KV7.1 channel blockade inhibits neonatal renal autoregulation triggered by a step decrease in arterial pressure

KV7, the voltage-gated potassium channels encoded by KCNQ genes, mediate heterogeneous vascular responses in adult rodents. Postnatal changes in the functional expression of KV7 channels have been reported in rodent saphenous arteries, but their physiological function in the neonatal renal vascular bed is unclear. Here, we report that, unlike adult pigs, only KCNQ1 (KV7.1) out of the five members of KCNQ genes was detected in neonatal pig renal microvessels. KCNQ1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. Activation of the renal vascular smooth muscle cell (SMC) KV7.1 stimulated whole-cell currents, inhibited by HMR1556 (HMR), a selective KV7.1 blocker. HMR did not change the steady-state diameter of isolated renal microvessels. Similarly, intrarenal artery infusion of HMR did not alter the mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) in the pigs. An approximately 20 mmHg reduction in the MAP evoked effective autoregulation of the RBF, which HMR inhibited. We conclude that 1) The expression of KCNQ isoforms in porcine renal microvessels is dependent on kidney maturation, 2) KV7.1 is functionally expressed in neonatal pig renal vascular SMCs, 3) a decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs, and 4) SMC KV7.1 does not control basal renal vascular tone but contributes to neonatal renal autoregulation triggered by a step decrease in arterial pressure.

P0549ESSENTIAL ROLES OF TESTOSTERONE IN MALE KIDNEY MATURATION AND REPAIR AFTER INJURY

Background and Aims The newborn kidneys are structurally and functionally immature. They mature after birth to adapt to extra-uterine life, yet the comprehensive changes of the kidney have not been defined. We investigated the structural and functional changes in the kidney after weaning and examined the effect of testosterone in this maturation process in male mice. Method We performed phenotypic analysis of male mouse kidneys as well as those subjected to castration utilizing a combination of histological analysis, bulk transcriptome analysis, charged metabolite analysis by CE-TOFMS and brush border membrane vesicles LC-MS/MS analysis. Results Bulk transcriptome analysis of postnatal 3 and 8 week male kidneys showed the gene sets enriched in 8w included those associated with metabolic process and transporters in proximal tubule, and structural analysis revealed proximal tubule elongation and hypertrophy in 8 weeks. Most differentially expressed genes (DEGs) identified above were induced between postnatal day 28 and 40, a period of testosterone surge, and were closely correlated with DEGs between adult male and female kidneys. These structural and gene expression changes during maturation were mostly cancelled by castration before testosterone surge. Proximal tubules expressed androgen receptor, and some of these DEGs were upregulated or downregulated by the administration of testosterone in cultured proximal tubules. Consistent results with DEGs were also confirmed at protein levels and related metabolites in vivo. For instance, induction of cystine transporter SLC7A13 expression as well as the increase in cysteine and cysteine-glutathione disulphide-Divalent in the kidney were confirmed during this period. Fatty acid β-oxidation enzymes were also increased during this period. Some of the down-regulated genes across puberty were reactivated after injury and returned to the basal levels with repair. Conclusion Proximal tubules are the main site of maturation after weaning, and acquire the abilities to absorb and metabolize a variety of filtered substances and to increase energy metabolism, which were mainly driven by testosterone.

Adenosine A1 receptor-operated calcium entry in renal afferent arterioles is dependent on postnatal maturation of TRPC3 channels

Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca2+ concentration ([Ca2+]i) elevation in smooth muscle cells (SMCs) via activation of its cognate A1 receptors (A1Rs). Mechanisms that underlie A1R-dependent [Ca2+]i elevation in renal vascular SMCs are not fully resolved. Whether A1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of A1Rs by 2-chloro- N6-cyclopentyladenosine (CCPA) does not stimulate store-operated Ca2+ entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca2+]i elevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca2+]i was unchanged in afferent arterioles isolated from newborn (0-day-old) pigs compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca2+]i in afferent arterioles from 20-day-old pigs. A1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0- vs. 20-day-old pigs. However, the TRPC3 channel protein expression level was ~92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of A1Rs elicits receptor-operated Ca2+ entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of A1Rs.

Dietary-induced gestational iron deficiency inhibits postnatal tissue iron delivery and postpones the cessation of active nephrogenesis in rats

Gestational iron deficiency (ID) can alter developmental programming through impaired nephron endowment, leading to adult hypertension, but nephrogenesis is unstudied. Iron status and renal development during dietary-induced gestational ID (<6 mg Fe kg–1 diet from Gestational Day 2 to Postnatal Day (PND) 7) were compared with control rats (198 mg Fe kg–1 diet). On PND2–PND10, PND15, PND30 and PND45, blood and tissue iron status were assessed. Nephrogenic zone maturation (PND2–PND10), radial glomerular counts (RGCs), glomerular size density and total planar surface area (PND15 and PND30) were also assessed. Blood pressure (BP) was measured in offspring. ID rats were smaller, exhibiting lower erythrocyte and tissue iron than control rats (PND2–PND10), but these parameters returned to control values by PND30–PND45. Relative kidney iron (µg g–1 wet weight) at PND2-PND10 was directly related to transport iron measures. In ID rats, the maturation of the active nephrogenic zone was later than control. RGCs, glomerular size, glomerular density, and glomerular planar surface area were lower than control at PND15, but returned to control by PND30. After weaning, the kidney weight/rat weight ratio (mg g–1) was heavier in ID than control rats. BP readings at PND45 were lower in ID than control rats. Altered kidney maturation and renal adaptations may contribute to glomerular size, early hyperfiltration and long-term renal function.

Postnatal kidney maturation regulates renal artery myogenic constriction

Intravascular pressure-induced vasoconstriction (myogenic constriction) is central to renal blood flow autoregulation. At term, kidney maturation is functionally incomplete. Premature neonates are at risk of kidney dysfunction. However, it is unclear whether renal artery myogenic constriction is altered after preterm birth. Here, we compared renal artery myogenic constriction in full-term and preterm pigs during the first week of life.We investigated myogenic constriction in small interlobular arteries isolated from the kidneys of pigs delivered at term and at 91% of term (with and without 96 h of neonatal intensive care).Cross-sectional area, media/lumen ratio, and luminal diameter measured under passive conditions were similar in arteries from full-term and preterm pig kidneys. An acute elevation in intravascular pressure from 20 to 100 mm Hg increased arterial wall tension and induced steady-state constriction of the arteries. However, arteries isolated from newly born preterm pigs (within 24 h) developed greater myogenic tone and lower active wall tension compared with arteries from full-term and 4-day-old preterm neonates. Pressure-induced elevation in intracellular CaMyogenic constriction is elevated in newly born preterm pigs. Our data also suggests that postnatal kidney maturation may modulate renal blood flow autoregulation.

Pressor and renal regional hemodynamic effects of urotensin II in neonatal pigs

Renal expression of the peptide hormone urotensin II (UII) and its receptor (UTR) are dependent on kidney maturation and anatomical regions. However, renal regional hemodynamic effects of UII in neonates are unclear. Here, we investigated regional hemodynamic responses to acute intrarenal arterial administration of UII in newborn pigs. Western immunoblotting and immunofluorescence confirmed UTR expression and membrane localization in newborn pig renal afferent arterioles and afferent arteriolar smooth muscle cells respectively. Intrarenal arterial bolus injections of human UII (hUII; 1–100 ng/kg) resulted in a dose-dependent decrease in total renal blood flow (RBF) and an increase in mean arterial pressure (MAP) and renal vascular resistance (RVR) in newborn pigs. Moreover, hUII dose dependently reduced cortical blood flow (CBF) but increased medullary blood flow (MBF) in the piglets. hUII-induced MAP elevation and hemodynamic changes were inhibited by urantide, a UTR antagonist, but not losartan, a type 1 angiotensin II receptor antagonist. U-73122, a phospholipase C (PLC) inhibitor, and 2-aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate (IP3) receptor antagonist, attenuated hUII-induced MAP and RVR elevations, RBF and CBF reductions, but not MBF increase. These findings indicate that intrarenal arterial administration of hUII elevates blood pressure and induces region-selective renal hemodynamic changes in newborn pigs. Our data also suggest that the PLC/IP3signaling pathway contributes to hUII-induced alterations in MAP, RBF, RVR, and CBF but not MBF in newborn pigs.

Galectin-3 Modulates Ureteric Bud Branching in Organ Culture of the Developing Mouse Kidney

Galectin-3 is a mammalian β-galactoside—specific lectin with functions in cell growth, adhesion, and neoplastic transformation. On the basis of expression patterns in humans, it is proposed that galectin-3 modulates fetal collecting duct growth. This article provides evidence that galectin-3 can modulate branching morphogenesis of the mouse ureteric bud/collecting duct lineage. With the use of immunohistochemistry, galectin-3 was not detected in early metanephrogenesis but was upregulated later in fetal kidney maturation when the protein was prominent in basal domains of medullary collecting ducts. Addition of galectin-3 to embryonic days 11 and 12 whole metanephric cultures inhibited ureteric bud branching, whereas galectin-1 did not perturb morphogenesis, nor did a galectin-3 mutant lacking wild-type high-affinity binding to extended oligosaccharides. Exogenous galectin-3 retarded conversion of renal mesenchyme to nephrons in whole metanephric explants but did not affect nephron induction by spinal cord in isolated renal mesenchymes. Finally, addition of a blocking antiserum to galectin-3 caused dilation and distortion of developing epithelia in embryonic day 12 metanephroi cultured for 1 wk. The upregulation of galectin-3 protein during kidney maturation, predominantly at sites where it could mediate cell/matrix interactions, seems to modulate growth of the ureteric tree.

Expression of Angiopoietin-1, Angiopoietin-2, and the Tie-2 Receptor Tyrosine Kinase during Mouse Kidney Maturation

The Tie-2 receptor tyrosine kinase transduces embryonic onic endothelial differentiation, with Angiopoietin-1 (Ang-1) acting as a stimulatory ligand and Ang-2 postulated to be a naturally occurring inhibitor. Expression of these genes was sought during mouse kidney maturation from the onset of glomerulogenesis (embryonic day 14 [E14]) to the end of nephron formation (2 wk postnatal [P2]), and during medullary maturation into adulthood (P8). Using Northern and slot blotting of RNA extracted from whole organs, these three genes were expressed throughout the experimental period with peak levels at P2 to P3. By in situ hybridization analysis at E18, P1, and P3, Ang-1 mRNA was found to localize to condensing renal mesenchymal cells, proximal tubules, and glomeruli in addition to maturing tubules of the outer medulla. In contrast, Ang-2 transcripts were more spatially restricted, being detected only in differentiating outer medullary tubules and the vasa recta bundle area. Using in situ hybridization and immunohistochemistry, Tie-2 was detected in capillaries of the nephrogenic cortex, glomerular tufts, cortical interstitium, and medulla including vessels in the vasa recta. Using Western blotting of protein extracted from whole organs, Tie-2 protein was detected between E14 and P8 with tyrosine phosphorylated Tie-2 evident from E18. These data are consistent with the hypothesis that Tie-2 has roles in maturation of both glomeruli and vasa rectae.