Soline Bourgeois

Regulated acid-base transport in the collecting duct

The renal collecting system serves the fine-tuning of renal acid–base secretion. Acid-secretory type-A intercalated cells secrete protons via a luminally expressed V-type H+-ATPase and generate new bicarbonate released by basolateral chloride/bicarbonate exchangers including the AE1 anion exchanger. Efficient proton secretion depends both on the presence of titratable acids (mainly phosphate) and the concomitant secretion of ammonia being titrated to ammonium. Collecting duct ammonium excretion requires the Rhesus protein RhCG as indicated by recent KO studies. Urinary acid secretion by type-A intercalated cells is strongly regulated by various factors among them acid–base status, angiotensin II and aldosterone, and the Calcium-sensing receptor. Moreover, urinary acidification by H+-ATPases is modulated indirectly by the activity of the epithelial sodium channel ENaC. Bicarbonate secretion is achieved by non-type-A intercalated cells characterized by the luminal expression of the chloride/bicarbonate exchanger pendrin. Pendrin activity is driven by H+-ATPases and may serve both bicarbonate excretion and chloride reabsorption. The activity and expression of pendrin is regulated by different factors including acid–base status, chloride delivery, and angiotensin II and may play a role in NaCl retention and blood pressure regulation. Finally, the relative abundance of type-A and non-type-A intercalated cells may be tightly regulated. Dysregulation of intercalated cell function or abundance causes various syndromes of distal renal tubular acidosis underlining the importance of these processes for acid–base homeostasis.

A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility

The kidney has an important role in the regulation of acid–base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH3 diffusion coupled to H+ secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading—a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg-/- acid-loaded mice show reduced apical permeability to NH3 and impaired transepithelial NH3 transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

PTH-independent regulation of blood calcium concentration by thecalcium-sensing receptor

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

Defective ENaC Processing and Function in Tissue Kallikrein-deficient Mice

An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK–/–) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of γ-ENaC in TK–/– mice. Urinary Na+ excretion after amiloride injection was blunted in TK–/– mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK–/– mice, whereas amiloride-sensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of γ-ENaC was increased, indicating that its absence in TK–/– mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK–/– mice with TK resulted in the appearance of the 70-kDa band of the γ-ENaC, indicating that TK was able to promote γ-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na+ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon.

NHE4 is critical for the renal handling of ammonia in rodents

Ammonia absorption by the medullary thick ascending limb of Henles loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na+/H+ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na+/H+ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4-/- mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4-/- mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH3/NH4+ needed to excrete an acid load, contributing to systemic metabolic acidosis.

Sulfatides are required for renal adaptation to chronic metabolic acidosis

Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na+-K+/NH4+-2Cl− cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid–base homeostasis.

Tamm-Horsfall protein translocates to the basolateral domain of thick ascending limbs, interstitium, and circulation during recovery from acute kidney injury.

Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidneys thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross talk between the two functionally distinct tubular segments. However, the extent, timing, specificity, and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is downregulated at the peak of injury, it is significantly upregulated 48 h after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium, and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of Na(+)-K(+)-2Cl cotransporter (NKCC2) and basolateral targeting of Na(+)-K(+)-ATPase. Compared with the wild-type, THP(-/-) mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of proinflammatory cytokines and chemokines such as monocyte chemoattractant protein-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.

The rhesus protein RhCG : a new perspective in ammonium transport and distal urinary acidification.

Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H(+)-ATPases and requires the parallel movement of NH₃, and its protonation to NH₄(+), to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/μm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid-base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid-base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels.

NKCC2 Surface Expression in Mammalian Cells DOWN-REGULATION BY NOVEL INTERACTION WITH ALDOLASE B

Apical bumetanide-sensitive Na+-K+-2Cl- co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

The Proton-activated G Protein Coupled Receptor OGR1 Acutely Regulates the Activity of Epithelial Proton Transport Proteins

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H+-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl2 and CuCl2 that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.