Access Type

Open Access Dissertation

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Pablo A. Ortiz

Abstract

In humans, inactivating mutations in the Alström syndrome 1 (ALMS1) gene cause obesity, insulin resistance and hypertension (metabolic syndrome). More so, SNPs in the ALMS1 gene have been associated with kidney disease and cardiovascular disease. The

mechanisms causing these alterations are unclear. We recently found that the ALMS1 is expressed in the kidney thick ascending limb (TAL) where it mediates endocytosis of the renal Na/K/2Cl cotransporter termed NKCC2. To study the role of ALMS1 in renal

physiology we generated ALMS1 knockout (KO) rats in a Dahl salt-sensitive genetic background via zinc-finger nuclease targeting. We previously found that the amount of NKCC2 in the apical surface is higher in the TALs from ALMS1 KO rats compared to WT

(Dahl) SS (WT) rats. We hypothesized that deletion of ALMS1 will increase SBP and enhance salt-sensitivity of BP, in part due to higher NKCC2-mediated Na reabsorption. We used noninvasive tail cuff measurements and invasive radio-telemetry to study the

effects of dietary sodium (normal salt: .22% and high salt: 4% Na chow) on the systolic blood pressure (SBP) of the KO rats. The tail cuff measurements revealed with 0.22% Na chow the ALMS1 KO rats had a higher SBP than the WT rats (KO: 136±3 and WT: 125±3

mmHg, p<0.05). Then radio-telemetry confirmed the KO rats have a higher baseline SBP on .22% Na chow (KO: 145±2 and WT: 134±1 mmHg, p<0.025). After 2 weeks on high Na intake (4% Na chow) the SBP in ALMS1 KO rats increased to 181±1 mmHg, a 35±3

mmHg increase, whereas the SBP increased to 159±2 mmHg in WT rats, a 25±1 mmHg increase (p<0.025 vs ALMS1 KO). Then upon giving a daily dose of bumetanide (3mg/kg), an NKCC2 inhibitor, the SBP decreased in both groups (KO: -23±4 and WT: -30±6 mmHg,

p=0.40). After 6 days of treatment with bumetanide, the SBP was normalized only in the WT rats while the SBP remained elevated in the ALMS1 KO rats (KO: 131±3 and WT: 115±4 mmHg, p<0.025). We hypothesize that ALMS1 deletion leads to hypertension due

to a decrease ability to excrete a salt load secondary to enhanced NaCl transport by the TAL and other nephron segments. Using conscious rats in metabolic cages, it took longer for KO rats to excrete the Na load overall in which at all time points (6, 9, 24 hour) the

urinary Na excretion was lower in KO rats (cumulative Na, KO: 4251±590 vs. WT: 8788±994 μmols/24h, p<0.025). To study the role of different nephron segments in Na reabsorption we used a maximal single dose of segment-specific diuretic and measured

urine Na and volume excretion. The TAL diuretic bumetanide 20mg/kg, induced a higher natriuretic response in KO rats (KO: 485.2±37.1 vs. WT: 221.8±32 μmols/8h, p<0.05). Contrarily, acetazolamide, HCTZ and benzamil resulted in a lower natriuretic response in the KO rats; suggesting in absence of the inhibition with these diuretics there is no increase Na reabsorption along the proximal tubule, distal convoluted tubule and collecting duct. Our data showed that KO have a decreased ability to excrete a salt load,

and also the deletion of ALMS1 increases the sensitivity to TAL diuretics but not to acetazolamide, HCTZ or benzamil which is in part due to enhanced Na transport by the TAL. Furthermore, we found that deletion of ALMS1 in rats causes progressive obesity and insulin resistance evidenced by the growth charts and glucose tolerance tests obtained in our previous protocols. So it is unclear whether increases in blood pressure occur prior or after the development of metabolic alterations in the KO. So we studied the

effect caloric restriction (CR) on body weight (BW), SBP, glucose tolerance and leptin levels. A control-fed group of KO and WT rats received 30 g/day regular chow (0.4% Na). A caloric restricted (CR) group received 20% less food (24 g/day of a modified regular

chow (0.4%). We hypothesized that hypertension in the KO rats is primarily caused by a renal defect and not secondary to the metabolic syndrome. For the control-fed group we used young KO and WT rats (5-7 weeks old) with 6 male rats per group. At 5-7 weeks of age KO rats had similar body weights (KO: 239±16.18 g vs. WT: 230±16.64 g, N.S.); the baseline glucose was similar with a similar AUC. However, SBP was already higher in KO rats (KO: 156±5 vs. WT: 143±3 mmHg, p<0.025). Four weeks later the body weight began to diverge between strains (ALMS1 KO: 326±10 g vs. WT: 282±17 g, p<0.05). Baseline glucose was higher in KO (KO: 84±2 vs. WT: 71±3 mg/dL, p<0.005) and glucose tolerance test produced a larger AUC (KO: 11915±1349 vs. WT: 9176±9155 mg*min/dL). The SBP continued to be elevated in the KO rats (KO: 160±3 vs. WT: 144±3 mmHg, p<0.05). The 20% caloric restricted group had 7 male rats per group. At 7 weeks of age, the rats had a similar BW (KO: 209±13 g vs. WT: 182±15 g), but the baseline glucose was higher (KO: 86±3 vs. WT: 75± mg/dL, p<0.05), as well as the GTT. More importantly, SBP was higher in ALMS1 KO rats (KO: 156±5 vs. WT: 143±3 mmHg, p<0.05). Caloric restriction for 4 weeks prevented the increase in BW (KO: 359±8 vs. WT: 340±3 g, N.S.) but did not influence SBP or glucose intolerance. Therefore we conclude that the hypertension in the KO rats occur independently of obesity and insulin resistance. Furthermore, the caloric restriction prevents the increase in BW but does not prevent hypertension or improve metabolic alterations in this rat model. We conclude that the ALMS1 KO rats are hypertensive and salt sensitive in part due to enhanced Na reabsorption along the TAL via the NKCC2 cotransporter which is independent of obesity and insulin resistance. To our knowledge this is the first rat model of metabolic syndrome with established hypertension that does not require a high salt diet. Ongoing experiments with this rat model will explore the other physiological systems involvement in blood pressure regulation/elevation in the ALMS1 KO rats.

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