Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.

The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, β-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)—induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes. Conclusions: In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.