American Journal of Physiology-Renal Physiology

Renal cysts are categorized as non-pathogenic simple cysts and pathogenic malignant cysts based on their pathophysiological status. Cyst formation is divided by cyst initiation and cyst progression/promotion. Pathogenic cysts are thought to be developed through continuous initiation followed by progression until pathogenic status is achieved. Although many genetic and environmental factors are identified to cause pathogenic cyst formation, the mechanisms that discriminate cyst initiation and progression are poorly understood. Using genetic mutation models of ETV transcription factors, ETV1, ETV4, and ETV5, and a pharmacological inhibitor of hedgehog signaling, cyclopamine, we identified one of the mechanisms regulating cyst initiation and progression. Nephron specific deletion of ETV4 and ETV5 initiated cyst formation. However, cyst initiation did not continue as animals grow, and a limited number of the initial cysts underwent further growth. Additional deletion of ETV1 was required for continuous initiation in addition to promotion of cyst growth. Furthermore, administration of cyclopamine attenuated promotion of cyst progression but had little effect on cyst initiation. Therefore, we provide evidence that cyst initiation and progression is genetically and molecularly distinct and can be modulated. This information provides new insight into how to control renal cyst initiation and progression and can be used to suppress pathogenic cyst growth.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Spinal cord injury (SCI) disrupts innervation to the lower urinary tract, resulting in bladder dysfunction that predisposes to urinary infections and renal impairment. While inflammation is central to bladder pathology after SCI, the molecular events linking acute to chronic remodeling are poorly defined. We hypothesized that early treatment with pirfenidone, an anti-inflammatory and anti-fibrotic drug, would attenuate bladder pathology after SCI. Adult female C57BL/6J mice underwent contusive SCI or sham laminectomy, and bladders were collected at 2, 7, 16, and 45 days later. SCI induced bladder hypertrophy, edema, hemorrhage, neutrophil infiltration, cell proliferation and loss of voiding function in the first 48 hours. Transcriptomic profiling at this timepoint was characterized by activation of inflammatory and cytokine pathways including TNFalpha, IL-6, the complement cascade, and TGFbeta. Although bladder function partially recovered by day 7, inflammatory pathways persisted and extracellular matrix (ECM) remodeling programs emerged. By day 16, robust activation of ECM-remodeling pathways was evident in all bladders. Treatment with pirfenidone during the acute inflammatory phase (day 2-7) reduced bladder hypertrophy and suppressed expression of pro-fibrotic, inflammatory, and neuroplasticity-associated genes including Bdnf and Chrm2 that encodes muscarinic receptor 2 (M2). Mechanistically, pirfenidone attenuated TGFbeta signaling as shown by downregulation of phosphoSmad2 protein in whole bladders and decreased M2 receptor expression in the urothelium. These molecular changes correlated with improved function in pirfenidone-treated mice as shown by fewer voiding events with larger urine volumes up until 45 days after SCI. Early treatment with pirfenidone limits inflammation and fibrosis, normalizes neural signaling, and improves bladder function after SCI.

Modulation of renal nerves to improve blood pressure (BP) control has become a topic of intense investigation over the last 10-15 years. Given that renal innervation is composed of mixed nerve fibers containing both afferent (sensory) and efferent (sympathetic) fibers, subsequent preclinical studies have been investigating their respective roles in hypertension pathobiology in different genetic and salt-sensitive rat models. Here we set out to investigate how renal afferent and efferent nerves regulate hypertension development in the chronic mild stress model (CMS). We show that in male CMS rats, ablation of afferent renal nerves (ARDNx) and all renal nerves (TRDNx) resulted in similar BP (104 {+/-} 2 mmHg vs. 101 {+/-} 3 mmHg, respectively), both reduced compared to the SHAM group (118 {+/-} 1 mmHg, p = 0.003 and p < 0.001, respectively) arguing for a prominent role of afferent renal nerves in CMS hypertension. Additionally, we show a reduction of vasopressin (AVP) V1b but not V1a receptor abundance in ARDNx CMS males but not females, suggesting that afferent renal nerves are involved in increase in BP via V1b AVP receptor. We additionally show that despite normal BP, female CMS rats display increased renal sympathetic nerve activity (RSNA; 2.39 {+/-} 0.23 bursts/beat vs. 1.44 {+/-} 0.12 bursts/beat, p < 0.005) measured directly with implanted telemetry in conscious rats over one week and aortic stiffness, as evidenced by increased aortic pulse wave velocity (173.2 {+/-} 50.9 mm/s vs. - 10.7 {+/-} 54.6 mm/s in controls, p = 0.0393). NEW & NOTEWORTHYWe show that renal denervation mitigates the rise in blood pressure (BP) in a model that is not genetic nor diet-dependent, the chronic mild stress model (CMS). Specifically, we demonstrate the role of afferent, rather than efferent, renal nerves in mediating the rise in BP in male CMS rats. Finally, we report that renal sympathetic nerve activity, but not BP, is elevated in female CMS rats measured by telemetry over seven days in conscious rats.

Background: Neuropsychiatric comorbidities are highly prevalent in chronic kidney disease (CKD), yet the underlying neural mechanisms remain poorly defined. Methods: We established multiple mouse models of CKD and identified an adenine-induced model as the most suitable platform to study neurobehavioral alterations. Anxiety susceptibility was operationalized as the emergence of anxiety-like behavior after subthreshold unpredictable stress (SUS) and was assessed using the SUS paradigm combined with behavioral assays. Region-focused c-Fos mapping, fiber photometry, and chemogenetic manipulation were used to interrogate neural circuit activity. Pharmacological and genetic approaches were applied to investigate the role of angiotensin II (Ang II) signaling. Finally, hypothalamic paraventricular nucleus (PVN) activation was used to explore brain-to-kidney feedback by using in vivo multiphoton microscopy imaging techniques. Results: CKD mice showed no consistent baseline anxiety-like phenotype across standard assays but developed robust anxiety-like behavior after subthreshold unpredictable stress. Region-focused c-Fos profiling and fiber photometry identified the central amygdala (CeA) as a stress-sensitized limbic node in CKD. Chemogenetic inhibition of CeA GABAergic neurons attenuated anxiety-like behavior, supporting a functional role for CeA activity. Mechanistically, CKD elevated circulating Ang II and enhanced CeA accumulation of peripherally administered FAM-Ang II-associated signal. CeA-specific Agtr1a knockdown attenuated anxiety-like behavior and exaggerated stress evoked CeA calcium responses. Exploratory experiments further showed that sustained PVN glutamatergic activation aggravated early renal injury markers in a mild renal injury model. These findings support a kidney-to-brain model in which CKD primes CeA stress circuits, while local Ang II AT1R signaling contributes to the behavioral expression of stress-induced anxiety-like behavior, with a potential brain to kidney feedback component. Conclusions: CKD promotes stress-induced anxiety susceptibility through a CeA-centered mechanism involving local Ang II AT1R signaling. These findings identify CeA Ang II AT1R signaling as a potential contributor to CKD-associated stress-related affective vulnerability.

The Notch signaling pathway plays a central role in development and cell fate determination. Its function depends on tightly regulated intracellular trafficking of the Notch receptor and the Notch intracellular domain (NICD) after cleavage by {gamma}-secretase. Notch signaling is essential for principal cell differentiation within the renal collecting duct and for proximal-distal patterning during kidney development. Notch activity has also been shown to influence the trafficking of several membrane proteins, including nephrin in kidney cells and monocarboxylate transporter 1 in brain endothelial cells. Aquaporin-2 (AQP2) is the key vasopressin-regulated water channel in the collecting duct, and proper AQP2 trafficking and recycling are required for physiologically appropriate urine concentration. To determine whether and, if so, how Notch signaling modulates AQP2 trafficking, we performed studies using LLCPK1 renal epithelial cells stably expressing AQP2 (LLCPK1-AQP2). Exposing cells to 35 M DAPT (which inhibits y-secretase, preventing cleavage and activation of Notch receptor signaling) for 30 min significantly increased AQP2 membrane accumulation in LLCPK1-AQP2 cells as revealed by immunofluorescence staining. Using a rhodamine-transferrin internalization assay, we found that DAPT reduced clathrin-mediated endocytosis by 60%. This blockade increases AQP2 membrane accumulation by preventing the reinternalization of AQP2 that is delivered to the plasma membrane by exocytosis during its constitutive recycling pathway. Using an F-actin polymerization assay, we then found that Notch inhibition decreases F-actin polymerization by de-activating the small GTPase RhoA, using GSTRBD, a substrate that binds to active RhoA, as seen by western blotting using phospho-specific antibodies. Because actin polymerization is required for AQP2 endocytosis, RhoA inhibition by DAPT would result in the decreased internalization of AQP2 that we observed by immunofluorescence. While the mechanism by which DAPT inhibits RhoA activity remains to be determined, our study shows that AQP2 trafficking is regulated by the Notch signaling pathway in vitro and suggests that modulation of Notch signaling may represent a novel strategy to address water balance disorders that involve defects in the AQP2 trafficking process.

Introduction: C3 glomerulopathy (C3G) is driven by fluid-phase alternative complement pathway dysregulation, with emerging evidence linking glomerular neutrophil infiltration to disease severity. Neutrophil extracellular traps (NETs) are implicated in other forms of glomerulonephritis. However, their participation in the pathogenesis of C3G remains undefined. Methods: Kidney biopsies from 33 patients with C3G (15 with dense deposit disease [DDD] and 18 with C3 glomerulonephritis [C3GN]) were compared with 15 anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) biopsies as a neutrophil-rich disease control in this retrospective cross-sectional study. Glomerular neutrophils and NETs were identified using immunofluorescence, staining for myeloperoxidase, citrullinated histone H3, peptidyl arginine deiminase-4, and DNA. Supervised machine learning was used to quantify glomerular NET formation, and the data were correlated with kidney function at time of biopsy using linear regression. Results: Intraglomerular NETs were abundant and detected in the majority of glomeruli in C3G biopsies. Compared with AAV, C3G showed a significantly higher fraction of neutrophils forming NETs, despite similar neutrophil counts per glomerulus. NET abundance was similar in DDD and C3GN. In exploratory analyses, a greater proportion of glomeruli containing NETs was associated with lower kidney function (estimated glomerular filtration rate) at biopsy, and this association remained significant after adjustment for age, C3G subtype, and interstitial fibrosis. Conclusions: These observations demonstrate that intraglomerular NETs are a common and prominent observation in C3G and are associated with reduced kidney function at biopsy. These findings raise the possibility that NET deposition in glomeruli is a previously unrecognized driver of glomerular injury in C3G.

Introduction: Autosomal-dominant tubulointerstitial kidney disease (ADTKD) is characterized by chronic kidney disease (CKD) with an average age of end-stage renal disease (ESRD) of approximately 45 years, bland urinary sediment, the absence of proteinuria and autosomal dominant inheritance. While several causative genes have been found, there remain families in whom no molecular diagnosis has been identified (ADTKD-NMD). Methods: We identified BICC1 truncating variants in several families with ADTKD-NMD in the Wake Forest Rare Inherited Kidney Disease Registry and then screened families in our database and other referred families for BICC1 truncating variants. We performed segregation analysis and characterized affected individuals for clinical and histopathologic phenotypes. We analyzed oligomer formation of BICC1 mutants with wild-type BICC1-, ANKS3- and ANKS6 proteins through co-immunoprecipitation and Western blotting, and we tested for posttranscriptional regulation of the BICC1 target mRNA, Dand5, in a Luciferase reporter assay. Results: We found 6 heterozygous truncating mutations in BICC1 segregating with the ADTKD phenotype in 8 independent pedigrees worldwide. Affected individuals developed kidney failure in the 6th to 7th decade of life that was characterized pathologically by tubular atrophy and interstitial fibrosis. The truncated gene products localized to cytoplasmic bodies and demonstrated various degrees of self-association or binding to the known interaction partners, ANKS3 and ANKS6. While the wild-type BICC1 gene product acts as a posttranscriptional repressor of target mRNAs, all truncation variants exhibited increased expression of substrate mRNA. Conclusions: Truncating variants in BICC1 are a novel cause of ADTKD, segregating with the disease phenotype and upregulating BICC1 target gene expression through a dominant-negative- or a gain-of-function mode of action.

Recurrent urinary tract infection represents a substantial unmet public health in women. Local administration of estradiol has been shown to reduce recurrence, however in vitro models of the female urinary tract remain limited and the mechanisms underlying the effects of estradiol are incompletely understood. Here, we describe a novel iPSC organoid differentiation protocol and its application to establish a multilayered transwell barrier culture model. Estradiol treatment resulted in reduced expression of innate antimicrobial peptides and cytokines, together with increased expression of demannosylation pathways. Treatment of transwell cultures with a combination of female sex hormones reduced endogenous CXCL8 signaling, independently of a 24-hour uropathogenic Escherichia coli (UPEC) challenge. To our knowledge, this is the first iPSC organoid-derived model of the urinary tract, which provides a platform for investigating interactions between the urothelium, urobiome and hormonal environment.

Background: Chronic inflammation is the most common histological feature in Benign Prostatic Hyperplasia (BPH), and T cells are a key component of immune infiltrate. Advanced BPH is commonly associated with the formation of nodules, but it remains unclear whether a link exists among T cell infiltration, nodular development, and BPH progression. Using a Toxoplasma gondii (T. gondii) model and human specimens, we characterize the subtypes of T cells present during prostatic hyperplasia and their association with nodular development of the prostate. Methods: Male CBA/j mice were intraperitoneally infected with T. gondii parasites, and flow cytometry was performed on the prostate to quantify the number of CD4+ and CD8+ T cells. Histology was used to score microglandular hyperplasia (MGH), and immunofluorescence was used to quantify and examine the locality of CD4+ and CD8+ T cells and compared that to human BPH tissue. Results: We found that infecting male mice with T. gondii resulted in an increase of both CD4+ and CD8+ T cells in the prostate acutely and that CD8+ cells remained sustained at chronically. We also established the presence of glandular nodule formation at this timepoint through hematoxylin and eosin (H&E) staining. Immunofluorescence revealed that CD8+ cells were found proximal to forming glandular nodules relative to non-nodular glands. We also found more CD8+ cells localized to non-nodular glands in nodular BPH tissue versus non-nodular BPH tissue. Finally, we discovered a higher prevalence of CD8+ cells in T. gondii IgG+ patients than in IgG- patients. All T. gondii IgG+ patients exhibited nodular BPH, whereas all but one IgG- patient exhibited non-nodular BPH. Conclusions: This study is the first to investigate the presence and location of CD4+ and CD8+ T cells within nodular and non-nodular BPH glands. We found an association of the presence of CD8+ T cells with nodular progression. This association held true in human prostate tissue. Translationally, CD8+ T cells may enhance nodular BPH progression, and T. gondii infection may promote this CD8+ T cell-mediated response.

How water intake is initiated and maintained following V2 vasopressin receptor antagonism remains poorly understood. To elucidate the role of the V1b receptor in managing dehydration stress induced by V2 antagonism, we used deep learning-based computer vision to analyze drinking behavior in V1b knockout (V1bKO) and wild-type (WT) mice. While total water access and intake volume were comparable between genotypes, V1bKO mice exhibited distinct temporal dynamics. Modeling cumulative intake with the Hill equation revealed that the time required to reach 50\% of maximal water access was significantly shorter in V1bKO mice than in WT mice. This accelerated drinking effectively mitigated increases in serum osmolality and body weight loss. A reduced Hill's coefficient in V1bKO mice indicates a reduction of the rapid, cooperative-like water accumulation seen in WT mice. Furthermore, elevated basal hemoglobin levels in V1bKO mice were independent of dehydration, as confirmed via bone marrow transplant. Analysis of movement trajectories revealed that V1bKO mice exhibit a lower proportion of vertical movement (required for nozzle access) despite similar total distances traveled. Collectively, our results demonstrate that the V1b receptor critically regulates water-seeking behavior and osmotic homeostasis.

HNF1B is a transcription factor required for proximal tubule (PT) specification during kidney development, but whether it is also required to maintain PT identity after differentiation remains unknown. Using PT-specific genetic deletion in mice, we found that loss of Hnf1b in differentiated PT cells causes cyst formation and early postnatal lethality. PT-specific transcriptomic analysis revealed downregulation of PT-specific gene programs, including Hnf4a and PT-enriched transport and metabolic genes. Strikingly, Hnf1b-deficient PT cells ectopically activated podocyte-specific genes, including Wt1 and Nphs1, demonstrating that PT cells retain the capacity to engage alternative nephron segment programs when identity-stabilizing mechanisms are disrupted. In addition, loss of Hnf1b disrupted epithelial integrity, as evidenced by reduced epithelial adhesion gene expression and induction of mesenchymal markers. Wnt/{beta}-catenin signaling was also aberrantly activated, suggesting broader dysregulation of epithelial homeostasis. These findings establish HNF1B as a critical post-specification regulator of PT identity that sustains PT-specific transcriptional programs and actively suppresses alternative segmental identity programs.

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13BV358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated two independent Arl13bV358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13BV358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of GPR161 and TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently-derived, mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses. Summary for ReviewersThis study examined how excluding the protein ARL13B from primary cilia affects kidney epithelial cells. The researchers created two independent cell lines carrying a modified form of ARL13B,along with matched rescue cell lines. The findings show that ciliary ARL13B helps maintain normal ciliary composition. By comparing the cell lines, the researchers identified a high-confidence set of genes associated with loss of ciliary ARL13B. By highlighting the importance of using independent gene-edited clones and rescue-based controls, these results advance understanding of how cilia regulate kidney cell function and provide guidance for designing robust transcriptomic analyses.

Background: Fibrosis, characterised by excessive accumulation of collagen type I (COL1), is a common feature of chronic diseases, including liver diseases (LDs), chronic kidney disease (CKD) and heart failure (HF). COL1 degradation products can be detected in urine by proteomics/ peptidomics analyses and may serve as non-invasive biomarkers of fibrosis. We aimed to identify a common molecular signature of fibrosis across these diseases that may ultimately guide interventions to slow disease progression and prevent organ damage. Methods: Using capillary electrophoresis coupled to mass spectrometry (CE-MS), naturally occurring COL1 degradation products (peptides) in the urine of patients with fibrotic disease, LDs (n=127), CKD (n=263) or HF (n=187), were investigated and compared with the same number of matched controls. Disease-associated COL1 peptides were identified separately for each condition, and peptides showing consistent associations across the three diseases were selected to define a common fibrosis signature. A support vector machine model based on the selected peptides was developed and validated in independent cohorts of patients with LDs (n=110), CKD (n=93), HF (n=32) and controls (n=643). Results: We identified a common fibrotic signature consisting of 50 COL1 degradation products, mainly downregulated in fibrosis. A model based on these peptides achieved a strong performance, with an area under the receiver operating characteristic curve (AUC) of 0.935 (95% confidence interval (CI) 0.917-0.953, p<0.0001) in an external validation cohort comprising pooled disease groups (LDs, CKD, and HF) and controls. Performance was maintained in LDs, CKD and HF, with AUCs of 0.917 (95% CI 0.890-0.944, p<0.0001), 0.951 (95% CI 0.931-0.971, p<0.0001) and 0.950 (95% CI 0.903-0.997, p<0.0001), respectively. The model scores were significantly associated with fibrosis stage in LDs (p=0.0097) and with interstitial fibrosis and tubular atrophy in CKD (p=0.045). Conclusion: A model of urinary COL1 peptides captures a shared collagen degradation signature across organs and diseases, enabling the non-invasive assessment of fibrosis irrespective of its origin. As these peptides exclusively reflect collagen degradation, the findings suggest impaired collagen degradation as a driver in fibrosis. Future clinical studies are warranted to evaluate the utility of this model for early fibrosis detection and earlier implementation of anti-fibrotic interventions.

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by [~]50{+/-}12% and calculated fluid volume displacement by each contraction by [~]35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7{micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9nL versus BODIPY cholesterol: 3.3{+/-}1.2nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11{micro}m, volume displacement: 5.5{+/-}2.4nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

Intracranial aneurysms (IA) and their rupture (subarachnoid haemorrhage, SAH) are a rare but serious complication of autosomal dominant polycystic kidney disease (ADPKD). Both genetic and environmental risk factors contribute to the pathogenesis of IA and SAH, but specific information on risk factors in the ADPKD population are limited and international clinical guidelines mainly recommend screening for ADPKD patients with a family history of IA or SAH. We assessed the associations of monogenic variants, polygenic risk, and clinical factors with the diagnosis of IA or SAH in 2,200 adult ADPKD patients from three cohorts: the Irish Kidney Gene Project (IKGP, n=475), FinnGen (n=826), and Genomics England (GEL, n=899). Polygenic risk scores (PRS) for IA, hypertension, and smoking intensity were derived from previously published GWAS summary statistics and additionally conditioned using mtCOJO to account for their genetic correlation. IA or SAH was diagnosed in 158 of 2,200 patients (7.2%; mean age at diagnosis 51.1 years). Female sex (HR 2.21, 95% CI 1.50-3.25, p=6.52x10^-5) and smoking (HR 2.06, 95% CI 1.43-2.96, p=9.49x10^-5) were associated with IA or SAH in univariate Cox proportional hazards models, while diabetes was protective (HR 0.39, 95% CI 0.22-0.70, p=1.37x10^-3). Monogenic variant status was not found to associate with increased risk of IA or SAH. Polygenic score for IA was associated with increased risk of IA or SAH, with a 1 standard deviation increase in PRS associated with a pooled hazard ratio (HR) of 1.30 (95% CI 1.09-1.57, p=4.66x10^-3), and the association of the conditioned IA PRS remained in multivariate Cox proportional hazards models accounting for clinical factors and monogenic variants (HR 1.26, 95% CI 1.03-1.55, p=0.02). The addition of polygenic scores added significant prognostic information for IA or SAH beyond clinical risk factors in FinnGen (p=3.61x10^-3) and GEL (p=8.84x10^-3) but not in IKGP, as assessed by the likelihood ratio test. Overall, our study shows that the strongest risk factors for IA or SAH in ADPKD patients are smoking history, female sex, and polygenic risk for IA. Hypertension was not associated with IA (HR 0.50, 95% CI 0.24-1.00, p=0.051), likely due to the high prevalence of hypertension in this population across cohorts (69.6-85.3%). While family history may be considered a proxy of genetic risk, this study suggests that family history itself may be of limited utility in discriminating individuals with ADPKD at risk of IA or SAH. Keywords: Intracranial Aneurysms, subarachnoid haemorrhage, Autosomal Dominant Polycystic Kidney Disease, Polygenic Risk Scores, Hypertension, Family History

A subset of untreated primary prostate cancer (PCa) contain substantial focal T-cell infiltrates, but whether these reflect antitumor responses that could potentially be enhanced by immune checkpoint blockade (ICB) remains unclear. We used immunohistochemistry, immunofluorescence, whole-slide spatial analysis, bulk RNA sequencing, and immune-cell deconvolution to characterize immune infiltrates in untreated primary PCa. Absolute CD8 T-cell density generally increased with total CD3 T-cell density, but the CD8/CD3 ratio decreased as overall T-cell density increased, indicating a preferential increase in CD4 T cells. Highly infiltrated tumors also had lower GZMB abundance relative to CD8 T-cell abundance. Multiplex analysis showed trends toward greater TIM3 and LAG3 expression among PD1CD8 T cells and increased regulatory T-cell features in highly infiltrated tumors. TIGIT cell density and the TIGIT/CD3 ratio increased with T-cell infiltration, whereas PD1/CD3 was not associated with overall CD3 T-cell density. Both TIGIT/CD3 and PD1/CD3 ratios were enriched within lymphoid aggregates compared with matched tumor and benign regions, consistent with these structures being checkpoint-rich immune niches. Transcriptomic analyses supported a shift in relative immune composition toward CD4 T cells and selective increases in immune checkpoints. Together these findings suggest that effective immune responses in a subset of primary PCa with increased T-cell infiltration are being repressed by several mechanisms and may respond to therapies targeting specific immunosuppressive mechanisms.

APOL1 risk variants are the strongest genetic determinants of HIV-associated nephropathy (HIVAN), yet the mechanisms linking inflammation to APOL1-mediated podocyte injury remain poorly understood because authentic patient-derived human disease models are lacking. Using urine-derived podocytes established from children with HIVAN and endogenous APOL1 reporter cell lines derived from these cells, we identified a previously unrecognized pathway of inflammatory, cathepsin-dependent APOL1 proteolysis. Endogenous APOL1 cleavage was detected in patient-derived podocytes, whereas reporter cell lines enabled the identification and functional characterization of N-terminal and C-terminal APOL1 fragments with distinct intracellular localization and pathogenic functions. The nuclear N-terminal fragment activated inflammatory transcriptional programs and promoted podocyte injury, whereas the membrane-associated C-terminal fragment mediated membrane toxicity and remained susceptible to pharmacologic inhibition by inaxaplin. Cathepsin S directly cleaved APOL1 in vitro, linking inflammatory signaling to APOL1 fragmentation. These findings identify inflammatory APOL1 proteolysis as a mechanism that partitions APOL1 toxicity into distinct pathogenic programs and nominate APOL1 processing as a therapeutic target for HIV-associated and other APOL1-mediated kidney diseases.

BackgroundHypertension, the persistent elevation of blood pressure (BP), is characterized by chronic low-grade inflammation and systemic cytokine release. Circulating cytokines contribute to the development of hypertension and end-organ damage. However, the specific immune profile associated with the progression of hypertension remains unclear. We hypothesize that a plasma cytokine signature reflects early BP changes in older adults. MethodsSeventy participants aged 57-81 years were categorized as normotensive (n = 17), elevated BP (n = 10), or hypertensive (n = 43) based on 24-hour ambulatory BP monitoring and antihypertensive treatment status. Plasma IL-1{beta}, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, and TNF- were quantified using immunoassays. Partial Pearson correlations adjusted for demographic and biochemical covariates were used to assess associations between cytokines, BP, and cytokine-cytokine networks. ResultsIn untreated hypertensive individuals, plasma IL-23 was positively correlated with 24-hour diastolic BP. Antihypertensive treatment was associated with reduced IL-17A concentrations, which are negatively associated with 24-hour systolic BP. In the elevated BP group, IL-21 concentrations were higher than in normotensive individuals. To further characterize the cytokine signature, cytokine-cytokine correlations were examined. IL-23 and IL-17A were positively correlated with most interleukins, whereas TNF- showed few associations. IL-1{beta} exhibited strong correlations with both IL-23 and IL-17A, particularly in untreated participants. ConclusionIL-23 and IL-17A are associated with BP status and are broadly interconnected with other inflammatory cytokines, highlighting the potential importance of the IL-23/IL-17A axis in the hypertension of development. Early alterations in IL-21 in elevated BP may reflect immune changes that precede the onset of hypertension.

Left ventricular hypertrophy, common in cardiometabolic and renal disease, is a major risk factor for cardiovascular morbidity and mortality. Left ventricular mass is a highly heritable, polygenic trait. Linkage studies in WKY and SHRSP rats have identified a quantitative trait locus for left ventricular mass index on chromosome 14. Congenic strains, where trait-associated genetic loci are introduced into a control strain, can identify causal genetic mediators relevant to human disease. Chromosome 14 congenic (WKY.SPGla14a), WKY, and SHRSP strains underwent cardiac phenotyping and transcriptome profiling at; 1-3 days (neonate), 5 weeks, and 16-weeks. Compared to WKY, LVMI was significantly increased in SHRSP and WKY.SPGla14a at 5 weeks (LVMISHRSP-WKY=0.26g/kg, LVMIWKY.SPGla14a-WKY=0.30g/kg), prior to measured hypertension in this model. SHRSP blood pressure was significantly greater than WKY.SPGla14a, and WKY from 12-20 weeks (AUCdiff=497 vs WKY, AUCdiff=412 vs WKY.SPGla14a). Cardiac transcriptome analysis of neonate, 5-week, and 16-week hearts identified significantly increased expression of secreted phosphoprotein 1 (Spp1/osteopontin) in SHRSP and WKY.SPGla14a compared to WKY, which is positioned within the transferred congenic region. Overexpression of Spp1 mRNA significantly increased H9c2 cell size and was shown to be transferred in small extracellular vesicles (sEV). Overexpression of Spp1 in neonatal chromosome 14 congenic and SHRSP strains preceded development of increased cardiac mass and onset of hypertension. The congenic strategy identified Spp1 as a positional and functional candidate gene determining increased LVMI in the SHRSP model of human cardiovascular disease.