Secretin: a hormone for HCO3- homeostasis.

Secretin is a key hormone of the intestinal phase of digestion which activates pancreatic, bile duct and Brunner gland HCO3- secretion. Recently, the secretin receptor (SCTR) was also found in the basolateral membrane of the beta-intercalated cell (B-IC) of the collecting duct. Experimental addition of secretin triggers a pronounced activation of urinary HCO3- excretion, which is fully dependent on key functional proteins of the B-IC, namely apical pendrin and CFTR and the basolateral SCTR. Recent studies demonstrated that the SCTR knock-out mouse is unable to respond to an acute base load. Here, SCTR KO mice could not rapidly increase urine base excretion, developed prolonged metabolic alkalosis and exhibited marked compensatory hypoventilation. Here, we review the physiological effects of secretin with distinct focus on how secretin activates renal HCO3- excretion. We describe its new function as a hormone for HCO3- homeostasis.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Loss of the Secretin Receptor Impairs Renal Bicarbonate Excretion and Aggravates Metabolic Alkalosis in Mice during Acute Base-Loading.

SIGNIFICANCE STATEMENT: During acute base excess, the renal collecting duct ß -intercalated cells ( ß -ICs) become activated to increase urine base excretion. This process is dependent on pendrin and cystic fibrosis transmembrane regulator (CFTR) expressed in the apical membrane of ß -ICs. The signal that leads to activation of this process was unknown. Plasma secretin levels increase during acute alkalosis, and the secretin receptor (SCTR) is functionally expressed in ß -ICs. We find that mice with global knockout for the SCTR lose their ability to acutely increase renal base excretion. This forces the mice to lower their ventilation to cope with this challenge. Our findings suggest that secretin is a systemic bicarbonate-regulating hormone, likely being released from the small intestine during alkalosis. BACKGROUND: The secretin receptor (SCTR) is functionally expressed in the basolateral membrane of the ß -intercalated cells of the kidney cortical collecting duct and stimulates urine alkalization by activating the ß -intercalated cells. Interestingly, the plasma secretin level increases during acute metabolic alkalosis, but its role in systemic acid-base homeostasis was unclear. We hypothesized that the SCTR system is essential for renal base excretion during acute metabolic alkalosis. METHODS: We conducted bladder catheterization experiments, metabolic cage studies, blood gas analysis, barometric respirometry, perfusion of isolated cortical collecting ducts, immunoblotting, and immunohistochemistry in SCTR wild-type and knockout (KO) mice. We also perfused isolated rat small intestines to study secretin release. RESULTS: In wild-type mice, secretin acutely increased urine pH and pendrin function in isolated perfused cortical collecting ducts. These effects were absent in KO mice, which also did not sufficiently increase renal base excretion during acute base loading. In line with these findings, KO mice developed prolonged metabolic alkalosis when exposed to acute oral or intraperitoneal base loading. Furthermore, KO mice exhibited transient but marked hypoventilation after acute base loading. In rats, increased blood alkalinity of the perfused upper small intestine increased venous secretin release. CONCLUSIONS: Our results suggest that loss of SCTR impairs the appropriate increase of renal base excretion during acute base loading and that SCTR is necessary for acute correction of metabolic alkalosis. In addition, our findings suggest that blood alkalinity increases secretin release from the small intestine and that secretin action is critical for bicarbonate homeostasis.

Unrecognized role of claudin-10b in basolateral membrane infoldings of the thick ascending limb.

Claudin-10b is an important component of the tight junction in the thick ascending limb (TAL) of Henle's loop and allows paracellular sodium transport. In immunofluorescence stainings, claudin-10b-positive cells exhibited extensive extra staining of basolateral, column-like structures. The precise localization and function have so far remained elusive. In isolated cortical TAL segments from C57BL/6J mice, kidney-specific claudin-10 knockout mice (cKO), and respective litter mates (WT), we investigated the localization and protein expression and function by fluorescence microscopy and electrophysiological measurements. Ultrastructural analysis of TAL in kidney sections was performed by electron microscopy. Claudin-10b colocalized with the basolateral Na+ -K+ ATPase and the Cl- channel subunit barttin, but the lack of claudin-10b did not influence the localization or abundance of these proteins. However, the accessibility of the basolateral infolded extracellular space to ouabain or fluorescein was increased by basolateral Ca2+ removal and in the absence of claudin-10b. Ultrastructural analysis by electron microscopy revealed a widening of basolateral membrane infoldings in cKO in comparison to WT. We hypothesize that claudin-10b shapes neighboring membrane invaginations by trans interaction to stabilize and facilitate high-flux salt transport in a water-tight epithelium.

Dietary K+ acts as a genuine diuretic.

K+ balance in mammals relies on regulated renal K+ excretion matching unregulated fluctuating K+ intake. Upon a K+ rich meal, rapid and powerful K+ excretion is needed. Renal K+ secretion is stimulated by the increased tubular flow. We speculated that high K+ intake acutely increases urinary flow to stimulate K+ excretion. METHODS: Mice were K+ challenged through diets or gavage. Post K+ loading urinary output, osmolarity, [K+ ]u , [Na+ ]u , plasma osmolarity, [copeptin]p , [K+ ]p , and [Na+ ]p were measured. To locate the mechanism of K+ -induced diuresis in the glomerular/tubular system we measured creatinine excretion and assessed functional transport in isolated perfused TALs and CDs during an acute [K+ ]bl switch from 3.6 to 6.5 mM. Molecular adaptations of transport proteins involved in water reabsorption were investigated by immunoblotting. RESULTS: (1) Mice switched from a 1% to 2% K+ diet increased diuresis within 12 hours and reciprocally reduced diuresis when switched from 1% to 0.01% K+ diet. (2) A single K+ gavage load, corresponding to 25%-50% of daily K+ intake, induced 100% increase in diuresis within 30 minutes. This occurred despite augmented plasma osmolarity and AVP synthesis. (3) K+ gavage did not change GFR. (4) In isolated perfused TALs, shifting [K+ ]bl from 3.6 to 6.5 mM did not affect AVP-induced NaCl transport. (5) In sharp contrast, in isolated perfused CDs, shifting [K+ ]bl from 3.6 to 6.5 mM markedly reduced CD AVP sensitivity, ie inhibited water absorption. CONCLUSION: Dietary K+ loading induces a rapidly on-setting diuresis. The mechanism of K+ -induced diuresis involves desensitization of the CD to AVP.

The molecular mechanism of CFTR- and secretin-dependent renal bicarbonate excretion.

This review summarizes the newly discovered molecular mechanism of secretin-stimulated urine HCO3- excretion and the role of cystic fibrosis transmembrane conductance regulator (CFTR) in renal HCO3- excretion. The secretin receptor is functionally expressed in the basolateral membrane of the HCO3- -secreting ß-intercalated cells of the collecting duct. Here it activates a fast and efficient secretion of HCO3- into the urine serving to normalize metabolic alkalosis. The ability to acutely increase renal base excretion is entirely dependent on functional pendrin (SLC26A4) and CFTR, and both proteins localize to the apical membrane of the ß-intercalated cells. In cystic fibrosis mice and patients, this function is absent or markedly reduced. We discuss that the alkaline tide, namely the transient urine alkalinity after a meal, has now received a clear physiological explanation.

Impaired renal HCO3- secretion in CFTR deficient mice causes metabolic alkalosis during chronic base-loading.

AIM: Cystic fibrosis patients have an increased risk of developing metabolic alkalosis presumably as a result of altered renal HCO3- handling. In this study, we directly assess the kidneys' ability to compensate for a chronic base-load in the absence of functional CFTR. METHODS: Comprehensive urine and blood acid-base analyses were done in anaesthetized WT mice or mice lacking either CFTR or pendrin, with or without 7 days of oral NaHCO3 loading. The in vivo experiments were complemented by a combination of immunoblotting and experiments with perfused isolated mouse cortical collecting ducts (CCD). RESULTS: Base-loaded WT mice maintained acid-base homeostasis by elevating urinary pH and HCO3- excretion and decreasing urinary net acid excretion. In contrast, pendrin KO mice and CFTR KO mice were unable to increase urinary pH and HCO3- excretion and unable to decrease urinary net acid excretion sufficiently and thus developed metabolic alkalosis in response to the same base-load. The expression of pendrin was increased in response to the base-load in WT mice with a paralleled increased pendrin function in the perfused CCD. In CFTR KO mice, 7 days of base-loading did not upregulate pendrin expression and apical Cl- /HCO3- exchange function was strongly blunted in the CCD. CONCLUSION: CFTR KO mice develop metabolic alkalosis during a chronic base-load because they are unable to sufficiently elevate renal HCO3- excretion. This can be explained by markedly reduced pendrin function in the absence of CFTR.

Impaired Renal HCO3- Excretion in Cystic Fibrosis.

BACKGROUND: Patients with cystic fibrosis (CF) do not respond with increased urinary HCO3- excretion after stimulation with secretin and often present with metabolic alkalosis. METHODS: By combining RT-PCR, immunohistochemistry, isolated tubule perfusion, in vitro cell studies, and in vivo studies in different mouse models, we elucidated the mechanism of secretin-induced urinary HCO3- excretion. For CF patients and CF mice, we developed a HCO3- drinking test to assess the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in urinary HCO3-excretion and applied it in the patients before and after treatment with the novel CFTR modulator drug, lumacaftor-ivacaftor. RESULTS: ß-Intercalated cells express basolateral secretin receptors and apical CFTR and pendrin. In vivo application of secretin induced a marked urinary alkalization, an effect absent in mice lacking pendrin or CFTR. In perfused cortical collecting ducts, secretin stimulated pendrin-dependent Cl-/HCO3- exchange. In collecting ducts in CFTR knockout mice, baseline pendrin activity was significantly lower and not responsive to secretin. Notably, patients with CF (F508del/F508del) and CF mice showed a greatly attenuated or absent urinary HCO3--excreting ability. In patients, treatment with the CFTR modulator drug lumacaftor-ivacaftor increased the renal ability to excrete HCO3-. CONCLUSIONS: These results define the mechanism of secretin-induced urinary HCO3- excretion, explain metabolic alkalosis in patients with CF, and suggest feasibility of an in vivo human CF urine test to validate drug efficacy.

Viewing Cortical Collecting Duct Function Through Phenotype-guided Single-Tubule Proteomics.

The revolution of the omics technologies has enabled profiling of the molecules of any sample. However, the heterogeneity of the kidney with highly specialized nephron segments like the cortical collecting duct (CCD) poses a challenge regarding integration of omics data and functional analysis. We examined function and proteome from the same single CCDs of C57Bl6 mice by investigating them in a double-barreled perfusion system before targeted mass spectrometry. Transepithelial voltage (Vte), transepithelial resistance, as well as amiloride-sensitive voltage (ΔVteamil) were recorded. CCDs were of 400-600 µm of length, showed lumen negative Vte between -8.5 and -32.5 mV and an equivalent short circuit current I'sc between 54 and 192 µA/cm2. On a single-tubule proteome level, intercalated cell (IC) markers strongly correlated with other intercalated cell markers and negatively with principal cell markers. Integration of proteome data with phenotype data revealed that tubular length correlated with actin and Na+-K+-ATPase expression. ΔVte(amil) reflected the expression level of the ß-subunit of the epithelial sodium channel. Intriguingly, ΔVte(amil) correlated inversely with the water channel AQP2 and the negative regulator protein NEDD4L (NEDD4-2). In pendrin knockout (KO) mice, the CCD proteome was accompanied by strong downregulation of other IC markers like CLCNKB, BSND (Barttin), and VAA (vH+-ATPase), a configuration that may contribute to the salt-losing phenotype of Pendred syndrome. Proteins normally coexpressed with pendrin were decreased in pendrin KO CCDs. In conclusion, we show that functional proteomics on a single nephron segment scale allows function-proteome correlations, and may potentially help predicting function from omics data.

P2X Receptors Inhibit NaCl Absorption in mTAL Independently of Nitric Oxide.

Activation of basolateral P2X receptors markedly reduces NaCl absorption in mouse medullary thick ascending limb (mTAL). Here we tested the role of nitric oxide (NO) in the ATP-mediated (P2X) transport inhibition. We used isolated, perfused mTALs from mice to electrically measure NaCl absorption. By microelectrodes we determined the transepithelial voltage (Vte) and transepithelial resistance (Rte). Via these two parameters, we calculated the equivalent short circuit current, I'sc as a measure of the transepithelial Na+ absorption. Basolateral ATP (100 µM) acutely induced reversible inhibition of Na+ absorption (24 ± 4%, n = 10). Addition of L-arginine (100 µM) had no apparent effect on the ATP-induced transport inhibition. Acute reduction of extracellular [Ca2+] to either 100 nM or 0 nM by addition of EGTA had no effect on the ATP-induced transport inhibition. In the presence of the NO synthase (NOS) inhibitor L-NAME (100 µM) and/or ODQ to inhibit the guanylyl cyclase, the ATP effect remained unaffected. Increasing the concentration and incubation time for L-NAME (1 mM) still did not reveal any effect on the ATP-mediated transport inhibition. Acute addition of the NO donors SNAP (100 µM) and Spermine NONOate (10 µM) did not alter tubular transport. High concentrations of L-NAME (1 mM) in itself, however, reduced the transepithelial transport significantly. Thus, we find no evidence for nitric oxide (NO) as second messenger for P2X receptor-dependent transport inhibition in mTAL. Moreover, Ca2+ signaling appears not involved in the ATP-mediated effect. It remains undefined how P2X receptors trigger the marked reduction of transport in the TAL.