Donna L. Ralph

Considerations when quantitating protein abundance by immunoblot

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.

Sexual Dimorphic Pattern of Renal Transporters and Electrolyte Homeostasis

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) α and γ 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.

Potassium Supplementation Prevents Sodium Chloride Cotransporter Stimulation During Angiotensin II Hypertension

Angiotensin II (AngII) hypertension increases distal tubule Na-Cl cotransporter (NCC) abundance and phosphorylation (NCCp), as well as epithelial Na+ channel 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-hour 2% potassium meal would lower NCCp in Sprague–Dawley rats after 14 days of AngII (400 ng/kg per minute). The potassium-rich meal neither decreased NCCp nor increased K+ excretion. AngII-infused rats exhibited lower plasma [K+] versus controls (3.6±0.2 versus 4.5±0.1 mmol/L; P<0.05), suggesting that AngII-mediated epithelial Na+ channel 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. Epithelial Na+ channel subunit abundance and cleavage increased 1.5- to 3-fold in both A1K and A2K; ROMK (renal outer medulla K+ channel abundance) abundance was unaffected by AngII 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 epithelial Na+ channel stimulation.

Short‐term nonpressor angiotensin II infusion stimulates sodium transporters in proximal tubule and distal nephron

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.

Renal Collectrin Protects against Salt-Sensitive Hypertension and Is Downregulated by Angiotensin II

Collectrin, encoded by the Tmem27 gene, is a transmembrane glycoprotein with approximately 50% homology with angiotensin converting enzyme 2, but without a catalytic domain. Collectrin is most abundantly expressed in the kidney proximal tubule and collecting duct epithelia, where it has an important role in amino acid transport. Collectrin is also expressed in endothelial cells throughout the vasculature, where it regulates L-arginine uptake. We previously reported that global deletion of collectrin leads to endothelial dysfunction, augmented salt sensitivity, and hypertension. Here, we performed kidney crosstransplants between wild-type (WT) and collectrin knockout (Tmem27Y/- ) mice to delineate the specific contribution of renal versus extrarenal collectrin on BP regulation and salt sensitivity. On a high-salt diet, WT mice with Tmem27Y/- kidneys had the highest systolic BP and were the only group to exhibit glomerular mesangial hypercellularity. Additional studies showed that, on a high-salt diet, Tmem27Y/- mice had lower renal blood flow, higher abundance of renal sodium-hydrogen antiporter 3, and lower lithium clearance than WT mice. In WT mice, administration of angiotensin II for 2 weeks downregulated collectrin expression in a type 1 angiotensin II receptor-dependent manner. This downregulation coincided with the onset of hypertension, such that WT and Tmem27Y/- mice had similar levels of hypertension after 2 weeks of angiotensin II administration. Altogether, these data suggest that salt sensitivity is determined by intrarenal collectrin, and increasing the abundance or activity of collectrin may have therapeutic benefits in the treatment of hypertension and salt sensitivity.

Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling

Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na(+)/H(+) exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na(+)-dependent intracellular pH recovery. We found that 10(-7) M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization.

Paradoxical activation of the sodium chloride cotransporter (NCC) without hypertension in kidney deficient in a regulatory subunit of Na,K‐ATPase, FXYD2

Na,K‐ATPase generates the driving force for sodium reabsorption in the kidney. Na,K‐ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na+ retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K‐ATPase activity was documented in the Fxyd2−/− mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K‐ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide‐sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na+ reabsorption in this segment. Specifically, NCC total increased 30% and NCC phosphorylated at T53 and S71, associated with activation, increased 4‐6 fold. The phosphorylation of the closely related thick ascending limb (TAL) apical NKCC2 (Slc12a1) increased at least twofold. Abundance of the total and cleaved (activated) forms of ENaC α‐subunit was not different between genotypes. Nonetheless, no elevation of blood pressure was evident despite the fact that NCC and NKCC2 are in states permissive for Na+ retention. Activation of NCC and NKCC2 may reflect an intracellular linkage to elevated Na,K‐ATPase activity or a compensatory response to Na+ loss proximal to the TAL and DCT.