can result in lifelong renal impairment, often culminating in kidney failure and transplantation. Nephric tubule formation requires coordinated epithelial processes, including polarity, adhesion, signaling, and vesicle transport; however, how these processes are integrated during kidney morphogenesis remains unclear. Dynamin binding protein (Dnmbp) is a multi-domain scaffolding protein expressed in human kidneys that is involved in several cellular processes. Using the Xenopus embryonic kidney, we previously demonstrated that Dnmbp is essential for nephrogenesis, yet the mechanisms by which it influences nephron development remain undefined. Here, we identify Dnmbp as a novel interacting partner of the Wnt/planar cell polarity effector Daam1. The interaction between Daam1 and Dnmbp was independently identified in two yeast two-hybrid screens, biochemically verified, and supported by structural modeling predictions of a Daam1-Dnmbp complex. In developing Xenopus laevis kidneys, Dnmbp localized to punctate structures associated with E-cadherin-rich cell-cell contacts. Dnmbp depletion significantly reduced junctional E-cadherin localization in both epithelializing and mature nephric tubules without affecting total E-cadherin levels, indicating a role in E-cadherin recruitment or stabilization at adherens junctions. Furthermore, expression of human DNMBP rescued the junctional defects, confirming the specificity of the loss-of-function phenotype. Together, these findings identify Dnmbp as an essential regulator of kidney development and support a model in which Dnmbp provides a mechanistic link between Wnt/PCP signaling, Cdc42 activation, and adherens junction formation during nephrogenesis.

Background: Early renal function decline is often accompanied by bothersome urological symptoms, yet effective early-stage nutritional interventions remain limited. L-Ergothioneine (EGT), a diet-derived antioxidant concentrated in renal tissue via the OCTN1 transporter, has shown renoprotective potential preclinically, but human interventional data are sparse. Methods: In this single-center, open-label, self-controlled trial, 31 adults (aged 45-70 years) with early renal function decline and persistent urological symptoms ([&ge;]3 months) received oral EGT (120 mg/day) for 90 days; 27 completed the study. Participants served as their own controls. The primary outcome was the within-subject change in eGFR (CKD-EPI 2021 creatinine); secondary outcomes included cystatin C-based eGFR, serum creatinine, UACR, a 10-item voiding diary, and a low-back-pain visual analogue scale (VAS). Within-subject changes were assessed by paired t-test or Wilcoxon signed-rank test. Results: Creatinine-based eGFR increased from 86.04 {+/-} 17.89 to 93.25 {+/-} 19.00 mL/min/1.73 m2 (+8.4%; p = 0.0016) and serum creatinine fell by 7.0% (p = 0.015). However, cystatin C-based eGFR and serum cystatin C were unchanged (p = 0.31 and p = 0.99), so the filtration signal was not corroborated by an independent, muscle-mass-independent marker. UACR showed a non-significant downward trend. Patient-reported outcomes improved most robustly: the total voiding diary score decreased by 57.2% (p < 0.0001) and low-back-pain VAS by 67.2% (p = 0.0002), with significant relief of urgency, frequency, and voiding difficulty. No product-related adverse events occurred. Conclusions: In this uncontrolled study, 90-day EGT supplementation was associated with marked improvement in urological symptoms and in creatinine-based eGFR, although the latter was not confirmed by cystatin C. These changes cannot be attributed to EGT alone and may substantially reflect placebo and natural-history effects. The findings are hypothesis-generating and warrant confirmation in a randomized, placebo-controlled trial using validated symptom instruments. Trial Registration: ChiCTR2500108897; Prospectively registered on 2025-09-08.

Fibrotic extracellular matrix (ECM) is not merely a structural scaffold but an instructive signaling interface that shapes epithelial cell state. However, the molecular cues by which matrix remodeling controls tubular metabolism during kidney fibrosis remain poorly defined. Here, we identify Fibulin-2 (FBLN2) as a fibroblast-derived matrix cue that transduces fibrotic ECM remodeling into tubular mitochondrial metabolic reprogramming. Using fibroblast-selective deletion of Smoothened (Smo) across distinct fibroblast subpopulations, we found that loss of fibroblast Smo preserved kidney function and attenuated fibrosis in mouse models of chronic kidney injury. Multi-omics profiling revealed coordinated remodeling of the fibrotic matrisome, highlighted by suppression of FBLN2, an ECM glycoprotein genetically linked to kidney function in humans. Mechanistically, FBLN2 engaged EGFR in tubular epithelial cells and activated EGFR-AKT signaling in a non-canonical ligand-like manner. This signaling axis suppressed acetyl-CoA acetyltransferase 1 (ACAT1), a mitochondrial regulator of fatty acid oxidation and amino acid metabolism. Disruption of fibroblast Smo-FBLN2 signaling restored ACAT1-dependent oxidative metabolism and reduced tubular fibrotic activation. Spatial lipidomics revealed compartment-specific lipid remodeling associated with altered mitochondrial fatty acid metabolism, including acylcarnitine and phospholipid changes linked to reduced fibrotic injury. Together, these findings define a Fibulin-2-EGFR-ACAT1 matrix-to-metabolism signaling axis that couples fibrotic ECM remodeling to tubular mitochondrial metabolism during kidney fibrosis.

The adrenal cortex is a critical for life endocrine system. It manages metabolic homeostasis, electrolyte balance, stress response, and sex development. This is accomplished through the release of various steroids from a dynamically changing landscape of concentric cellular zones/layers. Adrenocortical dysfunction is implicated in pathologies ranging from adrenal insufficiency to hypertension. The in-depth investigation of adrenal gland biology, pathology and drug discovery has been hampered by a lack of human, experimentally tractable models. Particularly missing are models that recapitulate the cellular diversity of the adrenal cortex with representation of all fetal and adult cell layers and the capsule. Here, we employ human pluripotent stem cells (hPSCs) to generate cells of all three cortex zones alongside capsular cells in a single 2D platform. This platform mimics the cellular diversity expected from an organoid, yet it provides the simplicity of monolayer cultures, that are better suited for drug discovery and high-throughput settings. These cultures secrete zone-specific steroids (cortisol, aldosterone, and DHEA-S) and exhibit robust, physiologically relevant ACTH stimulation responses. Transcriptomic analysis revealed sequential acquisition of profiles consistent with adrenocortical development, zonation, and signaling programs consistent with zone maintenance. Our platform is validated by a literature meta-analysis that defines transcriptomic signatures for each human cortical cell type, spanning fetal and adult stages. Together, this novel adrenocortical platform enables investigation of adrenal development, disease mechanisms, and therapeutic strategies. Significance StatementWe describe a hPSC-based 2D differentiation strategy with the cell type complexity of an organoid and the technical simplicity necessary for high-throughput assays. The platform contains all cortical subtypes that mediate electrolyte regulation, stress response, sex development and self-maintenance of the tissue. This co-differentiation offers a unique opportunity to study human adrenal development, biology, pathology and enables drug discovery.

Nephrons are the functional units of the kidney; within each nephron, the glomerulus is the initial site of selective filtration that allows removal of waste products while preserving proteins in the bloodstream. Each glomerulus consists of a network of capillaries surrounded by specialized epithelial cells, podocytes, which mediate selective filtration. Abnormalities in glomerular structure impair renal function, resulting in proteinuria and kidney disease. Although several microscopy-based approaches exist to characterize glomerular architecture and structural abnormalities, quantitative analysis is often limited by labor-intensive image segmentation. In this study we present a semi-automated approach for segmentation and analysis of glomerular architecture from three-dimensional confocal microscopy data. Using mTmG transgenic mice that express membrane-associated EGFP in podocytes and membrane-associated tdTomato across all other cell types, we reconstruct podocyte processes and glomerular capillaries from volumetric renal images. This semi-automated approach reduces manual segmentation effort and supports more efficient, standardized analysis of glomerular architecture in three-dimensional confocal microscopy datasets.