Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common monogenic kidney disease worldwide. Tolvaptan is currently the only approved intervention to slow kidney cyst growth, but its indication is limited to rapidly progressing ADPKD and use compromises quality of life. Hence, new therapeutic options are of high clinical importance. Metabolic dysregulation is a hallmark of kidney cyst growth, including reprogramming of arginine metabolism and polyamine production. Sustained increases in polyamine synthesis drive pathological epithelial cell proliferation, tissue remodeling, and activation of M2-like macrophages, all known drivers of PKD. Difluoromethylornithine (DFMO) is an FDA approved irreversible inhibitor of ornithine decarboxylase (ODC1), the rate limiting enzyme in the arginine-polyamine pathway. Here, we treated C57Bl6/J p.R3277C ( Pkd1 RC/RC ) mice for five months with DFMO-supplemented drinking water to test if DFMO treatment can slow kidney cyst growth in an orthologous model of ADPKD that mimics the disease pathophysiology seen in patients. At study end, Pkd1 RC/RC mice treated with DFMO presented with significantly reduced percent kidney weight normalized to body weight as well as kidney cystic index and cyst number compared to control. Kidney RNA-seq analyses revealed correction of many genes and pathways perturbed in PKD post DFMO treatment, as well as overall heathier kidney cellular architecture as inferred by RNAseq-base cell type deconvolution. Our data highlight significant potential to repurpose DFMO for the treatment of patients with ADPKD and investigate the role of polyamines in modulating epithelial as well as myeloid cell fate in the setting of PKD.

KEY POINTS:High concentrations of tubular phosphate directly contribute to kidney mineral retention, tubular injury/inflammation, and cyst growth in mice. Human polycystic kidney disease kidneys exhibit subclinical calcium-phosphate microcrystals that may be promoting kidney pathology in this setting. Phosphaturia and kidney mineral deposition may be novel and modifiable therapeutic targets to slow polycystic kidney disease progression. BACKGROUND:Accumulating evidence supports a direct contribution of abnormalities in phosphate homeostasis to progression of polycystic kidney disease (PKD); however, the mechanisms for these relationships remain undefined. A universal feature of nephron loss in all forms of kidney disease is increased urinary phosphate excretion by residual functional nephrons that helps to maintain systemic phosphate balance. To date, no studies have examined the direct contribution of urinary phosphate to kidney pathology in PKD. METHODS:We first examined the impact of dietary phosphate on kidney outcomes in Pkd1RC/RC mice, an orthologous murine model of PKD. Next, to more specifically delineate the direct contribution of urinary phosphate to kidney pathology, we characterized kidney phenotypes in phosphaturic Slc34a1-/- mice. We then investigated how increasing urinary phosphate excretion through Slc34a1 deletion affected cystic kidney disease progression in Pkd1RC/RC mice. Finally, to define the relevance of our findings to human PKD, we conducted high-resolution imaging and trace mineral analysis of human PKD and control kidney specimens to assess the prevalence and composition of subclinical mineral deposition. RESULTS:Pkd1RC/RC mice fed a high phosphate diet exhibited kidney injury with activation of early fibrosis pathways, accelerated cyst growth, and phosphate-based crystal deposition that spatially colocalized with macrophages. Slc34a1-/- mice demonstrated extensive nephrocalcinosis, tubular microcyst formation, and evidence of early kidney injury, inflammation, and fibrosis. Moreover, compound mutant Pkd1RC/RC , Slc34a1-/- mice had increased kidney cyst burden accompanied by reduced kidney function compared with Pkd1RC/RC , Slc34a1+/+ controls. Finally, micro-computed tomography and trace mineral analyses of human kidneys revealed consistent deposition of calcium-phosphate microcrystals in PKD kidneys. CONCLUSIONS:Increased urinary phosphate excretion directly contributes to kidney injury, cyst formation/growth, inflammation, and fibrosis. Urinary phosphate excretion and tubular mineral solubility may represent novel therapeutic targets for slowing PKD progression in humans.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), with acute kidney injury (AKI) contributing substantially to morbidity and mortality in those patients. To determine whether APAP-induced AKI depends on hepatic CYP2E1-mediated bioactivation, we used CYP2E1^flox/flox^ mice treated with AAV8-TBG-Cre to selectively delete hepatic CYP2E1 while preserving renal metabolism. Male and female mice received APAP (600 mg/kg) and were evaluated up to 48 h for liver and kidney injury. Liver-specific CYP2E1 deletion reduced APAP hepatotoxicity, confirming the reduction of hepatic NAPQI formation. Despite this protection, both male and female mice treated with AAV8-TBG-Cre and APAP developed progressive renal injury, with marked increases in blood urea nitrogen (BUN) and creatinine, tubular vacuolation, and strong induction of KIM-1 and osteopontin, along with cell death at 48 h. Notably, female mice lacking renal CYP2E1 and showing no detectable renal protein adducts still progressed to AKI, demonstrating that kidney injury can occur through CYP-independent mechanisms. Given that APAP-induced AKI is a delayed injury, we further considered p-aminophenol (PAP), a deacetylation product of APAP, as a potential CYP-independent contributor. These findings support the concept that non-CYP pathways, including PAP formation, may contribute to kidney injury during the later phase of toxicity, although this pathway likely represents only one component of a multifactorial injury process. Together, these results demonstrate that APAP-induced AKI is a kidney-intrinsic process that can develop independently of both hepatic and renal CYP2E1 activity, emphasizing the need for kidney-specific therapeutic strategies for preventing APAP-induced renal injury.

Antiretroviral therapy (ART) has decreased HIV-1 associated morbidity. However, despite ART, immune cells remain latently infected and slowly release viral proteins, leading to chronic inflammation and HIV-1 associated comorbidities. New strategies are needed to target viral proteins and inflammation. We found activation of Notch3 in several renal cells of the HIV-1 mouse model (HIV-Tg26) and in patients with HIV associated Nephropathy. We hypothesized that targeting Notch3 activation constitutes an effective therapy for HIV-related chronic kidney diseases (HIV-CKD). We generated HIV-Tg26 mice with Notch3 knocked out (Tg-N3KO). Compared to HIV-Tg26 mice at 3 months, HIV-Tg-N3KO mice showed a marked reduction in renal injury, skin lesions and mortality rate. Bulk RNA sequencing revealed that N3KO not only reduced renal infiltrating cells but significantly reduced the expression of HIV genes. Moreover, Notch3 activated the HIV- promoter and induction of HIV-1 resulted in increased Notch3 activation indicating a feedback mechanism. Further, bone marrow derived macrophages (BMDMs) from HIV-Tg26 mice showed activation of Notch3 indicating systemic effects. Consistent with that, systemic levels of TNF-α, MCP-1 and other inflammatory chemokines and cytokines were reduced in Tg-N3KO mice. Thus, Notch3 inhibition/deletion has a dual therapeutic effect in HIV-CKD and may extend to other HIV-related pathologies.

e12582 Background: The goal of our study is to improve the diagnosis and treatment of DCIS through the identification of the factors driving its invasion and metastasis. Methods: We utilized the Mouse INtraDuctal (MIND) model to investigate the mechanism(s) by which some DCIS become invasive while others remain indolent. This model closely replicates the human condition by injecting non-invasive epithelial cells from patients into mouse mammary ducts to form in situ lesions. In some cases, these cells will display invasive behavior by overcoming the natural barriers of myoepithelium. To compare these two processes, we performed single-cell (sc) RNA-sequencing analysis of 60,000 cells from the pre-transplant time-point representing 17 DCIS (11 became invasive, 6 remained non-invasive). We also performed scATAC/RNA sequencing of a DCIS with associated IDC. Results: Our analysis revealed that the proportion of endothelial cells was higher in cases that became invasive, while the proportion of plasma and T cells was higher in those that remained non-invasive (t-test; P<0.05). This finding suggested that invasive cells reprogrammed the immune microenvironment to exclude immune cells as a means of evading immune surveillance. Additionally, we performed cell-cell interaction (CCI) analysis by CellPhoneDB. This analysis revealed highly ranked CCIs in invasive cases, including luminal cell interactions with pericytes via Wnt5A-SFRP1. On the other hand, highly ranked CCIs in non-invasive cases included HR-positive luminal cell interactions with T cells via CD226-Nectin-2 and CD96-Nectin1. Notably, CD226 and CD96 are T cell receptors that enhance CD8+ T cell activity. These results support the potential role of T-cell interactions with DCIS luminal cells in preventing their invasive transition. We conducted additional analysis of sc-RNA-sequencing data using a stem cell-based gene signature algorithm called CytoTRACE. Our analysis revealed that invasive cells were enriched in cell clusters with significantly higher stem cell scores than non-invasive cells. In another analysis of a patient DCIS with associated IDC using sc-ATAC and RNA sequencing, we discovered that the open chromatin regions in the stem cell cluster were enriched for FOXA1 binding motifs. This cluster also had the highest expression of two pioneer factors (FOXA1 and GRHL2), NEAT1 (paraspeckle protein), ERBB2, 3,4, and ANKRD30A (cell surface protein). Our findings suggest that the pioneer factors FOXA1 and GRHL2 play a crucial role as transcriptional drivers of stemness. Additionally, the ANKRD30A could be used to isolate stem cell clusters to validate their self-renewal potential. Conclusions: DCIS progression is facilitated by interactions between stromal, immune, and epithelial cells, which lead to epigenetic and transcriptional changes. This results in the formation of stem-like cells that are characterized by their plasticity and invasive capacity.

AbstractOsteopontin (OPN) is a multi‐functional glycoprotein that coordinates the innate immune response, prevents nanocrystal formation in renal tubule fluid, and is a biomarker for kidney injury. OPN expression is markedly increased in cystic epithelial cells of polycystic kidney disease (PKD) kidneys; however, its role in PKD progression remains unclear. We investigated the in vitro effects of recombinant OPN on the proliferation of tubular epithelial cells from PKD and normal human kidneys and in vivo effects of OPN deletion on kidney cyst formation, fibrosis, and mineral metabolism in pcy/pcy mice, a non‐orthologous model of autosomal‐dominant PKD. In vitro studies revealed that OPN enhanced the proliferation of PKD cells but had no effect on normal kidney cells. Deletion of OPN in pcy/pcy mice significantly reduced kidney cyst burden; however, this was accompanied by increased fibrosis and no change in kidney function. The loss of OPN had no effect on kidney macrophage numbers, cyst epithelial cell proliferation, or apoptosis. Furthermore, there was no difference in kidney mineral deposition or mineral metabolism parameters between pcy/pcy mice with and without OPN expression. Global deletion of OPN reduced kidney cyst burden, while paradoxically exacerbating kidney fibrosis in mice with cystic kidney disease.

Supplementary Figures 1-2 from Loss of Betaglycan Expression in Ovarian Cancer: Role in Motility and Invasion

Background: A large fraction of human DCIS (>50%) may not need the multimodality treatment options currently offered to all patients. More importantly, while we may be overtreating many, we cannot identify those most at risk for invasion/metastasis. Revealing the cellular and molecular mechanisms by which some DCIS remain indolent while others advance to invasive and metastatic breast cancers is currently a clinical unmet need. Methods: To address this gap, we developed the Mouse-INtraDuctal (MIND) model, by which patient-derived (PDX) DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Single cell RNA-sequencing (scRNA-seq) was utilized to profile the DCIS epithelial and stroma cells in progressors vs. non-progressors. To distinguish between stromal (diploid) cells and tumor (aneuploid) cells, we calculated Copy Number Aberration (CNA) profiles from RNA using CopyKAT. Cell-type specific differential gene expression analysis of DCIS epithelial cells and microenvironment cell types in progressors and non-progressors was performed. We also predicted putative ligand:receptor interactions between the tumor cells and cell types in the microenvironment by CellPhoneDB. Results: Among 37 PDX DCIS MIND models followed for a median of 9 months, 20 (54%) grafted into 95 glands, showed in vivo invasive progression (progressed) while 17 (46%), injected into 107 glands, remained non-invasive (non-progressed). ScRNA-seq was performed on 13 DCIS samples including 10 progressors and 3 non-progressors. Aneuploid cells were further analyzed to identify deferentially expressed genes that were upregulated in progressors compared to non-progressors (log2 fold=1, FDR p< 0.05).. Notable genes included NEAT1, EIF4EBP1, SCGB2A2, TFF1 and TFF3 that were upregulated in the progressors. NEAT1, the core structural component of the paraspeckles, is frequently overexpressed in human cancers and its expression is correlated with worse survival in cancer patients. NEAT1 drives tumor progression by regulating genes involved in cellular growth, migration, invasion, metastasis, EMT, stemness, radio- and chemoresistance, supporting its role as a potential biomarker and therapeutic target. TFF1/TFF3 mRNAs show increased expression in metastatic breast cancers. EIF4EBP1 is located on chrom 8p11-p12 which is frequently amplified in breast cancer and is associated with poor clinical prognosis. Further analysis using Cancer Hallmarks identified mitotic spindle, interferon signaling, DNA repair, oxidative phosphorylation and P53 pathway among the top signatures that were upregulated in the progressors. CellPhoneDB identified expression of several receptor/ligand interactions including CD74/MIF involved in epithelial/stromal and stromal/stromal cross talks that may play a role in DCIS invasive progression. Conclusions: Future studies will validate our findings using patient DCIS samples with known long-term outcome and in vivo MIND models to further refine risk associated biomarkers for invasion/metastasis and to identify more effective treatments. Citation Format: Aditi Rastogi, Fariba Behbod, Nicholas Navin, Jerome Lin, Linheng Li, Hua Li, Andrew K. Godwin, Alastair M. Thompson, Timothy Fields, Yan Hong. Epithelial/stromal cross talks that induce malignant transition of human ductal carcinoma in situ. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-08.

Autosomal dominant polycystic kidney disease (ADPKD) involves the development and persistent growth of fluid filled kidney cysts. In a recent study, we showed that ADPKD kidney cyst epithelial cells can stimulate the proliferation and differentiation of peri-cystic myofibroblasts. Although dense myofibroblast populations are often found surrounding kidney cysts, their role in cyst enlargement or fibrosis in ADPKD is unclear. To clarify this, we examined the effect of myofibroblast depletion in the Pkd1RC/RC (RC/RC) mouse model of ADPKD. RC/RC;αSMAtk mice that use the ganciclovir-thymidine kinase system to selectively deplete α-smooth muscle actin expressing myofibroblasts were generated. Ganciclovir treatment for four weeks depleted myofibroblasts, reduced kidney fibrosis and preserved kidney function in these mice. Importantly, myofibroblast depletion significantly reduced cyst growth and cyst epithelial cell proliferation in RC/RC;αSMAtk mouse kidneys. Similar ganciclovir treatment did not alter cyst growth or fibrosis in wild-type or RC/RC littermates. In vitro, co-culture with myofibroblasts from the kidneys of patients with ADPKD increased 3D microcyst growth of human ADPKD cyst epithelial cells. Treatment with conditioned culture media from ADPKD kidney myofibroblasts increased microcyst growth and cell proliferation of ADPKD cyst epithelial cells. Further examination of ADPKD myofibroblast conditioned media showed high levels of protease inhibitors including PAI1, TIMP1 and 2, NGAL and TFPI-2, and treatment with recombinant PAI1 and TIMP1 increased ADPKD cyst epithelial cell proliferation in vitro. Thus, our findings show that myofibroblasts directly promote cyst epithelial cell proliferation, cyst growth and fibrosis in ADPKD kidneys, and their targeting could be a novel therapeutic strategy to treat PKD.

Journal of the American Society of Nephrology 34(11S):p 688, November 2023. | DOI: 10.1681/ASN.20233411S1688b

Journal of the American Society of Nephrology 34(11S):p 1124, November 2023. | DOI: 10.1681/ASN.20233411S11124a

The PKD1 gene, encoding protein polycystin-1 (PC1), is responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 has been shown to be present in urinary exosome−like vesicles (PKD−ELVs) and lowered in individuals with germline PKD1 mutations. A label−free mass spectrometry comparison of urinary PKD−ELVs from normal individuals and those with PKD1 mutations showed that several proteins were reduced to a degree that matched the decrease observed in PC1 levels. Some of these proteins, such as polycystin-2 (PC2), may be present in a higher-order multi-protein assembly with PC1—the polycystin complex (PCC). CU062 (Q9NYP8) is decreased in ADPKD PKD−ELVs and, thus, is a candidate PCC component. CU062 is a small glycoprotein with a signal peptide but no transmembrane domain and can oligomerize with itself and interact with PC1. We investigated the localization of CU062 together with PC1 and PC2 using immunofluorescence (IF). In nonconfluent cells, all three proteins were localized in close proximity to focal adhesions (FAs), retraction fibers (RFs), and RF-associated extracellular vesicles (migrasomes). In confluent cells, primary cilia had PC1/PC2/CU062 + extracellular vesicles adherent to their plasma membrane. In cells exposed to mitochondrion-decoupling agents, we detected the development of novel PC1/CU062 + ring-like structures that entrained swollen mitochondria. In contact-inhibited cells under mitochondrial stress, PC1, PC2, and CU062 were observed on large, apically budding extracellular vesicles, where the proteins formed a reticular network on the membrane. CU062 interacts with PC1 and may have a role in the identification of senescent mitochondria and their extrusion in extracellular vesicles.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked β-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.