Conditional Deletion of Hsd11b2 in the Brain Causes Salt Appetite and Hypertension

Supplemental Digital Content is available in the text.


Fixed salt intake
. The brain was removed after cervical dislocation and the hind-brain was cut away from the forebrain and the cerebellum removed. The top half of the medial section of the hind-brain, containing the NTS was collected for extraction of total RNA. A) Hsd11b2 mRNA abundance expressed in arbitrary units (AU) was normalised to that of hypoxanthine guanine phosphoribosyl transferase (hprt) in the same sample. B) The percentage reduction in Hsd11b2 expression in Hsd11b2.BKO mice was calculated by normalizing to the mean expression of the control group. Individual points are shown with the group median and interquartile range. The comparisons were made using the Mann-Whitney test. .

B) Relative Hsd11b2 expression
Supplemental Figure 2. Expression of 11HSD2 imunoreactivity in fixed sections from A) control and B) Hsd11b2.BKO mouse kidney. 11HSD2 expression was restricted to the collecting duct segments. Images (x200 magnification) of cortical collecting ducts are shown and in both genotypes, 11HSD2 was expressed in principal cells, but not in intercalated cells. C) Hsd11b2 mRNA abundance and D) 11HSD2 enzyme activity in whole kidney homogenates. There were no differences between genotype analysed by Mann-Whitney test and unpaired t-test, respectively.
Supplemental Figure 3. A) Systolic blood pressure; B) diastolic blood pressure; and C) heart rate in control (open symbol; n=6) and Hsd11b2.BKO (black symbol; n=6) mice. Recordings were made by radiotelemetry over 7 consecutive days during which all mice had ad libitum access to standard rodent diet and dH 2 0. The diurnal variability was assessed in each mouse over the final 4 days of recording and data combined to give a group average. Bar indicates subjective night. Data are shown as mean ± SEM.
Supplemental Figure 4. The effect of salt on mesor and amplitude SBP in control (open circles; n=6) and Hsd11b2.BKO (black squares; n=6) mice. Group mean and SEM are shown in Supplemental Table 2. In this figure, data points (with group means ± SEM) from individual mice are shown for A) mesor and B) amplitude of SBP. Two-way ANOVA with repeated measures was used to assess the main effects of salt diet and genotype and the interaction between these two. For mesor, there was a significant effect of diet (P=0.016) and genotype (P=0.008), but not of interaction (P=0.161). For amplitude there was no significant effect of salt diet (P=0.623), but the effect of genotype was different (P=0.039) as was the interaction (P=0.015). Planned comparisons were made within genotype, as indicated.
Supplemental Figure 5. Heart rate in control (open circle; n=6) and hsd11b2.BKO (black square; n=6) mice. Recordings were made by radiotelemetry and mice had ad libitum access to standard rodent diet and two drinking bottles containing dH 2 0 and 1.5% NaCl, respectively. Bottles were rotated every 24 hours. The diurnal variability was assessed in each mouse over the final 4 days of recording and data combined to give a group mean ± SEM. The bar indicates subjective night Hsd11b2.BKO mice (n=6, black squares). Mice were fed a gel diet delivering a fixed sodium intake per day. For the first 5 days, mice received 0.1% Na diet before being fed 1% Na diet for the next 8 days. Data, normalized to body weight, are mean ± SEM. Two-way ANOVA with repeated measures was used to assess the main effects of salt diet and genotype and the interaction between these two. For sodium excretion, the effect of diet was significant (P<0.0001), the effect of genotype not significant (P=0.063) and the interaction significant (P=0.048). For urine flow rate the effect of diet (P<0.0001), genotype (P=0.004) and the interaction (P<0.0001) was significant. For potassium excretion the effect of diet (P<0.0001 and genotype (P=0.035) was significant; the interaction was not significant (P=0.837). No planned or post-hoc comparisons were made. Figure 7. Baroreceptor reflex function under high salt conditions. The baroreflex was measured pharmacologically in anaesthetized Hsd11b2.BKO mice (black squares; n=5 mice/59 responses) and controls (white circles; n=6 mice; 71 responses) mice after 7 days of ad libitum access to 2.5% salt diet. A) the baroreflex curve showing individual data points for the change in heart rate (∆HR) in response to induced changes in systolic blood pressure (∆SBP). There was a significant difference (P<0.0001) between genotypes bylLinear regression analysis. B) the baroreflex gain during intravenous injection of sodium nitroprusside (tachycardic gain) and during C) intravenous injection of phenylephrine (bradycardic gain); individual data points are shown, with the median and IQR. Comparisons were by Mann-Whitney test, with P values as indicated.