bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel CRISPR/Cas9 Knock‐in Rat Model Defines A Polymorphic Variant of Secreted Phosphoprotein 2 as A Quantitative Trait Nucleotide Linked to The Heritability of Blood Pressure
Authors: Chakraborty Saroj , Nie Ying , Cheng Xi , Mell Blair , Galla Sarah , Joe Bina
Published: November 2023

Hypertension is a complex polygenic disease caused by a combination of inherited (genetic) and environmental factors. Rat models of inherited hypertension serve as tools to dissect and prioritize genetic factors as candidate genes causing hypertension. One such candidate gene prioritized through systematic linkage and substitution mapping is Spp2 or Secreted Phosphoprotein 2. A single non‐synonymous G/T polymorphism between the Dahl Salt‐ Sensitive (S) rats and Spontaneously Hypertensive Rats (SHR) at the Spp2 locus was hypothesized to cause a reduction in blood pressure (BP) observed in the S.SHR congenic strain spanning the Spp2 locus. To test this hypothesis, a novel rat model was generated using the CRISPR/Cas9 precision‐engineering technology, whereby the ‘G’ allele at the Spp2 locus of the S rat was replaced by the ‘T’ allele of the SHR rat. Protein modeling prediction by SWISSPROT indicated a significantly altered protein structure of the Spp2 protein in the resultant Spp2 knock‐ in rescue model. Following transgenesis, pups born were genotyped and grouped into founders and non‐founders. The founder and non‐founder rats were isogenic except for the Spp2 locus, wherein the founder S rats contained the ‘G’ allele and the non‐founder S rats contained the ‘T’ allele. Both founder and non‐founder rats (n=12 male rats/group) were fed a high salt (2% NaCl) containing diet and their BP was monitored by radiotelemetry. Both systolic and diastolic BP of the Spp2 knock‐in rescue model were significantly lower compared to that of the non‐founder S rats. These data provide conclusive evidence for a single nucleotide polymorphism within the Spp2 gene as a quantitative trait nucleotide (QTN) responsible for the inheritance of blood pressure

Support or Funding Information

Funding for this work to BJ from the NHLBI/NIH (HL020176) is gratefully acknowledged
Activation of collecting duct NOS1β during high salt feeding is critical for down‐regulation of Na+ and K+ transporters, in part, via down‐regulation of aldosterone
Authors: Hyndman Kelly A , Collins Jessika D , Pollock Jennifer S
Published: November 2023

We determined that deletion of nitric oxide synthase‐1β (NOS1β) from the collecting duct (CD) results in inappropriate ENaC activation during high salt diet. Given that sodium influx via ENaC is a driving force for potassium (K) secretion, we hypothesized that CD NOS1β regulates potassium handling. Male control and CDNOS1KO mice were placed on either 1) 0.4% NaCl + 1% K diet, NSNK, 2) 4% NaCl + 1% K diet, HSNK, 3) 0.4% NaCl + 0% K diet, NSLK, or 4) 4% NaCl + 0% K diet, HSLK, for 3 – 4 days. Food and water intake was similar between genotypes. As expected, plasma potassium was similar between NSNK (control 4.8 ± 0.3; KO 4.6 ± 0.2 mM) and HSNK (control 4.6 ± 0.4; KO 4.6 ± 0.1 mM), regardless of the genotype; however it was significantly reduced in the NSLK (control 3.9 ± 0.2; KO 3.5 ± 0.1 mM) and HSLK treatments (control 3.5 ± 0.1; KO 4.1 ± 0.2 mM). Plasma aldosterone failed to be suppressed in the CDNOS1KO mice on HSNK diet (control 74 ± 5; KO 422 ± 64 pg/ml, P < 0.05). Interestingly, plasma renin concentration was appropriately suppressed in both genotypes on HSNK diet. However, plasma aldosterone was appropriately suppressed in both genotypes on the potassium deficient diets (NSLK: control 81 ± 41, KO 177 ± 48 pg/ml; HSLK: control 95 ± 25, KO 100 ± 32 pg/ml). CDNOS1KO mice given spironolactone pellets (50 mg/kg b.w.) for 7 days while on a HSNK diet presented with significantly higher plasma potassium compared to controls with similar treatment (4.2 ± 0.07 mM vs. 3.9 ± 0.1 mM, P = 0.04). Western blot analysis of renal cortical lysates determined that during HSNK feeding CDNOS1KO mice display increased Kir4.1 potassium channel expression, increased NCC activation, increased NKCC2 expression, and ENaC activation compared to control cortical lysates. These data suggest that activation of NOS1β in the CD during high salt feeding modulates not only sodium handling but also potassium handling, in part, via down‐regulation of aldosterone.

Support or Funding Information

Research reported in this publication was supported by NIH NIDDK under award number K01DK105038 to K.A.H. and NIH NHLBI under award number P01HL95499 to J.S.P.
Cyclophosphamide Treatment Attenuates Hypertension in an Experimental Model of Systemic Lupus Erythematosus
Authors: Wolf Victoria L. , Ryan Michael J.
Published: November 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women during their reproductive years, leading to speculation that estrogens factor in the pathogenesis of the disease. However, published data from our laboratory show a complex temporal relationship with estrogens likely serving a permissive role for SLE disease early in life, but acting to protect against cardiovascular risk factors like hypertension during adulthood. Hypertension, a major cardiovascular risk factor and independent predictor of mortality in women with SLE, is prevalent in these patients for reasons that remain unclear. Our laboratory showed that autoimmunity is an important factor driving the pathogenesis of hypertension and that immunosuppressive therapies can attenuate the development of hypertension in an experimental mouse model of SLE. Cyclophosphamide is a common immunosuppressive treatment for patients with SLE that works by inhibiting DNA synthesis via the alkylation of nucleic acids in rapidly dividing cells. While CYC‐based treatment regimens have significantly improved survival rates, they also contribute to an increased prevalence of premature ovarian failure among patients treated for advanced lupus nephritis that could ultimately impact estrogen status of these women. We hypothesized that the administration of CYC (25 mg/kg, once/week, IP injection) for four weeks would blunt the development of hypertension and renal disease in an established female mouse model of SLE with hypertension (adult NZBWF1 female mice at 30 weeks of age), and that the estrogen status of the mice would be impaired as a side effect of the treatment. Mean arterial pressure (MAP) was measured by indwelling carotid catheters at the conclusion of the study in conscious freely moving mice. Results suggest that MAP was lower in CYC treated SLE mice (n=4) compared to vehicle treated SLE mice (n=4) [119.7±2.0 vs 135.1±5.1 mmHg, p<0.05]. Preliminary data suggest that treatment with CYC attenuates renal injury assessed by urinary albumin excretion [0.0858±0.0657 (n=4) vs 0.4539±0.2565 (n=6) mg/day] and may have had an effect to reduce uterine weight [0.13±0.01 (n=5) vs 0.20±0.03 (n=6) % of body weight], a surrogate marker for estrogen status. CYC treatment also did not significantly affect body weight, with a decrease of 2.68±0.02% (n=5) in CYC treated SLE mice compared to a decrease of 2.88±0.01%(n=6) in vehicle treated SLE mice, suggesting that the change in MAP was not caused by the loss of body weight that sometimes accompanies cytotoxic drugs. Additional studies are required to determine the impact of CYC on hormonal status in mice with SLE and whether hormone therapy can be safely used in SLE patients using CYC that may be at risk of losing ovarian function.

Support or Funding Information

VA Merit Award (BX002604‐01A2) NIH (PO1HL051971, P20GM104357, T32HL105324‐05)
Is estrogen an inhibitor of sodium appetite and an enabler of sodium skin reservoir?
Authors: Mecawi Andre Souza , Menezes Veronica Cristina Lopes , Dos‐Santos Raoni Conceição , Monteiro Livia Rocha Natalino , Paes‐Leme Bruno , Casartelli Evelton Alves , Cortes Wellington Silva , Vivas Laura , Reis Luis Carlos
Published: November 2023

It was demonstrated that estradiol could modulate neuroendocrine and behavioral responses concerning body fluid and cardiovascular homeostasis. Not entirely consensual data support that estrogen may influence sodium appetite and food intake in cycling female and estrogen‐replaced ovariectomized (OVX) rats as well. The key question herein is whether differences in the baseline or stimulated water and sodium intake are a function of circulating estrogen in OVX rats and if there are any relationship with a conceivable skin sodium storage. Female Wistar (~230 g) rats were bilaterally ovariectomized and injected for 7 days with oil (OVX) or estradiol cypionate, 40 μg/kg (OVX+40). We evaluated spontaneous and induced water and sodium intake, plasma and urine electrolyte concentration. In addition, sodium skin level was determined in replete and sodium‐depleted OVX and OVX+40 rats. OVX rats displayed higher food intake compared to estrogen‐replaced rats. OVX+40 rats also showed a significant increase in sodium intake (7%) and normalized plasma sodium 24 h after sodium depletion, while OVX rats still maintained hyponatremia (−4%). However, OVX+40 rats expressed reduced sodium intake induced by sodium depletion compared with OVX rats. Estrogen‐replaced rats presented higher skin stored sodium load both at baseline (24%) and at 24 h after sodium depletion (52%). The osmotically active sodium load stored on the skin was higher (3.5%) in the estrogen‐replaced rats at baseline, but decreased (8%) after sodium depletion in this same group. The osmotically inactive sodium was not changed at any condition. The sodium/water ratio was significantly higher in the group of estrogen‐replaced rats after sodium depletion, compared to the OVX rats without replacement. Our data clearly show that estrogen influence behavioral ingestive response after sodium depletion in OVX rats. Regarding the higher plasma levels in our experimental paradigm, we may suggest that estrogen replacement makes them less vulnerable to volume depletion. This condition is possible based on, at least in part, on the ability of estrogen replacement to establishing higher levels of sodium stored in the skin.

Support or Funding Information

CNPq, FAPERJ
Assessment of Hydration State by Combining Urine Color and Void Number
Authors: Adams J.D. , Johnson Evan C , Jansen Lisa T , Mauromoustakos Andy T , Perrier Eric , Kavouras Stavros A
Published: November 2023

PURPOSE

To evaluate the diagnostic ability of two combined practical markers for elevated urine osmolality in free‐living adults.

METHODS

One hundred and one healthy adults (females N=52, 1.64±0.06 m, 72.0±16.6 kg, 26.9±6.1 kg/m2; males N=49, 1.77±0.07 m, 81.5±16.0 kg, 26.0±4.7 kg/m2) collected urine for 24 h on two occasions day 1 (D1) and day 2 (D2), separated by 1 week. Urine was analyzed for urine osmolality (UOsm), color (UC), while the number of voids (Void) was also recorded. Receiver Operating Characteristic (ROC) analysis was performed for UC, Void, and combination of UC and Void, to determine markers' diagnostic ability for detecting high urine osmolality (UOsm >500 mmol·kg−1).

RESULTS

For the first and second day, UOsm was 415±216 & 457±225 mmol·kg−1, UC was 2±1 & 2±1, and Void number was 8±3 & 8±3. ROC analysis results for UC for UOsm>500 mmol·kg−1 both D1 and D2 had good overall classification ability (area under the curve, AUC 81–85%), with good sensitivity (77–81% and good specificity (76–80%). Threshold analysis revealed a UC > 3 for both D1 and D2. ROC analysis results for Void for D1 and D2 had good overall classification ability (AUC both 72%), with good sensitivity (78–80%), but moderate specificity (67–74%). Threshold analysis revealed a void number > 8 for D1 and > 7 D2. Based on the ROC analysis, combining both UC and Void number displayed good overall classification ability for D1 and D2 (AUC 84–86%) with moderate sensitivity (69–78%), and good specificity (89–90%).

CONCLUSION

Both urine color alone and the combination of urine color with void number can be used as a valid and simple diagnostic measure to detect elevated urine osmolality.

Support or Funding Information

The study was supported by a grant by Danone Research
ALDOSTERONE INFUSION INTO THE 4TH VENTRICLE PRODUCES ACTIVATION OF HSD2 NEURONS IN THE NTS AND SODIUM APPETITE
Authors: Gasparini Silvia , Melo Mariana Rosso , Andrade‐Franze Glaucia Maria Fabricio , Menani Jose Vanderlei , Colombari Eduardo
Published: November 2023

Aim

Aldosterone sensitive neurons characterized by the presence of the enzyme HSD2 are present in the nucleus of the solitary tract (NTS) near to the 4th V. These neurons are activated under various conditions that stimulate sodium intake, suggesting that they may have a role in sodium homeostasis. A recent study showed that chronic infusion of low dose of aldosterone into the 4th ventricle (4th V) induces sodium intake. Indeed, in the present study, we sought to investigate if the dose of aldosterone that induces sodium intake when infused into the 4th V also activates HSD2 neurons in the NTS. In addition, it was also analyzed the activation of other areas of the brain important for the control fluid‐electrolyte balance and directly or indirectly connected with the NTS, like the lateral parabrachial nucleus (LPBN), central amygdala (CeA), supraoptic nucleus (SON), paraventricular nucleus (PVN) and medial preoptic nucleus (MPO).

Material and Methods

Male Holtzman rats with stainless steel cannulas implanted into the 4th V were used. Rats had free access to water and food and also to 1.8% NaCl during 2 hours/day. Water and 1.8% NaCl intake was measured in the first 5 days of infusion of vehicle or aldosterone (100 ng/μl/h) into the 4th V. On the sixth day of infusion, the brains were removed and the number of neurons expressing c‐Fos or c‐Fos and the enzyme HSD2 were quantified in different areas of the brain by immunohistochemistry.

Results

Chronic infusion of aldosterone (100 ng/h) into the 4th V increased 1.8% NaCl intake (10.3 ± 3, vs. vehicle: 2.3 ± 1 ml/2 h), without changing water intake. The infusion of aldosterone into the 4th V increased c‐Fos expression in HSD2 neurons in the intermediate NTS (3 ± 1, vs. vehicle: 0 ± 0 cells/section, p < 0.05) and rostral NTS (7 ± 2 vs. vehicle: 0 ± 0 cells/section, p < 0.05). The expression of c‐Fos also increased in non HSD2 neurons in the commissural NTS (22 ± 6, vs. vehicle: 5 ± 3 cells/section, p < 0.05). Aldosterone into the 4th V also increased c‐Fos expression in the SON (21 ± 2, vs. vehicle 7 ± 2 cells/section, p < 0.05), CeA (29 ± 9, vs. vehicle: 0 ± 0 cells/section, p < 0.05), without changing c‐Fos expression in the LPBN (14 ± 4, vs. vehicle: 13 ± 5 cells/section, p > 0.05). PVN (23 ± 5, vs. vehicle: 10 ± 4 cells/section, p > 0.05) or MPO (22 ± 5, vs. vehicle 7 ± 7 cells/section, p > 0.05.

Conclusion

The activation of HSD2 neurons in the NTS by aldosterone infused into the 4th V probably activates a central circuitry involved in the stimulation of sodium intake that may include one or more areas like SON and CeA.

Support or Funding Information

PROPE‐UNESP; CAPES; CNPq; FAPESP
Hypotestosteronemia‐induced hypertension in male Sprague‐Dawley rats is renin‐ angiotensin system‐dependent
Authors: Hanson Andrea E. , McKenna Joshua M. , Perusquia Mercedes , Stallone John N.
Published: November 2023

Acutely, testosterone (TES) and other androgens are efficacious vasodilators, both in vitro and in vivo; however, long‐term effects of the androgens on arterial blood pressure (BP) are unclear. Thus, long‐term effects of endogenous and exogenous TES on BP were studied in male Sprague‐ Dawley rats. 12–13 wk old rats remained intact (InT) or were castrated (CsX). Weekly measurements of systolic BP (tail cuff plethysmography) revealed a progressive rise in BP over 10 wks in CsX (108 ± 0.9 vs. 139 ± 2 mmHg), while BP remained stable in InT (109 ± 3 vs. 113 ± 0.3). During the next 5 weeks, half of the CsX received TES replacement therapy (CsX+TES‐ enanthate‐replaced; 1.75 mg/kg, given 2X/wk). BP gradually declined to normal in CsX+TES replaced rats (118 ± 1), while BP remained elevated in CsX (141 ± 1) and normal in InT (112 ± 3). Seminal vesicle and body weights of InT (1.212 ± 0.038 g and 441 ± 9 g, respectively), CsX (0.065 ± 0.006 g and 420 ± 4 g) and CsX+TES (1.203 ± 0.080 g and 433 ± 3 g) revealed that TES replacement produced physiological levels of TES. In a separate group of CsX rats, treatment with Losartan (LST; 250 mg/L drinking water) prevented development of hypertension at 10 wks (94 ± 2 CsX+LST vs. 131 ± 3 CsX). During the next 5 weeks with TES replacement therapy, BP declined in CsX+TES (114 ± 2) and remained lower in CsX+LST (101 ± 1). These data suggest that: 1) endogenous androgens (TES) exert antihypertensive effects in male SD rats; and 2) these antihypertensive effects may involve TES‐induced reductions in renin‐ angiotensin system function. (State of Texas)
DNA origami supported precision measurements of biomolecular interactions and structure
Authors: Dietz Hendrik
Published: November 2023

Programmable self‐assembly with DNA origami allows creating custom‐shaped nanoscale objects. Through this capacity, DNA origami enables constructing custom instruments to perform precision measurements of molecular interactions and structure, with enhanced control over positioning, orientating and manipulating the molecules under study. In my presentation I will report about a series of experiments in which we exploited this capacity to dissect the weak stacking forces between individual basepairs (1) and the forces that act between pairs of nucleosomes (2).

In experiment (1), we directly measured at the single‐molecule level the forces and lifetimes of DNA base‐pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual‐beam optical tweezers with DNA origami components to allow positioning of blunt‐end DNA helices so that the weak stacking force could be isolated. Base‐pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free‐energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from −0.8 to −3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA‐based nanoscale devices according to user specifications.

In experiment (2), we performed a direct measurement of inter‐nucleosomal interactions by integrating two nucleosomes into a DNA origami‐based force spectrometer, which enabled sub‐nanometer resolution measurements of nucleosome‐nucleosome distance frequencies via single particle electron microscopy imaging. From the data we derived the Boltzmann‐weighted distance‐dependent energy landscape for nucleosome pair interactions. We find a shallow but long‐ranged (~6 nm) attractive nucleosome pair potential with a minimum of −1.6 kcal/mol close to direct‐contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Due to the weak and shallow pair potential, higher‐order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional co‐factors. Our results contribute to a more accurate description of chromatin, while our force spectrometer provides a powerful tool for the direct and high‐resolution study of molecular interactions using imaging techniques.

Support or Funding Information
Playing a tug of war with integrin and Notch receptors
Authors: Ha Taekjip
Published: November 2023

It is now widely appreciated that cancer cells and stem cells can change their cell fate (differentiation, metastasis, etc.) depending on their mechanical environment. Mechanical sensing is likely to be initiated by individual membrane receptor proteins that are in direct contact with the mechanical environment. In order to understand how molecular level mechanical events trigger a cellular response, we need to examine the forces applied across individual cellular proteins during mechanical signaling.

To define the single molecular forces required to activate signaling through a ligand‐receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor‐ligand tension. Using a range of tethers with tunable tension tolerances, we showed that cells apply a peak tension of about 40 picoNewtons (pN) to single integrin‐ligand bonds during initial adhesion. This tension value was consistent across different cell types. We then presented a mixture of weak and strong TGTs (12 pN and 54 pN nominal tension tolerance) to the cells in order to better mimic native cellular environments that are likely heterogeneous. Surprisingly, just one or two strong TGTs were sufficient to allow the cells to adhere as long as there is a high density of weak tethers. Therefore, mechanical tugging through one or two integrins is enough to convince the cell that the substrate is stiff enough for adhesion. Even more surprisingly, when we presented a mixture of two different weak TGTs (12 pN and 23 pN), cells adhered to the surface despite the fact that individually neither of the two tethers could support cell adhesion. Furthermore, we found that the 23 pN tether can exert its influence even at very high dilution, down to as few as two tethers per cell. This phenomenon of ‘ultrasensitivity in differential force sensing through integrin’ can be explained by a model where simultaneous rupture of weaker bonds exerts a transient high tension across a single integrin to activate that integrin.

Notch signaling, involved in development and tissue homeostasis, is activated at the cell‐cell interface through ligand‐receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. In our original TGT paper, we showed that Notch signaling is activated even for the weakest TGT (12 pN), suggesting that if Notch activation requires force, it must be less than 12 pN. Subsequently, the Blacklow lab show that the exposure of a Notch cleavage site required for its activation occurs at a tension of ~5 pN39, consistent with our estimate of a 12 pN upper limit. We recently developed LTGT (a low tension gauge tether) utilizing the low unbinding force of ~ 4 pN between single‐stranded (ss)DNA and E. coli ssDNA binding protein (SSB) and showed that Notch activation requires forces between 4 and 12 pN.
A 3D code in the human genome
Authors: Aiden Erez Lieberman
Published: November 2023

Stretched out from end‐to‐end, the human genome – a sequence of 3 billion chemical letters inscribed in a molecule called DNA – is over 2 meters long. Famously, short stretches of DNA fold into a double helix, which wind around histone proteins to form the 10nm fiber. But what about longer pieces? Does the genome's fold influence function? How does the information contained in such an ultra‐dense packing even remain accessible?

In this talk, I describe our work developing ‘Hi‐C’ (Lieberman‐Aiden et al., Science, 2009; Aiden, Science, 2011) and more recently ‘in‐situ Hi‐C’ (Rao & Huntley et al., Cell, 2014), which use proximity ligation to transform pairs of physically adjacent DNA loci into chimeric DNA sequences. Sequencing a library of such chimeras makes it possible to create genome‐wide maps of physical contacts between pairs of loci, revealing features of genome folding in 3D.

Next, I will describe recent work using in situ Hi‐C to construct haploid and diploid maps of nine cell types. The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1 kb resolution. We find that genomes are partitioned into contact domains (median length, 185 kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify □10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind the protein CTCF. The CTCF motifs at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs “facing” one another.

Next, I will discuss the biophysical mechanism that underlies chromatin looping. Specifically, our data is consistent with the formation of loops by extrusion (Sanborn & Rao et al., PNAS, 2015). In fact, in many cases, the local structure of Hi‐C maps may be predicted in silico based on patterns of CTCF binding and an extrusion‐based model.

Finally, I will show that by modifying CTCF motifs using CRISPR, we can reliably add, move, and delete loops and domains. Thus, it possible not only to “read” the genome's 3D architecture, but also to write it.
N‐acetylcysteine Effects on Renal Ischemic Injury Associated to Chronic High Sodium Intake
Authors: Pereira Rafael Canavel , Romão Carolina , Gracioli Fabiana , Shimizu Maria , Oliveira Ivone , Furukawa Luzia
Published: November 2023

Introduction

Acute episodes of severe renal ischemia result in acute kidney injury. These episodes are followed by a characteristic recovery and repair response, which renal function appear completely restored within one mouth. However, the chronic effects of such an injury associated to high sodium intake and N‐acetylcysteine (NAC) have not been well investigated.

Aim

To evaluate the NAC effects associated with high sodium diet intake on rats submitted to bilateral ischemia and reperfusion (IR).

Material and Methods

Eight weeks old male Wistar rats were fed normal sodium diet (1,3% NaCl, 25,0% protein – NSD) or high sodium diet (8% NaCl, 25,0% protein ‐ HSD). Also, at same time, both diet groups received or not NAC (600 mg/L) in the drink water. The animals were submitted or not (SHAM) to bilateral IR for 45 minutes at 10 weeks old. At 22 weeks old, the animals had blood pressure, water intake, urine volume, right kidney and heart masses, plasma renin activity (PRA); serum uric acid, total antioxidant status and thiobarbituric acid reactive species (TBARS) and renal interstitial fibrosis measured.

Conclusion

The chronic NAC treatment associated to high sodium intake in renal injury, prevented the increase of blood pressure, kidney and heart masses. Also, it decreased lipid peroxidation and renal interstitial fibrosis, showing that it has beneficial effects in rats fed with high sodium diet.

Support or Funding Information

CAPES and FAPESP

Results

Variables from male Wistar rats fed normal (NSD) or high (HSD) sodium diet submitted or not (SHAM) to ischemia and reperfusion (IR) and received or not N‐acetylcysteine (NAC) at 22 weeks old.

SHAM
IR
IR+NAC

Variable
NSDn=8
HSDn=7
NSDn=9
HSDn=7
NSDn=10
HSDn=10

Blood‐pressure (mmHg)
147,5±11,2
179,0±10,0*
144,0±12,3
177,0±11,1#
157,8±11,0
170,8±9,8

Water‐intake (mL/24h)
30,0±18,5
87,1±19,8*
30,3±9,0
93,0±45,0#
31,1±16,7
118,6±46,0&

Urine volume (mL/24h)
13,5±3,7
68,9±17,75*
25,2±13,3*
96,0±30,6#
16,0±7,7
71,5±42,3&

Right kidney mass (g/femur)
0,5±0,05
0,6±0,1
0,5±0,2
0,8±0,
0,5±0,2
0,7±0,2

Heart mass (g/femur)
0,4±0,04
0,4±0,1
0,4±0,03
0,69±0,2
0.4±0,04
0,4±0,1

PRA (ngANG/mL/hr)
14,4±7,7
0,09±0,01*
5,6±2,9
0,06±0,1#
9,4±2,0
0,9±1,1&

Uric acid (mg/dL)
1,4±0,2
1,0±0,1#
1,2±0,3
1,8±0,5$
1,5±0,2
2,25±1,4

Total antioxidant status (mmol/L)
1,3±0,1
1,3±0,1
1,3±0,1
1,4±0,03
1,2±0,1
1,3±0,1

TBARS (mmol/L)
2,7±0,65
3,2±0,4
3,7±0,8
3,2±0,7
0,9±0,5*#
0,6±0,4&$

Renal interstitial fibroses (%)
0,6±0,2
0,5±0,2
1,8±0,7*
2,3±1,8#
1,5±0,6
1,5±0,5

Data are expressed as mean ± SD.

p<0.05 vs. Sham‐NSD;

p<0,05 vs. IR‐NSD;

p<0,05 vs. IR+NAC ‐ NSD;

p<0,05 vs. Sham‐HSD.
Selective cannabinoid 2 receptor stimulation reduces tubular epithelial cell injury following renal ischemia‐reperfusion injury
Authors: Pressly Jeffrey , Mustafa Suni , Moore Bob M , Park Frank
Published: November 2023

Acute kidney injury (AKI) is an increasingly common clinical problem and has been studied for its association with chronic kidney disease progression. Currently, therapies to treat AKI are not available, so further insight into new therapeutic targets are essential in the treatment of this disease. In this study, a novel cannabinoid receptor type 2 (CB2) agonist, SMM‐295, was designed and synthesized for testing as a potential therapeutic to treat acute kidney injury. Molecular docking of SMM‐295 into a CB2 active state model showed that SMM‐295 interacts well with key amino acids to stabilize the active state. In cell culture, SMM‐295 was shown to exhibit ~100‐fold selectivity for CB2‐ versus CB1‐expressing 293 cells. Moreover, the CLogP values were consistent with a more aqueous solubility for SMM‐295 (CLogP=4.96) compared with a commonly used CB2 agonist, JWH‐133 (CLogP=5.72). Subsequent in vivo testing of the CB2 agonist was performed using a mouse model of AKI. Bilateral ischemia‐reperfusion injury was performed in mice, which is a common model to mimic human AKI, and SMM‐295 (6 mg/kg) was immediately administered IP to the mice upon reperfusion of the kidneys following the ischemic episode and every 24 hours until the end of the experiment. Histological damage assessment after 48 hours following reperfusion demonstrated around a 50% reduction (P<0.05) in tubular damage in the presence of SMM‐295 (n=5) compared to vehicle‐treated mice (n=4). This was consistent with a significant reduction (P<0.05) in both plasma creatinine and NGAL by approximately 60% with SMM‐295 administration (n=5) versus vehicle alone (n=7). SMM‐295 also showed better therapeutic results compared to the commercially available CB2 agonist JWH‐133 which also led to a drop in both serum creatinine levels and tubular injury, but did not reach statistical significance (P>0.05). Conversely, CB2 inverse agonist, SMM‐189, showed significant increases (P<0.05) in both serum creatinine (152%; n=4) at 24 hours and tubular injury (47%; n=3) at 48 hours following IRI. SMM‐295 treated mice (n=5) also showed significant reduction (32%; P<0.05) in PCNA‐positive cells in the outer medulla compared to the vehicle treated group (n=4) 48 hours following IRI. Western blot analysis of kidney lysates in SMM‐295 treated mice (n=5) at 48 hours following IRI demonstrate increased levels of cell survival proteins including total and active forms of Akt as well as Bcl‐2 compared to vehicle treated mice (n=3). These data would suggest that selective CB2 activation can play a beneficial role in protecting tubular epithelial cells following transient injury stimulus application to the kidneys, and may open the door to new small molecule activators of the cannabinoid systems for renal‐directed therapies.

Support or Funding Information

This study was partially funded by NIH RO1 DK090123 (F.P.) and institutional funds (F.P.).
Consumption of Gamma‐oryzanol prevents kidney disease and maintains redox balance in a model of obesity induced by high sugar‐fat diet
Authors: Francisqueti Fabiane , Mangegale Maria Clara , Garcia Jessica , Ferron Artur , Moreto Fernando , Silva Vanessa , Ferreira Ana Lucia , Correa Camila
Published: November 2023

Aim

to investigate the effect of Gamma‐oryzanol (yOz) on renal disease, redox state and agressor factors of animals submitted to high sugar‐fat diet (HSF).

Methods

32 wistar male rats were divided into 4 groups (n=8 animals/group): control (C), control + yOZ (CyOZ), HSF and HSF + yOZ (HSFyOZ). C and CyOZ received standard diet (60% complex carbohydrate‐dextrin, 4% fat‐soybean oil); HSF and HSFyOz received HSF diet (53% simple carbohydrate‐sucrose and fructose,16% fat‐lard) + 25% of sucrose in drinking water. yOz was added to the feed at a dose of 0.5%. During 30 weeks, body weight was measured weekly and feed intake, daily. After the period, it was analysed plasm glucose (mg/dL), HOMA‐IR and systolic blood pressure‐SBP(mmHg). Obesity was characterized by adiposity index (% of body fat) and renal tissue was used to analyse antioxidant protection (%protection/g protein). Renal disease was analyzed by glomerular filtration rate (GFR‐ml/min)) and proteinuria (protein/creatinin ratio).

Results

HSF induced renal disease and risk factors, such as obesity, increased glucose, HOMA‐IR, SBP and renal oxidative stress. yOz was efficient to reduce adipogenesis, improves renal antioxidant protection and prevent renal disease. Table shows the results.

Conclusion

Gamma‐oryzanol prevented renal disease and oxidative stress. Therefore, the compound can be a strategy to prevent these complications related to obesity.

Support or Funding Information

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP‐2015/10626‐0)

Nutritional and metabolic profile and renal function

C
CyOz
HSF
HSFyOz

food intake (g/day)
20 ± 3
19 ± 3
8 ± 1#
8 ± 2$

caloric intake (kcal/day)
71 ± 10
68 ± 11
71 ± 6
61 ± 22

final body weight (g)
493 ± 51
497 ± 54
583 ± 76#
494 ± 75&

adiposity index (%)
5 ± 1
4 ± 1
9 ± 2#
6 ± 2&,$

plasm glucose (mg/dl)
71 ± 20
78 ± 21
106 ± 18#
89 ± 7

HOMA‐IR
6 ± 3
7 ± 4
22 ± 22#
26 ± 17$

SBP (mmHg)
121 ± 6
127 ± 9
140 ± 12#
145 ± 13$

GFR (ml/min)
2.5 ± 1
3 ± 0*
1.6± 1#
2.3 ± 1&,$

protein/creatinin ratio
0.0022 ± 0.005
0.0027 ± 0.002
0.0031 ± 0.005#
0.0025 ± 0.002&

renal antioxidant protection (%/g protein)
51 ± 7
52 ± 5
41 ± 6#
54 ± 18&

compares C to CyOZ

compares C to HSF

compares HSF to HSFyOZ

compares CyOz to HSFyOz
Pretreatment with estrogen protects HK‐2 cells from cisplatin‐induced cytotoxicity
Authors: Chen Yu‐Tong , Fu Yaw‐Syan , Shih Hou‐Yu , Huang Bin , Lu Tzongshi , Wu Chia‐Chun
Published: November 2023

Introduction and hypothesis

Estrogen is a major sex hormone in females, and exerts multiple physiological functions. Cisplatin is a platinum‐containing anti‐cancer drug that is widely used as a chemotherapeutic agent. Nephrotoxicity of cisplatin is a dose‐limiting side effect and that can cause significant morbidity. HK‐2 cell line is an immortalized proximal tubule epithelial cell line from normal male adult human kidneys. In this study, we investigated the protective roles of estrogen on HK‐2 cells using a cisplatin‐induced nephrotoxicity model.

Methods

HK‐2 Cells were treated at time 0 with: 1) 17β‐estradiol (0~20 nM); 2) estrogen receptor agonists; 3) estrogen receptor antagonists for 12 hours respectively. Then, the all groups of cells were treated with cisplatin (0–15 mg/ml) for 24 hours. The cell survival rate, proliferation, morphology were analyzed by ELISA and histology studies and, cell apoptosis/necrosis were analyzed by Annexin V/PI staining using flow cytometry. Estrogen receptors and apoptosis related proteins expressions were further confirmed by Western blotting.

Results

Our data indicates that HK‐2 cells express all 3 types of estrogen receptors. HK‐2 cells pretreated with 10 nM 17β‐estradiol have higher cell survival rate (87%) in comparison with untreated cells (60%). And, both Annexin V positive apoptotic cells and caspase 3 expressions are significant lower in 17β‐estradiol pretreated HK‐2 cells. All 3 types of estrogen receptors exert the protective effects in cisplatin‐induced cytotoxicity, and the G protein‐coupled estrogen receptor (GPER) is the most effective estrogen receptor. In addition, GPER antagonist also significantly accentuates cytotoxicity of cisplatin treated HK‐2 cells.

Conclusions

Our data suggested that estrogen pretreatment could protect HK‐2 cells from cisplatin induced cytotoxicity, and this protective effect occurs mainly through the GPER pathway. It may offer a novel therapeutic strategy for cisplatin‐induced nephrotoxicity. Further investigation into the molecular mechanisms and clinical applications are needed.

Support or Funding Information

KMU‐TP104E34
Docosahexaenoic acid attenuates renal injury and inflammation in LPS‐induced acute kidney injury via increased resolvin D2
Authors: Katary Mohamed , Elsherbini Nehal M. , Ibrahim Ahmed S. , Al‐Shabrawey Mohamed , Elmarakby Ahmed A.
Published: November 2023

The polyunsaturated omega‐3 fatty acid, docosahexaenoic acid (DHA), has a promising role in preventing acute kidney injury (AKI); however; DHA reno‐protective mechanism remains to be elucidated. Our aim is to explore how DHA attenuates lipopolysaccharide (LPS)‐induced AKI. Four groups of wild type C57BL/6 (WT) mice were used in the current study (n=4); control, LPS injected (4 mg/kg i.p), LPS treated with the 12/15‐lipoxygenase (12/15‐LO) inhibitor baicalein (20 mg/kg i.p for 6 days) and LPS treated with DHA (50 mg/kg i.p for 6 days). LPS injection in WT mice showed a significant elevation in markers of renal injury compared to WT control mice (albuminuria was 112±4 vs. 8±1 mg/day and podocalyxin was 3.5±0.6 vs. 0.6±0.1 mg/day in LPS injected mice vs. WT control, P< 0.05). The elevation in renal injury markers were also associated with increases in oxidative stress and inflammation as evident by increased urinary thiobarbituric acid reactive substance (TBARs) and monocyte chemoattractant protein‐1 (MCP‐1), respectively, in LPS injected mice. Treatment of LPS injected mice with either DHA or baicalein significantly reduced markers of renal injury and inflammation. Although DHA or baicalein treatment significantly reduced the elevation in renal intercellular adhesion molecule 1 (ICAM‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression levels in LPS injected mice, DHA was superior over baicalein in reducing Interleukin‐6 (IL‐6) and IL‐1β and in increasing the anti‐inflammatory IL‐10 in LPS injected mice. DHA reduced renal tubular necrosis and vaculated cells and lowered renal expression of CD45, a marker of leucocyte adhesion, in LPS injected mice. DHA also restored the decrease in plasma resolvin D2 in LPS injected mice. Finally, injection of resolvin D2 decreased markers of renal injury, oxidative stress and inflammation and reduced renal tubular degeneration in LPS‐induced AKI. In conclusion, our data suggest that DHA is a superior treatment over 12/15‐LO inhibitor in preventing LPS‐induced AKI, at least in part, via increased resolvin D2.

Support or Funding Information

Augusta University intramural Funding
Mitochondrial Function in Long‐term Consequences of Renal Ischemia‐Reperfusion Injury
Authors: Hepokoski Mark , Bullen Alexander , Li Ying , Hall Elanore , Pham Hai , Singh Prabhleen
Published: November 2023

Background

Renal tissue hypoxia is a common link between AKI and CKD. The kidney has high mitochondrial oxidative metabolism, and is exquisitely sensitive to hypoxic injury. Mitochondria play a central role in cell injury and their dysfunction has been implicated in early AKI, but limited information is available regarding the long‐term consequences of AKI.

Methods

We performed 10min and 15min of bilateral renal artery clamping (IR) in mice and then serial GFR measurement by FITC inulin kinetics at days 1, 3, 7, 14 days, 4 weeks and 14 weeks after ischemia and protein expression of mitochondrial oxidative phosphorylation complexes (Ox phos), fission, fusion and AMPK at the same time points.

Results

Serial GFRs

In the 10min group, GFR was significantly lower at days 1 and 3 but improved after that to levels similar in shams. In the 15min group, GFR was dramatically reduced with partial recovery over 4 and 14 weeks.

Ox Phos expression

Significant reduction in expression for both 10 and 15 min IR at 1–2 weeks for most complexes, with recovery in expression at 4 weeks for 10min, but persistent reduction for 15min IR. Mitochondrial Proteins at 4weeks, 15min IR showed significant reduction in Mfn2 and increase in Fis1 expression. Total AMPK expression was increased at day1, but no increase in pAMPK was seen in the 15min IR kidney. In 10min IR, pAMPK was increased at 24 hrs and persisted at 4 weeks.

Conclusions

Our findings show duration of IR corresponding with severity of injury and impaired recovery in GFR, and mitochondrial dysfunction with decreased Ox phos proteins and altered dynamics with impaired AMPK activation in IR kidneys, corresponding to severity of injury, but in some instances despite functional recovery. These may be important in transition of AKI to CKD. Additional studies to compare these two models when manipulated with maneuvers to worsen or improve recovery from IR are ongoing.

Support or Funding Information

NIDDK O'Brien Center for AKI Research P30DK079337.

Veterans Affairs (VA) Merit Award BX002175 (P. Singh)

NIDDK R03 DK101841 (P.Singh)

NIDDK R01 DK107852 (P.Singh)
Automated Glomerular Localization in Bright‐field, Trichrome‐stained Rat Kidneys
Authors: Bukowy John D , Evans Louise C , Dayton Alex , Cowley Allen W
Published: November 2023

Histological examination of fixed renal tissue is widely used to assess kidney morphology and the progression of renal disease. Common renal metrics include glomerular number, cortical area/volume, protein cast area, and glomerular damage. Evaluation of these metrics is a time consuming process requiring a trained observer and manual recording/counting. The purpose of this study is to implement an automated pipeline to localize glomeruli in trichrome‐stained, bright‐field, whole renal slices resulting in minimization of human‐hours required to process experimental data groups, elimination of human observer variability, and expansion of glomerular‐specific renal metrics.

The dataset for this study consisted of a population of histologically prepared rat kidney slices with varying degrees of protein casts and glomerular damage. All kidney slices were prepared from formalin fixed and paraffin embedded kidneys cut in 4 μm slices and mounted to glass slides. Slices were then stained with trichrome and imaged in bright‐field on a Nikon Ni‐E automated microscope with a resolution of 0.68 μm/pixel. Feature based thresholding and selection was performed over images of whole renal slices to nominate glomerular candidates which were subsequently cropped into individual images. From 14 separate kidney slices, initial filtering produced over 6,500 images (observations) containing >1,400 glomeruli. From this dataset, manual separation of glomerular positive images was performed creating a training dataset for supervised learning methods to classify glomerular candidates. The trained classifier was tested on a naïve dataset of 619 observations summarized by:

Sensitivity ~ 98% | Ratio of the number of images with glomeruli correctly identified by algorithm to number identified by human observer.

Specificity ~ 94% | Ratio of the number of images without glomeruli correctly identified by algorithm to number detected by human observer.

Precision ~ 79% | Ratio of the number of images with glomeruli correctly identified by algorithm to total number of images with glomeruli identified by the algorithm.

False Discovery Rate ~ 21% | Ratio of the number of images without glomeruli incorrectly identified by algorithm to number of glomeruli detected by algorithm.

Successful localization of glomeruli provides a framework for future workflows targeted to reduce processing time including semi‐automated scoring of identified glomeruli. Given the widespread usage of trichrome staining in renal morphology/pathology, this technique has the potential to greatly reduce the time and effort spent on renal dataset processing.

Support or Funding Information

American Heart Association | 16PRE29700006 ‐‐ National Institutes of Health | HL‐116264
Diminished TRPV4 activity contributes to compromised [Ca2+]i homeostasis in human ADPKD cells
Authors: Tomilin Viktor N , Zaika Oleh , Mamenko Mykola , Reif Gail , Wallace Darren , Pochynyuk Oleh
Published: November 2023

Polycystic kidney disease (PKD) is a devastating clinically relevant pathology causing graduate decline in kidney function due to development and growth of cysts filled with fluid. No effective pharmacological treatments exist for PKD patients. At the cellular level, defective flow‐mediated [Ca2+]i responses and disrupted [Ca2+]i homeostasis have been repeatedly associated with the development of PKD. Our previous work in rodents demonstrated that the activity of the Ca2+‐permeable TRPV4 channel is imperative for flow‐mediated [Ca2+]i responses in the distal renal tubule. Moreover, TRPV4 function is dramatically decreased in isolated cyst monolayers and systemic stimulation of TRPV4 interferes with PKD progression in PCK 453 rats, an autosomal recessive PKD model. To probe translational implication of this mechanism, we used primary cultured normal human kidney (NHK) and autosomal dominant PKD (ADPKD) cells isolated from patients and determined TRPV4 function and its role in [Ca2+]i signaling. As expected, ADPKD cells failed to respond to elevated flow and had significantly lower basal [Ca2+]i levels of 43±1 nM, when compared to NHK cells (91±1 nM). Application of a selective TRPV4 activator, GSK1016790A (30 nM) elicited more than two times higher [Ca2+]i response in NHK than in ADPKD cells. GSK1016790A‐mediated responses were precluded upon pretreatment with a specific TRPV4 inhibitor, HC‐067047 (4 μM) or a Ca2+‐free media, suggesting a direct TRPV4‐dependent Ca2+ influx. While total TRPV4 levels were comparable in NHK and ADPKD cells, we detected a marked decrease in TRPV4 glycosylation. Consistently, treatment with tunicomycin for 24 hours to prevent glycosylation significantly inhibited TRPV4 activity pointing to a mechanism of reduced TRPV4 function in ADPKD cells. Finally, acute application of TRPV4 antagonist, HC‐067047 significantly reduced basal [Ca2+]i levels in NHK cells but had no measurable effect in ADPKD cells. Overall, we demonstrate that similarly to studies in rodents, TRPV4 function and glycosylation are greatly diminished in primary cultured human ADPKD cells and this is at least partially responsible for their distorted [Ca2+]i signaling. We speculate that TRPV4 stimulation might be beneficial in restoring [Ca2+]i homeostasis in cyst cells thereby counteracting ADPKD progression in humans.

Support or Funding Information

This research was supported by NIH‐NIDDK DK095029 (to O. Pochynyuk), AHA‐15SDG25550150 (to M. Mamenko)
Chitooligosaccharide inhibits cyst formation and enlargement through calcium restoration and AMPK activation
Authors: Tuangpholkrung Phattharaphon , Muanprasat Chatchai , Chatsudthipong Varanuj
Published: November 2023

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disease. It is developed from mutation in renal Pkd1or/and Pkd2 gene leading to decreased intracellular calcium and consequently cysts formation. These renal cysts replace normal parenchyma cells disrupting its function that could lead to end‐stage renal failure. Currently, no effective therapy is available for PKD treatment. Recently, activation of AMP‐activated protein kinase (AMPK) by Metformin was found to inhibit cell proliferation and also reduce fluid secretion via CFTR in Madin‐Darby Canine Kidney (MDCK) cell. Interestingly, our group has shown that chitooligosaccharide or COS, the derivative of chitosan, could activate AMPK via the mechanism involved an elevation of intracellular calcium in colon cancer cell. Therefore, we hypothesized that COS‐activated AMPK may inhibit cyst formation and growth in MDCK cyst model. The results of toxicity test indicated that COS at the concentrations of 50, 100, 200 and 500 μg/ml produced no toxic effect to cells. In contrast, it was found that the treatments with COS at concentrations of 100, 200 and 500 μg/ml markedly decreased both cyst formation and cyst growth. Minimum effective dose of COS (100 μg/ml) was selected to study for its underlying mechanisms. Western blot analysis showed that COS at concentration of 100 μg/ml could activate AMPK and its downstream protein, acetyl Co‐A carboxylase (ACC). COS also induced an elevation in intracellular calcium. These data indicated that COS was able to inhibit cyst formation and retard cyst enlargement, in part, by the restoration of intracellular calcium and activation of AMPK.

Support or Funding Information

This work was supported by Development and Promotion of Science and Technology Talents Project (DPST), Institute for the Promotion of Teaching Science and Technology (to PT), and Faculty of Science, Mahidol University, Thailand.