Less potassium coming out, less sodium going in: Phenotyping ROMK knockout rats

The Dahl salt-sensitive rat represents a widely studied model of salt-sensitive hypertension¹. In the current work, Zhou et al² phenotype a renal outer medullary potassium channel (ROMK) homozygous (−/−) and heterozygous (+/−) knockout rat on a Dahl salt-sensitive background. The authors performed a series of hemodynamic and metabolic studies to address the effects of genetically manipulating ROMK in the Dahl salt-sensitive rat. This model represents a major advance in tools available to study the role of ROMK in renal development, Na homeostasis and blood pressure regulation. It is another of a growing number of −/− rats generated by zinc finger nucleases that will eventually replace the need to study −/− mice. For this reason alone the study should be considered a major accomplishment as the literature on renal physiology and blood pressure regulation in rats and our ability to study rats is far more sophisticated than it is for mice.

ROMK is expressed in the thick ascending limb of the loop of Henle (TALH) and principal cells of the collecting duct³. Both segments play a critical role in salt homeostasis and therefore blood pressure. In the TALH, both transcellular and paracellular pathways are involved in salt reabsorption. Transcellular absorption occurs when Na⁺, K⁺ and Cl⁻ enter the cells via the apical Na/K/2Cl cotransporter (NKCC2) and exits via Na⁺/K⁺ ATPase. ROMK, located in the apical membrane of the TALH, recycles K⁺ back to the lumen. The lumen-positive voltage created when K⁺ is recycled drives Na⁺ reabsorption through the paracellular pathway. Thus ROMK plays important roles in both NaCl absorption and K⁺ secretion in this segment (figure 1). In the collecting duct ROMK is also located in the apical membrane where it secretes K⁺. In this segment transcellular salt absorption occurs when Na⁺ enters the principal cells via the apical epithelial Na⁺ channel (ENaC) and exits via Na⁺/K⁺ ATPase, and Cl⁻ is absorbed via the paracellular pathway by the lumen negative voltage and through intercalated cells (figure 1). In the collecting duct factors that increase Na⁺ absorption such as aldosterone also increase K⁺ secretion. However, transport of these ions can be regulated independently⁴. Although ROMK is expressed in both the TALH and principal cells, Zhuo et al.² attribute essentially all of the phenotype of the ROMK homozygous knockout (−/−) and heterozygous knockout (+/−) rats to alterations in thick ascending limb function.

Figure 1.

Schematic representation of a thick ascending limb of the loop of Henle cell and a collecting duct principal cell.

The importance of sodium transport in the TALH is best evidenced by the so-called loop diuretics and by loss-of-function mutations. The loop diuretic furosemide inhibits the activity of NKCC2 and has been largely used to treat hypertension and to manage clinical situations of volume overload⁵. Loss-of-function mutations in NKCC2 and ROMK cause Bartter’s syndrome type I and type II respectively^{6, 7}. Among other metabolic features, these syndromes are characterized by polyuria, salt wasting and hypotension. On the other hand, abnormally elevated reabsorption of salt by the TALH has been implicated with the development of salt-sensitive hypertension^{8, 9}.

On a normal-salt diet ROMK −/− pups showed similar characteristics to ROMK −/− mice including severe volume depletion and high mortality rate after day 14¹⁰. In contrast, ROMK +/− and ROMK +/+ pups did not exhibit significant differences in body and organ weights and blood electrolytes on a normal diet. On a low-salt diet, ROMK +/− have a slightly reduced blood pressure compared to ROMK +/+, but the difference was not statistically significant. Although the difference was not significant, this may have been due to the small numbers of rats studied due to their limited availability. When ROMK +/− rats were challenged with either 4 or 8% salt diet, the increase in blood pressure was smaller compared to their ROMK +/+ littermates. Together these data demonstrate that ROMK plays an important role in salt excretion. Given the strong link between K recycling and salt absorption in the TALH, the authors imply that this is likely due to effects on this segment. Similarly, studies in humans showed that individuals with ROMK heterozygous mutations have reduced systolic and diastolic blood pressure and are less likely to develop hypertension¹¹. Thus the ROMK +/− rat appears to be a good model of human disease.

While the development of the ROMK −/− rat is of considerable interest in terms of salt wasting and blood pressure regulation, its real value may come in the area of renal development where it may open up new paradigms. ROMK −/− rats have a defective renal structure, and only a small percentage of them reach adulthood. These data show that ROMK is not only important in NaCl absorption by the TALH, but it is required for proper development of the kidney. The influence of ROMK on development may be related to its role in NaCl absorption and/or K secretion, or it may be completely independent of its function as a transporter. This could be due to changes in either the TALH or collecting ducts, a possibility not raised by the authors. While the role of ROMK in renal development is novel, the fact that K transport can influence development has been shown in other tissues¹².

In contrast to −/− rats, ROMK +/− rats have normal kidneys. Such data indicate that a single copy of ROMK is adequate for normal renal development. These animals also develop smaller increases in blood pressure and less renal injury when fed high salt indicating that ROMK heterozygous mutations in Dahl salt-sensitive rats exert a protective effect. The results concerning renal damage are interesting. While it may be that ROMK +/− kidneys were protected due to the reduction in blood pressure, a potentially more interesting interpretation exists. That is that the role ROMK plays in development is also responsible for the renal protection. This latter hypothesis can only be tested in the −/− rats that reach adulthood and +/− animals.

The fact that the heterozygous mutation does not seem to cause any disturbance in kidney development but antagonizes salt-sensitivity may indicate that expression and activity of ROMK can change over time, be modified by different stimuli/environment or that ROMK plays distinctly separate roles in renal development and NaCl absorption/K secretion. Collectively, these findings strongly suggest that ROMK expression and activity might not be only regulated by genetic mechanisms.

Previous investigations in mice and humans suggested the physiological importance of ROMK on salt reabsorption and blood pressure regulation^{7, 10, 11}. The current findings are of particular interest not only because they add to previous knowledge but also because they show for the first time that disruption of ROMK channels lead to attenuation of salt-sensitive hypertension in the Dahl salt sensitive rat model. The study by Zhou et al will surely contribute to the development of new pharmacological strategies to manage hypertension and other diseases caused by electrolyte disturbances, and may open new areas of research in renal development.