Prostaglandin-E2 Mediated Increase in Calcium and Phosphate Excretion in a Mouse Model of Distal Nephron Salt Wasting

<div><p>Contribution of salt wasting and volume depletion to the pathogenesis of hypercalciuria and hyperphosphaturia is poorly understood. Pendrin/NCC double KO (pendrin/NCC-dKO) mice display severe salt wasting under basal conditions and develop profound volume depletion, prerenal renal failure, and metabolic alkalosis and are growth retarded. Microscopic examination of the kidneys of pendrin/NCC-dKO mice revealed the presence of calcium phosphate deposits in the medullary collecting ducts, along with increased urinary calcium and phosphate excretion. Confirmatory studies revealed decreases in the expression levels of sodium phosphate transporter-2 isoforms a and c, increases in the expression of cytochrome p450 family 4a isotypes 12 a and b, as well as prostaglandin E synthase 1, and cyclooxygenases 1 and 2. Pendrin/NCC-dKO animals also had a significant increase in urinary prostaglandin E2 (PGE-2) and renal content of 20-hydroxyeicosatetraenoic acid (20-HETE) levels. Pendrin/NCC-dKO animals exhibit reduced expression levels of the sodium/potassium/2chloride co-transporter 2 (NKCC2) in their medullary thick ascending limb. Further assessment of the renal expression of NKCC2 isoforms by quantitative real time PCR (qRT-PCR) reveled that compared to WT mice, the expression of NKCC2 isotype F was significantly reduced in pendrin/NCC-dKO mice. Provision of a high salt diet to rectify volume depletion or inhibition of PGE-2 synthesis by indomethacin, but not inhibition of 20-HETE generation by HET0016, significantly improved hypercalciuria and salt wasting in pendrin/NCC dKO mice. Both high salt diet and indomethacin treatment also corrected the alterations in NKCC2 isotype expression in pendrin/NCC-dKO mice. We propose that severe salt wasting and volume depletion, irrespective of the primary originating nephron segment, can secondarily impair the reabsorption of salt and calcium in the thick ascending limb of Henle and/or proximal tubule, and reabsorption of sodium and phosphate in the proximal tubule via processes that are mediated by PGE-2.</p></div

The effect of indomethacin treatment on urine calcium and phosphate excretion, and NKCC2 isotype expression of WT and pendrin/NCC-dKO mice.

<p>Effect of indomethacin treatment (25mg/kg BW/day) on <b>(A)</b> urinary PGE2 levels, <b>(B)</b> calcium and phosphate excretion and <b>(C)</b> the expression levels of NKCC2 isotypes was determined (results are the average+/-standard deviation of n = 6 animals/genotype/treatment for parts A and B; and the average +/- SEM of n = 4 animals/genotype/treatment for part C).</p

Examination of the effect of simultaneous ablation of pendrin and NCC on renal function and histology of double knockout (pendrin/NCC-dKO) mice.

<p>The urinary (top panels) and serum (bottom panels) calcium <b>(A)</b> and phosphate <b>(B)</b> levels of WT and pendrin/NCC-dKO animals were compared. The results are average +/- SEM of 3 animals/genotype, similar results were obtained in two independent studies. <b>(C)</b> Histological examination of the H&E stained kidney sections of WT (panels <b>a</b> and <b>b</b>, 200X and 400X magnification respectively) and pendrin/NCC-dKO (panels <b>c</b> & <b>d</b>, 200X and 400X magnification respectively; arrows point to calcium deposits), NCC KO (panel <b>e</b>, 200X magnification) and pendrin KO (panel <b>f</b>, 200X magnification) mice. Von Kossa staining of kidneys of pendrin/NCC-dKO mice indicates that the deposits contain calcium (panel <b>g</b>, 400X magnification, arrow points to Von Kossa stain positive calcium deposit).</p

Comparison of the expression of Na-K-2Cl co-transporter 2 (NKCC2) isoforms in the kidneys of WT and pendrin/NCC-dKO mice.

<p><b>A)</b> The NKCC2 isoform profiles of WT and pendrin/NCC-dKO mice were compared using qRT-PCR (the results represent the average +/- SEM of n = 4 animals/genotype). <b>B)</b> Immunofluorescence microscopic examination of NKCC2 expression in the renal cortex and medulla of WT and pendrin/NCC-dKO mice.</p

The expression of arachidonic acid pathway enzymes and production of PGE2 are elevated in pendrin/NCC-dKO mice.

<p><b>(A)</b> The expression of Cox1 (panels <b>a-d</b>), Cox2 (panels <b>e-h</b>) and Ptges1 (panels <b>i-l</b>) were compared in the kidneys of WT and pendrin/NCC-dKO mice. Arrows point to positive staining tubules <b>(B)</b> The expression of Cox1, Cox2 and Ptges1 were examined in the kidneys of pendrin/NCC-dKO and WT mice (n = 4) by western blot analysis (immunofluorescence microscopy results are representative of the staining observed in the kidney sections from n = 3 animals/genotype). <b>(C)</b> Urinary PGE-2 levels in WT and pendrin/NCC-dKO mice were compared (results are the average+/-SEM of time matched urine samples from n = 6 animals/genotype).</p

Comparison of the expression of NaPi-II isoforms in the kidneys of WT and pendrin/NCC-dKO mice.

<p><b>(A)</b> Northern blot images of NaPi-IIa in kidneys of WT and pendrin/NCC dKO mice (left panel) and NaPi-IIb in kidney medulla of WT and dKO mice (right panel) show a significant downregulation in NaPi-IIa expression and an unexpected induction of NaPi-IIb transcripts in kidneys of pendrin/NCC-dKO mice. Depiction of immunofluorescence microscopic images demonstrating a significant reduction in the expression of NaPi-IIa and NaPi-IIc in the proximal tubule <b>(B & C)</b> of pendrin/NCC-dKO mice (vs. WT animals), and the induction of NaPi-IIb (arrows) in the kidney cortex <b>(D</b>, right top panel<b>)</b> and medulla <b>(D</b>, right bottom panel<b>)</b> of pendrin/NCC-dKO animals. Co-localization (middle panel) of NaPi-IIb <b>(red</b>, left panel<b>)</b> with AQP2 <b>(green</b>, right panel<b>)</b> in the medullary collecting duct (MCD)-cells of pendrin/NCC-dKO mice is shown <b>(E)</b>. (immunofluorescence microscopy results are representative of the staining observed in the kidney sections from at least n = 3 animals/genotype).</p

Comparison of renal Cyp4a12a and 20-HETE levels in WT and pendrin/NCC-dKO mice.

<p><b>(A)</b> The expression of Cyp4a12a in the kidneys of pendrin/NCC-dKO and WT mice (n = 3) were examined by northern blot analysis. <b>(B)</b> Renal 20-HETE levels in WT and pendrin/NCC-dKO mice were compared (n = 3 mice/genotype).</p

The effect of high salt diet on calcium and phosphate excretion, and the expression of NKCC2 isotype in WT and pendrin/NCC-dKO mice.

<p>Effect of provision of liquid normal and high salt diets for 7 days <b>(A)</b> on urinary PGE-2 levels, <b>(B)</b> calcium and phosphate excretion and <b>(C)</b> the expression levels of NKCC2 isotypes in WT and pendrin/NCC-dKO mice was examined in WT and pendrin/NCC-dKO mice (results are the average+/- SEM of n = 6 animals/genotype/treatment for parts A and B; and the average +/- SEM of n = 4 animals/genotype/treatment for part C).</p

The non-diuretic hypotensive effects of thiazides are enhanced during volume depletion states

<div><p>Thiazide derivatives including Hydrochlorothiazide (HCTZ) represent the most common treatment of mild to moderate hypertension. Thiazides initially enhance diuresis via inhibition of the kidney Na⁺-Cl^- Cotransporter (NCC). However, chronic volume depletion and diuresis are minimal while lowered blood pressure (BP) is maintained on thiazides. Thus, a vasodilator action of thiazides is proposed, likely via Ca²⁺-activated K⁺ (BK) channels in vascular smooth muscles. This study ascertains the role of volume depletion induced by salt restriction or salt wasting in NCC KO mice on the non-diuretic hypotensive action of HCTZ. HCTZ (20mg/kg s.c.) lowered BP in 1) NCC KO on a salt restricted diet but not with normal diet; 2) in volume depleted but not in volume resuscitated pendrin/NCC dKO mice; the BP reduction occurs without any enhancement in salt excretion or reduction in cardiac output. HCTZ still lowered BP following treatment of NCC KO on salt restricted diet with paxilline (8 mg/kg, <i>i</i>.<i>p</i>.), a BK channel blocker, and in BK KO and BK/NCC dKO mice on salt restricted diet. In aortic rings from NCC KO mice on normal and low salt diet, HCTZ did not alter and minimally decreased maximal phenylephrine contraction, respectively, while contractile sensitivity remained unchanged. These results demonstrate 1) the non-diuretic hypotensive effects of thiazides are augmented with volume depletion and 2) that the BP reduction is likely the result of HCTZ inhibition of vasoconstriction through a pathway dependent on factors present in vivo, is unrelated to BK channel activation, and involves processes associated with intravascular volume depletion.</p></div

Intravascular volume depletion in Pendrin/NCC dKO mice.

<p>(a) Northern hybridization shows a significant enhancement in mRNA expression levels of renin in kidneys of pendrin/NCC dKO mice compared to WT, pendrin KO and NCC KO. (b) Immunofluorescent microscopic analysis of kidney sections indicates that the expression of renin is significantly up regulated in pendrin/NCC dKO, but not in WT mice. (c) Western blots confirmed the significant increase in renin expression in pendrin/NCC dKO kidney vs WT. (d) Olmesartan (1 mg/kg) treatment causes a more significant reduction in the systolic BP of pendrin/NCC dKO mice compared to WT mice, (n = 4 each group); paired t-test * P<0.05.</p