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.

Extracellular fluid (ECF) [K+] is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+ deficient diet (0K) versus 1% K+ diet (1K) for 10 days (n=5-6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporters' adaptations in 0K vs. 1K fed mice, assessed by immunoblot, included decreased sodium pump a2-b2 subunits, decreased K+- Cl-- cotransporter isoform 3, and increased phosphorylated (p) Na+-K+-2 Cl- cotransporter isoform 1 (NKCC1p), Ste20/SPS-1 related proline-alanine rich kinase (SPAKp) and oxidative stress responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters' adaptations, effecting a 98% reduction in K+ excretion, included 2-3-fold increased phosphorylated Na+-Cl- cotransporter (NCCp), SPAKp and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 mmol K+ into the urine and muscle shifts ~47 mmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency.

Raising blood pressure stimulates pressure natriuresis (P-Nat). In males (M) Sprague Dawley rats (SDR), Na + reabsorption (T Na ) is acutely reduced by retraction of proximal tubule (PT) NHE3 to microvillar base and NaPi2 internalization. In females (F), at baseline PT NHE3 is already at microvillar base and NaPi2 is less abundant than in M. We AIM to determine characteristics and mechanisms of P-Nat in F (vs M) rats. Methods: Inactin anesthetized F and M SDR (n=5/group) were provoked by vasoconstriction (or sham). Mean arterial pressure (MAP) was recorded via carotid artery, urine collected via bladder, Na + transporters’ abundance assessed via immunoblot and localization by immunohistochemistry. Results (Fig 1A): Baseline MAP (mmHg) was lower in F vs. M (91 ± 5 vs.105 ± 3, P =0.04) while urine volume (UV) and electrolyte excretion (UNaV, UKV) were similar. After celiac and mesenteric bed constriction, MAP rose to 128 ± 3 mmHg in both sexes; UNaV, UV and C Na increased 12 to 15-fold in F (all P <0.01) vs 6-fold in M ( P >0.08). Constriction of abdominal aorta further raised UNaV in F with less impact in M. Na + transporters . In F, NHE3 remained at PT microvillar base and NaPi2 was internalized with vasoconstriction. NHE3P (indicating inactivation) abundance increased 29% in F, P =0.058. Lithium clearance, measure of volume flow leaving early nephron, increased 9-fold in F ( P =0.02) vs. 5-fold in M ( P =0.07). F mTAL NHE3, NKCC2p, and SPAKp (co-transporter kinase) abundances were 22, 24, and 43% lower vs shams ( ANOVA P <0.0001). Summary: F vs M SDR exhibit more robust P-Nat associated with less T Na in early nephron and reductions in PT-mTAL Na + transporters, consistent with higher UNaV at any given BP (Fig 1B).

When chronic kidney disease progresses to end stage renal disease, the optimal treatment is living kidney donation (LKD). A 10 yr prospective study of 1214 donors (PMID 27006347), reported that LKD increased risk of hypertension (HTN), by 3.64-fold, as well as HTN induced albuminuria. Our objective was to test whether a DASH-style diet (DD: 0.74% NaCl, 3%K with 1% each: Cl - , citrate, HCO 3 - ) would blunt hypertension and albuminuria following UNX vs. a western style diet (WD: 2% NaCl, 1%KCl). Uninephrectomized (UNX) male SD rats (n=5/group) preequilibrated on diets were analyzed 12-14 wks post-UNX. Systolic BP (SBP, mmHg) was similar pre-UNX in DD (126 ± 8) and WD (132 ± 8); SBP increased post-UNX by 7 ± 2 (DD) and 21 ± 5 (WD) mmHg, p=0.04. Urine injury markers (albumin, angiotensinogen, KIM-1 and plasminogen) increased in both DD and WD (all p<0.01). Kidney weight increased similarly from 1.5 ± 2 (pre-UNX) to 2.3 ± 0.2 g (post-UNX) in pooled DD, WD. By IHC, glomeruli diameters increased 1.6-fold pre-vs. post-UNX. Transporter profiles were generated from both pre-and post-UNX kidneys. Pre-UNX, DD (vs. WD) exhibited lower abundance of proximal tubule (PT) NHE3, NaPi2 and NHERF1, and higher abundance of NKA α1, NCC, NCCp, claudin 7, SPAK, ENaC subunits and AQP2 (all p < 0.01). Post-UNX (vs. pre-UNX) abundance, normalized to equivalent protein, exhibited lower claudin2, megalin, AQP1 and AQP2 in both DD and WD (all p < 0.01), and, specifically to DD post-UNX: 1.4-fold increases in NCCp, SPAKp and 0.7-fold decreases in cleaved α and γ ENaC (all p<0.05). By IHC, NHE3 localized within the apical microvilli in DD and WD pre- and post-UNX. Lithium clearance (CLi, ml/min/Kg BW) increased post-UNX in DD (p=0.006) not WD; post UNX CLi was higher in DD (0.075 ± 0.013) vs WD (0.042 ± 0.009), p=0.036. In conclusion, the phenotype of HTN and proteinuria observed following LKD in humans is evident in this pilot SD rat study. Compared to the 2% NaCl/1% KCl “Western style” diet, the 3% K + and alkali rich “DASH style diet” lowered baseline PT transporter levels, increased CLi (indicator of flow out of PT), elevated distal Na + transporters (likely due to flow from PT) and blunted the rise in BP post-UNX. The findings suggest that DASH style dietary electrolytes may improve outcomes following LKD.

Experimental AngII hypertension is reported to be blunted in female (F) vs. male (M) mice and in mice lacking the inflammatory cytokine IL17A (time point dependent). This study aimed to determine how sex impacts renal Na+ transporters, Na+ and K+ excretion, and markers of renal injury during AngII hypertension (A) in both C57Bl/6 (WT) and IL17A−/− (IL17−/−) mice.11–16 wk old F and M mice were infused with AngII (490 ng/kg/min) or sham treated for 3 wk (n=6/group). Rate of excretion of a saline load (10% b.wt. subcutaneous) was slowed in all AngII infused groups, indicating Na+ reabsorption activation. Blood pressure (BP), by tail cuff, increased similarly across groups (in Δ mmHg): WTMA: 40 ± 1.6, WTFA: 37 ± 10, IL17−/−MA: 37 ± 3.8, IL17−/−FA: 46.4 ± 4.7. Thus, no blunting of hypertension was detected at 3 wk time point by tail cuff in F or in IL17−/−.In both M and F WT and IL17−/−, AngII provoked activation (‐phosphorylation) of NKCC2 and NCC, and activation of ENaC (more ‐cleaved (Cl), less full length (FL)). These distal anti‐natriuretic responses were balanced by natriuretic responses including: decreased abundance of proximal NHE3, NaPi2, and decreased medullary thick ascending limb NHE3, NKCC2 and NaK‐ATPase (see Figure). We previously proposed that increased ‐NCCp is a secondary compensation, driven by AngII activation of ENaC, to minimize K+ secretion (PMID27600183). Providing further support for this idea, urinary Na/K ratio (UNa/K), collected overnight (during fasting +water), was significantly reduced by AngII (WTM: from 0.71 ± 0.1 at baseline to 0.41 ± 0.9 with AngII, WTF: 0.80 ± 0.1 to 0.28 ±0.05, IL17−/−M: 0.99 ±0.13 to 0.41 ± 0.041, IL17−/−F: 1.05 ± 0.07 to 0.46 ± 0.46). UNa/K fell due to decreased o/n UNa as o/n UK was unaltered by AngII. Urine volume (V), unaltered by AngII treatment, was 2‐fold higher in M vs. F of both genotypes, and 2‐fold higher in IL17−/− vs. WT within each sex.During AngII, the injury marker U albumin increased across groups. Renal cortical angiotensinogen was lower in IL17−/− vs. WT (by 50% in F and 25% in M). KIM1 also tended to be lower in IL17−/− vs. WT in both sexes during AngII.From these results, we propose: AngII stimulation of ENaC drives UK secretion which activates NCCp (more in WT than IL17−/− males), thus reducing UNa to limit K secretion and lowering UNa/K which may contribute to BP elevation. IL17−/− mice exhibit higher UV and lower intrarenal angiotensinogen and KIM1 which suggests less tissue injury.Support or Funding InformationAHA GIA (15GRNT23160003) and an NIH NIDDK R01 (DK083785) to A.A. McDonough, NIH NHLBI K08 award (HL121671), Gilead Cardiovascular Scholars Grant, and NIH DP2 award (HL137166) to M.S. MadhurThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.