bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxic Regulation of Vascular Proteasomal Degradation of MLCK
Authors: Adeoye Olayemi Olufikayo , Sorensen Dane William , Williams James , Pearce William
Published: November 2023

Chronic exposure of in‐utero fetuses to severe hypoxia induces maladaptive changes in vascular structure and function, and elevates postnatal susceptibility to metabolic, endocrine, cardiovascular diseases. Correspondingly, multiple studies have demonstrated that chronic hypoxia depresses vascular contractility through mechanisms that remain to be fully elucidated. Our recent studies have shown that in fetal arteries chronic hypoxia decreases the abundance and activity of myosin light chain kinase, which is the rate‐limiting enzyme for force generation in smooth muscle cells. Here we hypothesize that chronic hypoxia decreases vascular contractility via age dependent increases in proteasomal degradation of MLCK protein. To test this hypothesis we used a well established chronic hypoxia model involving pregnant ewes maintained at sea level or at altitude (3280m) during the last 110 days of gestation. In vitro contractility experiments revealed that chronic hypoxia decreased maximum myogenic stress by 56%. Chronic hypoxia also significantly decreased MLCK abundance, and moreso in fetal (90%) than adult (47%) arteries. Conversely, qPCR measurements of MLCK mRNA revealed similar abundances in adult and fetal arteries, suggesting that increased loss of MLCK was not due to inhibition of transcription. To more directly elucidate mechanisms of MLCK degradation, the effects of MG132 (a potent inhibitor of proteasomal degradation) were quantified organ cultured fetal and adult arteries. Rates of MLCK degradation averaged 0.83 ± 0.21 in fetal and 0.99 ± 0.38 in adult normoxic arteries. With chronic hypoxia, degradation rates increased by 53% and 61% in fetal and adult arteries respectively. Together these data suggest that depressed contractile activity during hypoxic acclimatization occurs via hypoxic promotion of proteasomal degradation of MLCK slightly more in adult than fetal arteries.

Support or Funding Information

These studies were supported by an NIH Program Project grant from NICHD (HD31266,) and an NIH R01 grant from NINDS (NS076945).
Passive Heat Therapy as a Novel Approach for Inducing Angiogenesis in Humans: Roles of Nitric Oxide
Authors: Brunt Vienna E , Needham Karen Wiedenfeld , Comrada Lindan N , Francisco Michael A , Minson Christopher T
Published: November 2023

Chronic passive heat therapy is a novel health intervention which may have widespread cardiovascular benefits. Studies in animals suggest chronic heat exposure promotes angiogenesis via NO, independently of VEGF. We sought to determine whether the same occurs in humans using an endothelial tubule formation assay to quantify serum angiogenic balance.

METHODS

Serum was collected from ten young (23±2 years), healthy human subjects before and after 8 weeks of passive heat therapy (36 sessions of hot water immersion sufficient to maintain rectal temperature ≥38.5°C for 60min per session; post‐heat therapy serum was collected at least 36h after the last session). All analyses were performed in duplicate using human sera collected pre and post heat therapy. To assess serum angiogenic balance, human umbilical vein endothelial cells (HUVECs) were plated onto serum‐free Matrigel, exposed to human sera, and incubated for 10h, after which total tubule length per frame was determined by two blinded investigators. To investigate whether potential improvements in serum angiogenic balance following heat therapy were NOS‐dependent, angiogenesis experiments were repeated in which HUVECs were additionally treated with 1mM L‐NNA, a competitive inhibitor of NOS. In separate experiments, HUVECs were exposed to human sera for 24h and eNOS protein was determined using Western blot, normalized to vinculin loading control. Lastly, serum VEGF concentration was determined by ELISA.

RESULTS

Serum angiogenic balance was improved following 8 weeks of heat therapy (total tubule length: 71.8±1.0 to 74.3±1.1mm, p=0.03) and this improvement was prevented by L‐NNA (73.1±1.7 to 73.9±1.1, p=0.56). eNOS protein expression increased by 18.6±3.4% (p=0.05) in endothelial cells exposed to human sera following heat therapy relative to pre‐heat therapy. Heat therapy decreased serum VEGF concentration (370±77 to 331±69 pg/ml, p=0.05).

CONCLUSIONS

Chronic passive heat therapy in humans upregulates circulating factor(s) which promote angiogenesis in a NOS‐dependent and VEGF‐independent manner, possibly through increasing eNOS expression. These results are consistent with those reported in animal studies and suggest chronic passive heat therapy could be a viable treatment option for improving vascularization in at‐risk populations.

Support or Funding Information

Supported by AHA Grant #14PRE20380300 and the Eugene and Clarissa Evonuk Memorial Foundation
DPPI Deficiency Enhances Both Angiogenesis and Arteriogenesis and Improves Cardiac Function after Myocardial Infarction
Authors: Kolpakov Mikhail A , Hooshdaran Bahman , Guo Xinji , Wang Tao , Rafiq Khadija , Vlasenko Liudmila , Qi Zhao , Garikipati Venkata NS , Kishore Raj , Houser Steven R , Sabri Abdelkarim
Published: November 2023

Study objective

Myocardial infarction (MI) induces both hypoxia and inflammation that trigger angiogenesis. Numerous proangiogenic and proarteriogenic factors are released during the inflammatory reaction post‐MI. However, the overall effect of the various factors of the inflammatory component depends on the local environment. This study examined the potential role of inflammatory serine protease (ISP) inhibition on angiogenesis after MI.

Methods and results

We subjected wild‐type (WT) and mice lacking Dipeptidyl peptidase I (DPPI), a lysosomal enzyme involved in the cleavage and activation of major ISPs, to MI up to 4 weeks and assessed the role of DPPI gene deletion on angiogenesis and cardiac remodeling and function.

DPPI deficient mice show markedly reduced activity of neutrophil‐ and mast cell‐derived serine proteases after MI compared to WT mice, along with a reduction in infarct size and improved cardiac function. DPPI deficiency also increased the number of capillaries and mature vessels in infarcted hearts by upregulating the expression of angiogenic cytokines such as vascular endothelial growth factor (VEGF) and angiopoietin‐1. Investigation of the mechanisms involved show reduced levels of soluble VEGF receptor 1 (sVEGFR1) accumulation in DPPI KO infarct together with increased phosphorylation of VEGFR2. The negative role of inflammatory serine proteases on angiogenesis was further demonstrated in vitro in tube formation assay. Treatment of human umbilical vein endothelial cells with the neutrophil‐derived serine protease cathepsin G (CG) led to up‐regulation of sVEGFR1 and its interaction with VEGFA, decreased VEGFR2 signaling and reduced the number and length of endothelial cell tubes. In contrast, preincubation of endothelial cells with neutralizing anti‐sVEGFR1 antibodies attenuated endothelial cell tube disorganization, improved VEGFR2 signaling and preserved new vessels formation.

Conclusions

Our findings reveal the role of ISPs as key components of a molecular mechanism that negatively modulates angiogenesis through regulation of the relative levels of sVEGFR1 in endothelial cells. Our study also point to DPPI inhibition as a potential therapeutic target for stimulation of angiogenesis and the maintenance of cardiac function post‐MI.

Support or Funding Information

This work was supported by the National Institute of Health (HL088626 and HL076799) and American Heart Association (15SDG22650004 and 14PRE20380518).
Angiotensin‐(1–7) Requires both Mas1 and AT1 Receptors to Restore Angiogenesis
Authors: Stodola Timothy , Exner Eric , Hoffmann Brian , Didier Daniela , Greene Andrew
Published: November 2023

The Renin‐Angiotensin System plays an important role in many physiological processes such as sodium homeostatis, regulating vascular tone and angiogenesis. The octapeptide Angiotensin II (AngII) acting through the AT1 receptor has long been known to promote vasoconstriction, fibrosis, cell proliferation and skeletal muscle angiogenesis in response to exercise or electrical stimulation. A second vasoactive peptide of the Renin‐Angiotensin System, Angiotensin‐1–7 (Ang‐(1–7)), acts through the Mas1 receptor and is generally thought to have many physiological actions that are antagonistic to the actions of the AngII‐AT1 axis, such as vasodilation, anti‐fibrotic and anti‐proliferative effects. We hypothesized Ang‐(1–7) acting through the Mas1 receptor could restore skeletal muscle angiogenesis in response to electrical stimulation. Little is known about the signaling events initiated by Mas1 receptor activation by Ang‐(1–7) relative to AngII‐AT1 activity. Here we show in Sprague Dawley rats with Renin suppressed through high dietary salt intake, which suppresses angiogenesis, that infused Ang‐(1–7) acting through the Mas1 receptor restores angiogenesis in vivo in skeletal muscle in response to electrical stimulation and in vitro in a tube formation assay using rat microvascular endothelial cells, in both cases independent of AngII. Using advanced proteomic methodology to characterize the components of Mas1 receptor signaling we identified signaling pathways that may play a role in the observed angiogenic response, such as nascent protein synthesis, exocytosis, cellular reorganization and regulators such as NO, NF‐kappaB and ERK1/2 signaling. Inhibition of ERK1/2 signaling inhibited tube formation by endothelial cells stimulated by AngII or Ang‐(1–7). We further assessed angiogenesis in response to electrical stimulation promoted by Ang‐(1–7) in SS‐Mas1em1Mcwi (Mas1 mutant knockout) and SS‐Agtr1aem5Mcwi (AT1 mutant knock out) strains, and compared to wild‐type SS/Mcwi. We found reduced angiogenesis to electrical stimulation after Ang‐(1–7) or orally active Ang‐(1–7) analog AVE0991 treatment in both the Mas1 mutant and the AT1 mutant knock outs relative to the SS/Mcwi. The RAS is a complex physiological system that can affect a wide range of processes in the cardiovascular system. Even though the AT1 and Mas1 receptor axes largely have antagonistic functions, we have shown that capillary angiogenesis can both be restored by AngII‐AT1 or Ang‐(1–7)‐Mas1 activity, and the restoration depends on the presence of functioning Mas1 and AT1 receptors.

Support or Funding Information

NIH Heart, Lung and Blood Institute 082798
Hypoxia and Ischemia Regulate Placental Syndecan Expression and Shedding
Authors: George Eric M
Published: November 2023

Preeclampsia, a hypertensive disorder of gestation, affects ~5% of the population, and is a leading cause of fetal and maternal morbidity and mortality. While the origins of the disease are unclear, it is commonly accepted that placental ischemia/hypoxia is a central causative factor. In response, the placenta secretes soluble factors which cause the maternal manifestation of the disorder. Among the pathways believed to be important in this response is activation of innate immune mechanisms, such as inflammatory cytokines. It has been widely reported in sepsis that vascular inflammation induces remodeling and shedding of the vascular endothelial glycocalyx, in particular cleavage and shedding of members of the integral membrane syndecan family. It is now known that these shed fragments have biological activity and act as potent inducers of the innate immune and inflammatory response. What is less well known is the presence of a functional glycocalyx in the placenta. Here we hypothesize that hypoxia will induce changes in placental syndecan production and shedding in vitro, and that chronic placental ischemia will cause syndecan shedding from the placenta in vivo. To examine the effects of hypoxia on placental cells, we cultured BeWo cytotrophoblast cells in oxygen tensions mimicking healthy (8%) and ischemic (1%) placentas, and examined syndecan1–4 mRNA and soluble SDC1 in the media. In response to hypoxia, SDC1,2, and 4 mRNA increased significantly (14, 6.5, and 2.5 Fold, respectively, p<0.05 each), as assayed by TaqMan qRT‐PCR. SDC3, in contrast, exhibited no significant difference in expression. Soluble SDC1 in the media, meanwhile was significantly increased by ~50% compared to normoxic control as measured by ELISA (41 ± 4 vs 63 ± 4 pg/ml, p<0.05). To determine the effects of chronic placental hypoxia/ischemia on SDC, we utilized the reduced uterine perfusion pressure (RUPP) rodent model. RUPP rats exhibited significantly increased blood pressure on gestational day 19 when compared to their control counterparts (105 ± 4 vs 122 ± 6 mmHg, p<0.05). Interestingly, soluble circulating SDC1 in the maternal circulation was significantly increased in RUPP rats when compared to their control counterparts (11 ± 4 vs 99 ± 20 pg/ml, p<0.05). These data suggest that hypoxia/ischemia differentially regulate SDC isoforms in vitro. Furthermore it is possible that isoform specific or pan‐syndecan circulating levels of syndecans could serve as a biomarker for preeclampsia. The importance of syndecan fragments in the inflammatory cascade during preeclampsia remains to be determined, as do the mechanisms regulating syndecan production and cleavage in these tissues.

Support or Funding Information

This work was supported in part by P01HL51971, P20GM104357 (EMG), and R00HL116774 (EMG).
Angiogenic Imbalance and Hypertension during Pregnancy Results in Intrauterine Growth Restriction and Developmental Delays in Rodent Offspring
Authors: Bowles Teylor , Golden Leia , Spencer Shauna‐Kay , Owens Michelle Y , Wallace Kedra
Published: November 2023

Introduction

In the current study our objective was to determine if rat pups born to dams experiencing angiogenic imbalance and hypertension during pregnancy had intrauterine growth restriction (IUGR) and developmental delays. To test this objective we infused sFlt‐1 and sEndoglin into timed pregnant rats. This animal model creates pathological symptoms similar to what is seen in women with Hemolysis Elevated Liver enzyme Low Platelet (HELLP) syndrome and severe preeclampsia (sPreE). Additionally these pregnant rats development hypertension and have increased production of TNF‐alpha, IL‐6 and IL‐17 which is also experienced by women with HELLP and sPreE.

Methods

On gestational day (GD) 12, mini osmotic pumps infusing sFlt‐1 and sEndoglin (4.7 and 7 ug/kg respectively) were implanted into timed pregnant rats (n=4) to create HELLP or sPreE. Within 12hrs of delivery 4 pups from each litter of HELLP rats and normal pregnant (NP; n=4) rats were cross‐fostered with a set of lactating SHAM rats (n=4) so that the final litter size per SHAM rat was 16 pups. Rat pups underwent the standard Irwin battery of developmental milestones beginning on post‐natal day (PND) 2 – PND16.

Results

There was not a difference in the gestational day of delivery between the groups, but pups in the HELLP group did display IUGR compared to pups in the NP group (6.16 ± 0.10 vs. 6.69 ± 0.13g in NP; p=0.004). There was a significant interaction (p<0.0001) in surface righting with pups displaying the largest delay in this task at PND2; 35.06 ± 9sec in HELLP rats vs. 18.41 ± 3.73sec in NP rats. Significant interactions between the days of testing were seen in negative geotaxis (p<0.0001), cliff avoidance (p<0.0001) and open circle (p<0.0001). Rats in the HELLP group had a significant delay in achieving the forelimb grasp test compared to NP rats (p<0.0001), with up to 39% of the NP having achieved this task by PND5 compared to only 15% of the HELLP group. Pups born to dams with HELLP had a significant delay in eye opening compared to NP pups (p=0.0043).

Conclusions

These data support the hypothesis that IUGR is associated with developmental delay. The angiogenic imbalance during pregnancy in this model may serve as one mechanism whereby IUGR occurs in the absence of pre‐term delivery, thereby mediating the developmental delay in the resulting offspring

Support or Funding Information

This work was funded by an Institutional Development Award (IDeA) from the NIGMS under grant number P30GM103328.
Early life stress (ELS) Induces Increased Interleukin 1 beta in Renal Tubular Epithelial Cells in Adult Rats
Authors: Obi Ijeoma , De Miguel Carmen , Ho Dao , Loria Analia , Pollock Jennifer
Published: November 2023

ELS is an independent risk factor in the development of cardiovascular disease and hypertension. ELS is also associated with enhanced biomarkers of inflammation in adults. There is substantial evidence that inflammation contributes to the development of hypertension and/or hypertension‐induced renal injury. Utilizing a rat model of ELS, maternal separation (MatSep), we previously reported that ELS induces an exaggerated blood pressure response and increased renal vascular injury to pro‐hypertensive stimuli in male adult rats. We hypothesized that ELS induces a systemic and pro‐inflammatory state in adult male rats. MatSep involves separating rat pups from the dam for 3hrs/day from postnatal day 2 (PD2) to PD14. Normally‐reared littermate rats were used as controls. Immune cells were determined by FACS analysis, magnetic bead isolation, and immunohistochemical staining. Renal cytokine expression was assessed by ELISA and immunohistochemical staining. Circulating and renal mononuclear cells (PBMC) and T‐cell absolute numbers were similar between control and Matsep rats (control vs. MatSep; circulating PBMC: 41.1±6.9 vs. 49.3±2.5 x 106 cells/ml; renal PBMC: 52.9±9.2 vs. 56.5±8.3 x 106 cells/kidney; circulating T cells: 3.0±0.3 vs. 3.4±0.4 x 106 cells/ml; renal T cells: 7.3±1.2 vs. 6.7±1.2 x 106 cells/kidney; p < 0.05). In addition, both groups presented similar percentages of circulating macrophages (control vs. MatSep; n=4/group: 33.1±2.5 vs. 33.5±0.7%) and T‐lymphocyte subpopulations (TH cells: 29.9±.3 vs. 29.4±0.9%; TC cells: 15.8±0.9 vs. 13.5±0.6%; Treg cells: 4.2±0.2 vs. 4.9±0.2%); however, B cell numbers were significantly decreased in MatSep rats (23.7±1.2 vs. 20.1±0.7%; p<0.05). Renal expression of interferon gamma, interleukin 6, and interleukin 4 were similar in control and MatSep rats. Renal interleukin 1 beta (IL‐1β) was significantly increased in MatSep rats compared to control (control: 4.4±0.5 pg/mg protein, MatSep: 7.9±1.0 pg/mg protein; p<0.05). Immunohistolocalization showed increased IL‐1β immunoreactivity in MatSep rats, specifically in renal cortical tubular cells. Proximal tubules of MatSep rats showed increased IL‐1β positive staining on the apical membrane as compared to control rats. These findings indicate that MatSep induces increased renal expression of IL‐1β in cortical tubular cells without a change in numbers of renal mononuclear or T cells. Future studies will determine whether increased IL‐1β in MatSep adult rats is linked to the exaggerated blood pressure phenotype in response to a hypertensive stimuli.

Support or Funding Information

Funded by APS Porter Fellowship to IEO, T32DK007545 to CDM, K99/R00HL111354 to ASL, F32HL116145 to DHH, and P01HL69999 to JSP
Effect of a dietary and exercise intervention during pregnancy and lactation on white adipose tissue gene profiles and adiposity with maternal obesity
Authors: Symonds Michael , Bloor Ian , Galvez Fernandez , Domfeh Emmanuel , Maicas Blasco , Poston Lucilla , Taylor Paul
Published: November 2023

Background and aims

The recent surge in maternal obesity and its associated outcomes has led to an increase in intervention studies designed to attenuate the potential adverse effects on both the mother and offspring. Pregnancy and lactation are periods of great maternal metabolic flux and regulation of adipose tissue metabolism which is one potential determinant of the adverse outcome. This study focuses on the gene expression profile of the inguinal white adipose depot with maternal obesity and its response to an intervention designed to alleviate obesity.

Methods

Sprague Dawley dams (n=17) were randomized to control (C; n=6), obesity (Ob; n=5) or obesity intervention (ObDEx; dietary (low glycaemic index diet) and physical exercise (n=6)) groups. At late gestation (G20) all dams were euthanized and adipose tissues were weighed and then stored in RNA later. The study was then repeated but all dams were taken through to lactation day 14 before tissue collection. To analyze gene expression for those genes involved in energy sensing, inflammation, lipid handling and beiging, QPCR assays were applied and mRNA abundance was calculated using geNorm.

Results

Expression of genes involved in energy sensing (e.g. Akt, INS‐R1/2, LPL, mTOR), inflammation (e.g. TLR4, TNFα) and lipid handling (e.g. CD36, FATP4, LPL, SREBP) pathways were all significantly higher in pregnancy compared with lactation for controls, whereas beiging (e.g. UCP1) was unaffected. These differences were further increased in the presence of obesity, but attenuated with the intervention during pregnancy but not lactation. ObDex also reduced both fat mass (C: 5.4±0.8; Ob: 8.3±0.7; ObDEx; 4.4±0.3 g (P<0.01)) and serum leptin (C: 4.2±0.7; Ob: 11.9±1.7; ObDEx; 5.0±0.9 ng/ml (P<0.01)) in pregnancy whilst fetal weight was unaffected.

Conclusions

A combined dietary and exercise intervention improved the maternal metabolic gene profile within white fat and prevented excess adiposity. This effect appeared to be confined to pregnancy due to the pronounced metabolic adaptations within fat through lactation. The longer term consequences for the offspring are currently being examined.

Support or Funding Information

Acknowledgements: The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007–2013), project EarlyNutrition under grant agreement no. 289346.
INTRARENAL ANGIOTENSINOGEN PRODUCTION INDUCED BY CHRONIC ANGIOTENSIN II AND HIGH SALT INTAKE IS AUGMENTED IN TUMOR NECROSIS FACTOR‐ALPHA RECEPTOR TYPE 1 KNOCKOUT MICE
Authors: Mehaffey Eamonn P. , Castillo Alexander , Navar L. Gabriel , Majid Dewan S. A.
Published: November 2023

Chronic angiotensin II (AngII) treatment increases TNF‐α generation in the kidney and also enhances intrarenal angiotensinogen (AGT) formation. However, the link between TNF‐α increase and AGT formation in the kidney remains unclear as it has also been demonstrated in‐vitro that TNF‐α suppresses AGT production in cultured human proximal tubule cells. These findings may indicate differential activation of TNF‐alpha receptors type 1 (TNFR1) and type 2 (TNFR2) signaling pathways induced by chronic AngII infusion. We examined the hypothesis that chronic elevation in AngII levels coupled with high salt (HS) intake reduces TNFR1 activity but enhances TNFR2 activity that induces cellular responses leading to increased formation of intrarenal AGT. We assessed the renal and systemic responses to chronic infusions with AngII (25 ng/min; implanted minipump) with high salt (HS; 4% NaCl) diets for 4 weeks in mice lacking TNFR1 receptors (TNFR1KO; n=7) and in mice lacking TNFR2 (TNFR2KO; n=6) and compared these responses to those in wild‐type (WT; C57BL6 strain, n=6) mice. Systemic blood pressure (SBP) was measured by tail‐cuff plethysmography and 24‐hour urine collections were done using metabolic cages at baseline and at 4 week periods. The urinary excretion rate of AGT (uAGT) was measured as a reflection of intrarenal generation of AGT using ELISA. Urinary excretion rates of sodium (UNaV) and potassium (UKV) were determined by analyzing urinary concentration of these electrolytes using flame photometry. The increase in mean SBP in response to AngII + HS for 4 weeks was greater in TNFR1KO mice (77±2 to 115±3 mmHg; P<0.05) compared to WT mice (76±1 to 102±2 mmHg; P<0.05), while the change in TNFR2KO (78±2 to 99±5 mmHg; P<0.05) had no difference from WT. Interestingly, the increase in uAGT after 4 weeks treatment period was also greater in TNFR1KO mice (6±2 to 167±75 ng/24hr; P<0.05) compared to WT mice (6±3 to 46±16 ng/24hr; P<0.05), while the changes in TNFR2KO mice (8±7 to 65±44 ng/24hr; P=0.11) were not significantly different from the changes in WT mice. All three groups showed similar increases in UNaV (WT, 118±13 to 883±121 mM/24hr; TNFR1KO, 96±15 to 942±86 mM/24hr; TNFR2KO, 94±8 to 733±195 mM/24hr) while no group experienced a significant change in UKV. The results suggest that TNFR1 activity mitigates the hypertensive response to chronic AngII infusion with high salt intake, likely by attenuating the increase in intrarenal AGT formation.

Support or Funding Information

Grant support: National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337) and Tulane Bridge Fund (DSAM).
Deletion of Rag1 prevents high dietary protein‐induced exacerbation of hypertension and renal injury in Dahl SS rats
Authors: Abais‐Battad Justine M , Lund Hayley , Mattson David L
Published: November 2023

The role of immune mechanisms in the amplification of hypertension has long been appreciated. Previous studies in our laboratory demonstrated how the amount as well as source of dietary protein has a profound influence on the development of hypertension and the renal infiltration of immune cells, specifically T lymphocytes. The current study hypothesized that Dahl SS rats lacking T and B lymphocytes via mutation of recombination‐activating gene 1 (Rag1) will confer protection against hypertension and renal injury exacerbated by high dietary protein intake. Immediately after weaning, Dahl SS rats and Rag1 mutant rats were placed on modified AIN‐76A, isocaloric, 0.4% NaCl diets (Dyets Inc.) custom formulated to contain either low, normal, or high protein (6%, 18%, or 30%, respectively). At 9 weeks of age, rats were switched to a 4.0% NaCl diet containing the same amount of dietary protein, and maintained on this high‐salt diet for 3 weeks. Urine was collected during the low salt period, and after 7, 14, and 21 days of high salt. Blood pressure was measured via femoral arterial catheters which were implanted at 10 weeks of age, with blood pressure recording occurring between 11 and 12 weeks of age. In Dahl SS rats, there was a protein‐dependent elevation in blood pressure and urinary albumin excretion as the amount of dietary protein was increased. Dahl SS rats fed the high protein diet had the highest pressure and albumin excretion (159±3 mmHg and 140.8±16.0 mg/day, respectively) after 21 days on high salt compared to rats fed either normal protein (139±4 mmHg, 69.4±11.3 mg/day) or low protein (113±1 mmHg, 0.4±0.1 mg/day) diets. Compared to SS rats, Rag1 mutant rats on the high protein diet were protected from salt‐induced hypertension and renal damage (143±6 mmHg, 66.2±10.8 mg/day). Flow cytometric analysis demonstrated a corresponding increase in total leukocyte infiltration (CD45+) in the kidneys of both Dahl SS rats and Rag1 mutants as dietary protein was increased. As expected, Rag1 mutants on the high protein diet had fewer CD3+ T lymphocytes and CD45R+ B lymphocytes infiltrating the kidney (2.10 x 105 CD3+ cells/kidney, 0.74 x 105 CD45R+ cells/kidney) versus their Dahl SS counterparts (7.97 x 105 CD3+ cells/kidney, 4.99 x 105 CD45R+ cells/kidney). There was, however, no change in the infiltration of CD11b/c+ antigen presenting cells, suggesting that the protective effects observed in the Rag1 mutants were specific to the reduction of lymphocytes. Together, these results provide evidence that T and B lymphocytes importantly contribute to the exacerbation of salt‐induced hypertension and renal injury in Dahl SS rats caused by high dietary protein intake.

Support or Funding Information

DK96859, HL116264, 5T32HL007792, 15SFRN2391002
Lactate dehydrogenase activity, a novel renal cortical imaging bio‐marker of tubular injury
Authors: Nielsen Per Mose , Laustsen Christoffer , Qi Haiyun , Nørlinger Thomas , Mikkelsen Emmeli , Norregaard Rikke , Stødkilde‐Jørgensen Hans
Published: November 2023

Renal ischemia/reperfusion injury (I/R‐I) is the leading cause of acute kidney injury (AKI) in several disease states, including hypovolemic shock, sepsis, surgery. Imbalance in energy metabolism and mitochondrial function is a hallmark in I/R‐I which can be caused by mechanisms like oxidative stress, apoptosis and inflammation. Lactate dehydrogenase (LDH) activity has previously been suggested as a renal tubular injury marker, but has a major limitation in the sense that it can only be measured in terminal kidneys. Recent advances in magnetic resonance technology, has allowed hyperpolarized [1‐13C]pyruvate magnetic resonance imaging (MRI) to monitor metabolic changes in real time in situ without ionizing radiation. Thus this procedure allows for investigation of renal tissue injury in concern with I/R‐I, by probing the renal metabolism after I/R‐I and especially the LDH activity.

Sixteen Wistar Rats were included in the study. Rats were subjected to unilateral (or bilateral) renal ischemia for 60 min followed by reperfusion for 24 hours (n=8). Sham operated rats were prepared in parallel (n=8). A tail vein catheter was inserted for injection of hyperpolarized [1‐13C]pyruvate before the MRI scanning. Temperature, arterial oxygen saturation and respiration rate were monitored throughout the experiment. After MRI scanning, kidneys were excised and stored for further biochemical analyses.

I/R‐I resulted in statistically significantly increased plasma creatinine, BUN and decreased creatinine clearance, indicating AKI. Additionally kidney injury molecule 1 (KIM‐1) was significantly increased in rats subjected to I/R‐I, indicating cellular injury. Hyperpolarized [1‐13C]pyruvate showed a reduced LDH activity in the post‐ischemic kidneys, calculated from pyruvate‐to lactate conversion. This was verified by activity measurements of LDH in terminal kidneys. Using a bilateral I/R‐I model increased LDH activity was also observed in urine samples. Like LDH activity a reduced ALT and PDH activity was also found in the I/R‐I model, calculated from pyruvate‐bicarbonate conversion, and pyruvate‐to‐alanine conversion. An increased LDH mRNA transcription, together with a reduced NADH/NAD+ ratio indicates a preference for anaerobic glycolysis in the I/R‐I kidney. This is supported by MR results when comparing lactate‐to‐bicarbonate formation, which individually describes the anaerobic and aerobic glycolysis.

In conclusion, a decreased LDH activity is observed in the I/R‐I kidney, and this is correlated with tissue injury. Although the general LDH activity in the I/R‐I kidney is lowered, a weighted preference towards anaerobic metabolism was evident in the I/R‐I kidney.

Support or Funding Information

Karen Elise Jenens Foundation
Proteolytic processing of the catalytic subunit of protein kinase A (PKA C) by meprin β in ischemia/reperfusion‐induced acute kidney injury
Authors: Niyitegeka Jean‐Marie Vianney , Sedighi Rashin , Harris Regine S , Ongeri Elimelda Moige
Published: November 2023

Ischemic/reperfusion (IR) is associated with acute kidney injury. Meprin metalloproteases are the most abundant proteins in the brush border membranes (BBMs) of proximal kidney tubules. Presence of meprins was previously shown to enhance kidney injury in IR. However, the mechanisms by which meprins modulate kidney injury in IR are not fully understood. We are gaining insights from identification of kidney meprin targets, which include extracellular matrix (ECM) proteins, cytoskeletal proteins, tight junction proteins, and members of the AGC kinase family of proteins (e.g protein kinase A and protein kinase C). Meprins are redistributed from the BBM to the cytosol and basolateral membranes in ischemia/reperfusion bringing them in close contact with proteins in these cell compartments. We recently showed that meprins cleave the catalytic subunit of protein kinase A (PKA C) in an isoform specific manner. While both meprin A and meprin B could cleave PKA Cβ1, only meprin B cleaved PKA Cα and PKA Cβ2. In mice subjected to IR‐induced acute kidney injury, fluorescent microscopy showed a decrease in total PKA C in the proximal tubules (which express meprins) but not in distal tubules (which lack endogenous meprins). In fact the levels of PKA C in distal tubules increased at 6h post‐IR, suggesting that the PKA pathway plays a role in the hypoxia response associated with IR.

The objective of the current study was to determine how proteolytic processing of PKA C by meprin β affects the levels PKA C isoforms in mice with IR‐induced acute kidney injury. To this end, surgical procedures were used for unilateral nephrectomy coupled with clamping of renal arteries for 27 minutes to induce IR‐associated injury in the contralateral kidneys of 12‐week old male mice on a C57BL/6 background (n=6 meprin β knockout; n=5 wild‐type). Blood samples were collected pre and post IR injury to phenotype kidney. The mice were sacrificed at 6h post IR and kidney tissues processed for proteomic analysis. Serum creatinine levels were determined by ELISA to confirm kidney failure. Western blot analysis with anti‐PKA C and anti‐PKA Cα specific antibodies was used to evaluate the levels of total PKA C and the PKA Cα isoform in kidney proteins. The intensities of the PKA C protein bands were quantified using a calibrated densitometer. Statistical analysis utilized 2‐way ANOVA. Our data showed a decrease in the kidney levels of total PKA C and PKA Cα in WT kidneys subjected to IR. This decrease was not observed in meprin β knockout kidneys. The data suggests increased in vivo proteolytic processing of PKA C present in kidneys by meprin β in IR. Meprin β may thus enhance renal tubular damage via cleaving of PKA C. We will evaluate the impact on downstream targets of the PKA pathway and hypoxia response genes.

Support or Funding Information

NIH/NIGMS Grant # SC3GM102049
The Involvement of Iron on Erythropoietin Expression
Authors: Ikeda Yasumasa , Oshima Keisuke , Horinouchi Yuya , Izawa‐Ishizawa Yuki , Kihira Yoshitaka , Ishizawa Keisuke , Tsuchiya Koichiro , Tamaki Toshiaki
Published: November 2023

Background

Renal anemia is often seen in the development of chronic renal failure (CRF). Therefore, Iron supplementation, as well as erythropoiesis‐stimulating agents, are used for treatment of renal anemia. Meanwhile, iron is shown to cause oxidative stress through toxic hydroxyl radical production via iron catalyzed‐Fenton reaction. Therefore, iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CRF. In the present study, we studied iron action on erythropoietin expression.

Methods and Results

In mice with unilateral ureter obstruction (UUO), EPO gene was reduced, and renal iron content was elevated in kidney of UUO side. Similarly, EPO gene was suppressed in mice with iron treatment. The expression of hypoxia‐inducible factor‐2 alpha (HIF‐2a), a positive regulator of EPO gene, was also diminished in both UUO‐ and iron‐treated kidney. In in vitro experiments using HepG2 cells, iron stimulation reduced the expression of EPO gene, as well as HIF‐2a, in a dose‐dependent manner. Iron also suppressed CoCl2‐induced EPO gene upregulation under hypoxia‐mimicked condition. Moreover, iron treatment augmented oxidative stress production, and iron‐induced reduction of EPO and HIF‐2a expression was restored by an anti‐oxidant agent treatment.

Conclusion

These findings suggested that Iron supplementation reduced EPO gene expression through oxidative stress‐HIF‐2a‐dependent manner.
Kidney decapsulation reduces ischemia‐reperfusion‐induced proteinuria
Authors: Nensén Oskar , Hansell Peter , Fasching Angelica , Palm Fredrik
Published: November 2023

Ischemia‐reperfusion injury (IRI) alters the integrity of the glomerular barrier integrity resulting in proteinuria. Decapsulation reduces interstitial pressure in the kidney; a procedure which we have previously reported to reduce diabetes‐induced glomerular protein leakage. Therefore, the present study determined if the same beneficial effects of decapsulation on proteinuria is achieved during the acute phase following IRI in rats.

Measurements of urinary protein leakage, tubular pressures and renal blood flow (RBF) were determined before and immediately after thirty minutes of warm complete unilateral ischemia in normal and decapsulated adult male Sprague Dawley rats.

Decapsulation per se completely prevented the IRI‐induced proteinuria. Decapsulation concomitantly reduced both baseline tubular free flow pressure (Pff; 14.0±0.6 versus 11.9±0.8 mmHg) and tubular stop flow pressure (Psf; 32.1±0.8 versus 30.2±0.7 mmHg) resulting in similar net filtration pressures (Pnet) in both groups (18.1±0.5 and 18.3±0.7 mmHg). IRI resulted in increased Pff in both groups (+5.0±1.0 and +4.9±0.8 mmHg for controls and decapsulated, respectively). Psf was unaltered by IRI resulting in similar reduction in Pnet after IRI in both groups. Interestingly, decapsulation reduced baseline RBF (7.8±1.1 versus 6.6±0.5 ml/min/kidney) but significantly mitigated the IRI‐induced reduction in RBF (−3.6±1.6 versus −1.9±0.7 ml/min/kidney for control and decapsulated, respectively).

These data demonstrate that decapsulation prevents IRI‐induced proteinuria, possibly by reducing interstitial hydrostatic pressure. It is tempting to speculate that therapies lowering renal interstitial pressure could potentially be used to prevent IRI‐induced proteinuria and protect long‐term kidney function.
Low salt diet does not protect kidney function in normotensive rat model of chronic kidney disease
Authors: Patinha Daniela , Pihl Liselotte , Fashing Angelica , Palm Fredrik
Published: November 2023

Intrarenal hypoxia has been shown to cause renal damage. Na+‐restriction in normotensive rats reverses the normal cortical‐medullary oxygen gradient, an alteration that may compromise long‐term kidney function. Indeed, two weeks of low salt (LS) diet to rats induces both glomerular and tubulointerstial fibrosis. We hypothesized that chronic LS diet may also aggravate kidney damage in chronic kidney disease (CKD).

Male Sprague Dawley rats were subjected to a two‐step surgery with right nephrectomy and surgical ablation of the upper and lower poles of the left kidney (5/6Nx). This produces a normotensive salt‐sensitive animal model of CKD. After one week recovery, animals where fed normal (NS, 0.4%) or low (LS, 0.01%) salt diet during the following four weeks. Thereafter, catheters were placed in the carotid artery and femoral vein for blood pressure measurement and infusion of saline containing 3H‐inulin, respectively, in thiobutabarbital anesthetized animals. Oxygen consumption (QO2) was estimated from the arterio‐venous difference in O2 content multiplied by renal blood flow (RBF, transonic flow probe). Renal cortical and medullary partial pressures of oxygen (PO2) were measured using Clark‐type electrodes. At the end of the experiment, a bolus injection of angiotensin II AT1 receptor blocker candesartan (1 mg/kg) was given intravenously in order to determine the degree of renin‐angiotensin system (RAS) activation.

Mean arterial blood pressure, glomerular filtration rate and RBF were similar in healthy control and 5/6Nx. RAS activation was confirmed by the more pronounced decrease in MAP following systemic angiotensin II AT1 receptor blockade in LS. 5/6Nx on both diets had incerased QO2 and lower Na+ transport efficiency when compared with controls. Reduced cortical PO2 was also observed in 5/6Nx animals on both diets, whereas medullary PO2 was increased only in 5/6Nx on LS. Increased TBARS were not improved and total protein excretion was further increased in 5/6Nx animals by LS

Taken together, these results indicate that LS diet does not normalize renal QO2, prevents cortical hypoxia or TBARS in the 5/6Nx model of CKD. Actually, LS diet to 5/6Nx magnified the already existing proteinuria, which may imply that LS and subsequent RAS activation may have detrimental effect on long term kidney function in CKD.

Support or Funding Information

This work was supported by the Swedish Heart and Lung Foundation and the Swedish Research Council.
Renal cortical hydrogen peroxide (H2O2) stimulates renin release from juxtaglomerular (JG) cells and increases blood pressure: Role of JG cell NOX4
Authors: Hypertension Mariela Mendez. , Division Vascular Reasearch
Published: November 2023

Renin plays an essential role in blood pressure control and the development of hypertension. Enhanced hydrogen peroxide (H2O2) levels in the kidney are associated with hypertension. This is thought to be due to direct stimulation of tubular salt transport and enhanced vascular reactivity. However, we previously found that H2O2 can directly stimulate renin release from renal juxtaglomerular (JG) cells suggesting that this pathway may also be responsible for the hypertension. However, it is not clear whether enhanced H2O2 in the renal cortex stimulates renin release and increases blood pressure. We hypothesized that enhancing renal cortical H2O2 stimulates renin release and increases blood pressure. To enhance renal cortical H2O2 in mice, we placed a subcapsular renal catheter connected to an osmotic minipump to reach a concentration of 1 μM, and measured blood pressure by radio telemetry and plasma renin concentration (PRC). Two days after infusion of H2O2, systolic blood pressure increased by 22±2 mmHg (p<0.05) and PRC doubled (from 110± 7 to 214±39 ngAngI/hr/ml, p<0.05). In control mice, with renal cortical infusion of saline, blood pressure and PRC were not different from baseline, indicating that renal cortical H2O2 increased renin release and blood pressure. In vivo, H2O2 produced by surrounding cells or endogenously produced, could enhance renin release. We found that in freshly isolated mouse JG cells, decreasing endogenous H2O2 with catalase (100U/ml) decreased baseline renin release by 45±9% (n = 6; p < 0.05) indicating that endogenously produced H2O2 tonically stimulates renin release. To start dissecting the enzymatic sources of H2O2 production, we studied the NADPH oxidase isoforms present in JG cells. By Western blot, we found that NOX1, NOX2 and NOX4 are expressed in JG cell lysates (n=3). By immunofluorescence and confocal imaging, we observed that NOX4 isoform is highly expressed in JG cell renin granules (n=3). Treating JG cells with apocynin (120 μM), a general inhibitor of NADPH oxidases, decreased renin release by 28±7% (p<0.04). To identify whether NOX4 mediates the endogenous H2O2 production, we used adenovirus mediated gene silencing of NOX4. Silencing NOX4 in JG cells decreased renin release by 34 ±4% (p < 0.05). We concluded that renal cortical H2O2 is a potent stimuli for renin release in vivo. Endogenously produced H2O2 from NOX4 expressed in mouse JG cells stimulates renin release. Our data suggest that enhanced renal cortical reactive oxygen species may induce hypertension by enhancing renin release. NOX4 inhibition in JG cells might be a candidate for renin release and blood pressure control.

Support or Funding Information

SDG12070304; RO3DK105300‐01
Lithium Modulates Cilia Length in Renal Collecting Duct Cells
Authors: Mehran Nikki A. , Mallow John F. , Himmel Nathaniel J. , Blount Mitsi A.
Published: November 2023

Cilia are important regulators of multiple cell processes throughout the body. Optimal cellular function can depend on cell‐specific regulation of cilia length; however, the mechanisms that control cilia length are not well understood. Defects in ciliary length appear to be related to ciliopathic diseases, which often present with renal cysts. Lithium chloride (LiCl) has been shown to affect cilia in multiple cell types, but its effects have not been explored in inner medullary collecting duct (IMCD) cells. We found that following a 24 hr LiCl (25 mM) treatment of IMCD cells in vitro, cilia are significantly elongated by 1.5 fold. Although LiCl can inhibit GSK3β, addition of the GSK3β‐specific inhibitor TDZD‐8 did not increase IMCD cilia length, indicating that LiCl‐mediated cilia elongation is independent of GSK3β. Instead we found that the SQ‐22536‐inhibtion of adenylate cyclases in IMCD cells mimics LiCl‐mediated cilia elongation, whereas addition of a cAMP analog in the presence of LiCl presence prevents elongation. In addition, we observed a similar increase in cilia length in renal collecting duct cells from rats fed dietary lithium for 2 wk coinciding with a decrease in urinary cAMP. We conclude that lithium increases cilia length in IMCD cells by dampening cellular cAMP in a manner independent of GSK3β. Our data provide new insights into the effects of LiCl on renal cilia and suggest that lithium may have therapeutic benefits in the treatment of some ciliopathies.

Support or Funding Information

Emory SOM‐Renal Division
Monocyte Mitochondrial Function as a Marker of Calcium Oxalate Stone Formers
Authors: Mitchell Tanecia , Williams Jennifer , Holmes Ross P , Assimos Dean G
Published: November 2023

The prevalence of kidney stones (KS) is 8.8% in the US and contributes significantly to health care costs. The most common type of KS is the calcium oxalate kidney stone (CaOx‐KS). Both inflammation and oxidative stress have been suggested to play an integral role in KS pathogenesis. The purpose of this study was to investigate whether inflammation is elevated and cellular bioenergetics is suppressed in immune cells from calcium oxalate (CaOx) stone formers (SF) compared to healthy subjects (HS). Adult age‐matched HS (n=18) and CaOx SF (n=15) were included in the study. Data collection included demographic and clinical values from electronic medical records. Pro‐inflammatory Interleukin‐6 (IL‐6) plasma values were measured using ELISA. Bioenergetic function was assessed in monocytes, lymphocytes, and platelets using the Seahorse XF96 Analyzer. Fifty‐three percent of the CaOx SF had a family history of KS and 60% had a KS event previously. Plasma IL‐6 levels were significantly elevated (p=0.0024) and monocyte mitochondrial function was decreased in CaOx‐SF compared to HS. Specifically, maximal respiration (p=0.0001) and reserve capacity (p<0.0001) were significantly lower. In contrast, lymphocyte and platelet mitochondrial function was similar to monocytes from HS. The bioenergetic health index (BHI), an integrated value of mitochondrial function, was also determined to be significantly lower in CaOx SF monocytes compared to HS (p=0.0191). These data suggest that impaired monocyte mitochondrial function may play a role in CaOx KS pathogenesis and the BHI could potentially serve as a marker in CaOx SF.

Support or Funding Information

Supported by NIH grants DK054468, UL1TR001417, P30 DK079337, and the 2015–2016 UAB Faculty Development Grant Program (TM). TM is also supported by DK054468‐S1.
Low Birth Weight Impairs Murine Kidney Development and Function
Authors: Ratliff Brian , Pawar Rahul , Aldumuahdhi Wasan , Nnoli Oluwadara , Zullo Joseph , Rabadi May
Published: November 2023

Background

The low birth weight (LBW) neonate is at greater risk for morbidity and mortality after birth and during adulthood.

Methods

Using immunohistochemistry, immunofluorescence, PCR, Laser‐Doppler flowmetry, and ELISA assay, we examined survival, kidney development and function, and circulating levels of pro‐inflammatory cyto‐/chemokines in the LBW offspring of malnourished (caloric and protein restricted) pregnant mice.

Results

Maternal malnourishment during gestation led to LBW newborns (40% reduced body weight) with only 46% of these babies surviving the first week after birth. The LBW neonate had elevated serum creatinine, while kidney blood perfusion was reduced by 26%. During gestation the LBW offspring suffered a severe impairment in nephrogenesis, due to a 76% drop in renal vesicle formation that was characterized by a 2.2‐fold increase in apoptosis, a 75% decrease in six2 positive nephron progenitor cells, and a 27% decrease in expression of the renal mesenchymal‐to‐epithelial signaling factor Wnt9b. At the conclusion of nephrogenesis, the LBW neonate had 36% fewer nephrons than controls. At birth, the LBW neonate had elevated plasma levels of IL‐1, IL‐12(p70) and GM‐CSF, further impairing six2 positive progenitor cell competence and nephrogenesis.

Conclusion

The kidneys of LBW neonates are underdeveloped, thereby predisposing the neonate to adverse health problems.

Support or Funding Information

Studies were supported by AHA grant 12SDG9080006, ASN grant 010973‐101 and The New York Community Trust‐Renal Clinical Fund (BBR)