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Background: Dietary potassium (K+) has emerged as a modifiable factor for cardiovascular and kidney health in the general population, but its role in people with chronic kidney disease (CKD) is unclear. Here, we hypothesize that CKD increases the susceptibility to the negative effects of low and high K+ diets., Methods: We compared the effects of low, normal and high KChloride (KCl) diets and a high KCitrate diet for 4 weeks in male rats with normal kidney function and in male rats with CKD using the 5/6th nephrectomy model (5/6Nx)., Results: Compared with rats with normal kidney function, 5/6Nx rats on the low KCl diet developed more severe extracellular and intracellular K+ depletion and more severe kidney injury, characterized by nephromegaly, infiltration of T cells and macrophages, decreased estimated glomerular filtration rate and increased albuminuria. The high KCl diet caused hyperkalemia, hyperaldosteronism, hyperchloremic metabolic acidosis and severe hypertension in 5/6Nx but not in sham rats. The high KCitrate diet caused hypochloremic metabolic alkalosis but attenuated hypertension despite higher abundance of the phosphorylated sodium chloride cotransporter (pNCC) and similar levels of plasma aldosterone and epithelial sodium channel abundance. All 5/6Nx groups had more collagen deposition than the sham groups and this effect was most pronounced in the high KCitrate group. Plasma aldosterone correlated strongly with kidney collagen deposition., Conclusions: CKD increases the susceptibility to negative effects of low and high K+ diets in male rats, although the injury patterns are different. The low K+ diet caused inflammation, nephromegaly and kidney function decline, whereas the high K+ diet caused hypertension, hyperaldosteronism and kidney fibrosis. High KCitrate attenuated the hypertensive but not the pro-fibrotic effect of high KCl, which may be attributable to K+-induced aldosterone secretion. Our data suggest that especially in people with CKD it is important to identify the optimal threshold of dietary K+ intake., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

Background: Metabolic acidosis accelerates progression of chronic kidney disease, but whether this is also true for autosomal dominant polycystic kidney disease (ADPKD) is unknown., Methods: Patients with ADPKD from the DIPAK (Developing Interventions to halt Progression of ADPKD) trial were included [n = 296, estimated glomerular filtration rate (eGFR) 50 ± 11 mL/min/1.73 m2, 2.5 years follow-up]. Outcomes were worsening kidney function (30% decrease in eGFR or kidney failure), annual eGFR change and height-adjusted total kidney and liver volumes (htTKV and htTLV). Cox and linear regressions were adjusted for prognostic markers for ADPKD [Mayo image class and predicting renal outcomes in ADPKD (PROPKD) scores] and acid-base parameters (urinary ammonium excretion)., Results: Patients in the lowest tertile of baseline serum bicarbonate (23.1 ± 1.6 mmol/L) had a significantly greater risk of worsening kidney function [hazard ratio = 2.95, 95% confidence interval (CI) 1.21-7.19] compared with patients in the highest tertile (serum bicarbonate 29.0 ± 1.3 mmol/L). Each mmol/L decrease in serum bicarbonate increased the risk of worsening kidney function by 21% in the fully adjusted model (hazard ratio = 1.21, 95% CI 1.06-1.37). Each mmol/L decrease of serum bicarbonate was also associated with further eGFR decline (-0.12 mL/min/1.73 m2/year, 95% CI -0.20 to -0.03). Serum bicarbonate was not associated with changes in htTKV or htTLV growth., Conclusions: In patients with ADPKD, a lower serum bicarbonate within the normal range predicts worse kidney outcomes independent of established prognostic factors for ADPKD and independent of urine ammonium excretion. Serum bicarbonate may add to prognostic models and should be explored as a treatment target in ADPKD., (© The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA.)

Background: Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear., Methods: Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization., Results: Urine particle and creatinine concentrations were highly correlated in the water-loading study ( R 2 0.96) and in random spot urines from healthy subjects ( R 2 0.47-0.95) and patients ( R 2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs., (Copyright © 2021 by the American Society of Nephrology.)

ARNI [dual AT1 (angiotensin II type 1) receptor-neprilysin inhibition] exerts beneficial effects on blood pressure and kidney function in heart failure, compared with ARB (AT1 receptor blockade) alone. We hypothesized that ARNI improves cardiac and kidney parameters in diabetic TGR(mREN2)27 rats, an angiotensin II-dependent hypertension model. Rats were made diabetic with streptozotocin for 5 or 12 weeks. In the final 3 weeks, rats were treated with vehicle, irbesartan (ARB) or irbesartan+thiorphan (ARNI). Blood pressure, measured by telemetry in the 5-week group, was lowered identically by ARB and ARNI. The heart weight/tibia length ratio in 12-week diabetic animals was lower after ARNI compared with after ARB. Proteinuria and albuminuria were observed from 8 weeks of diabetes onwards. ARNI reduced proteinuria more strongly than ARB, and a similar trend was seen for albuminuria. Kidneys of ARNI-treated animals showed less severe segmental glomerulosclerosis than those of ARB-treated animals. After 12 weeks, no differences between ARNI- and ARB-treated animals were found regarding diuresis, natriuresis, plasma endothelin-1, vascular reactivity (acetylcholine response) or kidney sodium transporters. Only ARNI-treated rats displayed endothelin type B receptor-mediated vasodilation. In conclusion, ARNI reduces proteinuria, glomerulosclerosis and heart weight in diabetic TGR(mREN2)27 rats more strongly than does ARB, and this occurs independently of blood pressure., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy., (© 2015 Authors; published by Portland Press Limited.)

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Background: VTP-27999 is a renin inhibitor with an IC50 that is comparable to that of aliskiren, but with a higher bioavailability. Unexpectedly, VTP-27999, unlike aliskiren, did not unfold renin's precursor, prorenin, and increased the affinity of the antibodies applied in renin immunoassays., Methods: Here we verified to what degree these differences affect intracellular renin inhibitor accumulation in renin-synthesizing human mast cells (HMC-1), and (pro)renin's signaling via the (pro)renin receptor ((P)RR) in rat vascular smooth muscle cells. We also addressed the consequences of (P)RR knockdown by small-interfering (si) RNA on (pro)renin release. Finally, making use of FRET(Bodipy-FL)-labeled aliskiren, we studied, by subcellular fractionation, the cellular distribution pattern of this renin inhibitor., Results: VTP-27999 accumulated at higher levels in HMC-1 cells than aliskiren, allowing this inhibitor to block intracellular renin at approximately five-fold lower medium levels. Labeled aliskiren accumulated in mitochondria and lysosomes, and its distribution pattern was different from that of renin. Moreover, the intracellular accumulation of both inhibitors in nonrenin-synthesizing HEK293 cells was not different from that in HMC-1 cells, suggesting that it is renin synthesis-independent. VTP-27999, but not aliskiren, blocked renin's capacity to stimulate extracellular signal-regulated kinase 1/2 phosphorylation in vascular smooth muscle cells, whereas neither inhibitor interfered with prorenin-induced signaling. (P)RR knockdown greatly increased renin (and to a lesser degree, prorenin) release, without affecting the capacity of forskolin or cAMP to stimulate renin release., Conclusion: VTP-27999 differs from aliskiren regarding its level of intracellular accumulation and its capacity to interfere with renin signaling via the (P)RR, and the (P)RR determines prorenin-renin conversion and constitutive (but not regulated) (pro)renin release.

The efficacy of the ACE (angiotensin-converting enzyme) inhibitor perindopril in coronary artery disease [EUROPA (European trial on reduction of cardiac events with perindopril in stable coronary artery disease) study] is associated with the rs12050217 A/G single nucleotide polymorphism in the B1 receptor (bradykinin type 1 receptor) gene. To investigate the underlying mechanism, we examined the effect of this polymorphism on B1-receptor-mediated coronary artery dilation and peripheral blood mononuclear cell activation. Vasorelaxant responses of human coronary microarteries from subjects without coronary disease to des-Arg(9)-bradykinin and to bradykinin were studied in organ bath experiments. Des-Arg9-bradykinin responses were endothelium-dependent and exclusively mediated by B1 receptors, whereas responses to bradykinin were induced through B2 receptors (bradykinin type 2 receptors). The presence of the G allele reduced the response to 3 × 10(-8) mol/l des-Arg(9)-bradykinin by 29% [AA (n=13) compared with AG/GG (n=8); P<0.03], and tended to lower concentration-related responses (P=0.065) to this agonist, whereas the responses to bradykinin were unaffected by the rs12050217 genotype. In freshly obtained human mononuclear cells 1 μmol/l des-Arg(9)-bradykinin increased expression of the pro-inflammatory factors CXCL5 (CXC chemokine ligand 5) and IL6 (interleukin-6). These responses were not affected by genotype and exclusively occurred in blood cells from women, correlating (in the case of CXCL5) with their plasma 17β-oestradiol levels (r(2)=0.32, P=0.02; n=17). IL-1β (interleukin-1β) increased CXCL5 and IL6 expression in both genders, and this response was not associated with 17β-oestradiol levels. The gender difference in responses to B1 receptor stimulation in blood mononuclear cells implies possible gender differences in the response to ACE inhibitor therapy, which needs to be studied more comprehensively. The observed decrease in coronary vasodilator response might contribute to the impaired treatment response to perindopril of G allele carriers found in the EUROPA study.

Red wine polyphenols may preserve endothelial function during aging. Endothelial cell senescence enhances age-related endothelial dysfunction. We investigated whether RWE (red wine extract) prevents oxidative-stress-induced senescence in HUVECs (human umbilical-vein endothelial cells). Senescence was induced by exposing HUVECs to tBHP (t-butylhydroperoxide), and quantified by senescence-associated β-galactosidase staining. RWE (0-50 μg/ml) concentration dependently decreased senescence by maximally 33±7.1%. RWE prevented the senescence-associated increase in p21 protein expression, inhibited tBHP-induced DNA damage of endothelial cells and induced relaxation of PCAs (porcine coronary arteries). Inhibition of SIRT1 (sirtuin 1) by sirtinol partially reversed the effect of RWE on tBHP-induced senescence, whereas both the NOS (nitric oxide synthase) inhibitor L-NMMA (NG-monomethyl-L-arginine) and the COX (cyclo-oxygenase) inhibitor indomethacin fully inhibited it. Furthermore, incubation of HUVECs with RWE increased eNOS (endothelial NOS) and COX-2 mRNA levels as well as phosphorylation of eNOS at Ser1177. RWE protects endothelial cells from tBHP-induced senescence. NO and COX-2, in addition to activation of SIRT1, play a critical role in the inhibition of senescence induction in human endothelial cells by RWE.

Background: Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction., Methods and Results: In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1(d/-) and Xpd(TTD) mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1(d/-) mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1(d/-) mice, age-related endothelium-dependent vasodilator dysfunction in Xpd(TTD) animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity., Conclusions: Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes, with as yet not fully elucidated functions and roles, present in many tissues but also circulating in plasma. The enzyme also functions as an adhesion molecule, the vascular adhesion protein-1. In healthy humans, plasma SSAO activity is constant from birth until 16 years of age, when it drops to lower values, gradually increasing again at advanced ages. When measuring SSAO activity, care should be taken to ensure proper preparation and storage conditions, and it should be realized that quite a few drugs unintentionally are good inhibitors, and sometimes even substrates, of SSAO. Under normal conditions SSAO activity is constant and inter-individual variation is small. In various pathophysiological conditions plasma SSAO activities are increased, most notably in diabetes mellitus (both type I and type II), in congestive heart failure and in cirrhotic liver inflammation. In patients with other vascular and inflammatory diseases plasma SSAO is normal, while it is low in children with congenital lung diseases. Interpretation of these changes is speculative, since source and regulation of plasma SSAO are as yet unknown. However, in two situations where the disease-causing process was ended (transplantation, delivery), plasma SSAO returned to normal. Many questions remain to be answered.

Semicarbazide-sensitive amine oxidase (SSAO) (E.C. 1.4.3.6) is a group of enzymes with as yet poorly understood function which is widely present in nature. The variation in methodology for determination of activity, differences in substrates used and in nomenclature have made it difficult to compare SSAO in different species and tissues. Since SSAO is implicated in the pathophysiology of diabetes mellitus and congestive heart failure, our aim was to analyse the importance and abundance of SSAO in human plasma and tissues compared to other mammals. In plasma of ten different mammals, Vmax values were found to vary more than 10,000-fold, while KM differed much less; in human plasma SSAO activity is relatively low. In some species more than one SSAO entity was present in plasma. SSAO activity was ubiquitous in tissues of human, rat and pig, but varied considerably, both between species and between tissues. In human tissues, SSAO activity is higher than in tissues from rat and pig. Relative to monoamine oxidase-B there is also wide variation in SSAO, with much higher relative activities in human than in rat and pig tissues. We conclude that in plasma, SSAO activity is highest in ruminants, while in tissues, SSAO activity is more prominently present in human than in rat and pig.

Atrial natriuretic peptide (ANP) has become an important parameter for assessing the condition of patients with cardiac disease. Recently, attention has also focused on N-terminal pro-atrial natriuretic peptide (NtproANP) in this context. NtproANP circulates in plasma in higher concentration, is more stable ex vivo, and may be a better parameter for cardiac function over time. We have evaluated a new commercially available radiommunoassay kit for NtproANP and compared results and method with those of ANP measurements. The NtproANP kit was found to be reliable and easy to use (no plasma extraction step is necessary), with good reproducibility (coefficients of variation 7-15%). Normal values in 15 healthy laboratory workers, 25 healthy elderly subjects and 25 patients with heart failure were 207 +/- 70, 368 +/- 134 and 1206 +/- 860 pmol/l, respectively, 8.3, 11.8 and 13.0 times higher, respectively, than corresponding ANP concentrations. NtproANP correlated well with ANP (r 0.64-0.78). We conclude that plasma NtproANP measurement may be a good alternative to plasma ANP measurement: technically, it is easier to perform, and NtproANP is more stable in plasma. Whether NtproANP is a better diagnostic and prognostic parameter than ANP remains to be further established.

We have evaluated a new commercially available ELISA kit for determination of plasma chromogranin A with respect to its usefulness in the diagnosis of neuroendocrine tumors, mainly pheochromocytoma. Serum and differently anticoagulated plasmas gave different chromogranin A concentrations. Control values (n = 21) were 18.9 +/- 5.8 units/l. Chromogranin A values > 30.4 units/l (mean + 2 S.D.) were considered elevated. In 22 patients suspected of (but found not to have) pheochromocytoma and in 24 patients with renovascular hypertension, 18% were found to have elevated chromogranin A concentrations. In renovascular hypertension chromogranin A correlated positively with serum creatinine; chromogranin A was strongly elevated especially in chronic renal failure. In 45 patients with pheochromocytoma, 13 (29%) had chromogranin A concentrations within the normal range, as had 3 out of 11 patients with neuroblastoma (27%). In 13 pheochromocytoma patients with elevated chromogranin A, measurements were repeated after surgical removal of the tumor; values then all fell within the normal range. We conclude that measurement of chromogranin A adds little to already existing methods for the diagnosis of pheochromocytoma.