Immunohistochemical localization of receptor protein tyrosine phosphatase γ in mouse renal proximal tubule, thick ascending limb and distal convoluted tubule (2022)

Most normal physiological processes require that both intracellular (i) and extracellular (o) pH be maintained within a tight range. pH o is contingent on arterial (a) pH, which is determined by the ratio of the arterial carbon dioxide to the arterial bicarbonate concentration, [CO 2 ] a /[HCO 3 − ] a . Pulmonary ventilation controls [CO 2 ] a . The kidney controls [HCO 3 − ] a by adjusting HCO 3 − transport rates ( J HCO3 ) across the basal membranes of particular nephron epithelia into the blood to maintain normal pH a , or compensate for an acid‐base insult. In an effort to identify the sensor proteins for pH a , [CO 2 ] a , or [HCO 3 − ] a , our laboratory previously reported that, in isolated proximal tubules (PTs) from receptor protein tyrosine phosphatase γ (RPTPγ) knockout (KO) mice, J HCO3 is insensitive to changes in the basolateral [CO 2 ] or [HCO 3 − ]. Furthermore, RPTPγ‐KO mice have a deficiency in defending against a whole‐body metabolic acidosis. These data implicate RPTPγ as a major [CO 2 ] or [HCO 3 − ] sensor. RPTPγ possesses an extracellular catalytically inactive carbonic‐anhydrase‐like domain (CALD) and fibronectin type III (FNIII) domain, a single transmembrane helix, and intracellular domains D1 and D2. Dimerization of RPTPγ presumably allows the inhibitory D2 of one monomer to inhibit the otherwise catalytically active D1 of the other monomer. Thus, the dimerization/monomerization state controls downstream signaling. We hypothesize that the binding of CO 2 or HCO 3 − to the CALD alters the CALD conformation, thereby transmitting signals that control the dimerization status of RPTPγ and thereby regulate downstream effects on J HCO3 . We hypothesize that RPTPγ is localized in nephron segments important for regulating HCO 3 − transport/reabsorption, in the basal membranes that face the blood. To determine the location of RPTPγ in wild‐type mouse kidney cryosections, we use a chicken IgY anti‐RPTPγ antibody (Ab) raised against an epitope within the FNIII domain, and co‐stain with rabbit polyclonal Abs against the Na/HCO 3 cotransporter (NBCe1‐A) as a PT marker, the Na/K/Cl cotransporter 2 (NKCC2) as a thick ascending limb (TAL) marker, and the Na/Cl cotransporter (NCC) as a distal convoluted tubule (DCT) marker. Imaging sections by confocal microscopy, we observe in 3 WT mice that RPTPγ localizes primarily to the PT basal membrane. However, 54% of PTs (NBCe1‐A positive) also exhibit apical RPTPγ staining of comparable intensity to the RPTPγ staining observed in the basal membrane. We also observe both apical and basal RPTPγ staining in Bowman’s capsules. We stained additional sections from the same animals with a rabbit polyclonal primary Ab that targets the RPTPγ CALD domain. In these sections, co‐stained with the anti‐FNIII Ab, we similarly observe both apical and basal staining in PTs. We observe RPTPγ staining in a minority of TALs, and at much lower intensity than observed in the PT. We observe minimal to no RPTPγ staining in the DCT. We validated the specificity of the RPTPγ Abs on RPTPγ‐KO kidney sections, which exhibit low background levels of fluorescence for both the anti‐FNIII and anti‐CALD Abs. In conclusion, these data confirm RPTPγ expression in PT basal membranes, but also describe the first examples of apical RPTPγ expression in renal epithelia.