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The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.

Connexins (Cxs) form gap junctions for intercellular exchange of inorganic ions and messenger molecules. In the kidney, Cxs play essential roles within its compartments, but data on the precise cellular localization and cell type-related function of their isoforms are scarce. We have tested whether Cx43 distribution is restricted to vascular and interstitial cells and whether medullary fibroblasts express Cx43 to coordinate profibrotic signaling. Confocal immunofluorescence techniques, ultrastructural labeling, and functional studies in cell culture were performed. Cx43 was chiefly expressed in the vasculature, but absent from tubular epithelia. All arterial, arteriolar and lymphatic endothelia showed continous Cx43 signal along their borders. In the inner medulla, only the interstitium showed Cx43 signals which were assigned to fibroblasts and their processes. Cultured, Cx43-expressing medullary fibroblasts served to study the role of gap junctions in a profibrotic context. Using a dye spreading assay, Cx43-sensitive diffusion of Lucifer Yellow was dependent on gap junctional passage. Adding transforming growth factor-β1 (5 ng/ml for 48 h) activated Cx43 biosynthesis and caused Cx43-sensitive transformation of the fibroblasts into a myofibroblast phenotype. This suggested that Cx43 gap junctional channels enable the coordination of profibrotic signaling between cells of the medullary interstitium. In sum, we demonstrate the presence of Cx43-expressing gap junctions within the two major renal compartments, vasculature and interstitium. Endothelial Cx43 likely provides functions of an earlier defined 'electrical syncytium' within the vascular wall. Additionally, Cx43 facilitates profibrotic signaling between medullary interstitial fibroblasts.

Objective Calcineurin inhibitors such as cyclosporin A (CsA) are in use as immunosuppressive drugs to prevent rejection of transplanted organs. Despite positive outcomes, side effects such as decreased glomerular filtration rate and overall functional and structural deterioration may affect the kidney. Among the causes, interactions of vasoactive systems have been considered. We therefore studied how CsA may cause dysregulation of key juxtagomerular signaling components. Methods Wistar rats were divided into 4 groups receiving vehicle, 25mg CsA/kg/d, 5mg candesartan (Cand)/kg/d or CsA + Cand for 3 weeks. Organs were perfusion‐fixed and embedded for morphology, or removed for biochemical and physiological analysis. Cultured macula densa (MD) cells were treated with CsA (5 to 40 μM) and angiotensin II (Ang II; 1 to 5 μM) for 6 or 24 h. Calcineurin isoforms were silenced in MD cells and studied after 72h. Immunoreactive signals of cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2), nitric oxide synthase (NOS), nuclear factor of activated T‐cells (NFAT) subtypes 1 to 4, and activating p38 mitogen‐activated protein kinase (p38 MAPK) were evaluated. Inhibitor to p38 MAPK (SB203580; 10 μM) was applied in MD cells. Cell parameters were further recorded by quantitative PCR and Western blot. Results CsA caused activation of the renin angiotensin system (RAS) with hyperplasia of renin granular cells and upregulation of renin biosynthesis in the in vivo situation. Juxtaglomerular COX‐2 expression was suppressed. An assumed link between NFAT subtypes and COX‐2 signal of MD could not be established. Additional administration of Ang II receptor blocker, candesartan, reversed the CsA‐induced COX‐2 suppression in MD. COX‐1 and NOS expression levels were unaffected by CsA or candesartan. Contrastingly, in cultured MD cells CsA caused a rise in COX‐2 mRNA and protein abundance after 6 or 24 hours; p38 MAPK phosphorylation was increased in parallel by > 40%. Along with this, knockdown of calcineurin isoforms in MD cells increased the expression of COX‐2 by > 50%. Inhibition of p38 MAPK attenuated CsA‐induced COX‐2 upregulation by 40%. AngII substantially decreased the baseline expression of COX‐2 by 25% (6h) and blunted CsA‐induced COX‐2 stimulation by 40% (6h), likely via inhibition of p38 MAPK. All data were significant (minimum p<0.05). Conclusions In summary, calcineurin inhibition by CsA in vivo suppresses juxtaglomerular COX‐2 biosynthesis independently of NFAT signaling. Suppression is causally linked to a stimulated RAS. Contrastingly, cultured MD cells show substantial stimulation of COX‐2 biosynthesis upon CsA, involving p38 MAPK. AngII has an inhibitory effect on this cascade, since the effect was blocked by candesartan. This novel, MD cell‐specific regulatory synergism of calcineurin and angiotensin, governing COX‐2 biosynthesis, may serve to address juxtaglomerular dysregulation under CsA treatment. Support or Funding Information This study was funded by SFB (Sonderforschungsbereich 1365) in Germany.

Objective Connexin 43 (Cx43) is a gap junction‐forming protein which mediates the intercellular passage of molecules < 1000 Da. In the kidney, Cx43 was reported in glomerular vessels and mesangium. Own results prompted us to reevaluate the distribution of Cx43 and its phosphorylated form, phospho‐S368‐Cx43 (pCx43), within the different zones of the rat kidney and in cultured fibroblasts in more detail. Methods Cx43 and pCx43 were localized by immunofluorescence in rat kidney sections and cultured human fibroblasts. High‐resolution immunostaining of Cx43 was achieved by ultrastructural immunogold labelling on electron microscopy sections. Double‐labelings with anti‐aquaporin 1 (AQP1), aquaporin 2 (AQP2), zonula occludens‐1 (ZO‐1), and cyclooxygenase‐2 (COX‐2) were conducted to clarify Cx43 distribution in the rat renal medulla. Cultured human fibroblasts served to study intercellular transfer of fluorescent lucifer yellow. Results Cx43 was distributed in renal arterial and arteriolar endothelia, lymphatic vessel walls, peritubular capillaries, and glomerular compartments. In the interstitium, Cx43 was present as linear or punctate signals of the cell membrane of fibroblasts, in colocalization with pCx43. Cx43‐positive medullary fibroblasts co‐expressed COX‐2. No colocalization was detected between Cx43 and AQP1 or AQP2. Ultrastructural analysis showed Cx43 immunoreactivity at membrane contact zones between fibroblasts and in isolated cell processes of inner medullary interstitium. Cultured fibroblasts showed extensive, punctate Cx43 and pCx43 signals between adjacent cells. Signal was further distributed in non‐junctional areas of plasma membrane, suggesting Cx43 hemichannel functions. Lucifer yellow spread rapidly (10 min) between fibroblasts suggesting intercellular exchange via gap junctions, which was blocked by the gap junctional inhibitor, 18β‐glycyrrhetinic acid. Conclusions Cx43 in rat kidney shows prominent glomerular, vascular, and interstitial distribution, while epithelia, with exception of the podocytes, were negative. Focusing on interstitial fibroblasts, Cx43‐mediated information transfer was demonstrated. A constitutive nature of Cx43 phosphorylation was established in these cells. Our data suggest a cell‐specific heterogeneity of Cx43‐positive gap junctions mediating information exchange among renal cell types.