Distal Cells Research Articles

ATP Hydrolases Superfamily Protein 1 (ASP1) Maintains Root Stem Cell Niche Identity through Regulating Reactive Oxygen Species Signaling in Arabidopsis.

The maintenance of the root stem cell niche identity in Arabidopsis relies on the delicate balance of reactive oxygen species (ROS) levels in root tips; however, the intricate molecular mechanisms governing ROS homeostasis within the root stem cell niche remain unclear. In this study, we unveil the role of ATP hydrolase superfamily protein 1 (ASP1) in orchestrating root stem cell niche maintenance through its interaction with the redox regulator cystathionine β-synthase domain-containing protein 3 (CBSX3). ASP1 is exclusively expressed in the quiescent center (QC) cells and governs the integrity of the root stem cell niche. Loss of ASP1 function leads to enhanced QC cell division and distal stem cell differentiation, attributable to reduced ROS levels and diminished expression of SCARECROW and SHORT ROOT in root tips. Our findings illuminate the pivotal role of ASP1 in regulating ROS signaling to maintain root stem cell niche homeostasis, achieved through direct interaction with CBSX3.

#417 Targeting parietal epithelial cell activation with mineralocorticoid receptor antagonism controls FSGS and crescentic glomerulonephritis

Abstract Background and Aims We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury [1]. Accumulating evidence has indicated the potential contributions of the mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is expressed in endothelial cells, glomerular mesangial cells, podocytes, and distal tubular cells. Previous studies have shown that administering mineralocorticoid receptor antagonists has beneficial effects in various renal injury animal models, the role of the MR in extracapillary glomerulopathies is still elusive and mechanistically unclear. Methods To investigate the cell-specific role of the MR in PEC in the course of focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (GN), we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Maladaptive FSGS was induced by combining a high-salt diet and deoxycorticosterone acetate (DOCA) with unilateral nephrectomy (DOCA-UniN). Crescentic glomerulonephritis (GN) was induced using the anti-glomerular basement membrane nephrotoxic serum (NTS) model. Results At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged by DOCA-UniN, PEC-selective Nr3c2 targeting significantly inhibited the expression of PEC activation markers Ki67, CD44, and FSGS lesions. Importantly, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function compared to Pec Cre Nr3c2 wt/wt counterparts. Likewise, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function in the NTS-induced CGN model. Crescents were also more numerous and organised in Pec Cre Nr3c2wt/wt mice than in Pec Cre Nr3c2lox/lox animals. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less expression of the CD44 activation marker on PECs. Thus, genetic disruption of MR in PEC and pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC within FSGS and crescents. We next evaluated PEC migration under various conditions (DMSO, HB-EGF, Eplerenone, or HB-EGF+Eplerenone) for 24 hours. Eplerenone fully abrogated HB-EGF-induced PEC migration and Pcna, Snai1, and Vim mRNA expressions. Conclusion Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis, along with the recently discovered CD9/EGFR/PDGFR [1] and CLDN1 pathways. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in FGSS and, more surprisingly, in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

The Rac pathway prevents cell fragmentation in a nonprotrusively migrating leader cell during C. elegans gonad organogenesis

The C.elegans hermaphrodite distal tip cell (DTC) leads gonadogenesis. Loss-of-function mutations in a C.elegans ortholog of the Rac1 GTPase (ced-10) and its GEF complex (ced-5/DOCK180, ced-2/CrkII, ced-12/ELMO) cause gonad migration defects related to directional sensing; we discovered an additional defect class of gonad bifurcation in these mutants. Using genetic approaches, tissue-specific and whole-body RNAi, and invivo imaging of endogenously tagged proteins and marked cells, we find that loss of Rac1 or its regulators causes the DTC to fragment as it migrates. Both products of fragmentation-the now-smaller DTC and the membranous patch of cellular material-localize important stem cell niche signaling (LAG-2 ligand) and migration (INA-1/integrin subunit alpha) factors to their membranes, but only one retains the DTC nucleus and therefore the ability to maintain gene expression over time. The enucleate patch can lead a bifurcating branch off the gonad arm that grows through germ cell proliferation. Germ cells in this branch differentiate as the patch loses LAG-2 expression. While the nucleus is surprisingly dispensable for aspects of leader cell function, it is required for stem cell niche activity long term. Prior work found that Rac1-/-;Rac2-/- mouse erythrocytes fragment; in this context, our new findings support the conclusion that maintaining a cohesive but deformable cell is a conserved function of this important cytoskeletal regulator.

Active nuclear positioning and actomyosin contractility maintain leader cell integrity during gonadogenesis

Proper distribution of organelles can play an important role in a moving cell's performance. During C.elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility.

Immediate but Temporal Response: The Role of Distal Epithelial Cells in Wound Healing.

Efficient oral mucosal wound healing requires coordinated responses from epithelial progenitor cells, yet their spatiotemporal recruitment and activation remain unclear. Using a mouse model of palatal mucosal wound healing, we investigated the dynamics of epithelial cells during this process. Proliferation analysis revealed that, in addition to the expected proliferation center near the wound edge, distal cell populations rapidly activated post-injury by elevating their mitotic activity. These distal cells displayed predominant lateral expansion in the basal layer, suggesting roles beyond just tissue renewal. However, while proximal proliferation center cells sustained heightened proliferation until re-epithelialization was completed, distal cells restored basal turnover rates before wound closure, indicating temporally confined contributions. Lineage tracing of Wnt-responsive epithelial cells showed remarkable clone expansion in basal layers both proximally and distally after wounding, contrasting with gradual clone expansion in homeostasis. Although prioritizing tissue repair, epithelial progenitor cells maintained differentiation programs and barrier functions, with the exception of the leading edge. At the leading edge, we found accelerated cell turnover, but the differentiation program was suspended. In summary, our findings uncovered that oral wound re-epithelialization involves two phases: an initial widespread response with proliferation of proximal and distal cells, followed by proliferation confined to the wound proximal region. Uncovering these stage-specific healing mechanisms provides insights for developing targeted therapeutic strategies to improve wound care.

The effect of ginger essential oil (Zingiber officinale) on catalase in rat kidney tissue

We were aimed to investigate the effect of ginger essential oil (Zingiber officinale) on catalase release in rat kidney tissue by histopathological and immunohistochemically method. This study, 21 male Wistar albino rats were used. Rats were divided into three groups: control, 100 mg/kg ginger essential oil (G100), and 500 mg/kg ginger essential oil (G500). Hematoxylin-eosin staining method was used for histopathological evaluations. Immunohistochemically localization of catalase in kidney tissue was determined by streptavidin-biotin peroxidase method. As a result of histopathological evaluations, an increase in glomerulus diameter was observed in kidney tissues of G100 and G500 groups. In addition, vacuolar degeneration was observed in the proximal and distal tubule epithelial cells in the renal cortex of the G100 group. The immunoreactivity of catalase in the renal cortex region; In the control group, it is strong in the proximal tubules and weak in the mesangial cells. While moderate severity in the proximal tubules and weak in the mesangial cells in the G100 group, very weak catalase immunoreactivity was observed in the G500 group. Strong catalase immunoreactivity was detected in the proximal and collecting ducts of the kidney medulla regions of the rats in all groups. We think that ginger essential oil can be used in appropriate doses and times to reduce kidney damage caused by oxidative stress in the kidney.

Cell Membrane-Camouflaged Chitosan-Polypyrrole Nanogels Co-Deliver Drug and Gene for Targeted Chemotherapy and Bone Metastasis Inhibition of Prostate Cancer.

The development of functional nanoplatforms to improve the chemotherapy outcome and inhibit distal cancer cell metastasis remains an extreme challenge in cancer management. In this work, a human-derived PC-3 cancer cell membrane-camouflaged chitosan-polypyrrole nanogel (CH-PPy NG) platform, which can be loaded with chemotherapeutic drug docetaxel (DTX) and RANK siRNA for targeted chemotherapy and gene silencing-mediated metastasis inhibition of late-stage prostate cancer in a mouse model, is reported. The prepared NGs with a size of 155.8nm show good biocompatibility, pH-responsive drug release profile, and homologous targeting specificity to cancer cells, allowing for efficient and precise drug/gene co-delivery. Through in-vivo antitumor treatment in a xenografted PC-3 mouse tumor model, it is shown that such a CH-PPy NG-facilitated co-delivery system allows for effective chemotherapy to slow down the tumor growth rate, and effectively inhibits the metastasis of prostate cancer to the bone via downregulation of the RANK/RANKL signaling pathway. The created CH-Ppy NGs may be utilized as a promising platform for enhanced chemotherapy and anti-metastasis treatment of prostate cancer.

Abstract 2148: Lung Capillary Endothelium To Arterial Endothelium Transition In Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear. Method and Results: Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggests that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for distal arterial ECs in PH. General capillary EC lineage tracing approach using Plvap-DreERT2; Tdtomato mice demonstrated that general capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or Notch4 neutralized antibodies normalized the arterial programming in PH. Conclusion: In conclusion, our study demonstrates that general capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients. Acknowledgment: This work was supported in part by NIH grants R00HL138278, R01HL158596, R01HL62794, R01HL169509, R01HL170096, AHA Career Development Award 20CDA35310084, The Cardiovascular Research and Education Foundation, Arizona Biomedical Research Centre funding (RFGA2022-01-06), and University of Arizona institution funding to Z.D.

Cellular Mechanisms for pH Regulation and Energy Metabolism in Sharks and Rays

Similar to mammalian kidney distal tubule cells, specialized shark and ray gill epithelial cells maintain blood pH homeostasis. These shark and ray gill cells are either enriched with the Na+/K+-ATPase (NKA) and responsible for acid secretion or enriched for the vacuolar H+-ATPase (VHA) and responsible for base secretion. Moreover, both cell types contain abundant mitochondria and glycogen, and glycogen content decreases in active acid-base regulatory cells in vivo. Additional experiments with isolated gill cells in vitro show that glycogen coincides with the subcellular localization of NKA and VHA. Under control conditions, glycogen and NKA are at the cell membrane of acid-secreting cells while glycogen and VHA are in the cytoplasm of base-secreting cells. During exposure to alkalosis, NKA and glycogen localization remains unchanged in acid-secreting cells; however, both VHA and glycogen translocate to the cell membrane of base-secreting cells. Interestingly, this process is prevented by pharmacological inhibition of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC). This suggests a novel mechanism, a coupling of glycogen metabolism with acid-base regulation that sustains ATPase function during sudden periods of increased energy demand. Since VHA, sAC, and glycogen are evolutionarily widespread, this mechanism is likely to apply broadly to other organisms and systems, perhaps including chronic kidney disease. APS Porter Predoctoral Fellowship and National Science Foundation Postdoctoral Research Fellowship in Biology #1709911 to JR, National Science Foundation Grant IOS 1354181 to MT. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lung capillary endothelium to arterial endothelium transition in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear. Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggest that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for arterial ECs in PH. General capillary EC lineage tracing approach using Plvap-DreERT2;Tdtomato mice demonstrated that general capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or pharmacological inhibition of Notch4 normalized the arterial programming in PH. In conclusion, our study demonstrates that general capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients. This work was supported in part by NIH grant R00HL138278, R01HL158596, R01HL62794, R01HL169509, R01HL170096, AHA Career Development Award 20CDA35310084, The Cardiovascular Research and Education Foundation, Arizona Biomedical Research Centre funding (RFGA2022-01-06), and University of Arizona institution funding to Z.D. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Brain-Derived Extracellular Vesicles from Dahl Salt-Sensitive Rats with High Salt Diet Increase PVN and SON Vasopressin Levels in Sprague Dawley rats

Extracellular vesicles (EVs), naturally secreted by almost all cell types, encapsulate a broad spectrum of biological molecules, and ferry these contents to the local and distal recipient cells. Under disease conditions, EV-to-cell communication can lead to diverse cell responses and changes. Previously we have demonstrated that brain-derived EVs from hypertensive rats increased inflammation and oxidative stress in primary neurons and hypothalamic paraventricular nucleus (PVN) of the brain, however, the long-term effects of EVs are unclear. PVN is a key hub to mediate blood pressure via its endocrine and autonomic control. Increased vasopressin (AVP) levels have been found in Dahl salt sensitive hypertensive rats, which are produced by PVN and supraoptic nucleus (SON). In this study, we hypothesized that brain-derived EVs from hypertensive rats regulate blood pressure via AVP system. To test this hypothesis, we first labeled brain-derived EVs with Rhodamine dye (Rho-EVs) and studied their bio-distribution via intracerebroventricular (i.c.v.) injection into the normal Sprague Dawley (SD) rats. Rats were euthanized 24 hours after injection, and brain sections containing the PVN and SON regions were stained to test the colocalization of Rho-EVs with neurons (NeuN), astrocytes (GFAP) and microglia (Iba1). Then we injected brain-derived EVs obtained from hypertensive Dahl salt sensitive rats (DSS-EVs) and normotensive Sprague Dawley rats (SD-EVs) into the lateral ventricle (8 μg/rat) in the SD male rats, and the injection was repeated 2 weeks later. Blood pressure and heart rates of each rat were monitored each week using telemetry transducers. 4 weeks post first EVs injections, rats were euthanized, their blood were tested for plasma AVP and norepinephrine (NE) levels, and their brains were tested for AVP and Fos-related antigen 1 (Fra1) mRNA levels. Besides, we also performed biweekly PVN (800 ng/rat) and SON (800ng/rat) injections of DSS-EVs and SD-EVs and measured the AVP and/or Fra1 protein levels with immunostaining. Lastly, we determined the colocalization of Rho-EVs with AVP-containing cells in the PVN and SON. Results show that Rho-EVs are dominantly taken up by neurons in both PVN and SON regions. Biweekly i.c.v. injections of DSS-EVs do not increase blood pressure and heart rate. However, DSS-EVs significantly upregulate mRNA levels of AVP (2.4-fold) and Fra1 (2.5-fold) in the PVN compared to SD-EVs. There is an increasing trend of plasma AVP levels in DSS-EV-treated rats compared to control. Biweekly PVN injections of DSS-EVs significantly increase AVP (1.5-fold) protein levels relative to SD-EVs, and biweekly SON injections of DSS-EVs significantly increase AVP (1.5-fold) and Fra1 (2.6-fold) protein levels relative to SD-EVs. Lastly, we found that Rho-EVs are mainly colocalized with vasopressin-containing cells in the SON. These results suggest that brain-derived EVs cause SON and PVN neuron activation, which in turn may regulate AVP system during hypertensive development. R01HL163159 (Shan); R15HL150703 (Shan); AHA 1807047 (Bi). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aldosterone applied mucosally or serosally is equally effective in stimulating ENaC activity in rat distal colon

Background: Aldosterone (ALDO), a mineralocorticoid hormone, is essential for regulating blood volume and electrolyte homeostasis. ALDO acts via intracellular mineralocorticoid receptors to stimulate epithelial Na+ channel (ENaC)-mediated Na+ absorption and large conductance K+ (BK) channel-mediated K+ secretion in both kidney and distal colon. We recently showed that ALDO stimulates iron absorption in rat distal colon and SK-CO15 cells (human colon cancer cell line), raising the prospect that ALDO might be used as adjuvant therapy in iron-deficient patients refractory to oral iron. Since, parenteral ALDO (or Fludrocortisone, a synthetic mineralocorticoid agonist) would adversely affect cardiovascular function, an alternative route of administration is required. Parenteral ALDO gains access to target epithelial cells via their serosal (basolateral) membranes, and in vitro studies have always employed serosally applied ALDO to determine its effects on colonic ion transport, but it is not known if mucosally (apically) applied ALDO has similar effects. Hypothesis: We hypothesized that like serosal ALDO, mucosal ALDO would diffuse across apical cell membranes and induce ENaC activity in normal rat distal colon. Aim: To determine whether mucosally applied ALDO stimulates amiloride-sensitive electrogenic Na+ transport in rat distal colon. Methods: Muscularis stripped normal rat (Sprague-Dawley; 126 – 150 g) distal colon was mounted under voltage clamp condition in Ussing chambers. ALDO (10 mM) was added to either the mucosal or serosal bath. ENaC activity was measured as the change in short-circuit current (Isc) following the mucosal addition of amiloride (10 mM). ALDO in mucosal and serosal bath solutions was measured using mass-spectroscopy. Results: With serosal addition of ALDO, transcriptionally induced ENaC activity began to increase after 4 hr and reached maximum/plateau by 5 hr. By contrast, with mucosal addition of ALDO, ENaC activity began to increase after 8 hr and slowly reached maximum/plateau by 9.5 hr. Maximal amiloride-sensitive ENaC activities were similar irrespective of whether ALDO was applied mucosally or serosally. Following addition of ALDO to the mucosal or serosal bath, a similar amount (40%) of ALDO had moved from mucosa to serosa or serosa to mucosa after 24 hr. Conclusions: We conclude that: (1) ALDO induces ENaC activity to the same extent, whether applied mucosally or serosally, and (2) the delayed effect of mucosally applied ALDO may reflect slower diffusion across apical membranes than across basolateral membranes. We speculate that developing a colon-specific delivery system may avoid the adverse cardiovascular effects of oral mineralocorticoid agonists (e.g. Fludrocortisone) if used to enhance colonic iron absorption in iron-deficient patients refractory to oral iron. Transition GrantSupport; Offce of Research and Graduate Education, WVU Health Sciences Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Proximal Tubule Response to Low Potassium Is Mediated by mTOR/AKT Activation via Kir4.2

Background: The renal epithelium is highly sensitive to changes in blood potassium (K+) levels. We have a detailed understanding of distal K+-sensitive signaling pathways, yet proximal mechanisms are less well-defined. Results: Mice lacking the basolateral proximal tubule inwardly rectifying K+ channel, Kir4.2, (Kir4.2−/−) demonstrated normal blood and urine electrolytes at baseline. Upon removal of K+ from their diet, Kir4.2−/− animals decompensated as evidenced by increased urinary K+ excretion and development of a proximal renal tubular acidosis compared to control animals (Kir4.2+/+). Potassium wasting in Kir4.2−/− mice was not proximal in origin, but was dependent upon increased ENaC activity along the connecting segment. This was demonstrated by the correction of K+ wasting in knockouts treated with the ENaC inhibitor, amiloride. Using lithium clearance studies, we further demonstrated increased distal sodium delivery in Kir4.2−/− animals due to reduced proximal tubule sodium reabsorption. Optical clearing and three-dimensional confocal imaging reveal Kir4.2−/− mice failed to undergo proximal tubule hypertrophy when fed a low K+ diet, while the distal convoluted tubule response was exaggerated in knockouts compared to control animals. A phosphoproteomics mass spectrometry analysis identified increased mTOR/AKT signaling as mediating the proximal dietary response to K+ depletion in control animals, and changes to mTOR/AKT signaling was blunted in Kir4.2 knockouts. Lastly, we demonstrated in isolated renal tubules that AKT phosphorylation in response to low K+ depended upon mTORC2 activation by secondary reductions in intracellular Cl−. Conclusions: In this work we identify the basolateral inwardly rectifying K+ channel, Kir4.2, as a mediator of the proximal tubule response to K+ deficiency. Kir4.2 knockout mice increase their distal nephron transport burden to maintain electrolyte homeostasis in the face of deficient proximal tubule cell function. Data identify a conserved proximal role for cell Cl− which, as it does along the distal convoluted tubule, responds to extracellular K+ changes to activate intracellular kinase signaling. Along the proximal tubule, mTOR/AKT signaling is essential for the cell response to low K+. Proximal and distal nephron cell Cl− responses work in concert to preserve K+ homeostasis. These studies were supported by NIH grants DP5-OD033412 (AST), DK51265, DK95785, DK62794, DK7569, P30DK114809 (RCH, MZZ), and the Vanderbilt Center for Kidney Disease and Vanderbilt Diabetes Research and Training Center (DK020593) pilot and feasibility grant (JSD). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dietary potassium effects on renal calcium transport at single nucleus resolution

Calcium homeostasis is maintained by the coordination of the intestines, bones, and kidneys. Dietary calcium is absorbed in the intestines, enters the bloodstream, and eventually finds its place in the bone reservoir or undergoes filtration by the glomerulus before being reabsorbed throughout the nephron. The late distal convoluted tubule (DCT2) and connecting tubule (CNT) determine the final rate of urinary calcium excretion, since no calcium reabsorption takes place beyond the connecting tubule (CNT). When excess calcium is excreted in the urine, also known as hypercalciuria, it contributes to the development of osteoporosis and the formation of kidney stones. High dietary potassium intake is strongly associated with a lower risk of kidney stone formation. Yet, the precise mechanism by which potassium intake modulates calcium excretion is not clear. To examine the transcriptional changes linking high dietary potassium intake and calcium transport, we applied single-nucleus RNA-sequencing (snRNA-Seq) in enriched distal nephron cells. To achieve the enrichment, we crossed a Calbindin 1 (Calb1)-driven Cre mouse line with the INTACT (for Isolation of Nuclei TAgged in Specific Cell Types) reporter line, which allows the GFP reporter to be expressed at the nuclear envelope of all calbindin 1-expressing cells. As the Calb1-Cre is constitutively expressed, all cells that have calbindin 1 expression at any time point during development will express the reporter. We examined the GFP expression by immunofluorescence, and found that it is expressed along the entire distal nephron from the distal convoluted tubule (DCT), CNT, to the collecting duct (CD). Male Calb1-Cre-INTACT mice were provided either a normal (NK, 1.05%) or a high (HK, 2%) potassium diet for 4 days and kidneys were snap-frozen for targeted snRNA-Seq using 10X Chromium (n=3 mice per group, targeting 10,000 nuclei per mouse). Our snRNA-seq dataset showed 6 major clusters, including DCT (DCT1 and DCT2), CNT (3 subclusters), CD principal cells (3 subclusters), ɑ-intercalated cells (2 subclusters), β-intercalated cells (2 subclusters), and proliferating cells. We curated a calcium score from the expression of known calciotropic genes ( Slc8a1, Vdr, Trpv5, Calb1, S100g, Ryr2, Trpv6) and used it as an index of calcium handling capacity. The calcium score is the highest in DCT2 and CNT along the distal nephron. In all CNT subclusters of the HK-treated animals, the calcium score is higher compared to the NK-treated animals, suggesting more calcium transport in CNT. Given that HK stimulates aldosterone and causes CNT hypertrophy, we compared the results with mice subjected to low dietary potassium (LK) treatment and metolazone (thiazide) treatment. The aim was to delineate the effects of dietary potassium and aldosterone A comprehensive summary of plasma potassium levels, urinary calcium excretion, aldosterone levels, calcium scores, and CNT morphology has been presented in the table below. Notably, the comparative analysis reveals a significant correlation between aldosterone levels and CNT size, both of which exhibit an inverse relationship with urinary calcium excretion: [Formula: see text] Our results suggest that DCT2 and CNT are the major sites for calcium transport. Yet, CNT exhibit more heightened adaptability in response to physiological or pharmacological perturbations at both transcriptional and morphological levels. Calcium homeostasis coincides with the regulation of aldosterone levels and CNT remodeling. DK51496 and DK133220 to DHE; K01DK121737 and AHA 20CDA35320169 to JWN. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluating Extended Curettage and Adjuvant Therapy Against Wide Resection and Reconstruction in the Management of Distal Radius Giant Cell Tumors: A Systematic Review and Meta-analysis.

The management of distal radius giant cell tumors (GCTs) remains challenging, and the optimal approach is still a matter of debate. This systematic review and meta-analysis aimed to compare the outcomes of extended curettage and wide resection, the mainstays of treatment. Medline (via PubMed), Cochrane Library, Web of Science, Google Scholar, ClinicalTrials.gov, and Embase databases were searched for comparative studies that assessed extended curettage with adjuvant therapy and wide resection with reconstruction in patients with GCTs of the distal radius up to April 2023. Data were collected and analyzed on rates of local recurrence, metastasis, overall complications, and functional outcomes. The Newcastle-Ottawa scale was used to appraise the risk of bias within each study. Fifteen studies (n = 373 patients) were included and analyzed. Patients who underwent curettage were more likely to develop recurrence (risk ratio [RR] = 3.02 [95% confidence interval; CI, 1.87-4.89], P < .01), showed fewer complications (RR = 0.32 [95% CI, 0.21-0.49], P < .01), and showed greater improvement in Visual Analog Scale and lower Disabilities of the Arm, Shoulder, and Hand scores (P < .00001) than those who underwent wide resection. No significant difference was found regarding metastasis (RR = 1.03 [95% CI, 0.38-2.78], P = .95). Regarding the surgical approach to GCT of the distal radius, curettage with adjuvant therapy was associated with a higher likelihood of recurrence compared with wide resection with reconstruction. Nevertheless, the curettage approach resulted in significantly lower rates of operative complications, decreased pain scores, and better functional outcomes in comparison to the resection group.

Tumor-penetrating iRGD facilitates penetration of poly(floxuridine-ketal)-based nanomedicine for enhanced pancreatic cancer therapy

Efficient intratumoral penetration is essential for nanomedicine to eradicate pancreatic tumors. Although nanomedicine can enter the perivascular space of pancreatic tumors, their access to distal tumor cells, aloof from the vessels, remains a formidable challenge. Here, we synthesized an acid-activatable macromolecular prodrug of floxuridine (FUDR)—poly(FUDR-ketal), engineered a micellar nanomedicine of FUDR, and intravenously co-administered the nanomedicine with the tumor-penetrating peptide iRGD for enhanced treatment of pancreatic tumor. A FUDR-derived mono-isopropenyl ether was synthesized and underwent self-addition polymerization to afford the hydrophobic poly(FUDR-ketal), which was subsequently co-assembled with amphiphilic DSPE-mPEG into the micellar nanomedicine with size of 12 nm and drug content of 56.8 wt% using nanoprecipitation technique. The acetone-based ketal-linked poly(FUDR-ketal) was triggered by acid to release FUDR to inhibit cell proliferation. In an orthotopic pancreatic tumor model derived from KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) cells that overexpress neuropilin-1 (NRP-1) receptor, iRGD improved penetration of FUDR nanomedicine into tumor parenchyma and potentiated the therapeutic efficacy. Our nanoplatform, along with iRGD, thus appears to be promising for efficient penetration and activation of acid-responsive nanomedicines for enhanced pancreatic cancer therapy.

Abstract LB217: Novel organoid models of lineage plasticity in castration resistant prostate cancer

Abstract The development of next-generation AR-targeted drugs, such as abiraterone and enzalutamide, has improved survival of patients with castration-resistant prostate cancer (CRPC). However, many patients eventually progress to therapy-resistant lethal prostate cancer. CRPC exhibits variant cellular states, including AR-pathway active prostate cancer (ARPC), AR activity-low prostate cancer (ARLPC), neuroendocrine prostate cancer (NEPC), amphicrine prostate cancer co-expressing AR and neuroendocrine genes (AMPC), and double-negative prostate cancer (DNPC) lacking both AR expression and neuroendocrine differentiation, indicating that lineage plasticity facilitates therapeutic resistance. While molecular characteristics of ARPC and NEPC are well described, other common forms of CRPC, such as DNPC which occurs in 20-25% of CRPC patients, remain poorly understood. Therefore, characterization of pre-clinical models of DNPC and identification of novel regulators of lineage plasticity in DNPC are urgent necessities for the development of effective treatment for lethal prostate cancer.In our studies, we have generated a new mouse model of CRPC in which Pten and Smad4 are deleted in distal luminal cells upon tamoxifen administration (Nkx3.1CreERT2/+; Ptenfl/fl; Smad4fl/fl; R26R-eYFP/+, NPS). We observed a molecular transition from ARPC to ARLPC and DNPC in the prostate tumors derived from NPS mice during tumor progression. Moreover, a combination of castration and enzalutamide treatment promoted a partial epithelial-mesenchymal transition (EMT) in DNPC. To identify the evolutionary trajectories of cancer progression and drivers of lineage plasticity in CRPC, we have established organoid lines from the prostate tumor cells of NPS mice. Single-cell RNA-sequencing (scRNA-seq) and marker validation analyses shown that these organoid lines display overt lineage plasticity, corresponding to ARPC, ARLPC, partial EMT-absent DNPC, and partial EMT-present DNPC. Orthotopic grafts derived from the established NPS organoid lines show consistent molecular phenotypes and confirm that AR-targeted therapies facilitate partial EMT in vivo. Furthermore, cross-species bioinformatic analyses using scRNA-seq datasets indicate that the established NPS organoid lines partially recapitulate the spectrum of lineage plasticity in human CRPC.In summary, we have established prostate cancer organoid lines from a novel mouse model of CRPC, which not only provide large and reproducible sources of two distinct forms of DNPC (partial EMT-absent DNPC and partial EMT-present DNPC) for molecular analyses, but also can capture the regulatory complexity of lineage plasticity in human CRPC. Therefore, NPS mice provide an excellent pre-clinical model of CRPC, especially DNPC, to identify novel regulators that drive lineage plasticity and to pursue assays for development of candidate drugs that can inhibit or even possibly convert therapy-resistant forms of CRPC to therapy-sensitive ones. Citation Format: Jinqiu Lu, Michael M. Shen. Novel organoid models of lineage plasticity in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB217.

Acaulon chilense Larraín & M.J.Cano (Pottiaceae, Bryophyta), a new species from central Chile

ABSTRACT Introduction During exploration of the central Chilean bryophyte flora over the past 10 years, especially during the wet winter months, a species of Acaulon was collected that did not match any of the known species of the genus. Here, we present our arguments for recognising these plants as a new moss species. Methods The new taxon was compared morphologically with the species of Acaulon currently recognised. In addition, most of the type material of the taxa attributed to this genus was studied. Key results and conclusions Acaulon chilense is described and illustrated as a new moss species from Chile. It is characterised by its reddish coloration, papillose distal laminal cells, costa with homogeneous cells, strongly thickened dorsal cell walls, spores less than 30 μm in diameter, and rhizoautoicous sexuality. The morphological similarities with closely related species are discussed. A distribution map of the new species is provided. This taxon is relatively common, appearing during the wet winter months in the Mediterranean climate areas of the lowlands of central Chile.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.