Polycystic kidney diseases (PKD) are characterized by development of multiple cysts, dilations of nephron segments, which replace normal tissues and lead to kidney insuffciency. To identify new gene pathways affected by cyst development in collecting ducts, we used a bulk RNAseq approach comparing gene expression of normal microdissected cortical collecting ducts (n=3 mice) vs cysts microdissected from Pkd1 RC/RC mice (n=4). Bulk-RNA analysis identified 18,000 genes and allowed statistical comparison of over 15,000 genes. Our data reveals that although cysts originate from normal collecting ducts, cystic epithelium show 2692 down-regulated and 2278 up-regulated genes (p<0.05 pAdj. FDR). Ingenuity Pathways Analysis identifies the following intracellular mechanisms mostly affected by transition: Rac and Rho signaling, fibrosis signaling, epithelial-to-mesenchymal transition, cytoskeleton rearrangement and ERK/MAPK signaling. Several of the differentially expressed genes were identified in GWAS of PKD, chronic kidney diseases and hypertension. Our previous publication reported that development of cysts in an autosomal recessive model of PKD is associated with a shift of P2Y to P2X receptor abundance. In the current study we found that in the autosomal dominant Pkd1 RC/RC mice purinergic signaling undergoes similar remodeling. The most abundant ionotropic receptors with reduced expression were P2ry2 -2.25; P2ry4 -1.37, log2 fold change, whereas ionotropic receptors P2rx5 and P2rx7 increased expression (2.88 and 1.53, log2 fold change, respectively). Additionally, analysis detected elevated abundance of P2ry6 and P2ry12 RNA level. We used a model of cystogenesis to test how P2X signaling affects growth of cysts formed by mpkCCD cl4 cells in Matrigel. Stimulation of P2X receptors with α,βMe-ATP significantly increases cyst growth. We hypothesize that the physiological significance of the predominant P2X signaling in the cysts include their role in regulation of ATP release via pannexin-1 channels. Abnormal ATP accumulation in the cyst space was shown earlier to contribute in cystogenesis and we previously showed that pannexin-1 mediates ATP release to the cyst lumen. Co-immunoprecipitatation experiments in mpkCCD cl4 cells revealed that P2X5 and P2X7 receptors bind pannexin-1 whereas P2Y6 and P2Y12 do not interact with pannexin-1. Interestingly, stimulation of cystogenesis with α,βMe-ATP was accompanied by increased abundance of pannexin-1. Lack of functional P2X5 in humans prioritizes P2X7 receptors as a therapeutic target in ADPKD. We conclude that development of ADPKD cysts involves massive transcriptome remodeling of collecting ducts which include a shift in purinergic signaling that facilitates pathogenic pannexin-1 hyperactivity. DK123266 and DK131114. 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.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45 + , CD3 + CD4 + , and CD3 + CD8 + cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Development of autosomal dominant polycystic kidney disease (ADPKD) involves renal epithelial cell abnormalities. Cystic fluid contains a high level of ATP that, among other effects, leads to a reduced reabsorption of electrolytes in cyst-lining cells, and thus results in cystic fluid accumulation. Earlier, we demonstrated that Pkd1(RC/RC) mice, a hypomorphic model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. In the current study, we found that human ADPKD cystic epithelia have higher pannexin-1 abundance than normal collecting ducts. We hypothesized that inhibition of pannexin-1 function with probenecid can be used to attenuate ADPKD development. Renal function in male and female Pkd1(RC/RC) and control mice was monitored between 9 and 20 months of age. To test the therapeutic effects of probenecid (a uricosuric agent and a pannexin-1 blocker), osmotic minipumps were implanted in male and female Pkd1(RC/RC) mice, and probenecid or vehicle was administered for 42 days until 1 year of age. Probenecid treatment improved glomerular filtration rates and slowed renal cyst formation in male mice (as shown in histopathology). The mechanistic effects of probenecid on sodium reabsorption and fluid transport were tested on polarized mpkCCD(cl4) cells subjected to short-circuit current measurements, and in 3D cysts grown in Matrigel. In the mpkCCD(cl4) epithelial cell line, probenecid elicited higher ENaC currents and attenuated in vitro cyst formation, indicating lower sodium and less fluid retention in the cysts. Our studies open new avenues of research into targeting pannexin-1 in ADPKD pathology.

Background: Extracellular ATP is a key mediator of evolutionary conserved purinergic signaling, which regulates many biological effects (tissue homeostasis, immunity, inflammation, healing etc.) and is involved in the pathogenesis of diseases of the immune, nervous, respiratory, urinary and digestive systems, skeletal muscles etc. Important source of ATP in extracellular space is the active extracellular ATP release, conducted by Panx1, a mechanosensitive non-selective ion channel, expressed in various mammalian tissues (macrophages, neurons, glial, muscular, endothelial, renal cells etc.). In turn, ATP stimulation via purinergic receptors (for example, P2X7) activates Panx1 channel. While purinergic signaling and Panx1 are considered relevant pathogenic targets for potential treatment, and some Panx1 inhibitors were already clinically tested, intrinsic properties of Panx1 channel were not well characterized. Here, we estimated electrophysiology parameters of Panx1 protein and provided its I-V Characteristic Curve, and showed that purinergic stimulation of P2X7 receptor increases Panx1 current in vitro. Methods: Activity of Panx-1 channel was recorded using a cell-attached patch clamp in CHO cells, transfected with PANX1 cDNA. To confirm Panx1 channel activity, we used blocking with 75 μM probenecid by adding the drug in bath buffer flow. For drawing I-V curve, Panx1 currents were measured and averaged (mean±SE) at different voltages in 85 experiments. This cellular model mimics cells of different systems which are abnormally overexpress the Panx1 in various pathologic conditions. We also tested if Panx1 channel stimulated via purinergic P2X7 receptor with 100μM αβ-MeATP in the CHO cells, co-transfected with plasmids, encoding PANX1 and P2X7 proteins, by analyzing the channel’s NPO dynamics. Statistical significance of experimental effects on NPO was estimated with non-parametric paired Wilcoxon Signed Ranks test with using the Origin software (Northampton, MA). Results: Probenecid significantly inhibits an activity of the channels, overexpressed in PANX1-transfected CHO cells. I-V Characteristic Curve of Panx1 channel in the same cells was obtained. NPO decreased from 1.32±0.16 to 0.52±0.09 (n=28, p<<0.0001). The inhibitory effect of the drug was reversible; after washout with bath buffer flow, NPO reverted to 1.21±0.12. Treatment of CHO cells, co-transfected with PANX1 and P2X7 plasmids, with αβ-MeATP significantly stimulated an activity of Panx1 channels; NPO increased from 0.70±0.26 to 0.97±0.23, n=9, p=0.0012. The effect was probenecid-dependent (NPO=0.40±0.12, p=0.0039). Conclusion: Purinergic stimulation of cells, co-expressing Panx1 and P2X7 proteins, increases probenecid-dependent Panx1 current. These results support that Panx1 channel is targetable for pharmacological interventions in diseases with pathogenic involvement of purinergic signaling. Support: ASN Carl W. Gottschalk award, DK123266. This is the full abstract presented at the American Physiology Summit 2023 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.

Histamine is a nitrogenous compound crucial for the inflammatory response. The knowledge regarding the renal effects of histamine is very limited. We showed that renal epithelia exhibit expression of the components of the histaminergic system. Furthermore, we revealed that there was a shift in the histaminergic tone in salt-sensitive rats when they were challenged with a high-salt diet. These data support the notion that histamine plays a role in renal epithelial physiological and pathophysiological functions.

Introduction . Histamine is a regulatory agent involved in the immune response, neurotransmission, and gastric acid secretion, and is produced and released from the mast cells and basophils. Elevated histamine levels and increased expression of histamine metabolizing enzymes have been reported in kidney diseases. The goal of this study is to provide insight into the physiological importance of the histamine-related pathways in salt-sensitive hypertension (SSH), a disease accompanied with inflammation-driven kidney damage. Methods . SSH was induced in Dahl Salt-Sensitive (DSS) rats by a 3-week long high salt diet challenge (HS, 4% NaCl); age-matched normal salt diet (NS, 0.4% NaCl) fed rats were used as a control group. To characterize the expression of histamine receptors, mast cells markers, and histamine metabolism-related enzymes, Western blot or IHC were performed on renal tissues collected from DSS rats maintained on HS or NS diets for 3 weeks. In order to test the effects of histamine receptor 2 (HR2) inhibition, he DSS rats were injected with a single daily i.p. dose of an HR2 blocker, ranitidine (RAN, 25mg/kg in saline) or saline (VEH), for 3 consecutive days before the switch to a HS diet, and at the end of the 21-day long HS challenge. Urine and water consumption were assessed in metabolic cages during hours 0-8 (acute), and 8-24 post-injections. At the end of the protocol, GFR was measured, and tissues were harvested. Results . Western blots revealed similar expression of the mast cell markers’ (FCER1A and tryptase) in NS and HS diet fed rats, however, we observed significantly lower histamine decarboxylase (HDC, p=0.002), and higher histamine N-methyltransferase (HNMT, p=0.035) levels in HS diet fed rats. Renal IHC revealed that expression of HR2 was downregulated, while HR3 and HR4 were upregulated in the collecting ducts of HS diet fed rats. In vivo experiments showed that RAN-administered rats on NS diet exhibited an acute (8-24 hr period) decrease in urine output and 24 h water intake on day 1 of injections (p=0.01). We observed trends for an increase in urinary electrolytes, and an overall increase in the excretion of osmolesin RAN vs VEH animals 24 hours post day 1 injection on the NS diet. Post HS challenge, electrolyte excretion, urine flow and water intake were similar between RAN and VEH animals. At the endpoint on HS, GFR between the groups was similar on day 3 post-injection. Conclusions . Differential expression of HRs, as well as changes in the enzymes responsible for histamine synthesis and metabolism between HS and NS fed rats, indicate a shift in the renal histaminergic tone during SSH development. Similar mast cell levels suggest an alternate, perhaps intrarenal, source of histamine. Our in vivo data show that inhibition of HR2 has effects on urine output and water consumption in SSH. Further research is required to discern the molecular pathways downstream of specific HRs, as well as the consequences of chronic administration of HR-specific pharmacology, in renal pathophysiology.

Background ADPKD cysts contain high levels of ATP that contribute to cyst enlargement. Among other effects, ATP excess leads to a reduced reabsorption in cyst-lining cells and cyst fluid accumulation. We demonstrated that Pkd1RC/RC mice, a model of ADPKD, exhibit increased expression of pannexin-1, a membrane channel capable of ATP release. Probenecid, a uricosuric agent, is also used as a pannexin-1 blocker reducing ATP release. We studied therapeutic potential of probenecid in Pkd1RC/RC mice and its effect on sodium reabsorption. Methods : Pkd1RC/RC mice, a hypomorphic model of ADPKD, were aged till 10.5 months and osmotic minipumps were implanted to deliver probenecid for 42 days. After treatment, 1 year old conscious mice were subjected to a measurement of glomerular filtration rate (GFR) and kidneys were collected for histomorphological studies. Effect of probenecid on Na reabsorption was tested on mpkCCD cells seeded onto permeable supports with open-circuit current measurements. Results In vivo inulin clearance study demonstrates that Pkd1RC/RC mice have normal GFR 1.05±0.09 ml/min/100g at the 6 months old age (n=15) whereas GFR in C57BL/6 mice - 0.94±0.1 ml/min/100g (n=8). With disease progression, GFR in Pkd1RC/RC mice reduces to 0.36±0.08 ml/min/100g at 12 months age. 42 days long probenecid treatment (15.9 mg/kg/day) significantly improves GFR to 1.43±0.11 ml/min/100g (p<0.001). Probenecid treatment also reduced kidney hypertrophy: kidney/TBW ratio in vehicle group was 2.54±0.17% vs 1.76±0.05% probenecid (p<0.05). Histological study on sectioned kidneys revealed that probenecid significantly reduces cyst size. Cyst to total slice area ratio was 13.9±2.7% (vehicle) vs 3.4±0.8% (probenecid) (n=5 each group). Earlier we have shown that probenecid decreases luminal ATP release in immortalized CD cell culture. As ATP is capable of downregulating Na reabsorption via the epithelial sodium channel we tested if probenecid increases ENaC activity. We applied probenecid to mpkCCD cell monolayer and found that the drug causes a bell-shaped dose-dependent increase of amiloride-sensitive transepithelial flux with maximal effect at 50 μM. Conclusion : Probenecid demonstrates therapeutic potential against ADPKD cyst progression in a Pkd1RC/RC mouse model by reducing cyst size, renal hypertrophy and supporting GFR and reabsorption from the cyst space.

BackgroundEvery year thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. However, most of the studies report a cohort of patients who does not significantly respond to sodium restriction. Generally, most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying self‐sustaining character of hypertension. Unfortunately, hypertension causes multiple abnormalities in the body which stays even when the trigger, caused the initial blood pressure raise, is gone. We hypothesize that chronic hypertension leads to a significant renal damage and abnormally high sodium reabsorption.MethodsWe used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4% NaCl diet for 3 weeks to induce hypertension and then diet was switched back to normal (0.4% NaCl). Patch‐clamp analysis was performed on freshly isolated split‐open cortical collecting ducts and CHO cells to characterize activity of the epithelial sodium channel (ENaC), responsible for the sodium reabsorption in the aldosterone‐sensitive distal nephron.ResultsWe found that 4% sodium diet significantly increases blood pressure within 3 weeks (from 111±0.9 to 138±3.5 mmHg) and switching back to 0.4% NaCl diet slightly reduces blood pressure (to 133.6±7.6). Patch clamp analysis reveals that development of hypertension was accompanied with elevated ENaC activity which also stayed high (NPo=1.22±0.23, n=8) after the salt challenge. Subsequent blockage of ENaC with a selective inhibitor, benzamil, caused a dramatic drop in blood pressure indicating that ENaC activity contributes to maintain sustaining hypertension in absence of the high salt consumption. We also found significant renal damage caused by hypertension. As earlier studies report that inflammation and reactive oxygen species production are involved in improper renal function and setting high blood pressure, we performed a subset of whole‐cell patch clamp experiments on CHO cells overexpressing mENaC to study effect of hydrogen peroxide on ENaC activity. We found that treatment with 100μM H2O2 within a few seconds to 2 minutes stimulated the ENaC current of the cells (−144±12 pA/pF vs −190±22 pA/pF before and after treatment respectively, n=8, p=0.0078).Based on our earlier publications and the current data we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge and reactive oxygen species production can keep ENaC active.Support or Funding InformationAcknowledgementASN Carl W. Gottschalk award; R00 HL116603

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK. P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK. P2rx7 + littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

IntroductionBaroreceptor (BR) reflex is a central mechanism that underlies autonomic control of the circulation due to tight coordination between heart output and peripheral vasculature resistance. Abnormal and/or pathological sensitivity of the BR reflex (BRRS) is associated with many cardiovascular diseases. ATP acting as a co‐transmitter in sympathetic and parasympathetic terminals regulates excitability of the carotid sinus cells and therefore participates in BRR. The goal of the present study was to reveal the role of P2X7 receptors in the autonomic control of the cardiovascular system and BR reflex sensitivity.MethodWe used a constitutive CRISPR/Cas9‐mediated knockout of P2X7 receptor in the PCK rat background. PCK rats are a model of chronic autosomal recessive polycystic kidney disease which associated with cardiovascular complications. As PCK rats were originally derived from the Sprague Dawley strain, the latter was used as control. BRR was studied in vivo in a series of telemetry‐based subchronic experiments. DSI HD‐S11 transmitters were implanted for direct blood pressure collection from carotid artery in addition to lead II electrode placement for electrocardiogram. After a week‐long recovery period standard approach for BRR testing with bolus intravenous sodium nitroprusside (SNP 10μg/kg) injection was used before and after double autonomic blockade (DAB, atropine plus atenolol). Mean arterial pressure (MAP) and heart rate (HR) were collected from free‐moving conscious animals and BRRS was calculated as ΔMAP/ΔHR relation.ResultsNo significant difference in resting HR was observed in Sprague‐Dawley (SD) and WT or P2X7 KO PCK rats (378±15, n=6; 359±16, n=6 and 365±8, n=6 respectively). After DAB, HR reduction was similar in WT and KO groups (11.3±2.2% and 10.1±2.3%), but SD demonstrated a more pronounced effect (18.2±3.1%). The KO rats demonstrated significantly reduced BRRS in comparison to WT and SD animals (0.43±0.05 and 0.63±0.07, 0.57±0.06, respectively). Moreover, while SD and WT rats are characterized by negative slope of ΔMAP‐ΔHR relation, ΔHR in KO group demonstrates lack of dependence on ΔMAP. In addition, the time from the beginning to maximal HR elevation in response to SNP application was significantly shorter in KO (13.5±2.5 s) vs SD (32.4±3.6s).ConclusionAutonomic control of cardiac function in PCK rats is impaired in comparison to SD rats due to chronic kidney disease. P2X7 deficiency leads to impaired BR reflex sensitivity. P2X7 receptors involved in neurotransmission contribute to the sympathetic/parasympathetic balance of cardiac function control.Support or Funding InformationNIH R00 DK105160, P30 DK090868 via Baltimore PKD Center P&F Grant and APS Dean Franklin AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic predisposition is necessary for polycystic kidney disease (PKD) initiation, although there are other, incompletely identified downstream processes that are required for cyst growth. Their characterization may provide a unique opportunity for clinical interventions. One of the poorly studied phenomena in PKD is high ATP content in cysts. Unfortunately, neither origins of uncontrolled ATP release, nor consequences of abnormal purinergic signaling in relation to epithelial transport are well explored in the polycystic kidney. We tested the distribution of pannexin-1 (Panx1) and P2X7, two proteins potentially involved in ATP release, in the kidneys of the Pkd1RC/RC mice, a model of autosomal dominant PKD (ADPKD). Abundances of both proteins were abnormally increased in the cyst lining cells compared to non-dilated collecting ducts. To establish if pannexin-1 contributes to ATP release in the collecting ducts (CD), we measured luminal accumulation of ATP in M1 cell renal CD monolayers, and found that treatment with probenecid, a Panx1 blocker, prevents ATP release. Single channel patch clamp analysis of polarized M1 cells revealed that apical stimulation of P2X receptors with αβ-MeATP acutely reduces ENaC activity. We conclude that in ADPKD progression, an abnormal hyperexpression of both PANX1 and P2RX7 occurs in the cyst lining epithelial cells. High abundance of both proteins is not typical for non-dilated CDs but, when it happens in cysts, pannexin1/P2X7 cooperation elevates ATP release into the luminal space. High ATP level is a pathogenic factor facilitating cystogenesis by reducing ENaC-mediated reabsorption from the lumen.

Accumulating evidence suggests that the autocrine and paracrine effects of Adenosine‐3‐phosphate (ATP) could be detrimental for the progression of polycystic kidney diseases (PKD). P2X7 targeting in cell cultures and zebrafish was shown to decrease cyst progression. In this project, CRISPR/Cas9 approach was employed to knockout P2X7 receptor in the PCK rat strain, a model of autosomal recessive PKD (ARPKD), to study involvement of P2X7 in PKD progression in mammals. Mendelian distribution was observed in the litters showing no embryonic lethality. Adult knockout animals exhibited moderate hypertension (mean arterial blood pressure 123±6 and 132±5 mmHg at 9AM/9PM as registered with continuous DSI telemetry). General morphological evaluation of the kidneys revealed that total P2X7 knockout caused exacerbated cystogenesis compared to wild‐type littermates.Impaired renal function in the knockout group was also confirmed with metabolic studies and inulin clearance studies in conscious animals. Thirteen weeks old P2rx7−/+ and P2rx7−/− animals had higher body mass than P2rx7+/+ littermates (421±9g and 430±10.5g vs 392±12g; n=5–14). Plasma sodium and chloride (not potassium) were slightly but significantly lower in the knockout group. We observe significant proteinuria of 35–40 mg/day/100g body weight in both groups (at 13 weeks) but with aging (at 24 weeks), it reached 89±13 and 157±19 mg/day/100g in wild‐type and knockout animals, respectively. Glomerular filtration rate was also lower in the P2rx7−/− group (0.88±0.06 vs 1.22±0.11 ml/min/100g body weight) in young rats, however, with aging GFR significantly decreased in both groups and did not differ (0.42 and 0.49, respectively).Purinergic signaling is known as a powerful down‐regulator of ENaC‐mediated sodium reabsorption. Thus, patch clamp analysis revealed that acute basolateral application of 100μM α,β‐methylene‐ATP to M1 collecting ducts cell culture grown on permeable membranes decreases activity of apical ENaC (NPo from 0.5 ±0.15 to 0.19±0.12, n=10). At the next step we investigated if the P2X7 knockout affect ENaC activity. Patch clamp technique was also applied on three types of tubular epithelia isolated from knockout PCK rats and their wild‐type littermates: split‐opened non‐dilated collecting ducts, small developing cysts and large mature cysts. In these preparations we found that developing cysts exhibit increased ENaC activity whereas mature cysts had impaired channel activity (NPo= 0.79±0.12; 1.1±0.2 and 0.39±0.16). In all three tissue types P2X7 knockout increased ENaC activity (NPo=1.36±0.17; 2.3±0.36; 0.86±0.05), as expected.In conclusion, knockout of the P2rx7 gene in PCK rats increases cystogenesis and aggravates renal insufficiency. P2X7 deficiency also increases activity of ENaC in PCK rat collecting ducts and cysts.Support or Funding InformationSupported by K99/R00 HL116603, P30 DK090868 via Baltimore PKD Center P&F Grant and R24 HL114474 via MCW Gene Editing Rat Resource CenterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ultrashort femtosecond pulsed lasers may provide indispensable benefits for medical bioimaging and diagnosis, particularly for noninvasive biopsy. However, the ability of femtosecond laser irradiation to produce biodamage in the living body is still a concern. To solve this biosafety issue, results of theoretical estimations as well as the in vitro and in situ experiments on femtosecond biodamage should be verified by experimental studies conducted in vivo. Here, we analyzed photodamage produced by femtosecond (19, 42 and 100 fs) near-infrared (NIR; ~800 nm) laser pulses with an average power of 5 and 15 mW in living undissected Drosophila larvae (in vivo). These experimental data on photodamage in vivo agree with the results of theoretical modeling of other groups. Femtosecond NIR laser pulses may affect the concentration of fluorescent biomolecules localized in mitochondria of the cells of living undissected Drosophila larva. Our findings confirm that the results of the mathematical models of femtosecond laser ionization process in living tissues may have a practical value for development of noninvasive biopsy based on the use of femtosecond pulses.

Polarization of the centrosome and the Golgi apparatus in the T cell (TC) toward the antigen-presenting cell (APC) is essential for the specificity of the immune response on the cellular level. Previously we reported the existence of thin, long processes on the TC surface, which emanated predominantly from the area next to the Golgi apparatus. They appeared to be involved in the orientation of the TC during the initial phases of its attachment, which preceded the formation of the immunological synapse mediated by lamellipodia. Here we improve the visualization of the long, thin protrusions in the cultured TC and demonstrate using cytoskeleton inhibitors and immunofluorescence that microtubules form their cytoskeletal basis. The protrusions are seen prior to the attachment and the development of the broad lamellipodia (within a few minutes). We propose the term "tubulopodia" for this distinct type of cell appendage. Using an established experimental model that replaces the APC surface with a biomimetic substrate coated with antibodies against the TC receptor (TCR), we demonstrate that abrogation of the lamellipodium-mediated synapse formation does not impede the orientation of the TC Golgi apparatus and the centrosome to the contact area. Video microscopy reveals the spreading of the tubulopodia on the TCR-binding substrate, which results in the area of their emanation, and consequently the Golgi apparatus and the centrosome, being closely apposed (polarized) to the TCR-binding surface. Treatment with paclitaxel made the tubulopodia rigid, preventing their attachment to the TCR-binding surface and the reorientation of the cell body with the intracellular structures. We speculate that the motility and polarity of the TC in vivo may be mediated on a large scale by differential adhesion through the long, flexible tubulopodia.

An Yb fiber laser oscillator with sub-30 fs pulses compressed by MIIPS is tested for multiphoton microscopy. It leads to greatly improved third harmonic generation images. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.

The potential application of nonlinear optical imaging diagnosis and treatment using femtosecond laser pulses in humans accentuates the need for studies carried out in whole organisms instead of single cells or cell cultures. While there is a general consensus that in order to minimize the level of photodamage the excitation power has to be kept as low as possible, it has yet to be determined if shorter pulses have greater benefit than longer pulses. Here we evaluate the rate of death in Drosophila melanogaster as the integral parameter related to photodamage resulting from femtosecond near infrared (NIR) laser irradiation under conditions comparable to those used in two-photon excited fluorescence (TPEF) microscopy. We found that the lethality (resulting from photodamage) as a function of laser energy fluence fits a 3-region dose-response curve. The lethality was accompanied with development of necrosis and apoptosis in irradiated tissues. Quantitative analysis showed that the damage has a mostly linear character on energy fluence per pulse, and for a given TPEF signal, shorter (37 fs) pulse duration results in lower lethality than longer (100 fs) pulse duration. These results have important implications for the use of femtosecond NIR laser pulses in microscopy as well as in vivo medical imaging.