The Role Of (Pro) Renin Receptor In Fructose-induced Salt-sensitive Hypertension And Potassium Homeostasis

Background(Pro)renin receptor(PRR) as a newly discovered component of the renin-angiotensin system(RAS), was firstly discovered by Nguyen et al in 2002, which binds renin and prorenin to respectively increase renin activity and induce non-proteolytic activation of prorenin. Within the kidney, immunofluorescence demonstrated that PRR is detected in the renal vasculature, distal convoluted tubules(DCT), proximal tubules, connecting tubules, and collecting ducts. Three molecular forms of PRR have been identified: the full- length form(f PRR), a soluble form with a N-terminal region(s PRR), and a truncated transmembrane form with a C-terminal region(8.9 k Da called M8.9). Although the controversy mostly stems from the observations that PRR is associated with vacuolar H~+-ATPase and Wnt/β-catenin signaling pathway and involved in embryogenesis both in mammals and low vertebrates, increasing evidence favors the renin-regulatory role of PRR in the kidney and brain. Systemic PRR knockout mice are fatal, there are two PRR inhibitors, including the handle region decoy peptide(HRP, 10-19 amino acid residues of the prorenin prosegment, R10IPLKKMPSV19) and the first 20 amino acid residues of the prorenin prosegment(PRO20, L1PTDTASFGRILLKKMPSVR20). Compared to HRP, PRO20 is a newly developed PRR decoy inhibitor, which can specific bind to PRR and more effectively attenuate hypertension induced by Ang II or DOCA-salt via XII inhibiting the activation of the local renin-angiotensin systemSodium(Na~+) and potassium(K~+), the main intracellular cations, maintaining total body Na~+ and K~+ balance is essential to the survival of most species. Aldosterone is thought to primarily derive from the zona glomerulosa of the adrenal cortex and be critical for electrolyte homeostasis. Both low salt and high salt increased renal PRR expression as well as aldosterone secretion, overexpression of human PRR in transgenic rats elevates aldosterone production, these results implicat a role of PRR in regulation of renin-angiotensin–aldosterone system(RAAS). Resent studies sho wed renal PRR regulated epithelial Na~+ channel(ENa C) expression and activity. O verall, these results further suggest that a potential role of PRR in regulation of electrolyte metabolism. ObjectiveBasing on the background, we hypothesized that renal PRR may regulate electrolyte homeostasis through an influence on local RAS. Thus, in this study, we performed high fructose(HF) intake animal modle and high K~+(HK) intake modle, all of which affact electrolyte balance, the primary goal of this study was to te st whether HF/HK influenced PRR expression and renal RAS, and the function and possible molecular mechanism of PRR in Na~+ and K~+ homeostasis. MethodsPart I: The role of(pro)renin receptor in fructose-induced salt-sensitive hypertension(1) Normal Sprague-Dawley(SD) rats were treated with 20% fructose in drinking water, then we examined the expression of renal PRR using western blotting(WB) and real-time quantitative polymerase chain reaction(RT-q PCR), and the plasma and urinary s PRR using enzyme linked immunosorbent assay(ELISA).(2) We continued to treat normal SD rats with 20% fructose drinking water, and simultaneously treated with PRO20 by subcutaneous injection, then detected the expression of sodium/hydrogen exchanger 3(NHE3) and Na/K/2C l cotransporter(NKCC2) using WB and RT-q PCR. ELISA was performed to detect the level of plasma and urinary s PRR, Ang II, aldosterone, renin activity, and renin content. In vivo NKCC2 activity was determined as reflected by rapid diuresis and natriuretic responses to furosemide. Urine and plasma electrolytes(Na~+, K~+, and C l-) and osmolality were determined with an automatic analyzer.(3) Normal SD rats were fed with 20% fructose drinking water, and simultaneously treated with allopurinol in drinking water. Plasma and urinary uric acid(UA), s PRR, aldosterone, renin activity, and renin content were determined using ELISA. K idney PRR, N HE3, and NKCC2 expression were examined using WB and RT-q PCR. In vivo NKCC2 activity, urine and plasma electrolytes and osmolality were also detected as described above.(4) All rats, including PRO20 and allopurinaol treated rats, were implanted telemetry transmitters and treated with a high-salt diet(8% Na C l) along with normal drinking water or 20% fructose in drinking water fo r 14 days. Blood pressure was monitored continuously throughout the experimental period.(5) Human renal proximal tubule epithelial cells(HK2) were cultured and treated with fructose, allopurinol was empolyed. PRR protein expression were detected using WB, the levels of s PRR in medium were examined using ELISA.Part II: The role of(pro)renin receptor in potassium homeostasis(1) Normal SD rats were fed with HK diet(5%, 92 g normal diet ~+ 8 g KC l), then we examined the expression of renal PRR using WB and RT-q PCR, and the plasma and urinary s PRR and aldosterone using ELISA.(2) Next, we continued to treat normal SD rats with HK diet, and simultaneously treated with PRO20 by subcutaneous injection, then detected the expression of PRR, Cytochrome P450, family 11, subfamily B, polypeptide 2(CYP11B2), ENa C, Na~+-Clcotransporter(NCC), renal outer medullary K~+ channel(ROMK), large- conductance calcium- activated K~+ channel(BK) and Alpha Na~+-K~+-ATPase(α-NKA) using WB, the expression of NCC in DCT was also tested by immunofluorescence. ELISA was performed to detect the level of plasma and urinary s PRR, aldosterone, renin activity, and renin content. Urine and plasma electrolytes(Na~+, K~+, and C l-) were determined with an automatic analyzer.(3) To further verifed the regulation role of renal PRR in renal-derived aldosterone release and K~+ homeostasis, all rats were subjected to right uninephrectomy and then to bilateral adrenalectomy(ADX), and then treated with HK diet and PRO20 by intrarenal infusion. Renal PRR, CYP11B2, ENa C, NCC, ROMK, BK, and α-NKA expression, urine and plasma electrolytes, s PRR, aldosterone, renin activity, and renin content, were detected using the methods as above described, respectively.(4) To further confirm the existence and importance of intrare nal aldosterone in K~+ homeostasis, ADX rats were given HK diet and simultaneously treated with spironolactone, urine and plasma electrolytes and the expression of ion channels as above described were detected.(5) Collecting ducts were important for kidney regulating K~+ homeostasis, so we tested whether HK influenced PRR and CYP11B2 expression in primary rat inner medullary collecting duct(IMCD) cells. IMCD cells were treated with 10 m M K~+, PRO20 and PRR si RNA were employed, PRR and CYP11B2 protein express ion were detected using WB, the levels of s PRR, aldosterone, and renin in medium were examined using ELISA. ResultsPart I: The role of(pro)renin receptor in fructose-induced salt-sensitive hypertension(1) HF intake promoted renal f PRR and s PRR protein and m RNA expression, urinary s PRR excretion, and plasma s PRR concentration.(2) HF intake increased urinary Ang II and aldosterone excretion, upregulated urinary renin activity and renin content, promoted kidney NHE3 and NKCC2 protein and m RNA expression, enhanced in vivo NKCC2 activity as reflected by rapid diuresis and natriuretic responses to furosemide, both of them were attenuated by PRO20 treatment without affacting plasma Ang II, aldosterone, renin activity, and renin content.(3) Fructose feeding or a high-salt diet alone did not affect mean arterial pressure, but the combination of the two elevated it by about 10 mm Hg, which was blunted by PRO20 treatment(4) Fructose- induced endogenous UA production was inhibited by allopurinol, accompany with downregulation of PRR, NHE3 and NKCC2 expression, urinary Ang II and aldosterone excretion, urinary renin activity and renin content, and in vivo NKCC2 activity, without affacting plasma Ang II, aldosterone, renin activity, and renin content.(4) HF- induced salt-sensitive hypertension was completely blocked by al opurinol.(5) In cultured HK2 cells, HF promoted f PRR and s PRR protein expression in a time- and dose- dependent manner, in parallel with an increase of medium s PRR level.Part II: The role of(pro)renin receptor in potassium homeostasis(1) HK intake increased renal f PRR protein expression and urinary s PRR excretion, but decreased plasma s PRR concentration, without affecting renal PRR m RNA level.(2) In normal SD rats, administration of PRO20 in K~+- loaded animals elevated plasma K~+ level and decreased urinary K~+ excretion, accompanied with suppressed urinary aldosterone excretion. HK intake significantly upregulated renal CYP11B2, ENa C, ROMK, BK, and α-NKA protein expression and downregulation of NCC, all of which were blunted by PRO20.(3) In ADX rats, HK intake promoted renal f PRR expression and urinary s PRR excretion without affecting by intrarenal PRO20 infusion. Intrarenal PRO20 infusion blocked HK- induced urinary aldosterone excretion, upregulation of renal CYP11B2, ENa C, ROMK, BK, and α-NKA protein expression and downregulation of NCC, accompanied with a further increase of plasma K~+ level.(4) In ADX rats, spironolactone treatment further increased plasma K~+ and urinary Na~+ excretion, decreased plasma Na~+ and urinary K~+ excretion, accompanied with an inhibition of renal ENa C, ROMK, BK, and α-NKA protein expression, without affacting urinary aldosterone excretion.(5) In primary rat IMCD cells, high K~+ but not C l- augmented f PRR protein expression and s PRR release in a time- and dose- dependent manner, in parallel with an increase of medium renin activity. HK also upregulated aldosterone secretion in parallel with an increase of CYP11B2 protein expression, both of them were attenuated by PRR si RNA or PRO20. Prorenin upregulated aldosterone release and CYP11B2 expression, both of which were inhibited by PRR si RNA. A recombinant s PRR(s PRR-His) also stimulated aldosterone release and CYP11B2 expression. ConclusionsIn this study,(1) High fructose promoted renal PRR expression by increasing renal UA production, then activates renal RAS, which may regulate NHE3 and NKCC2 expression and activity, and in turn regulates Na~+ reabsorption that contributes to the development of salt-sensitive hypertension.(2) High K~+- induced renal PRR expression regulates renal CYP11B2 expression and aldosterone synthesize and release via the activaton of renal RAS, which in turn regulates ENa C, NCC, ROMK, BK, and α-NKA expression to maintain K~+ homeostasis.This finding is significant because it implicates the function and regulation mechanism of local RAS, and the role of PRR in regulation of local RAS and electrolyte homeostasis, which may be a novel concept of RAS and regulation of electrolyte homeostasis.