Compared with males, females have lower BP before age 60, blunted hypertensive response to angiotensin II, and a leftward shift in pressure natriuresis. This study tested the concept that this female advantage associates with a distinct sexual dimorphic pattern of transporters along the nephron. We applied quantitative immunoblotting to generate profiles of transporters, channels, claudins, and selected regulators in both sexes and assessed the physiologic consequences of the differences. In rats, females excreted a saline load more rapidly than males did. Compared with the proximal tubule of males, the proximal tubule of females had greater phosphorylation of Na/H exchanger isoform 3 (NHE3), distribution of NHE3 at the base of the microvilli, and less abundant expression of Na/Pi cotransporter 2, claudin-2, and aquaporin 1. These changes associated with less bicarbonate reabsorption and higher lithium clearance in females. The distal nephrons of females had a higher abundance of total and phosphorylated Na/Cl cotransporter (NCC), claudin-7, and cleaved forms of epithelial Na channel (ENaC) and subunits, which associated with a lower baseline plasma K concentration. A K-rich meal increased the urinary K concentration and decreased the level of renal phosphorylated NCC in females. Notably, we observed similar abundance profiles in female versus male C57BL/6 mice. These results define sexual dimorphic phenotypes along the nephron and suggest that lower proximal reabsorption in female rats expedites excretion of a saline load and enhances NCC and ENaC abundance and activation, which may facilitate K secretion and set plasma K at a lower level.

Aim: Sexual dimorphisms are evident along the nephron: Females (F) exhibit higher ratios of renal distal to proximal Na + transporters' abundance, greater lithium clearance (C Li ) more rapid natriuresis in response to saline infusion and lower plasma [K + ] vs. males (M). During angiotensin II infusion hypertension (AngII-HTN) M exhibit distal Na + transporter activation, lower proximal and medullary loop transporters, blunted natriuresis in response to saline load, and reduced plasma [K + ]. This study aimed to determine whether responses of F to AngII-HTN mimicked those in M or were impacted by sexual dimorphisms evident at baseline. Methods: Sprague Dawley rats and C57BL/6 mice were AngII infused via osmotic minipumps 2 and 3 weeks, respectively, and assessed by metabolic cage collections, tail-cuff sphygmomanometer, semi-quantitative immunoblotting of kidney and patch-clamp electrophysiology. Results: In F rats, AngII-infusion increased BP to 190 mm Hg, increased phosphorylation of cortical NKCC2, NCC and cleavage of ENaC two to threefold, increased ENaC channel activity threefold and aldosterone 10-fold. K + excretion increased and plasma [K + ] decreased. Evidence of natriuresis in F included increased urine Na + excretion and C Li , and decreased medullary NHE3, NKCC2 and Na,K-ATPase abundance. In C57BL/6 mice, AngII-HTN increased abundance of distal Na + transporters, suppressed proximal-medullary transporters and reduced plasma [K + ] in both F and M. Conclusion: Despite baseline sexual dimorphisms, AngII-HTN provokes similar increases in BP, aldosterone, distal transporters, ENaC channel activation and K + loss accompanied by similar suppression of proximal and loop Na + transporters, natriuresis and diuresis in females and males. K E Y W O R D S angiotensin II, ENaC, female, potassium, proteinuria, sodium transport 2 of 12 | VEIRAS Et Al.

The development of the immunoblot to detect and characterize a protein with an antisera, even in a crude mixture, was a breakthrough with wide-ranging and unpredictable applications across physiology and medicine. Initially, this technique was viewed as a tool for qualitative, not quantitative, analyses of proteins because of the high number of variables between sample preparation and detection with antibodies. Nonetheless, as the immunoblot method was streamlined and improved, investigators pushed it to quantitate protein abundance in unpurified samples as a function of treatment, genotype, or pathology. This short review, geared at investigators, reviewers, and critical readers, presents a set of issues that are of critical importance for quantitative analysis of protein abundance: 1) Consider whether tissue samples are of equivalent integrity and assess how handling between collection and assay influences the apparent relative abundance. 2) Establish the specificity of the antiserum for the protein of interest by providing clear images, molecular weight markers, positive and negative controls, and vendor details. 3) Provide convincing evidence for linearity of the detection system by assessing signal density as a function of sample loaded. 4) Recognize that loading control proteins are rarely in the same linear range of detection as the protein of interest; consider protein staining of the gel or blot. In summary, with careful attention to sample integrity, antibody specificity, linearity of the detection system, and acceptable loading controls, investigators can implement quantitative immunoblots to convincingly assess protein abundance in their samples.

The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.

Intrauterine and postnatal zinc restriction may result in an adverse environment for the development of cardiovascular and renal systems. This study evaluated the effects of moderate zinc deficiency during fetal life, lactation, and/or postweaning growth on systolic blood pressure, renal function, and morphology in adult life. Female Wistar rats received low (8 ppm) or control (30 ppm) zinc diets from the beginning of pregnancy up to weaning. After weaning, male offspring of each group of mothers were fed low or control zinc diet. Systolic blood pressure, creatinine clearance, proteinuria, renal morphology, renal apoptosis. and renal oxidative stress state were evaluated after 60 days. Zinc deficiency during pre- and postweaning growth induced an increase in systolic blood pressure and a decrease in the glomerular filtration rate associated with a reduction in the number and size of nephrons. Activation of renal apoptosis, reduction in catalase activity, glutathione peroxidase activity, and glutathione levels and increase in lipid peroxidation end products could explain these morphometric changes. Zinc deficiency through pre- and postweaning growth induced more pronounced renal alteration than postweaning zinc deficiency. These animals showed signs of renal fibrosis, proteinuria, increased renal apoptosis, and higher lipid peroxidation end products. A control diet during postweaning growth did not totally overcome renal oxidative stress damage, apoptosis, and fibrosis induced by zinc deficiency before weaning. In conclusion, zinc deficiency during a critical period of renal development and maturation could induce functional and morphological alterations that result in elevated blood pressure and renal dysfunction in adult life.

Ang II hypertension increases distal tubule Na-Cl cotransporter (NCC) abundance and phosphorylation (NCCp), as well as epithelial Na+ channel (ENaC) abundance and activating cleavage. Acutely raising plasma [K+] by infusion or ingestion provokes a rapid decrease in NCCp that drives a compensatory kaliuresis. The first aim tested whether acutely raising plasma [K+] with a single 3 hr 2% potassium meal would lower NCCp in Sprague Dawley rats after 14 days of AngII (400 ng/kg/min). The potassium-rich meal neither decreased NCCp nor increased K+ excretion. AngII infused rats exhibited lower plasma [K+] versus controls (3.6 ± 0.2 vs. 4.5 ± 0.1 mmol/L, p < 0.05) suggesting that Ang II mediated ENaC activation provokes K+ depletion. The second aim tested whether doubling dietary potassium intake from 1% (A1K) to 2% (A2K) would prevent K+ depletion during AngII infusion and, thus, prevent NCC accumulation. A2K fed rats exhibited normal plasma [K+] and 2-fold higher K+ excretion and plasma [aldosterone] versus A1K. In A1K rats, NCC, NCCpS71, and NCCpT53 abundance increased 1.5- to 3-fold versus controls (p< 0.05). The rise in NCC and NCCp abundance was prevented in the A2K rats, yet blood pressure did not significantly decrease. ENaC subunit abundance and cleavage increased 1.5- to 3-fold in both A1K and A2K; ROMK abundance was unaffected by Ang II or dietary K+. In summary, the accumulation and phosphorylation of NCC seen during chronic AngII infusion hypertension is likely secondary to potassium deficiency driven by ENaC stimulation.

In Sprague Dawley rats, 2-week angiotensin II (AngII) infusion increases Na+ transporter abundance and activation from cortical thick ascending loop of Henle (TALH) to medullary collecting duct (CD) and raises blood pressure associated with a pressure natriuresis, accompanied by depressed Na+ transporter abundance and activation from proximal tubule (PT) through medullary TALH. This study tests the hypothesis that early during AngII infusion, before blood pressure raises, Na+ transporters’ abundance and activation increase all along the nephron. Male Sprague Dawley rats were infused via osmotic minipumps with a subpressor dose of AngII (200 ng/kg/min) or vehicle for 3 days. Overnight urine was collected in metabolic cages and sodium transporters’ abundance and phosphorylation were determined by immunoblotting homogenates of renal cortex and medulla. There were no significant differences in body weight gain, overnight urine volume, urinary Na+ and K+ excretion, or rate of excretion of a saline challenge between AngII and vehicle infused rats. The 3-day nonpressor AngII infusion significantly increased the abundance of PT Na+/H+ exchanger 3 (NHE3), cortical TALH Na-K-2Cl cotransporter 2 (NKCC2), distal convoluted tubule (DCT) Na-Cl cotransporter (NCC), and cortical CD ENaC subunits. Additionally, phosphorylation of cortical NKCC2, NCC, and STE20/SPS1-related proline–alanine-rich kinase (SPAK) were increased; medullary NKCC2 and SPAK were not altered. In conclusion, 3-day AngII infusion provokes PT NHE3 accumulation as well as NKCC2, NCC, and SPAK accumulation and activation in a prehypertensive phase before evidence for intrarenal angiotensinogen accumulation.

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na reabsorption via activation of epithelial Na channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (PRR-KO). At basal conditions, PRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg·min), the increases in systolic BP and diastolic BP were mitigated in PRR-KO mice.PRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from PRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.