**3. Results**

Aged *Hyp* mice show a stimulation of the RAAS and a downregulation of renal αKlotho protein abundance, but they have normal kidney function.

As expected, 14-month-old male *Hyp* mice were characterized by an approximately 10-fold increase in serum intact FGF23 when compared to age- and sex-matched WT control animals (Figure 1A). The elevated circulating intact FGF23 levels in aged *Hyp* mice were associated with hypophosphatemia (Figure 1B). Serum calcium tended to be lower, whereas serum intact PTH tended to be higher in *Hyp* mice, but both effects did not reach statistical significance (Figure 1C,D). We reported earlier that 3-month-old *Hyp* mice were characterized by lower serum aldosterone levels relative to WT mice [13]. However, in aged *Hyp* mice, serum aldosterone was not down-, but rather up-regulated (Figure 1E). Aldosterone and FGF23 are known regulators of the sodium chloride cotransporter NCC activity in distal convoluted renal tubules [13]. Therefore, we quantified renal pNCC protein abundance by Western blotting and by immunofluorescence analysis. Similar to younger *Hyp* mice [13], renal pNCC levels were up-regulated and pNCC immunofluorescence tended to be higher in 14-month-old *Hyp* mice, relative to WT controls (Figure 1F,G). The abundance of αKlotho protein was distinctly down-regulated by about 50% in the kidneys of *Hyp* mice (Figure 1H), which may be a counter-regulatory mechanism to protect against the chronically elevated circulating FGF23 levels. However, renal function as evidenced by serum creatinine concentration and by renal creatinine clearance remained unchanged in aged *Hyp* mice (Figure 1I,J).

**Figure 1. Aged** *Hyp* **mice show a stimulation of the RAAS and a down-regulation of renal** α**Klotho protein abundance, but have normal kidney function.** (**A**) Serum intact FGF23 levels

(n = 14–15), (**B**,**C**) serum phosphate and calcium concentrations (n = 10–11), and (**D**,**E**) serum intact PTH and aldosterone levels (n = 9–14) in 14-month-old male wildtype (WT) and *Hyp* mice. (**F**) Left: representative images of immunofluorescent staining of kidney paraffin sections with an anti-pNCC antibody (original magnification 400×). Right: quantification of anti-pNCC immunofluorescence in the kidney in 14-month-old male WT and *Hyp* mice (n = 6). Western blot quantification of (**G**) pNCC and (**H**) α-Klotho from kidney cortex homogenates (n = 6–11), as well as (**I**) serum creatinine levels (n = 10–11) and (**J**) glomerular filtration rate per gram body weight as measured by renal creatinine clearance (n = 8–9) in 14-month-old male *Hyp* mice and WT controls. Bars in (**A**–**J**) represent mean ± SEM for WT and *Hyp* mice. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 by Student's *t*-test. ns, not significant.
