Abstract 17: Genetic Deletion Of Angiotensin Ii AT 1a Receptors Selectively In The Proximal Tubules Of The Kidney Attenuates Two-kidney, One-clip Goldblatt Hypertension In Pt- Agtr1a -/- Mice

The activation of the renin-angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two-kidney, one-clip Goldblatt hypertension (2K1C), but the roles of angiotensin II (Ang II) and AT 1a receptors in the proximal tubules has not been determined previously. The present study tested the hypothesis that genetic deletion of AT 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms. To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.2 mm internal diameter, on the left renal artery for 4 weeks in adult male wild-type (WT), global AT 1a receptor knockout ( Agtr1a -/- ), proximal tubule (PT)-specific Agtr1a -/- (PT- Agtr1a -/- ), or PT- Nhe3 -/- mice, respectively. In WT mice, systolic blood pressure increased in a time-dependent manner reaching a maximal response by Week 3 (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n=12, P <0.01). 2K1C Goldblatt hypertension in WT mice was associated with significant increases in renin mRNA expression in the clipped kidney (Control: 2366 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P <0.01) and decreases in renin mRNA expression in the nonclipped kidney (1738 ± 341 copies/ng RNA, P <0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C Goldblatt hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P <0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney ( P <0.01). In contrast to WT mice, the development of 2K1C hypertension was completely attenuated in Agtr1a -/- (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n=9, n.s .), PT- Agtr1a -/- mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 3 mmHg, n=12, n.s .) and PT- Nhe3 -/- mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n=12, n.s .). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a -/- mice, but it was decreased in the nonclipped kidney of PT- Agtr1a -/- mice ( P <0.05). Taken together, these data suggest that genetic deletion of AT 1a receptors selectively in the proximal tubules attenuates the development of 2K1C Goldblatt hypertension via AT 1a receptor-mediated, Na + /H + exchanger 3 (NHE3)-dependent mechanisms.

A role for tubular Na+/H+ exchanger NHE3 in the natriuretic effect of the SGLT2 inhibitor empagliflozin

Inhibitors of proximal tubular Na+-glucose cotransporter 2 (SGLT2) are natriuretic, and they lower blood pressure. There are reports that the activities of SGLT2 and Na+-H+ exchanger 3 (NHE3) are coordinated. If so, then part of the natriuretic response to an SGLT2 inhibitor is mediated by suppressing NHE3. To examine this further, we compared the effects of an SGLT2 inhibitor, empagliflozin, on urine composition and systolic blood pressure (SBP) in nondiabetic mice with tubule-specific NHE3 knockdown (NHE3-ko) and wild-type (WT) littermates. A single dose of empagliflozin, titrated to cause minimal glucosuria, increased urinary excretion of Na+ and bicarbonate and raised urine pH in WT mice but not in NHE3-ko mice. Chronic empagliflozin treatment tended to lower SBP despite higher renal renin mRNA expression and lowered the ratio of SBP to renin mRNA, indicating volume loss. This effect of empagliflozin depended on tubular NHE3. In diabetic Akita mice, chronic empagliflozin enhanced phosphorylation of NHE3 (S552/S605), changes previously linked to lesser NHE3-mediated reabsorption. Chronic empagliflozin also increased expression of genes involved with renal gluconeogenesis, bicarbonate regeneration, and ammonium formation. While this could reflect compensatory responses to acidification of proximal tubular cells resulting from reduced NHE3 activity, these effects were at least in part independent of tubular NHE3 and potentially indicated metabolic adaptations to urinary glucose loss. Moreover, empagliflozin increased luminal α-ketoglutarate, which may serve to stimulate compensatory distal NaCl reabsorption, while cogenerated and excreted ammonium balances urine losses of this “potential bicarbonate.” The data implicate NHE3 as a determinant of the natriuretic effect of empagliflozin.

Knockout of Na+-glucose cotransporter SGLT1 mitigates diabetes-induced upregulation of nitric oxide synthase NOS1 in the macula densa and glomerular hyperfiltration

Na+-glucose cotransporter (SGLT)1 mediates glucose reabsorption in late proximal tubules. SGLT1 also mediates macula densa (MD) sensing of an increase in luminal glucose, which increases nitric oxide (NO) synthase 1 (MD-NOS1)-mediated NO formation and potentially glomerular filtratrion rate (GFR). Here, the contribution of SGLT1 was tested by gene knockout (−/−) in type 1 diabetic Akita mice. A low-glucose diet was used to prevent intestinal malabsorption in Sglt1−/− mice and minimize the contribution of intestinal SGLT1. Hyperglycemia was modestly reduced in Sglt1−/− versus littermate wild-type Akita mice (480 vs. 550 mg/dl), associated with reduced diabetes-induced increases in GFR, kidney weight, glomerular size, and albuminuria. Blunted hyperfiltration was confirmed in streptozotocin-induced diabetic Sglt1−/− mice, associated with similar hyperglycemia versus wild-type mice (350 vs. 385 mg/dl). Absence of SGLT1 attenuated upregulation of MD-NOS1 protein expression in diabetic Akita mice and in response to SGLT2 inhibition in nondiabetic mice. During SGLT2 inhibition in Akita mice, Sglt1−/− mice had likewise reduced blood glucose (200 vs. 300 mg/dl), associated with lesser MD-NOS1 expression, GFR, kidney weight, glomerular size, and albuminuria. Absence of Sglt1 in Akita mice increased systolic blood pressure, associated with suppressed renal renin mRNA expression. This may reflect fluid retention due to blunted hyperfiltration. SGLT2 inhibition prevented the blood pressure increase in Sglt1−/− Akita mice, possibly due to additive glucosuric/diuretic effects. The data indicate that SGLT1 contributes to diabetic hyperfiltration and limits diabetic hypertension. Potential mechanisms include its role in glucose-driven upregulation of MD-NOS1 expression. This pathway may increase GFR to maintain volume balance when enhanced MD glucose delivery indicates upstream saturation of SGLTs and thus hyperreabsorption.

Lineage tracing aged mouse kidneys shows lower number of cells of renin lineage and reduced responsiveness to RAAS inhibition

Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.

Abstract P314: Urinary Renin and Kidney RAS Activation in Mice with Diabetic Kidney Disease

RAS is overactive in kidneys from patients with diabetic nephropathy (DN), but circulating plasma renin activity (PRA) is usually low. This is known as the renin-paradox. We evaluated juxtaglomerular (JGA), tubular and urinary renin, as a potential source of local RAS activation, to gain some understanding of this paradox. Mice with STZ induced diabetes were used which had mild albuminuria and glomerular mesangial expansion consistent with early DN. Renin expression in the JGA and in the collecting tubule (CT) was evaluated by immunohistochemistry. IF was used to localize renin within CT cells. Proximal tubular renin was evaluated by RT-real time PCR in microdissected proximal tubules (PT). Urinary renin and Ang II were measured by ELISA. Urinary Ang II was increased (37.8±11.4 vs. 99.0±21.6 pg/mg creat, p<0.05) reflecting an active kidney RAS. Urinary renin was also increased in STZ-treated as compared to controls (Table). In microdissected PTs there were no significant differences in renin mRNA between control and STZ-mice. By immunostaining, renin was localized to principal cells in the CT and the number of renin stained CTs was higher in STZ than in control mice. In sharp contrast, renin staining of the JGA of STZ-mice was significantly reduced as compared to controls. We conclude that in DN renin expression in the JGA, the physiologic site of renin secretion into the circulation is suppressed, whereas in the CT it is increased. Activation of the kidney RAS, as inferred from increased urinary Ang II, likely occurs as a result of renin of tubular origin rather than from JGA renin. Since PT renin is not increased, the CT may provide the source of tubular renin for RAS activation.

Abstract P187: Partial Renal Infarction Induces Hypertension

Renal infarction is an under-diagnosed and under-reported phenomenon. The U.S. incidence of renal infarction is estimated at 1.4%. Systemically thromboembolic originate from thrombus in the heart or aorta while renal infarction in situ typically involves the main renal artery or its branches. Acute or aggravated hypertension is commonly observed in previously normotensive or hypertensive patients with renal infarction. However, these pathophysiological mechanisms have not been elucidated. The goal of this study was to develop a hypertensive mouse model of renal infarction. Partial renal infarction was performed in C57BL/6 mice by ligating either the upper (LU) or lower (LL) branch of the renal artery in the left kidney while the right kidney remained intact. The mean arterial pressure (MAP) was continuously measured with a telemetry system in conscious mice fed 4 weeks of normal salt diet (NS) (0.4% NaCl) followed by 4 weeks of high salt diet (HS) (4% NaCl). Plasma renin concentration (PRC), renin mRNA in the kidney and TNF-α were measured. Body weight, salt and fluid intakes were similar in mice with LU and LL ligation compared with sham operated mice. The weight of the left kidney decreased by 16.3% in LU (118.1±8.9 mg) and 14.2% in LL (121.5±7.8 mg) compared with sham operated mice (141.0±9.5 mg) (n=6; p<0.05 vs sham). The right kidney weight increased by 41.5% in LU (201.3±15.6 mg) and 38.2% in LL (196.6±8.1 mg) compared with sham mice (142.2±8.8 mg) (n=6; p<0.01 vs sham). MAP in mice fed NS elevated by 25% in LU (119.4±12.9 mmHg) and 19.1% in LL (113.7±10.6 mmHg), compared with sham (95.4±4.7 mmHg) (n=4; p<0.05 vs sham). HS further increased the MAP to 124.2±17.4 mmHg in LU and 118.6±14.8 mmHg in LL mice. PRC decreased by 50.0% in LU (30.7±8.63 ng/ml) and 62.7% in LL (22.9±10.8 ng/ml), compared with sham operated mice (61.4±12.6 ng/ml) (n=6; p<0.05 vs sham). Expression of local renin mRNA in the left kidney was upregulated by 113.4% in LU and 64.1% in LL mice, compared with the sham. Inflammatory cytokines TNF-α was increased by 174.2% in LU and 106.3% in LL mice. In conclusion, we developed a mouse model of partial renal infarction with hypertension in C57BL/6 mice. The mechanism of hypertension may be due to the upregulation of local renin angiotensin system and inflammation.

Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice

The kidney is the primary organ ensuring K+ homeostasis. K+ is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K+ channel (Kir1.1) and by the Ca2+-activated K+ channel (KCa1.1). Here, we report a novel knockout mouse of the β2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K+ excretion. KCNMB2−/− mice displayed hyperaldosteronism, normal plasma K+ concentration, and produced dilute urine with decreased K+ concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2−/− mice. Renal K+ excretion rates were similar in the two genotypes; however, KCNMB2−/− mice required elevated plasma aldosterone to achieve K+ balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K+ excretion in KCNMB2−/− mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1−/− mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K+ secretion. KCNMB2−/− mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1−/− mice but were normotensive and displayed intact flow-induced K+ secretion. Despite elevated plasma aldosterone, KNCMB2−/− mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na+ diet, although plasma aldosterone remained elevated in KCNMB2−/− mice. In summary, KCNMB2−/− mice have a reduced ability to excrete K+ into the urine but achieve K+ balance through an aldosterone-mediated, β2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1−/− mice.

Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct

Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10−6 M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD.

PKC-α-dependent augmentation of cAMP and CREB phosphorylation mediates the angiotensin II stimulation of renin in the collecting duct

In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron.