Aging Impairs Renal Autoregulation in Mice

Abstract

Impaired renal autoregulation permits more transmission of disturbance in systemic blood pressure, which initiates barotrauma in intrarenal microvasculatures such as glomerular and tubulointerstitial capillaries, contributing to the development of kidney damage and deterioration in renal function, especially under the conditions with high blood pressure. Although it has been postulated that autoregulatory efficiency is attenuated in the aging kidney, direct evidence remains lacking. In the present study, we measured the autoregulation of renal blood flow (RBF), myogenic response of afferent arteriole (Af-Art), tubuloglomerular feedback (TGF) in vivo with micropuncture, as well as TGF in vitro in isolated perfused juxtaglomerular apparatus (JGA) in young and aged C57BL/6 mice. We found that RBF was not significantly changed in response to a defined elevation of renal arterial pressure in young mice, but significantly increased in aged mice. Additionally, myogenic response of Af-Art measured by microperfusion with a stepwise increase in perfusion pressure was significantly blunted in the aging kidney, which is associated with the attenuation of intraluminal pressure-induced intracellular calcium increases, as well as the reduced expression of integrin α5 in Af-Art. Moreover, both TGF in vivo and in vitro were nearly inactive in the aging kidney, which is associated with the significantly reduced expression of adenosine A1 receptor (A1AR) and suppressed vasoconstrictor response to adenosine in Af-Art. In conclusion, this study demonstrates that aging impairs renal autoregulation with blunted myogenic response and inhibited TGF response. The underlying mechanisms involve the downregulations of integrin α5 and A1AR in the Af-Art.

INTRODUCTION

With aging, the kidney exhibits a series of morphologic changes including nephron loss, arteriolar hyalinization, glomerulosclerosis and tubulointerstitial fibrosis, as well as a gradual decline in the clearance function.^1,2 In spite of extensive experimental and clinical research, the underlying mechanism for these structural and functional alterations in the aging kidney is not fully understood.

Autoregulation is a vital homeostatic mechanism, which maintains renal blood flow (RBF) and glomerular filtration rate (GFR) relatively constant over 80–180 mmHg in renal arterial pressure (RAP). Myogenic response and tubuloglomerular feedback (TGF) are the two primary mechanisms that largely control the autoregulatory efficiency by regulating the pre-glomerular vasomotor tone.³ Renal autoregulatory mechanism not only stabilizes the tubular fluid flow to keep electrolytes and water balance, but also prevents the transmission of fluctuation in systemic blood pressure to intrarenal vasculatures.^3,4 Impaired renal autoregulation permits more passing of the disturbance in renal perfusion pressure, which initiates barotrauma in micro-vasculatures such as glomerular and tubulointerstitial capillaries, contributing to the development of kidney damages and deterioration in renal function, especially under the conditions with high blood pressure.^3,5,6

Although it has been postulated that the autoregulatory efficiency is attenuated in the aging kidney, direct evidence is lacking. Therefore, in the present study, we measured autoregulation of RBF, myogenic response of afferent arteriole (Af-Art), TGF response in vivo with micropuncture, as well as TGF response in vitro in isolated perfused juxtaglomerular apparatus (JGA) in young and aged C57BL/6 mice.

METHODS

Data, analytical methods, and study materials are available from the corresponding author on reasonable request. The detail methods are available in the online supplement.

Animals

C57BL/6 mice (male) at the ages of 10–12 weeks and 22–24 months were purchased from Jackson Laboratory. All protocols were approved by the Institutional Animal Care and Use Committee at the University of South Florida, Morsani College of Medicine. All chemicals were purchased from Sigma (St. Louis, MO), except as otherwise indicated.

Statistics

Statistical analysis was performed using Prism 8 (GraphPad Software; San Diego, CA). The effects of interest were tested using t-test, or analysis of variance (ANOVA) followed by multiple comparisons post hoc test when appropriate. Data were presented as a mean ± SEM, and a p-value <0.05 was considered statistically significant.

RESULTS

Aging impairs autoregulation of renal blood flow

To determine the effect of aging on renal autoregulatory efficiency, we measured the change of RBF in response to a defined increase of RAP in young and aged mice (Figure 1A).^7–9 In young mice, there was no significant change in RBF following an increase of RAP by 20.2±4.5 mmHg. In aged mice, RBF increased by 33.8±8.1 % (from 1.09±0.06 to 1.46±0.06 ml/min) in response to a similar increase of RAP (Supplemental Table S3 and Figure 1B). The calculated autoregulatory index was significantly higher in aged mice (1.20±0.25) than young mice (0.09±0.06) (Figure 1C). These data demonstrate that aging impairs the autoregulation of RBF.

Figure 1. Age-related changes in autoregulation of renal blood flow.

A) The representative images of measurement of the changes of renal blood flow (RBF) in response to a defined increase of renal arterial pressure (RAP) in young and aged mice. B) Following the elevation of RAP in Stage II, RBF was not significantly changed in young mice, but rose by 33.8±8.1 % in aged mice. n=11, *p<0.01 vs Stage I. C) The calculated autoregulatory index was significantly higher in aged mice (1.20±0.25) than young mice (0.09±0.06). n=11, *p<0.01 vs Young mice.

Aging blunts myogenic response of afferent arteriole

To determine the effect of aging on renal myogenic response, we measured the diameter of Af-Art in response to a stepwise increase of 20 mmHg in perfusion pressure from 60 to 120 mmHg in young and aged mice (Figure 2A).^7,10–12 With a physiological calcium-containing solution, when the intraluminal pressure rose from 60 to 120 mmHg, the diameter of Af-Art decreased by 20.3±2.3 % in young mice but increased by 5.7±0.8 % in aged mice. With a calcium-free solution to abolish active tone, the passive increases in diameter of Af-Art in response to the rise of perfusion pressure were similar in young and aged mice (22.6±1.0 % and 21.4±1.3 %) (Figure 2B). These data demonstrate that aging impairs the myogenic response of Af-Art.

Figure 2. Age-related changes in myogenic response and pressure-induced intracellular calcium in afferent arteriole.

A) The representative images of measurement of myogenic response in isolated perfused afferent arteriole (Af-Art). B) Myogenic response was determined by the changes of luminal diameter in response to a stepwise increase of 20 mmHg in perfusion pressure from 60 to 120 mmHg. With a physiological calcium-containing solution, the diameter of Af-Art decreased by 20.3±2.3 % in young mice but increased by 5.7±0.8 % in aged mice. With a calcium-free solution (+EGTA), there were comparable passive increases in diameter of Af-Art in young and aged mice (22.6±1.0 % and 21.4±1.3 %). n=11–15, *p<0.01 vs Young mice. C) The representative images of measurement of pressure-induced intracellular calcium ([Ca²⁺]_i) changes in isolated perfused Af-Art. D) Following the elevation of perfusion pressure from 60 to 120 mmHg, [Ca²⁺]_i was increased by 41.8±11.6 % in young mice, but only 15.3±4.9 % in aged mice. n=6–7, *p<0.01 vs Young mice.

Aging attenuates pressure-induced intracellular calcium changes in afferent arteriole

Furthermore, to determine the effect of aging on [Ca²⁺]_i in myogenic response, we measured the intraluminal pressure-induced [Ca²⁺]_i changes in isolated perfused Af-Arts from young and aged mice (Figure 2C).^12–14 When the perfusion pressure rose from 60 to 120 mmHg, the [Ca²⁺]_i of Af-Art increased by 41.8±11.6 % (from 90.7±7.5 to 128.7±5.1 nM) in young mice but only 15.1±4.9 % (from 94.4±4.2 to 108.7±4.0 nM) in aged mice (Figure 2D). These data demonstrate that aging attenuates the pressure-induced [Ca²⁺]_i increases in Af-Art.

Aging decreases the expression of integrin α5 in afferent arteriole

To determine the effect of aging on the mechanotransduction in Af-Art, we measured the mRNA expression levels of the major mechanosensors including Trpc1, Trpc3, Trpc6, Itga5, Itgb1 and β-ENaC in isolated Af-Arts from mouse kidneys,^15–23 as well as human kidney biopsies.^24–30 Itga5 (Integrin α5) is the most down-regulated mechanosensitive initiating element in Af-Art with a 72.3±9.4 % reduction in aged mice compared with young mice, and a 53.6±11.7% reduction in aged subjects compared with young subjects (Table 1 and Table 2).

Table 1.

Age-related changes in mRNA expression of the major mechanosensors in Af-Arts isolated from young and aged mice.

Symbol	Age-related change in mRNA expression
Itga5	0.28±0.05*
Itgb1	NS
β-ENaC	NS
Trpc3	NS
Trpc6	0.76±0.07*
Trpc1	NS

Data (mean ± SEM) are presented as fold changes in mRNA expression. n=7;

^*p<0.05 vs young mice; NS, not significant vs young mice.

Table 2.

Age-related changes in mRNA expression of the major mechanosensors in Af-Arts isolated from young and aged subjects.

Symbol	Age-related change
Itga5	0.47±0.12*
Itgb1	NS
β-ENaC	NS
Trpc3	NS
Trpc6	NS
Trpc1	NS

Data (mean ± SEM) are presented as fold changes in mRNA expression. n=4;

^*p<0.05 vs young subjects; NS, not significant vs young subjects.

Aging inhibits tubuloglomerular feedback in vivo and in vitro

We measured TGF response in vivo with micropuncture in young and aged mice.^31,32 In young mice, when tubular perfusion rate of ATF rose from 0 to 40 nl/min, P_sf decreased from 36.6±2.8 to 31.9±2.6 mmHg (Figure 3A). TGF response indicated by ΔP_sf was 4.6±1.1 mmHg (Figure 3C). In aged mice, there was no significant change in P_sf when tubular perfusion rate of ATF increased from 0 to 40 nl/min (Figure 3B and 3C).

Figure 3. Age-related changes in tubuloglomerular feedback response.

The tubuloglomerular feedback (TGF) in vivo was determined by the change of stop-flow pressure (ΔP_sf) when the tubular perfusion rate increased from 0 to 40 nl/min. A) Stop-flow pressure (P_sf) decreased from 36.6±2.8 to 31.9±2.6 mmHg in response to the increase of tubular perfusion rate in young mice. n= 4 mice/11 tubules, *p<0.05 vs 0 nl/min. B) P_sf was not significantly changed in response to the increase of tubular perfusion rate in aged mice. n=5 mice/12 tubules. C) TGF response in vivo, as indicated by ΔP_sf, was 4.6±1.1 mmHg in young mice, but only 0.7±1.3 mmHg in aged mice. n=4–5 mice/11–12 tubules, *p<0.01 vs Young mice. TGF in vitro was determined by the change in diameter of Af-Art while switching the macula densa perfusate from 10 to 80 mM of NaCl. D) The diameter of Af-Art decreased from 14.7±0.7 to 10.7±1.1 μm in response to the increase of NaCl in young mice. n=14, *p<0.01 vs 10 mM of NaCl. E) The diameter of Af-Art was not significantly changed in response to the increase of NaCl in aged mice. n=12. F) TGF response in vitro, as indicated by the change in diameter of Af-Art, was 4.0±0.6 μm in young mice, but only 0.2±0.3 in aged mice. n=12–14, *p<0.01 vs Young mice.

To eliminate systemic confounding factors such as hormones and sympathetic activity, we also measured TGF response in vitro in isolated perfused JGAs.^31,32 In young mice, when NaCl concentration in tubular perfusate increased from 10 to 80 mM, the diameter of Af-Art decreased from 14.7±0.7 to 10.7±1.1 μm (Figure 3D). TGF response indicated by the change in luminal diameter of Af-Art was 4.0±0.6 μm (Figure 3F). In aged mice, there was no significant change in the diameter of Af-Art when tubular perfusate was switched from 10 to 80 mM NaCl (Figure 3E and Figure 3F). These data demonstrate that aging almost completely inhibits TGF response in vivo and in vitro.

Aging decreases the expression of adenosine A1 receptor in afferent arteriole

We measured the mRNA expression levels of A1AR in isolated Af-Arts from mouse kidneys^33,34, as well as human kidney biopsies.^35–37 The expression of A1AR in Af-Art was decreased by 90.2±4.5 % in aged mice compared with young mice (Figure 4A), and 76.1±11.1 % in aged subjects compared with young subjects (Figure 4B).

Figure 4. Age-related changes in the expression of adenosine A1 receptor and vasoconstrictor response to adenosine in afferent arteriole.

A) The mRNA expression level of A1AR in isolated Af-Arts from mouse kidneys was decreased by 90.2±4.5 % in aged mice compared with young mice. n=7, *p<0.01 vs Young mice. B) The mRNA expression level of A1AR in isolated Af-Arts from human kidneys was reduced by 76.1±11.1 % in aged subjects compared with young subjects. n=4, *p<0.01 vs Young subjects. C) The dose-response curve of adenosine (10⁻¹⁰ to 10⁻⁷ M) in isolated perfused Af-Arts from young and aged mice. n=5–7, *p<0.01 vs Young mice.

Aging inhibits the vasoconstrictor response to adenosine in afferent arteriole

Furthermore, we measured the dose-response curve of adenosine (10⁻¹⁰ to 10⁻⁷ M) in isolated perfused Af-Arts from young and aged mice. In young mice, successive addition of adenosine reduced the diameter of Af-Art to 92.3±1.2, 86.3±0.9, 81.7±0.8, and 79.8±1.1 % of baseline at the doses of 10⁻¹⁰, 10⁻⁹, 10⁻⁸ and 10⁻⁷ M, respectively. In aged mice, there was no significant change in the diameter of Af-Art in response to these doses of adenosine (Figure 4C). These data demonstrate that aging almost completely inhibits the vasoconstrictor response to adenosine in Af-Art.

DISCUSSION

The major findings of the present study are that aging impairs the autoregulation of RBF with blunted myogenic response and inhibited TGF response. These age-related changes in renal autoregulatory efficiency are associated with the downregulations of integrin α5 and A1AR in the Af-Art.

Autoregulation is a homeostatic mechanism in the kidney that maintains intra-renal dynamics relatively stable despite of the changes in systemic blood pressure within a defined range.^3,4 Impaired autoregulatory mechanism allows more intra-renal transmission of the fluctuation in systemic blood pressure and thereby initiates barotrauma in microvasculatures, such as glomerular and tubulointerstitial capillaries, which is associated with the development of renal damages, such as glomerulosclerosis and tubulointerstitial fibrosis, as well as the deterioration in renal clearance function.^3,5,6 The abnormalities in renal autoregulation have been reported in several animal models of diseases, including hypertension,³⁸ diabetes,^39,40 and chronic kidney disease.^41,42 It has also been postulated that the renal autoregulatory mechanism is impaired with aging. Hill et al.⁴³ suggested that the potential impediment in renal autoregulation may contribute to the morphologic changes in the aging kidney, including afferent arteriolar dilation, glomerular hypertrophy and subsequent focal segmental glomerulosclerosis. In addition, a micropuncture study by Tolbert et al.⁴⁴ speculated a weakened renal autoregulation in the aged animals, on the basis of the rise in intra-glomerular pressure and the decline in pre-glomerular vascular resistance. Nevertheless, the direct evidence showing the alterations in renal autoregulatory efficiency with aging remains lacking. In the present study, we provided the direct evidence by measurement of autoregulation of RBF, myogenic response of Af-Art and TGF response both in vivo and in vitro. We found that the autoregulatory efficiency was substantially decreased in the aged mice compared with young mice.

Renal autoregulatory mechanism encompasses two principal elements, myogenic response and TGF response, which coordinately regulate the pre-glomerular vasomotor tone primarily of the Af-Art.³ Myogenic response refers to an intrinsic vasoconstrictive reaction in response to an increase in perfusion pressure. The vasoconstriction is largely mediated by pressure-induced depolarization and subsequent activation of intracellular Ca²⁺ signaling pathway.^3,4 Aging-associated changes in renal vascular activities have been extensively studied. Tank et al. reported that the renal vascular responses to angiotensin II, endothelin-1 and L-NAME are significantly exaggerated in the aged animals, producing greater reductions in GFR and RBF.⁴⁵ Previous clinical studies also demonstrated that aged subjects exhibit excessive renal hemodynamic response to adrenergic stimulation⁴⁶ but less responsiveness to dopamine,⁴⁷ compared with young subjects. However, the effect of aging on renal myogenic response remains unknown. In the present study, we measured the myogenic response in isolated perfused Af-Arts with a stepwise increase in perfusion pressure from 60 to 120 mmHg in young and aged mice. We found that the myogenic response of Af-Art was significantly blunted in the aged mice compared with young mice. Furthermore, the pressure-induced [Ca²⁺]_i increases in Af-Art were significantly attenuated in the aged mice compared with young mice, which could be one of the major mechanisms that contribute to the impaired myogenic response in the aging kidney.

Although the underlying mechanisms for the pressure-induced rise in [Ca²⁺]_i are not yet fully understood, mechanotransduction is considered to be a key triggering process for the depolarization of the cell membrane that initiates Ca²⁺ signaling pathway.^3,48 Thus, we measured the mRNA expression levels of the major mechanosensors including both ion channels and extracellular proteins in isolated Af-Arts from both mouse kidneys and human kidney biopsies. We found that integrin α5 is the most down-regulated mechanosensitive initiating element in the aging kidneys. Integrins are a diverse family of transmembrane heterodimers composed of one α- and one β- subunit. Renal Af-Art was reported to express the α3-, α5-, αV-, β1-, and β3- subunits of integrin, and α5β1 was considered to be the major form of heterodimers.⁴⁹ Therefore, the reduced expression of integrin α5 subunit in the aging kidney may result in insufficient formation of the functional heterodimer integrin α5β1, thereby impeding the process of mechanotransduction in Af-Art. These results indicate that myogenic response is blunted in the aging kidney, which is associated with the attenuation of pressure-induced [Ca²⁺]_i increases, as well as the down-regulation of integrin α5 in Af-Art. Our finding in renal Af-Art is consistent with the results of a previous study on cerebral arteries.⁵⁰ The myogenic adaptation of middle cerebral arteries to pulsatile pressure was impaired in the aged animals, where the mechanisms were thought to be associated with the dysregulation of Kcna5 (Voltage-gated K⁺ channel, shaker-related sub-family, member 5) and Cacna1c (Voltage-gated Ca²⁺ channel, L-type), but the age-related changes in integrin were not assessed in this study.⁵⁰

TGF response is an important and unique mechanism for the autoregulation of renal hemodynamics. It describes a negative feedback between tubule and Af-Art where an increase in NaCl delivery to the macula densa promotes the release of adenosine, which then constricts the Af-Art and thereby induces a tonic inhibition of single nephron GFR.^3,51 The changes in TGF responsiveness may contribute to the alterations in renal hemodynamics, which is thought to occur in several physiological and pathological conditions such as high salt intake, volume expansion, unilateral nephrectomy, pregnancy, diabetes, hypertension and chronic kidney disease.^3,52,53 However, the effect of aging on TGF response remains to be determined. In the present study, we measured TGF response in vivo by micropuncture and in vitro by microperfusion in young and aged mice. We found that both TGF response in vivo and in vitro were almost completely lost in the aging kidney. TGF response is commonly considered to be mediated by adenosine via A1AR.^33,54,55 Therefore, we measured the mRNA expression levels of A1AR in isolated Af-Arts from both mouse kidneys and human kidney biopsies. We found that the afferent arteriolar A1AR expression is substantially decreased in the aging kidneys. Furthermore, we measured the dose-response curve of adenosine in isolated perfused Af-Arts from young and aged mice. We found that the vasoconstrictor response of Af-Art to adenosine was almost completely inhibited in the aged mice. These results indicate that TGF response is nearly inactive in the aging kidney, which is associated with the significantly reduced A1AR expression and suppressed vasoconstrictor response to adenosine in the Af-Art. Besides mediating the TGF vasoconstrictor response, adenosine is also shown to inhibit renin secretion via A1AR in JG cells.^54,56 Thus, with aging, the reduced expression and activity of A1AR in Af-Art, in particular JG cells, may promote the renin release and activate the renin-angiotensin system, which could lead to increases in post-glomerular vasoconstriction and thereby contribute to glomerular barotrauma in the aging kidneys.

PERSPECTIVES

This study demonstrates that aging impairs the renal autoregulation with blunted myogenic response of Af-Art and inhibited TGF response. The underlying mechanisms for these age-related changes in renal autoregulatory efficiency involve the downregulations of integrin α5 and A1AR in the Af-Art.

NOVELTY AND SIGNIFICANCE.

What Is New?

Using a variety of sophisticated techniques, we identified the age-related changes in renal autoregulation wherein aging impairs the autoregulatory efficiency with blunted myogenic response and inhibited tubuloglomerular feedback (TGF) response, which is associated with the downregulations of integrin α5 and adenosine A1 receptor in the afferent arteriole.

What Is Relevant?

Impaired renal autoregulation permits more transmission of disturbance in systemic blood pressure, which contributes to the development of kidney damages and deterioration in renal function, especially under the conditions with high blood pressure. Although it has been postulated that the autoregulatory efficiency is attenuated in the aging kidney, direct evidence remains lacking.

Summary

Aging impairs the renal autoregulation with blunted myogenic response and inhibited TGF response.