The most recently discovered carbonic anhydrase, CA XV, is expressed in the thick ascending limb of Henle and in the collecting ducts of mouse kidney

Abstract

Background: Carbonic anhydrases (CAs) are key enzymes for physiological pH regulation, including the process of urine acidification. Previous studies have identified seven cytosolic or membrane-bound CA isozymes in the kidney. Recently, we showed by in situ hybridization that the mRNA for the most novel CA isozyme, CA XV, is present in the renal cortex. CA XV is a unique isozyme among mammalian CAs, because it has become a pseudogene in primates even though expressed in several other species.

Methodology/principal findings: In the present study, we raised a polyclonal antibody against recombinant mouse CA XV that was produced in a baculovirus/insect cell expression system, and the antibody was used for immunohistochemical analysis in different mouse tissues. Positive immunoreactions were found only in the kidney, where the enzyme showed a very limited distribution pattern. Parallel immunostaining experiments with several other anti-CA sera indicated that CA XV is mainly expressed in the thick ascending limb of Henle and collecting ducts, and the reactions were most prominent in the cortex and outer medulla.

Conclusion/significance: Although other studies have proposed a role for CA XV in cell proliferation, its tightly limited distribution may point to a specialized function in the regulation of acid-base homeostasis.

Figure 1. Western blot of recombinant mouse CA XV identified with the new anti-CA XV antibody.

The antibody identifies a 34- to 36-kDa polypeptide and a smaller 31-kDa polypeptide that is suggested to be a non-glycosylated form of CA XV (left). Pre-immune serum showed no reaction (right).

Figure 2. Immunohistochemical staining of CA XV in the mouse kidney.

A sectional view of mouse kidney shows that CA XV is expressed in the cortex, and a weaker reaction is seen in the collecting ducts of the outer medulla (A). Intense staining is seen in the thick ascending limbs (B) and collecting ducts of the cortex (C). Original magnifications x100 (A), x630 (B,C).

Figure 3. Confirmation of CA XV immunostaining in the thick ascending limb of Henle.

Tamm-Horsfall glycoprotein (THP) antibody was used as a marker (B). CA XV (A) clearly labels the thick ascending limbs (arrows) and collecting ducts (arrowheads). Original magnifications x400.

Figure 4. Immunohistochemical staining of CA XV and CA II in parallel sections of mouse kidney.

In the cortex, the expression of CA XV (A) is more limited compared to CA II (B) that is mainly expressed in the collecting ducts (arrows) and proximal tubules (asterisks). Arrowheads show the thick ascending limbs of Henle, which are positively stained for CA XV. In the medulla, only a very faint reaction is seen for CA XV (C) in the collecting ducts (arrows), while the staining for CA II (D) is more intense. Original magnifications x400.

Figure 5. Immunohistochemical staining of CA XV and CA IV in mouse kidney.

The staining for CA XV (A) is more intense and restricted compared to CA IV (B). CA IV shows a reaction in the thick ascending limbs (arrowheads) like CA XV, and is also present in the proximal tubules, which are not stained for CA XV. Original magnifications ×400.

Figure 6. Immunohistochemical staining of CA XV, CA XII and CA XIV in mouse kidney.

CA XV (A) and CA XII (B) label the same cortical collecting ducts (arrows), even though the reaction for CA XV is notably more extensive and intense. CA XV is also present in the thick ascending limbs of Henle (asterisks), which are negative for CA XII. In the medulla, CA XIV (D) is expressed in the upper portion of the thin descending limbs, as described previously . These segments are negative for CA XV (C). Original magnifications ×400.

Figure 7. Immunohistochemical staining of CA XV in wild-type, Car4^−/− and Car14^−/− mouse kidneys.

In addition to the strong immunoreactions observed in all the cases in the collecting ducts and thick ascending limbs, weak positivity is also observed in the convoluted tubules of Car4^−/− kidney. Original magnifications ×400.

Figure 8. Quantitative real-time PCR analysis of Car15 mRNA levels in the kidney specimens of wild type and Car4^−/− and Car14^−/− knockout mice.

Three of five kidney samples from Car14^−/− knockout (KO) mice showed slightly increased expression of Car15 mRNA in comparison to the wild type (WT) and Car4^−/− kidneys, but the difference between the groups showed no statistical significance.