Effect of Losartan on Reflex Control of Sympathetic Nerve Activity in Chronic Kidney Disease

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The effect of losartan on differential reflex control of sympathetic nerve activity in chronic kidney disease

Online Supplement

Yimin Yao^a, Cara M. Hildreth^a, Melissa M. Farnham^a,b,

Manash Saha

^a

, Qi-Jian Sun

^a

,

Paul M.

Pilowsky^b & Jacqueline K. Phillips^a*

(a) Australian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia (b) Heart Research Institute and University of Sydney, Newtown, Sydney, NSW, Australia

* Correspondence to Prof. Jacqueline K Phillips, Australian School of Advanced Medicine, Macquarie University, Sydney, NSW 2109, Australia. E-mail: jacqueline.phillips@mq.edu.au

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SUPPLEMENTARY METHODS Baroreceptor  reflex  response  analysis  

The relationship between the active phase of the mean arterial pressure (MAP) change and the corresponding level of splanchnic sympathetic nerve activity (sSNA), renal sympathetic nerve activity (rSNA) and lumbar sympathetic nerve activity (lSNA) (% change) were fitted to a four- parameter sigmoid logistic function curve (Graph Pad software Inc., La Jolla, CA, USA) using the following equation:

y = A

1

/ {1+exp [A

2

(MAP-A

3

)]} +A

4

Curves with an R

²

less than 0.9 were not included in the data set. From each individual non- linear regression curve generated, the following parameters were obtained: range of the curve (A1), the gain coefficient (A2), midpoint of the curve (A3) and lower plateau (A4). Using these parameters, the upper and lower plateaus of the curve, range of the reflex, the gain of the reflex, mean arterial pressure at the midpoint of the curve (MAP

₅₀

), MAP threshold (MAP

_thr

), MAP saturation (MAP

_sat

) and operating range of reflex were calculated as described previously [1].

The SNA-MAP relationships were then plotted over a fixed range of 25–200 mmHg in the Lewis

and 50–250 mmHg in the LPK.

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SUPPLEMENTARY RESULTS FIGURE  S1  

Representative raw data traces, illustrating responses of splanchnic sympathetic nerve activity (sSNA), renal sympathetic nerve activity (rSNA) and lumbar sympathetic nerve activity (lSNA) to evoked changes in arterial pressure (AP) from a Lewis polycystic kidney (LPK) rat. In response to phenylephrine (PE), AP increased and SNA was silenced. When MAP is reduced by sodium nitroprusside (SNP), minimal change in SNA was observed.

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Table  S1  

Absolute values of baseline sympathetic nerve activity (µV) in urethane anaesthetised Lewis controls and Lewis polycystic kidney rats

Lewis n LPK n

sSNA (µV) 3.4 ± 0.4 14 5.7 ± 1 14

rSNA (µV) 3.7 ± 0.6 12 8.3 ± 1.2 15

lSNA (µV) 2.4 ± 0.3 12 5.0 ± 1.2 12

Absolute levels (microvolts µV) of baseline splanchnic sympathetic nerve activity (sSNA), renal sympathetic nerve activity (rSNA) and lumbar sympathetic nerve activity (lSNA). Results are expressed as mean ± SEM. n = number of animals per group.

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TABLE  S2  

Key defining parameters of sympathetic nerve baroreflex function curves in Lewis controls and Lewis polycystic kidney rats

Lewis LPK

sSNA n = 9 n = 9

Gain (%/mmHg) -2.0 ± 0.3 -0.7 ± 0.05*

Range (%) 79 ± 4 43 ± 3*

Upper Plateau (%) 103 ± 2 102 ± 1

Lower Plateau (%) 29 ± 5 59 ± 3*

MAP50 (mmHg) 119 ± 2 153 ± 9*

MAP_sat (mmHg) 132 ± 3 173 ± 9*

MAP_thr (mmHg) 105 ± 2 132 ± 10*

MAPoperating range (mmHg) 27 ± 2 41 ± 5*

rSNA n = 8 n = 10

Gain (%/mmHg) -2.1 ± 0.7 -0.6 ± 0.05*

Range (%) 84 ± 5 52 ± 4*

Upper Plateau (%) 106 ± 2 105 ± 2

Lower Plateau (%) 22 ± 4 53 ± 4*

MAP50 (mmHg) 116 ± 4 154 ± 7*

MAP_sat (mmHg) 133 ± 5 189 ± 7*

MAPthr (mmHg) 99 ± 4 125 ± 9*

MAPoperating range (mmHg) 35 ± 5 64 ± 7*^†

lSNA n = 7 n = 7

Gain (%/mmHg) -1.6 ± 0.5 -1.1 ± 0.1^{†, ‡}

Range (%) 60 ± 9 66 ± 6^†

Upper Plateau (%) 104 ± 1 104 ± 2

Lower Plateau (%) 42 ± 8 38 ± 7^†

MAP50 (mmHg) 110 ± 5 151 ± 6*

MAP_sat (mmHg) 128 ± 5 171 ± 6*

MAP_thr (mmHg) 98 ± 6 131 ± 8*

MAPoperating range (mmHg) 30 ± 4 41 ± 6^‡

sSNA, splanchnic sympathetic nerve activity; rSNA, renal sympathetic nerve activity; lSNA, lumbar sympathetic nerve activity; MAP₅₀, mean arterial pressure at the midpoint of the curve; MAP_thr, threshold MAP to trigger a change in SNA; MAP_sat, saturation MAP at which there is no further change in SNA. The baroreflex function curve parameters were obtained from the logistic function relating normalized SNA (%baseline) to MAP. Results are expressed as mean ± SEM. *P<0.05 vs. Lewis of the same nerve;

†P<0.05 vs. the same parameter in sSNA of the same strain; ^‡P<0.05 vs. the same parameter in rSNA of the same strain. Results are expressed as mean ± SEM. Statistical comparisons were determined using two-way ANOVA with strain and nerve as the independent variables, with Bonferroni’s post-hoc correction to identify specific group difference. n = number of animals per group.

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TABLE  S3  

Key defining parameters of baroreflex function curves in Lewis controls and Lewis polycystic kidney rats before and after losartan administration (3 mg/kg)

Lewis LPK

Before losartan

After losartan

Before losartan

After losartan

sSNA n = 6 n = 5

Gain (%/mmHg) -1.5 ± 0.1 -1.4 ± 0.1 -0.7 ± 0.07 -1.2 ± 0.1^δ

Range (%) 77 ± 4 71 ± 5 50 ± 4 51 ± 1

Upper Plateau (%) 105 ± 3 103 ± 1 102 ± 1 102 ± 0.1

Lower Plateau (%) 27 ± 5 33 ± 5 52 ± 4 51 ± 1

MAP₅₀ (mmHg) 121 ± 2 105 ± 6^δ 164 ± 7 135 ± 4^δ

MAPsat (mmHg) 135 ± 4 122 ± 5 189 ± 7 149 ± 4^δ

MAP_thr (mmHg) 105 ± 3 88 ± 7 142 ± 8 120 ± 4^δ

MAPoperating range (mmHg) 31 ± 4 35 ± 4 50 ± 3 29 ± 3^δ

rSNA n = 5 n = 6

Gain (%/mmHg) -2.1 ± 0.5 -1.7 ± 0.4 -0.6 ± 0.08 -0.71 ± 0.06

Range (%) 74 ± 4 68 ± 4 48 ± 6 48 ± 6

Upper Plateau (%) 101 ± 1 103 ± 2 104 ± 1 104 ± 1

Lower Plateau (%) 27 ± 3 35 ± 3 56 ± 6 55 ± 6

MAP50 (mmHg) 112 ± 3 91 ± 6^δ 155 ± 7 136 ± 4^δ

MAP_sat (mmHg) 126 ± 5 107 ± 9 180 ± 8 161 ± 6^δ

MAPthr (mmHg) 99 ± 2 75 ± 6^δ 129 ± 11 114 ± 5

MAPoperating range (mmHg) 28 ± 5 33 ± 8 56 ± 7 47 ± 8

lSNA n = 5 n = 5

Gain (%/mmHg) -2.1 ± 0.7 -2.6 ± 0.8 -1.2 ± 0.1 -1.5 ± 0.6

Range (%) 60 ± 14 69 ± 8 65 ± 10 56 ± 15

Upper Plateau (%) 103 ± 2 101 ± 1 104 ± 2 107 ± 2

Lower Plateau (%) 40 ± 13 38 ± 7 38 ± 11 41 ± 15

MAP₅₀ (mmHg) 114 ± 5 95 ± 7^δ 159 ± 5 135 ± 10^δ

MAPsat (mmHg) 135 ± 6 115 ± 8 177 ± 8 159 ± 18

MAP_thr (mmHg) 103 ± 4 81 ± 7^δ 141 ± 5 111 ± 11^δ

MAPoperating range (mmHg) 22 ± 4 21 ± 4 38 ± 6 38 ± 11

sSNA, splanchnic sympathetic nerve activity; rSNA, renal sympathetic nerve activity; lSNA, lumbar sympathetic nerve activity; MAP₅₀, mean arterial pressure at the midpoint of the curve; MAP_thr, threshold MAP to trigger a change in SNA; MAP_sat, saturation MAP at which there is no further change in SNA. The baroreflex function curve parameters were obtained from the logistic function relating normalized SNA (%

baseline) to MAP. Results are expressed as mean ± SEM. ^δSignificantly different between before and after losartan within same strain (P<0.05), as determined using two-way repeated measures of ANOVA followed by Bonferroni’s post-hoc correction. n = number of animals per group.

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Peripheral  chemoreflex  responses  to  100%  N

2

 for  12  seconds  and  the  effect  of  losartan  

The peripheral chemoreflex responses to 100% N

2

for 12 s were consistent to that observed with 10% O

2

in N

2

for 45 s. MAP and all sympathetic outflows were increased in the Lewis controls, but overall decreased in the LPK (Table S4). Administration of losartan did not alter responses in the Lewis (Figure S2). In the LPK, losartan ameliorated the MAP response to chemoreflex stimulation, such that while not significantly different to the LPK pre-losartan response (P> 0.99), it was no longer significantly different to the MAP response in the Lewis (P = 0.3757). After losartan, the SNA response to peripheral chemoreceptor stimulation with 100% N

2

was now sympathoexcitatory in all three sympathetic outflows, and not significantly different to the response seen in the Lewis rat (Figure S2).

TABLE  S4  

Peripheral chemoreflex, blood pressure and sympathetic nerve responses to 100% N

2

in Lewis controls and Lewis polycystic kidney rats

Lewis n LPK n

Δ MAP (mmHg) 12 ± 3 13 -10 ± 5* 14

% Δ sSNA 38.5 ± 7 8 -10 ± 5* 9

% Δ rSNA 58.4 ± 18 7 -18 ± 5* 10

% Δ lSNA 68 ± 19 7 -19.5 ± 6* 7

Parameters showing percentage (%) change (Δ) in mean arterial pressure (MAP) and sympathetic nerve activity in response to peripheral hypoxia evoked by 12 s 100% N₂ ventilation in Lewis and Lewis Polycystic Kidney (LPK) rats. sSNA, splanchnic sympathetic nerve activity; rSNA, renal sympathetic nerve activity; lSNA, lumbar sympathetic nerve activity. Results are expressed as mean ± SEM. *Significantly different to Lewis (P<0.05). MAP was compared using an unpaired two-tailed Student’s t-test. SNAs were compared determined using two-way ANOVA with strain and nerve as the independent variables, and Bonferroni’s post-hoc correction to identify specific group difference. n = number of animals per group.

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FIGURE  S2  

Effect of acute losartan administration (3mg/kg i.v.) on the peripheral chemoreflex response induced by 100% N₂ for 12 s. A: Representative traces showing recordings of mean arterial pressure (MAP), splanchnic sympathetic nerve activity (sSNA); renal sympathetic nerve activity (rSNA); lumbar sympathetic nerve activity (lSNA) from an LPK before (left panel) and after losartan administration (right panel, 3 mg/kg, i.v.). Absolute values of SNA (µV) are rectified and smoothed (t = 1s). B-E: The peak change in MAP (B) and % change from baseline in sSNA (C), rSNA (D) and lSNA (E) in response to peripheral hypoxia evoked by 12 s 100% N₂ ventilation. Lewis before losartan, Lewis after losartan, LPK before losartan and LPK after losartan. Results are expressed as mean ± SEM. *Significantly different between indicated groups (P<0.05), as determined using a two-way repeated measures of ANOVA followed by Bonferroni’s post hoc correction. Paired n values for Lewis and LPK data: MAP = 6/6, sSNA = 5/5, rSNA =

4/6 and lSNA = 5/4.    

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TABLE  S5  

Latency and amplitude of somatosensory reflex biphasic response in Lewis controls and Lewis polycystic kidney rats before and after losartan administration (3 mg/kg i.v.)

Lewis LPK

Latency (ms)

Before losartan

After losartan

Before losartan

After losartan

sSNA n = 6 n = 5

1^st peak 74 ± 1 77 ± 2 85 ± 3 84 ± 5

2^nd peak 156 ± 4 153 ± 9 139 ± 5 143 ± 4

Δ peak to peak 82 ± 2 75 ± 7 54 ± 4 60 ± 4

rSNA n = 5 n = 6

1^st peak 100 ± 2 101 ± 2 103 ± 5 100 ± 8

2^nd peak 192 ± 2 193 ± 4 166 ± 4 168 ± 5

Δ peak to peak 94 ± 2 93 ± 4 61 ± 5 69 ± 9

lSNA n = 5 n = 5

1^st peak 107 ± 3 103 ± 2 116 ± 10 116 ± 12

2^nd peak 202 ± 6 200 ± 3 164 ± 7 168 ± 7

Δ peak to peak 93 ± 4 98 ± 3 58 ± 12 53 ± 13 Peak amplitude

(% baseline)

Before losartan

After losartan

Before losartan

After losartan

sSNA n = 6 n = 5

1^st peak 114 ± 18 146 ± 34 59 ± 11 81 ± 15

2^nd peak 134 ± 21 135 ± 23 95 ± 15 99 ± 17

rSNA n = 5 n = 6

1^st peak 141 ± 28 106 ± 17 69 ± 17 75 ± 21

2^nd peak 103 ± 34 79 ± 29 81 ± 14 102 ± 28

lSNA n = 5 n = 5

1^st peak 171 ± 38 182 ± 20 102 ± 13 83 ± 18

2^nd peak 102 ± 31 97 ± 24 159 ± 49 138 ± 40

sSNA, splanchnic sympathetic nerve activity; rSNA, renal sympathetic nerve activity; lSNA, lumbar sympathetic nerve activity. Results are expressed as mean ± SEM. Statistical analysis performed using two-way repeated measures of ANOVA followed by Bonferroni’s post-hoc correction to compare the effect of losartan within each strain. n = number of animals per group.

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REFERENCES

1. Salman IM, Hildreth CM, Ameer OZ, Phillips JK. Differential contribution of afferent and central pathways to the development of baroreflex dysfunction in chronic kidney disease.

Hypertension 2014; 63:804-810.