bluuebunny/crossref_metadata_2025 huggingface dataset:
Expansion of Regulatory T cells using Low‐dose Interleukin‐2 Attenuates Hypertension in an Experimental Model of Autoimmune Disease
Authors: Taylor Erin B. , Ryan Michael J.
Published: November 2023

Systemic lupus erythematosus (SLE) is a prototypic multisystem autoimmune disorder that is characterized by prevalent hypertension, renal injury, and cardiovascular disease. In addition to a breakdown of tolerance to nuclear antigens that results in the activation of self‐reactive B and T lymphocytes, numerous studies report a low prevalence and/or impaired function of regulatory T cells (TREG) in both patients with SLE and in murine models. TREG are important for the maintenance of tolerance to self antigens and also secrete the anti‐inflammatory cytokines TGF‐β and IL‐10. Evidence suggests that TREG dysfunction in SLE results from a deficiency in interleukin (IL)‐2, a cytokine critical for maintenance of TREG cell populations. Recent studies suggest that low dose IL‐2 therapy expands TREG in mouse models of SLE, but whether expanding TREG protects against hypertension and renal injury during SLE is unclear. To examine this question, 30 week old female SLE (NZBWF1) and control (NZW) mice were injected intraperitoneally with phosphate buffered saline (PBS, vehicle) or 2 μg recombinant mouse IL‐2 in PBS (rIL‐2) three times in 24 hours followed by maintenance doses every five days for four weeks. Treatment with rIL‐2 effectively expanded the CD4+FoxP3+ TREG cell populations in the spleen (5.2±0.4 vs. 8.3±0.5%, p<0.0001), peripheral blood (0.66±0.05 vs. 0.94±0.13%, p<0.05), and kidneys (6.8±0.6 vs. 8.9±0.8%, p<0.05) when compared to vehicle treated SLE mice. Circulating levels of anti‐dsDNA IgG autoantibodies, a marker of SLE disease activity, were higher in SLE mice compared to control mice (1.53±0.17 vs. 0.39±0.10 OD450, p<0.01), but were unaffected by rIL‐2 treatment (SLE‐rIL‐2: 1.69±0.16 OD450, p=0.87). Similarly, urinary albumin excretion, an indicator of glomerular injury, was increased in SLE mice as compared to controls (8.2±4.6 vs. 0.032±0.01 mg/day), but was not different in SLE mice treated with rIL‐2 (SLE‐rIL‐2: 11.6±5.9 mg/day). As previously reported by our laboratory, mean arterial pressure (MAP; mmHg), measured in conscious mice by carotid catheter, was higher in SLE mice than in controls (136±3 vs. 115±3, p<0.05). MAP was significantly lower in rIL‐2 treated SLE mice when compared to vehicle treated SLE mice (SLE‐ rIL‐2: 125±4; p<0.05 vs. SLE‐vehicle), suggesting that expanding TREG using low dose IL‐2 attenuates the development of hypertension in female SLE mice. While the mechanism for the protection against hypertension is unclear, it does not appear to be related to the delay of SLE disease progression. Further studies are required to test whether anti‐inflammatory cytokines produced by TREG are responsible for the anti‐hypertensive effects of low‐dose IL‐2 therapy.

Support or Funding Information

Research supported by: VA Merit Award BX002604‐01A2, NIH PO1HL051971, P20GM104357, and T32HL105324‐05
Electrolyte Homeostasis in Migraine
Authors: Stanton Angela A
Published: November 2023

Migraine is considered to be a neurovascular disease. Most research is aimed at reducing migraine pain by medications that either limit the activity of an overstimulated brain or add neurotransmitters that are presumed to be below ideal levels in a migraine brain (1, 2). In this presentation, a new view is discussed. Taking a closer look at metabolic deficits in migraineurs (3–9), we can see that it is their metabolic processes that disrupt electrolyte homeostasis prior to migraine. Some of these metabolic deficiencies are genetic and others are caused by medicines. The reestablishing of electrolyte homeostasis can prevent migraine. Migraineurs have special sensory sensitivities with sensory neurons that have more receptor connections than regular brains (10). Neurons that are more sensitized and have more receptor connections require higher ionic concentration electrolytes to keep their overactive work function. The lack of proper electrolyte is likely the cause of cortical depression and the ensuing cortical spreading depression—the wave that reaches the pain sensors in the meninges. A solution is offered for preventing pain without medical intervention by reducing electrolyte disrupting elements in the diet of migraineurs to the minimum required, such as glucose, and ensuring a constant proper electrolyte balance by a potassium and magnesium heavy diet with more sodium than what the average population uses. In the past three years of my observation of migraineurs in the Facebook group I specifically designed for reaching electrolyte homeostasis, all migraineurs can stop their medicines and remain migraine free—provided they maintain this balance.

Support or Funding Information

no funding received

no conflict of interest
GPR83 function contributes to salt resistance
Authors: Patel Milan , Zheng Xiaoxu , Asico Laureano , Konkalmatt Prasad , Jose Pedro A , Armando Ines
Published: November 2023

The G protein‐coupled receptor 83 (Gpr83) is an orphan G protein‐coupled receptor that is expressed in the kidney but its function is unknown. We found that Gpr83 is expressed in mouse renal proximal and distal convoluted tubules, as well as in human renal proximal tubule cells (hRPTCs). High salt diet increased Gpr83 transcription by 2‐fold (P<0.05; n=4/group) in SJL/J and BALB/c salt‐resistant mice, relative to C57Bl/6J salt‐sensitive mice. In C57Bl/6J mice on normal salt diet, the lack of one (Gpr83+/−) or both Gpr83 (Gpr83−/−) alleles resulted in an increase in systolic blood pressure (SBP, ~20 mm Hg (P<0.05; n=4/group, measured under anesthesia) compared with Gpr83+/+ littermates, suggesting that Gpr83 is needed to keep a normal BP. Renal‐specific Gpr83 silencing by the renal subcapsular infusion of Gpr83 siRNA (3 μg/day; 7 days) increased SBP in C57Bl/6J mice on a normal salt diet relative to mice treated with non‐ silencing siRNA (120±5 vs 98±6 mmHg; P<0.05; n=4/group). In hRPTCs, forskolin (10 μM, 30 min) increased Gpr83 mRNA (3.5±0.06 vs 1.0±0.12‐fold; P<0.05; n=4–5/group), the effect was blocked by the protein kinase A (PKA) inhibitor H‐89 (20 μM, 1 h). In hRPTCs, phorbol myristate acetate (200 ng/mL, 30 min) which activates protein kinase C (PKC) decreased Gpr83 mRNA (0.43±0.2 vs 1.0±0.04‐fold, P<0.05; n=4–5/group), effect that was partially blocked by the PKC inhibitor GF109203× (1μM, 1h). Stimulation of hRPTCs with ZnCl2 (100 μM, 1 h), an activator of Gpr83, increased AKT (2.5±0.5 vs 1.0±0.06‐fold; P<0.05; n=4–5/group) and ERK1/2 (1.4±0.1 vs 1.0±0.08‐fold; P<0.05; n=4–5/group) phosphorylation and decreased p‐38 MAPK phosphorylation (0.1±0.05 vs 1.0±0.1‐fold; P<0.05; n=4–5/group). Our results suggest that Gpr83 may protect against the development of salt sensitivity. PKA positively while PKC negatively regulates Gpr83 expression. Gpr83 function may be mediated by the phosphorylation of AKT/ERK1/2 and dephosphorylation of MAPK. Thus, several pathways are involved in the Gpr83‐mediated regulation of salt‐sensitive hypertension.
Food restriction increases the cardiovascular response evoked by angiotensin‐[1–10] and angiotensin‐[1–7] in female Fischer rats
Authors: de Souza Aline Maria Arlindo , Pai Amrita , Ji Hong , Menezes Rodrigo , Sandberg Kathryn
Published: November 2023

Anorexia is associated with cardiovascular dysfunction including hypotension and cardiac arrhythmias. In a rat model of food restriction (FR) we previously demonstrated that the activity of endothelial α1 adrenergic receptors and brain type 1 angiotensin receptors were increased. The aim of this study was to determine the role of the angiotensin (Ang)‐[1–7] and Ang‐[I‐10] in the brain. Female Fischer rats (220g, 4 mo) were maintained for 14 days on a FR diet, that was 40% of the control group (C) food intake. Cannulas were implanted in the lateral ventricle (LV) on day 7. On day 14, the animals were catheterized with a polyethylene tube in the femoral artery for recording of mean arterial pressure (MAP) and heart rate (HR). After 14 days, MAP (mmHg) [C: 105±1, n=35 vs. FR: 102±2, n=36; p<0.05] and HR (bpm) [C: 386±5, n=35 vs. FR: 353±8, n=36, p<0.001] were reduced in the FR. Injection of Ang‐[1–7] caused a greater increase in MAP (Δ mm Hg) in the FR compared to C group (25pmol) [C: Δ −2±1, n=8 vs. FR: Δ 7±1, n=10; p<0.05]. Moreover, there was a greater increase in HR (Δ bpm) in the FR compared to the C group [C: Δ 9±3, n=8 vs FR: Δ 29±9, n=10; p<0.05]. Injection of Ang‐[1–10] caused a greater increase in MAP (Δ mm Hg) in the FR compared to C group (25pmol) [C: 5±1, n=7 vs. FR: 13±2, n=7; p<0.005]; however, there was no effect on HR. This Ang‐[1–10]‐induced increase in MAP was attenuated by administering captopril into the LV, in both the FR and C groups, suggesting this increase in MAP was mediated by angiotensin converting enzyme. These results suggest that increased Ang‐[1–7] and Ang‐[1–10] activity in the brain play a role in the increased sympathetic activity observed in anorexia.

Support or Funding Information

NIH 1R01HL119380 (KS)

UFOP (AS)

CAPES (AS)
Transcriptional Dynamics of Mfd
Authors: Wang Michelle
Published: November 2023

DNA damage is disruptive to both DNA replication and transcription and thus must be repaired in a timely fashion to ensure proper cell function and viability. The prokaryotic transcription‐repair coupling factor, Mfd, binds to RNA polymerase and is known to be a key player in transcription coupled repair. Yet due to a lack of structural information, interactions of Mfd with a transcription elongation complex (TEC) have been poorly understood. Here we have carried out comprehensive studies to examine Mfd‐TEC interactions using novel single molecule methods. We will discuss results on the dynamic and mechanical nature of these interactions and how Mfd can regulate the motor activities of RNA polymerase. These results provide a broader picture of how transcription may be regulated by Mfd.
The Effect of Renal pH on Ischemia‐Reperfusion‐Induced Acute Kidney Injury
Authors: Boone Jasmine E , Ruisheng Liu
Published: November 2023

Acute kidney injury (AKI) is a major public health issue associated with significant cost, extensive morbidity and increased mortality. However, no specific therapy to prevent or treat AKI is available, and the pathophysiological mechanisms of AKI remain unclear. Renal pH is related to dietary acid‐base load and affects the function of epithelial cells. Renal alkali intervention has been demonstrated to improve renal function and slow the decline in glomerular filtration rate (GFR) in the normal population and patients with chronic kidney disease. However, the role of renal pH in AKI is still not known.

In the present study, we hypothesized that elevations in renal pH protect against the development of ischemia reperfusion injury (IRI)‐induced AKI. AKI was induced in C57BL/6J mice by 18 minutes of ischemia bilateral clamping of renal pedicles. The mice that were performed same surgery without clamping pedicels were taken as sham group. The mice were then divided into three groups (n=6 per group) treated for 14 days with 0.5% NaCl, 0.72% NaHCO3 (same mEq of Na+ as in NaCl), or 0.46% NH4Cl (same mEq of Cl− as in NaCl).

Urine pH was significantly increased to 7.8±0.5 in the NaHCO3 treated group and decreased to 6.8±0.3 in the NH4Cl treated group, compared with the NaCl group (7.3±0.3). Plasma creatinine was measured 7 and 14 days after IRI to evaluate renal injury. The plasma creatinine was substantially increased in all three groups compared with the sham‐operated group (0.08±0.02 mg/dl) 7 days after IRI, with the NH4Cl group being the highest (0.98±0.15 mg/dl), followed by the NaCl group (0.71±0.11 mg/dl) and the NaHCO3 group (0.47±0.09 mg/dl). There was no significant difference in plasma creatinine between the three groups at 14 days after IR. To evaluate kidney function, GFR was measured by the clearance of plasma FITC‐insulin with a single bolus injection in conscious mice at baseline as well as 7 and 14 days after IRI. GFR decreased by 44.5% (244.7±9.1 to 135.8±13 μl/min) in the NH4Cl group, 31.8% (228.3±11 to 155.52±8.4 μl/min) in the NaCl group and 21.2% (237.6±7.5 to 187.3±14 μl/min) in the NaHCO3 group 7 days after IRI. The GFR returned to near baseline in all three groups at 14 days after IRI.

In summary, these results indicate that renal acidification aggravates whereas alkalization protects against the development of IR‐induced AKI.
Dietary oxalate induces monocyte mitochondrial dysfunction and crystalluria in healthy subjects: A role of spinach in kidney stone disease?
Authors: Mitchell Tanecia , Yarlagadda Vidush , Patel Mikita , Knight John , Assimos Dean , Holmes Ross
Published: November 2023

Dietary oxalate contributes significantly to urinary oxalate excretion and is linked to calcium oxalate kidney stone formation. Oxalate is a common component found in kidney stones and in plants such as spinach. We have previously shown that patients with calcium oxalate kidney stone disease have decreased mitochondrial function and increased IL‐6 levels compared to healthy subjects. The purpose of this study was to investigate whether a dietary oxalate load has any effect on monocyte mitochondrial function and urinary oxalate levels in healthy subjects. Nine healthy subjects between the ages of 21 and 31 with an average BMI of 24.6 ±1.2 were included in the study. Participants consumed a low oxalate diet for 3 days prior to drinking a high dietary oxalate load (spinach smoothie). Blood and urine was collected from participants prior to consuming a high oxalate spinach load (pre‐smoothie) and 5 hours following smoothie consumption (post‐smoothie). Monocyte mitochondrial function was measured using Seahorse XF96 technology. Urinary oxalate levels (both crystalline and soluble) were determined using ion chromatography‐mass spectrometry. The high dietary oxalate load caused a significant decrease in monocyte mitochondrial function in healthy subjects. These responses were similar to levels previously found in patients with calcium oxalate kidney stone disease. In addition, both crystalline and soluble urinary oxalate levels were significantly increased in post‐smoothie samples compared to pre‐smoothie samples. These findings suggest that a high dietary oxalate could affect monocyte mitochondrial function in patients. Future studies will investigate potential mechanisms responsible for these findings.

Support or Funding Information

This work was supported by NIH K01DK106284 and the UAB Department of Urology.
Knockout of Sphingosine Kinase 1 Attenuates Renal Interstitial Fibrosis in Unilateral Ureteral Obstruction (UUO) Model
Authors: Zhang Xiwen , Wang Fan , Wang Weili , Li Pin‐Lan , Comlekoglu Tien , Li Ningjun
Published: November 2023

Sphingosine‐1‐phosphate (S1P) is a bioactive sphingolipid metabolite and an important signaling molecule that plays a significant role in the differentiation of fibroblasts to myofibroblasts. S1P synthesis is catalyzed by sphingosine kinases (SPHK), which phosphorylate sphingosine into S1P. The present study tested the hypothesis that Sphk1‐S1P signaling pathway participates in the kidney damage in unilateral ureteral obstruction (UUO) model using Sphk1 knockout mice. UUO was created by ligation of the ureter on one kidney and the contralateral kidney is used as control. Wild type C57BL6 mice and Sphk1 KO mice were subjected to UUO for 14 days and four groups of kidneys were collected: wild type control group (WT‐C), wild type UUO group (WT‐UUO), knockout control group (KO‐C) and knockout UUO group (KO‐UUO). The mRNA level of Sphk1 in WT‐UUO was increased by 4 fold (p<0.05 vs. WT‐C) and there was no significant difference between other three groups. The protein levels of α‐smooth muscle actin (α‐SMA) and collagen I were upregulated in both UUO groups but the levels were significant lower in KO‐UUO group than in WT‐UUO group. The relative protein levels of α‐SMA were 1 ± 0.03 in WT‐C, 18.13 ± 2.55 WT‐UUO, 1.95 ± 0.67 KO‐C, and 9.54 ± 3.55 KO‐UUO, p<0.05 for KO‐UUO vs. WT‐UUO; collagen I levels were 1 ± 0.13 in WT‐C, 11.25 ± 1.74 WT‐UUO, 0.93 ± 0.58 KO‐C, and 3.09 ± 1.15 KO‐UUO, p<0.05 for KO‐UUO vs. WT‐UUO. Immunostaining showed that the percentage of lymphocyte marker CD43 positive cells was much higher in both UUO groups than in two control groups, but there was no significant difference between KO‐UUO and WT‐UUO, suggesting that the differences of renal fibrotic markers between WT‐UUO and KO‐UUO are not due to the change of immune regulation by knockout of Sphk1, rather, it is the direct effect of Sphk1 knockout on renal cells. Finally, the tubular injury score and glomerulosclerosis index were significantly lower in KO‐UUO than in WT‐UUO in kidney tissue slides with PAS staining (Tubular injury score: 0.07 ± 0.01 in WT‐C, 2.66 ± 0.17 WT‐UUO, 0.03 ± 0.02 KO‐C, and 1.21 ± 0.16 KO‐UUO, p<0.05 for KO‐UUO vs. WT‐UUO; Glomerulosclerosis index: 0.62 ± 0.12 in WT‐C, 2.24 ± 0.14 WT‐UUO, 0.43 ± 0.05 KO‐C, and 1.50 ± 0.10 KO‐UUO, p<0.05 for KO‐UUO vs. WT‐UUO). These results suggest that Sphk1‐S1P signaling pathway mediates kidney damage in UUO mice. Manipulating Sphk1‐S1P signaling pathway may be used as a therapeutic strategy in renal interstitial fibrosis.

Support or Funding Information

Support: NIH grant HL89563
Administration of Tempol Improves Renal Autoregulation in Ischemia‐reperfusion Induced Acute Kidney Injury
Authors: Guan Zhengrong , Ryoo Chan Woo , Inscho Edward W
Published: November 2023

Renal autoregulation is critical for protecting the kidney from fluctuations in arterial pressure by regulating renal vascular resistance to maintain stable renal perfusion and glomerular filtration rate (GFR). Renal ischemia‐reperfusion (IR) induced acute kidney injury (IR‐AKI) is characterized by reduced renal blood flow and a decline in GFR. We hypothesized that increased reactive oxygen species (ROS) contribute to impairment of renal autoregulatory capability in IR‐AKI. Afferent arteriole autoregulatory behavior was assessed using the in vitro blood‐perfused juxtamedullary nephron preparation. IR‐AKI was induced by 60 minutes of bilateral renal artery occlusion followed by 24 hours of reperfusion. We found that plasma creatinine was increased in IR rats (3.86 ± 0.2 mg/dl) compared to 0.93 ± 0.07 mg/dl in sham rats (n=6–7, p<0.05). Baseline afferent arteriole diameter averaged 13.0 ± 0.4 μm (n=5) in kidneys from sham rats and these arterioles exhibited normal autoregulatory behavior. Decreasing renal perfusion pressure from 100 to 65 mmHg increased arteriole diameter from 13.0 ± 0.4 to 15.1 ± 0.8 μm, corresponding to 116 ± 5% of the baseline diameter. Subsequent stepwise increases in perfusion pressure (15 mmHg) caused pressure‐dependent afferent arteriole vasoconstriction to 66 ± 2% of baseline diameter at 170 mmHg. In contrast, pressure‐mediated afferent arteriole responses were markedly attenuated in IR‐AKI rats. Baseline diameter averaged 11.7 ± 0.6 μm and remained between 91 and 101% of baseline over the 65–170 mmHg pressure range tested, indicating a loss of autoregulatory function. In another set of IR‐AKI rats, acute administration of the ROS scavenger, tempol (10−3 M), to afferent arterioles increased baseline diameter by 10 ± 2% from 11.3 ± 0.8 to 12.2 ± 0.9 μm (n=5). Furthermore, scavenging ROS production improved autoregulatory behavior of afferent arterioles from IR‐AKI rats. During exposure to tempol, decreasing renal perfusion pressure from 100 to 65 mmHg resulted in a diameter increase to 110 ± 2 % of the control diameter while increasing perfusion pressure to 170 mmHg decreased arteriole diameter to 78 ± 1% of control (P<0.05 vs IR). In conclusion, these results suggest that renal ischemia‐reperfusion leads to impaired autoregulatory behavior of afferent arterioles. Acutely scavenging endogenous ROS with tempol improves afferent arteriole autoregulatory responsiveness, suggesting that excessive ROS production contributes to impaired renal autoregulation in IR‐AKI.

Support or Funding Information

This study is supported by a Grant‐in‐Aid (15GRNT25240015) from the American Heart Association for ZG and by grants from NIH (DK044628, HL074167, HL098135 and HL095499) for EWI.
Role of carbonic anhydrase in acute recovery following renal ischemia reperfusion injury
Authors: Nensén Oskar , Hansell Peter , Palm Fredrik
Published: November 2023

Ischemia reperfusion injury is a common cause of acute kidney injury in the clinic. It has previously been reported that kidney oxygen consumption (QO2) in relation to glomerular filtration rate (GFR), and thus sodium load, is markedly increased following an ischemic insult. Since proximal tubular sodium transport (TNa) is a major contributor to overall QO2, we investigated how inhibition of TNa in this particular tubular segment impacted on oxygen metabolism and kidney function during the acute recovery following 45 minutes of warm ischemia by inhibiting carbonic anhydrase using acetazolamide.

Male Sprague Dawley rats were anesthetized and administered acetazolamide (50 mg/kg; n=11) or volume‐matched vehicle (n=12). Kidney function, hemodynamics and QO2 were determined before and after complete ischemia by clamping the renal artery.

Acetazolamide per se reduced GFR (−20%) and TNa (−22%), while it increased urine flow (+91%) and urinary sodium excretion (+3600%). Renal blood flow was reduced (−31%) as a result of increased renal vascular resistance (+37%) but without affecting QO2.

Ischemia reperfusion per se resulted in similar decrease in GFR, TNa, and TNa/QO2 independently of carbonic anhydrase status. However, the QO2/GFR ratio following ischemia‐ reperfusion was profoundly increased in the group receiving carbonic anhydrase inhibition indicating a significant contribution of basal oxygen metabolism to the total kidney QO2 following inhibition of proximal tubular function after an ischemic insult.

Support or Funding Information

Swedish Research Council ‐ Medicine and Health
C5aR2 Deficiency Improves Renal Regeneration after Renal Ischemia Reperfusion Injury and in Kidney Transplantation in Mice
Authors: Thorenz Anja , Chen Rongjun , Rong Song , Klemann Christian , Haller Hermann , Braesen Jan Hinrich , Klos Andreas , Gueler Faikah
Published: November 2023

Objective

The complement receptors C5aR1 and C5aR2 serve distinct roles in immune regulation. In kidney transplantation ischemia reperfusion injury (IRI) results in rapid complement activation. In this study C5aR1 and C5aR2 deficient mice were tested in a model of renal ischemia reperfusion injury (IRI) and in an isogenic cross over kidney transplantation (ktx) model.

Methods

IRI was induced by unilateral clipping of renal pedicle for 45 min in C5aR1, C5aR2 −/− and wild type mice (WT). Renal morphology, inflammation, peritubular capillary density and renal fibrosis were investigated by immunohistochemistry. FACS analysis was done at d7 after IRI to analyze infiltrating subsets of myeloid and lymphocytes. To study renal perfusion impairment due to IRI arterial spin labelling was done. In a second model isogenic ktx with prolonged cold ischemia time of 90 min was performed and C5aR1−/−, C5aR2−/− or WT mice served as donors or recipients.

Results

WT mice showed dramatic decrease of renal perfusion and severe inflammation resulting in interstitial fibrosis at three weeks after IRI with substantial loss of kidney volume. C5aR2 −/− mice had less inflammation and started to regenerate at 7 days after IRI. Renal fibrosis was attenuated in C5aR2−/− mice which had less rarefication of peritubular capillaries compared to WT IRI kidneys. In the ktx model C5aR2 −/− of the recipient showed the best renal morphology with attenuated renal ischemia reperfusion injury of the graft. By FACS analysis differential activation in γδ‐Tcells in C5aR2 deficient mice was observed and may have contributed to enhanced regeneration.

Conclusion

C5aR2 deficiency attenuates renal damage and fibrosis in renal IRI and ktx by better regeneration and protection of capillary integrity of the renal tissue.
Gut Microbial Metabolite Trimethylamine‐N‐Oxide Instigates NLRP3 Inflammasomes Activation and Podocyte Injury
Authors: Koka Saisudha , Zhang Yang , Li Ningjun , Hussain Tahir , Li Pin‐Lan , Boini Krishna M
Published: November 2023

Gut microbe derived metabolite, Trimethylamine‐N‐oxide (TMAO) is elevated in chronic kidney disease (CKD) and contributes to the progression of renal disease and increases the risk of mortality in CKD. However, it remains unknown how TMAO induces chronic kidney diseases. The present study tested whether TMAO induces NLRP3 Inflammasomes formation and activation and thereby contributes to podocyte injury. Confocal microscopic analysis showed that TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase‐1 in podocytes compared to control cells. Pretreatment with Nlrp3 siRNA or Caspase‐1 inhibitor YVAD abolished the TMAO‐induced NLRP3 inflammasome formation. Correspondingly, TMAO treatment significantly increased the caspase‐1 activity (2.5 fold) and IL‐1β production (2.4 fold) compared to control cells. The Nlrp3 siRNA transfection significantly attenuated the TMAO‐induced caspase‐1 activity and IL‐1β production. Further RT‐PCR and confocal microscopic analysis showed that TMAO treatment significantly decreased the podocin expression (podocyte damage) compared to control cells. However, pretreatment with Nlrp3 siRNA or YVAD attenuated the TMAO‐induced podocin reduction. Furthermore, Nlrp3 siRNA transfection was found to preserve podocyte morphology by maintaining the distinct arrangement of F‐actin fibers normally lost in response to TMAO. It also prevented podocyte dysfunction by restoring TMAO‐induced suppression of VEGF production and secretion. Based on these results, it is concluded that TMAO‐induced the NLRP3 inflammasome formation and activation in podocytes and thereby by resulting in podocyte injury.

Support or Funding Information

DK104031
Development of kidney‐specific resources for analysis and visualization of genomic data
Authors: Gonzalez‐Vicente Agustin , Hopfer Ulrich , Garvin Jeffrey L
Published: November 2023

The usefulness of modern genomic techniques which produce vast amounts of data is largely limited by our ability to analyze and interpret it. To manage big data, several digital platforms have been developed that use signaling and metabolic pathways to help with analysis and visualization. However, most of these pathways are based on human data, lack tissue/cell specificity and don't distinguish between isozymes. This makes it difficult to employ them to analyze genomic data from model systems. Thus, most available pathways are inadequate for kidney research. To fill this gap, we have been developing nephron segment‐specific pathways on the public depository Wikipathways using Rattus novergicus as a model organism. Wikipathways is an open platform, making it easy for anyone to edit and improve the pathways. Files in Wikipathways are also freely available to download and customize for publication. Before our contributions, there were only three pathways available directly related to renal physiology: Renin Angiotensin System (PW376), Vasopressin and Aquaporins (WP2662), and Glomerulosclerosis (WP2572). Thus far we have incorporated five new pathways. Two of these pathways list segment‐specific transporters from proximal tubules (WP3881) and thick ascending limbs (WP3882). The others summarize the enzymes involved in: 1) fructose and glucose metabolism in proximal tubules (WP3894); 2) lipid metabolism in proximal tubules (WP3901) and; 3) signaling cascades by which angiotensin II acutely affects transport in thick ascending limbs (WP3887). To create these pathways we drew data from multiple publications and databases. The enzymes, represented as nodes, are annotated using ENTREZ Gene ID. Annotations provide links to external databases for further research, and serve as an identifier for each node to merge the pathway with experimental data. In summary, the pathways we developed fill a specific need for the renal research community. Crowd contributions by renal physiologist to further develop these pathways and to create similar ones would provide a powerful and freely‐available tool for renal genomic research.

Support or Funding Information

This work was supported in part by a grant from the Heart, Lung and Blood Institute of the National Institutes of Health to J. L.G. (HL128053).
Identification of ezrin in a novel 3D in vitro model for ADPKD as a new target in cystogenesis
Authors: Dixon Eryn E , Woodward Owen M
Published: November 2023

The ciliopathy autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cystogenesis mediated by two ciliary proteins, polcystin‐1 (PC1) and polycystin‐2 (PC2/TRPP2). While the mechanism of cystogenesis in ADPKD is undefined, loss of function mutations in PKD1 or PKD2, which encode for transmembrane proteins PC1 and PC2/TRPP2, have been identified as initiators in cyst formation. Polycystins have been implicated in apical signal transduction facilitated by the primary cilium. Therefore, it is critical for understanding the downstream mechanism of cyst formation, including effects of aberrant proliferation, mislocalization of growth signals, and alterations in fluid secretion machinery. Our novel three dimensional (3D) cystogenesis model cultures primary renal cells from Pkd2fl/fl Pax8rtTA TetOCre +mTmG mice in Matrigel to investigate the effects of Pkd2 inactivation on tubulogenesis. Tubule formation in this 3D culture method is promoted by seeding cells between two layers of Matrigel and the manipulation of glial derived neurotrophic factor (GDNF) exposure. Following the induction of Cre, we observe a strong correlation between the mosaic mTmG reporter, the reduction in PC2 protein abundance, and the increase of cystic structures, strongly supporting the successful inactivation of Pkd2. Using this model, we explored possible disruption of the apical compartment and inappropriate recruitment of secretory machinery by looking closely at the critical scaffold and ERM family protein, ezrin. We found there is a significant decrease in apically localized ezrin in cells with inactivated Pkd2, a finding consistent with a disruption in the structure of the apical compartment. In nephrectomized human ADPKD cystic epithelia, ezrin localization is altered along the apical compartment of small cysts, where total ezrin and the phospho‐ezrin/radixin/moesin (p‐ERM) complex colocalize with basolateral markers. Interestingly, appropriate apical localization of ezrin is present in larger cysts, indicating that ezrin may be a critical player in the initiation of cystogenesis. Our 3D cystogenesis model for ADPKD demonstrates characteristics consistent with human ADPKD cysts and identifies important defects in membrane sorting mechanisms.

Support or Funding Information

NIDDK 5P30DK090868 Baltimore PKD Core Center and NIH/NIGMS T32 GM008181 Training Program in Integrative Membrane Biology
Lithium Administration Can Attenuate the Progression of Polycystic Kidney Disease
Authors: Kronk Trinity A. , Himmel Nathaniel J. , Mehran Nikki A. , Blount Mitsi A.
Published: November 2023

Polycystic kidney disease (PKD) is one of the most common genetic causes of renal decline and is characterized by the formation of fluid‐filled renal cysts that progressively expand to destroy normal kidney structure and function. Although PKD is the fourth leading cause of kidney failure in the US, there is no FDA‐approved pharmacological treatment; thus, many still regard PKD as an untreatable disease. Dysregulation of signaling pathways contribute to PKD including abnormally high production of renal cAMP. Because lithium can dampen renal cAMP synthesis and can be clinically administered with few complications, repurposing lithium as a treatment for the life‐long management of PKD is a viable and exciting new concept. Aged‐matched male Sprague‐Dawley (SD) and PCK rats (an orthologous model with physiological features that resemble human ADPKD) were pair‐fed a standard rodent diet supplemented with lithium carbonate (40 mmol/kg) for 2 weeks. SD and PCK rats pair‐fed an un‐supplemented, standard rodent diet were used as control groups. Following treatment, kidney/body weight, cyst number and cyst area were examined as indicators of cyst progression. Lithium‐treated PCK rats had lower kidney/body weight, fewer cysts and reduced average cyst area compared to untreated PCK rats. SD rats treated with lithium did not show a change in kidney weight compared to untreated SD rats. Compared to the SD untreated and SD lithium‐treated controls, GFR was slightly lower in untreated PCK rats; however, GFR was elevated in lithium‐treated PCK rats. A hallmark of renal damage, proteinuria, was observed in untreated PCK rats but the severity of proteinuria was dampened in lithium‐treated PCK rats. In conclusion, lithium treatment slows cyst progression in PCK rats and impedes the renal decline associated with progressing PKD, indicating that repurposing lithium may be an effective pharmacotherapy to attenuate advancing PKD.
Effects of endothelial‐specific interference with PPARγ activity in offspring born from AVP‐induced preeclamptic pregnancies
Authors: Nair Anand R , Mukohda Masashi , Agbor Larry N , Wu Jing , Sandgren Jeremy A , Grobe Justin L , Sigmund Curt D
Published: November 2023

PPARγ is a ligand‐activated transcription factor known to regulate metabolic and vascular function. Dominant‐negative (DN) mutations in PPARγ result in hypertension, and synthetic agonists of PPARγ have been shown to reduce blood pressure. Previously we found that mice expressing DN PPARγ driven by an endothelium‐specific promoter (E‐DN) exhibit vascular dysfunction. Preeclampsia (PE) is a hypertensive disorder of pregnancy which programs cardio/metabolic disease in offspring. PE is associated with vascular dysfunction, and we therefore hypothesized a role for endothelial PPARγ in the pathogenesis of PE and its sequelae. To test this, C57BL/6J dams were bred with E‐DN sires, and symptoms of PE were induced by the infusion of vasopressin (AVP, 24 ng/hr, sc) throughout gestation. We assessed phenotypes of PE first in pregnant dams, and then in offspring as adults. Compared to saline infusion (SAL), AVP induced significant elevations in maternal blood pressure (SBP in SAL 107±3 vs AVP 116±3, p<0.05) at gestational day 14–15 and urine protein (SAL 27±4 vs AVP 70±6 mg/mL, p<0.05) at gestational day 18. Offspring to these pregnancies were then genotyped and allowed to grow to adulthood to assess cardio‐metabolic function across three factors (offspring sex, offspring genotype, and maternal exposure to AVP vs SAL). A few notable findings of this ongoing, longitudinal study include: at 9 weeks of age, male NT mice born to AVP‐infused pregnancies had increased body mass compared to mice born from SAL‐infused pregnancies (21.3±0.1g in AVP‐WT vs 18.7±1.1 in SAL‐WT, p<0.05). In utero exposure to AVP significantly increased urinary protein levels in E‐DN (41±2.7 in AVP‐E‐DN vs 28±3.6 in SAL‐E‐DN, p<0.05) but not in NT mice (39±3.0 in AVP‐NT vs 29±2.0 in SAL‐WT). Exposure to AVP in utero significantly decreased proportional body fat in female NT (5.6±0.3% in AVP‐NT vs 8.6±1.5% in SAL‐NT, p<0.05) but not in E‐DN (5.6±0.4% in AVP‐E‐DN vs 7.3±0.6% in Sal‐E‐DN). E‐DN, but not WT, females born to pregnancies complicated by AVP infusion had increased proportional body fluid mass compared to E‐DN born to SAL pregnancies (6.8±0.1% in AVP‐E‐DN vs 6.0±0.0% in SAL‐E‐DN, p<0.05 and 6.5±0.1% in AVP‐NT vs 5.9±0.0% in SAL‐NT, n.s). These data highlight the impact of in utero exposure to elevated AVP upon cardiovascular function in the mother, and some of the consequences of this manipulation upon offspring. In addition, the data highlight the complex interactions that endothelial‐specific loss of PPARγ may have upon offspring health.

Support or Funding Information

NIH and AHA funding
Obese Melanocortin‐4 Receptor (MC4R)‐Deficient Pregnant Rats Have Increased Blood Pressure With Reduced Circulating Placental Growth Factor Levels
Authors: Spradley Frank Travis , Granger Joey P
Published: November 2023

Preeclampsia is a pregnancy‐specific disorder of hypertension, which significantly increases the immediate and long‐term morbidity of mother and offspring. Obesity is a major risk factor for preeclampsia, but the mechanisms linking obesity to this maternal disorder are unknown. Studies in humans have suggested that a greater angiogenic imbalance mediates this increased risk. Preeclampsia is thought to result from placental ischemia and release of anti‐angiogenic factors, such as sFlt‐1, into the maternal circulation where it reduces bioavailability of vascular protective and angiogenic factors, like placental growth factor (PlGF). We have shown previously, in two separate studies, the PlGF prevents placental ischemia‐induced hypertension in rats and that obese MC4R‐deficient rats have increased sFlt‐1 levels and blood pressure during pregnancy. Therefore, we tested the hypothesis that MC4R‐deficient pregnant rats have increased blood pressure with reduced circulating levels of PlGF. MC4R+/+ and MC4R+/− rats were maintained on a standard chow diet and timed‐pregnancies generated at 15 weeks of age using genotyped‐matched males. Carotid catheters were implanted on gestational day 18 for conscious mean arterial pressure (MAP) measurements and plasma collection on day 19. At day 19, absolute body weights were greater in MC4R+/− (n=13) over MC4R+/+ (n=10) pregnant rats (371±9 vs. 340±6 g, P<0.05), as was retroperitoneal adipose tissue mass (13±1 vs. 9±1 g, P<0.05). Circulating levels of leptin (6.7±1.0 vs. 3.5±0.4 ng/mL, P<0.05) were also greater in the obese pregnant rats. MAP was greater in the obese pregnant rats (108±2 vs. 101±1, P<0.05). In addition, circulating sFlt‐1 levels were doubled in the obese pregnant rats (440±128 vs. 252±106 ng/mL) but not in placental tissue (136±10 vs. 143±7 ng/mL). In contrast, the obese MC4R+/− pregnant rats had significantly reduced plasma levels of PlGF (45±10 vs. 121±34 pg/mL, P<0.05) but not in the placenta (496±152 vs. 508±183). In conclusion, these data suggest that the increased blood pressure found in obese MC4R‐deficient pregnant rats is associated with reduced circulating PlGF, and that therapeutic strategies to restore PlGF levels may be a way to reduce the increased incidence of preeclampsia in obese pregnancies.

Support or Funding Information

K99HL130577, HL51971, P20GM104357
Circulating Cell‐free DNA and Maternal Cardiovascular Responses In Healthy Pregnancy And Postpartum
Authors: Goulopoulou Styliani , Sprouse Marc , Phillips Nicole R , Jarvis Sara J , Okada Yoshiyuki , Fu Qi
Published: November 2023

Background

Pregnancy is an inflammatory state that is characterized by vast adaptations of the maternal cardiovascular system. Changes in maternal cardiovascular function ensure adequate blood flow to the fetoplacental unit and inflammation promotes embryo implantation, pregnancy maintenance, and delivery. Cell‐free nucleic acids are increased in various inflammatory conditions and cardiovascular diseases as well as in normal pregnant women. The temporal changes of cell‐free DNA (mitochondrial and nuclear) during normal pregnancy and their effects on maternal cardiovascular adaptations to pregnancy are unknown.

Objectives

To determine the levels of cell‐free DNA in the circulation of pregnant women at different gestational ages and postpartum, and to examine the relationship between circulating cell‐free nucleic acids and maternal cardiovascular parameters.

Methods

Maternal blood samples were collected at early (≤ 8 weeks of gestation) and late (32–36 weeks) pregnancy, and post‐partum (6–10 weeks after delivery) in healthy, normotensive, pregnant women (N=21). All participants were non‐smokers and had no overt history of gestational hypertensive disorders or chronic diseases. Absolute real‐time PCR quantification of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) was performed on whole genomic extracts from maternal serum using TaqMan® probes and chemistry.

Results

Circulating levels of mtDNA in late pregnancy were greater compared to early pregnancy (0.022 ± 1.2 pg/μL vs. 0.04 ± 1.2 pg/μL, p=0.04) and remained elevated post‐partum (0.031 ± 1.2 pg/μL). Circulating levels of nDNA did not significantly change from early to late pregnancy (p=0.14) but they were lower postpartum compared to late pregnancy (31.1 ± 1.2 ng/μL vs. 14.7 ± 1.2 ng/μL, p=0.007). There were no differences between nulliparous (n=10) and multiparous women (n=11) in concentrations of circulating cell‐free nucleic acids (reproductive stage × parity interaction, p=0.27). There were significant correlations between mtDNA and nDNA for each gestational age and postpartum (early pregnancy, r=0.64; late pregnancy, r=0.72; postpartum, r=0.67; p≤0.004). Both blood pressure and heart rate increased from early to late pregnancy and decreased postpartum (p<0.05). MtDNA and nDNA correlated neither with maternal cardiovascular parameters (systolic and diastolic blood pressure, heart rate) nor with neonatal parameters (neonatal length and weight).

Conclusion

Circulating levels of mtDNA and nDNA change with advancing gestational age and may reflect rates of placental cell apoptosis. It is unknown whether cell‐free DNA acts as a signaling molecule and if it plays a role in maintenance of pregnancy.

Support or Funding Information

AHA Scientist Development Grant (13SDG17050056); NIH R21 grant (HL088184); AHA Grant‐in‐Aid (13GRNT16990064); The Harry S. Moss Heart Foundation
20‐HETE Impedes Trophoblast Migration and Contributes to Hypertension in the RUPP Rat Model of Placental Ischemia
Authors: Murphy Sydney , Lee Nicole , Faulkner Jessica , Spencer Shauna‐Kay , Wallace Kedra , LaMarca Babbette
Published: November 2023

Few studies exist on cytochrome P450 (CYP450) metabolites of arachidonic acid (AA) pertaining to the pathophysiological events in pregnancy. Therefore, the goal of this study was to determine a role for 20‐Hydroxyeicosatrienoic acid (20‐HETE), a metabolite of the CYP450 enzyme, in trophoblast proliferation, migration, and hypertension during preeclampsia. Placental vascular CYP450 enzymatic activity and circulating eicosanoids were measured in normal pregnant (NP) and preeclamptic (PreE) patients. To determine a role for 20‐HETE on blood pressure we evaluated the effect of a 20‐HETE synthesis inhibitor, HET0016, on mean arterial pressure (MAP) in NP rats and the reduced uterine perfusion pressure (RUPP) rat model of PreE. PreE women displayed increased placenta 20‐HETE (0.1914 ± 0.025 pmol/min/mg, n=5, p<0.05) compared to NP women (0.1192 ± 0.017 pmol/min/mg, n=7) and increased ratio of circulating 20‐HETE:EETs compared to NP women (p<0.05). Importantly the increase in MAP in response to RUPP in rats (122 ± 3 mmHg) was attenuated with 20‐HETE blockade (112 ± 2 mmHg, p<0.05). To examine the role for CYP450 metabolites in trophoblast function, proliferation and migration assays were performed in the presence of exogenous 20‐HETE and HET0016. Trophoblast proliferation was increased with 20‐HETE (46%, p<0.05) and reduced with 20‐HETE blockade by HET0016 (75%, p<0.05). However, administration of exogenous 20‐HETE decreased trophoblast migration evaluated by scratch assay (p<0.05). In conclusion, alterations in the metabolism of AA, specifically increases in 20‐HETE, may contribute to pathophysiology of Pre by reducing trophoblast cell migration and causing hypertension in response to placental ischemia.

Support or Funding Information

14SDG20160020, 2RO1HD067541
βENaC is Required for Placental Vascular Remodeling in Mice
Authors: Warrington Junie Paula , Spradley Frank T , Chade Alejandro R , Ryan Michael J , Granger Joey P , Drummond Heather A
Published: November 2023

During normal pregnancy, spiral arteries of the maternal uterus remodel into large, low‐resistance vessels to accommodate increasing blood flow to the developing utero‐placental unit. During spiral artery remodeling, endothelial and vascular smooth muscle cells (VSMCs) are replaced by fetal‐derived cytotrophoblasts. We and others have shown that beta Epithelial Na+ Channel protein (βENaC) is required for normal trophoblast migration in vitro. However, whether βENaC mechanistically contributes to trophoblast migration underlying placental vascular remodeling is unknown. In this study, we tested the hypothesis that bENaC is required for normal placental vascular remodeling. To address this, we examined placental vascular morphology and trophoblast/VSMC localization in mice with reduced βENaC expression (βENaCm/m). Mutant and wild‐type (βENaC+/+) controls were mated to generate age‐matched, timed‐pregnant mice. Micro‐computed tomography analysis of gestational day 14.5 placental vascular casts (n = 7–8 placentas) showed reduced spiral artery density (0.4 ± 0.1 vs. 0.7 ± 0.1 per mm2) and mean diameters (185.1 ± 6.9 μm vs. 225.0 ± 8.3 μm; p<0.01) in βENaCm/m mice compared to wild‐type mice respectively. Additionally, the central canal vessel diameter was also reduced in βENaCm/m mice (318.6 ± 11.8 vs. 373.0 ± 19.6 μm). There was no difference in the intervillous space. On gestational day 18.5, another group of placentas were collected for immunofluorescent detection of trophoblast (cytokeratin‐13, CK‐13) and smooth muscle cell (desmin) presence in spiral arteries. In βENaC+/+ mice, we detected cytotrophoblast, but not VSMC marker expression. However, CK‐13 expression was reduced and desmin increased in βENaCm/m placentas. Although pup (0.82 ± 0.04 vs. 0.85 ± 0.06 g) and placenta weights (110 ± 5 vs. 110 ± 3 mg) were similar in mutant and wild‐type mice, respectively, βENaCm/m mice had fewer live pups (5 ± 1 vs. 9 ± 1, p=0.002) on gestational day 18.5. Taken together, these findings suggest that βENaC is required for normal utero‐placental vascular remodeling during pregnancy and mice with reduced βENaC may compensate by having smaller litter size.

Support or Funding Information

Funding: NIH grants P20GM104357, P01HL051971, K99HL129192, and the American Heart Association grant: 13POST16240000.