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. Author manuscript; available in PMC: 2026 Feb 25.
Published before final editing as: Hypertension. 2026 Jan 19:10.1161/HYPERTENSIONAHA.125.26077. doi: 10.1161/HYPERTENSIONAHA.125.26077

Neural upregulation of SGLT2-MAP17-PDZK1 complex in kidneys of rats with heart failure

Tapan A Patel 1, Hong Zheng 2, Kaushik P Patel 1
PMCID: PMC12930422  NIHMSID: NIHMS2135206  PMID: 41549941

Abstract

BACKGROUND:

Congestive heart failure (CHF) is characterized by the activation of neurohumoral drive concomitant with avid fluid retention. Renal denervation (RDN) alleviates this fluid retention. Sodium-glucose cotransporter-2 (SGLT2) inhibitors have shown remarkable improvement in patients with cardiovascular diseases. We have recently demonstrated a relationship between enhanced renal sympathetic nerve activity (RSNA) and SGLT2 expression as well as function during CHF, however, the precise molecular mechanisms involved in the expression and translocation of SGLT2 and associated scaffolding proteins to the luminal membrane remains to be examined.

METHODS:

CHF was induced by coronary artery ligation followed by bilateral RDN 4 weeks later, in rats. Western blot analysis and immunohistochemistry were performed to evaluate changes in the expression of SGLT2, MAP17, PDZK1 and activation of ERK/NF-KB in renal cortex. Human adult proximal tubular cells (HK2 cells) were used to determine the direct effect of norepinephrine (NE) on expression of SGLT2-MAP17-PDZK1 and activation of ERK/NF-KB pathway.

RESULTS:

Rats with CHF exhibited significantly enhanced expression of SGLT2, MAP17, PDZK1 with a concomitant significant activation of ERK and NF-KB in the renal cortex. In rats with CHF, RDN mitigated enhanced expression of SGLT2-MAP17-PDZK1 as well as activation of ERK and NF-KB. Direct action of NE on HK2 cells, triggered enhanced expression of SGLT2-MAP17-PDZK1 by the activation of the ERK/NF-KB pathway.

CONCLUSIONS:

Enhanced basal RSNA in CHF activates the ERK/NF-KB pathway, which in turn facilitates the enhanced expression and translocation of the SGLT2-MAP17-PDZK1 scaffolding protein complex to the luminal membrane, augmenting sodium reabsorption in CHF.

Keywords: Sodium-glucose cotransporter-2 (SGLT2), Membrane-associated protein-17 (MAP17), PDZ domain containing-1 (PDZK1), Renal denervation (RDN), Congestive heart failure (CHF), norepinephrine (NE), Renal sympathetic nerve activity (RSNA)

Graphical Abstract

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Introduction

One common characteristic of congestive heart failure (CHF) is increased neurohormonal activation with specific activation of the renal sympathetic nerve which leads to increased sodium and fluid retention1–6. Enhanced renal sympathetic nerve activity leads to an increased norepinephrine (NE) release which causes an increase in afferent arteriolar constriction, reduce glomerular filtration rate, increasing renin release and direct action on the renal tubules to increase sodium reabsorption7. Each of these actions of renal nerve activation leads to avid sodium and water retention, typically associated with CHF8,9. Consistent with this observation it has been shown that renal denervation (RDN)10, improves sodium excretion1, sympathoexcitation5 and cardiac function11 in rats with CHF. Clinically, RDN showed partial protection in patients with heart failure by improving hemodynamic alterations, increasing urine volume and reducing N-terminal pro–B-type natriuretic peptide levels (NT-proBNP) without any complications12–18.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have beneficial effects in patients with heart failure (HF) by significantly improving symptoms19–21 and reducing the hospitalization and mortality22–25. Recently, we have shown an increased expression of SGLT2 in the luminal membrane of the proximal tubular cells in the kidneys of rats with CHF10. Further, administration of SGLT2 inhibitor, dapagliflozin demonstrated greater excretion of sodium, indicating an increased functional activation of SGLT2 and sodium retention. This increased activation of SGLT2 and sodium retention by the kidneys in rats with CHF was mitigated by RDN. These results suggest that tonic basal renal nerve activity during CHF8,9,26 leads to overactivation of SGLT210and consequent sodium retention. One study indicates increased expression of SGLT2 in the proximal tubular cells in response to norepinephrine exposure27. However, the precise sequence of critical molecular mechanisms and pathways involved in the renal nerve mediated expression and luminal membrane localization of SGLT2 and associated scaffolding proteins remains to be examined. Such an investigation would lead to greater understanding into the precise molecular mechanism for the beneficial action of SGLT2 inhibition in CHF.

Structural examination of the SGLT2 demonstrated that transmembrane domain of SGLT2 physically interacts with membrane-associated protein-17 (MAP17)28,29. The expression of SGLT2 and MAP17 colocalize in luminal membranes of proximal tubular cells of human kidney30. MAP17 is associated with PDZ domain containing-1 (PDZK1), a scaffolding protein linked to Na+/H+ exchanger isoform 3 (NHE3)31–35. Interestingly, approximately 70% of all the filtered sodium is reabsorbed by the apical NHE3 in the proximal tubule of the kidneys36,37. MAP17 is also associated with various other trans-membrane proteins such as Na(+)/phosphate cotransporter (NaPi-IIa) in addition to SGLT238. Importantly, activity of SGLT2 is significantly enhanced by MAP17, indicating that MAP17 is crucial accessory protein for the function of SGLT239. Thus, SGLT2 is physically associated with NHE3 by MAP17-PDZK1 and forms a multi-protein complex SGLT2-MAP17-PDZK1-NHE340. MAP17 also enhances the production of ROS and inflammation under certain conditions41–44. This association imparts additional complexity to the regulation of SGLT2 in both normal and various disease conditions such as CHF. However, the molecular mechanism for the regulation of expression and membrane trafficking of SGLT2, MAP17 and PDZK1 remains to be examined. Additionally, there is no evidence to indicate a potential regulation of this complex by the sympathetic nervous system.

The actions of the sympathetic nervous system are known to be mediated intracellularly via the ERK (Extracellular signal-regulated kinase)-NF-KB (Nuclear factor-kappa B) pathway.45–48 Considering these observations together, we hypothesized that enhanced renal sympathetic activation triggers an enhanced expression/membrane translocation of SGLT2-MAP17-PDZK1 complex to the luminal membrane of the proximal tubule by the ERK/NF-KB signaling pathway in rats with CHF.

The present study was conducted to determine the contribution of enhanced renal sympathetic activity on expression/membrane localization of SGLT2 and MAP17 via ERK/NF-KB pathway in rats with CHF. We analyzed the expression/membrane localization of SGLT2, MAP17, PDZK1 and ERK/NF-KB pathway in the renal cortex of rats with and without RDN subjected to CHF. To further elucidate the specific molecular pathways in a circumscribed controlled manner, we evaluated the effect of ERK and NF-KB inhibitors on enhanced localization of functional SGLT2, MAP17 and PDZK1 to cell surface by direct action of NE in human proximal tubular cells (HK2 cells), in vitro.

Methods

All the detailed methodology used in the present study and all other supporting data are available in the Supplemental Materials.

General

In the present study, male Sprague-Dawley rats weighing 220–250g were procured from Charles River Laboratories, USA and housed with a 12:12hrs light:dark cycle at 22°C with 30% relative humidity. Laboratory chow and tap water were available ad libitum. After 1 week of acclimatization, rats were randomly assigned to four groups: Sham, CHF, Sham+RDN, and CHF+RDN. All the procedures on animals were approved by the University of Nebraska Medical Center (UNMC), Institutional Animal Care and Use Committees (IACUC).

Statistical Analysis

The data are expressed as mean ± SE. Statistical comparisons of different groups were assessed by one or two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test for post hoc analysis of significance (Prism 10; GraphPad Software) as appropriate. P<0.05 was considered indicative of statistical significance.

Results

General characteristics of the rats in the four groups

The general morphological characteristics of rats from all four groups are summarized in Table-S1. The heart weight and heart weight/body weight ratio were significantly higher in rats of CHF group compared with Sham group (CHF 1.44±0.06 g vs. Sham 1.02±0.02 g, P<0.05 and CHF 4.03±0.17 vs. Sham 3.02±0.07, n=10, p<0.05, respectively), whereas RDN did not exhibit any change in these parameters in Sham and CHF groups. Rats in Sham and Sham+RDN group had no visible myocardial damage. Average myocardial infarction was 38% of the left ventricle in the CHF group regardless of RDN. CHF group showed significantly higher left ventricular end-diastolic pressure (LVEDP) compared with Sham groups (CHF 24.70±2.65 vs. Sham 1.70±0.28 mmHg, p<0.05). RDN showed significant recovery of LVEDP in rats with CHF group. CHF group showed significant reduction in maximal slope of systolic pressure increment (+dP/dt) and maximal slope of diastolic pressure decrement (−dP/dt) compared with Sham groups (CHF 5033.30±241.81 vs. Sham 7116.80±360.36 mmHg/s, p<0.05 and CHF 3981.60±96.45 vs. Sham −6391.50±373.39 mmHg/s, p<0.05, respectively). RDN partially alleviated these parameters. All these parameters indicated that the rats of CHF group represent cardiac dysfunction, which were moderately recovered by RDN. There was no significant difference in kidney weights among the groups. Norepinephrine (NE) content of kidney was significantly higher in rats with CHF group compared with Sham (CHF 314.10±21.65 vs. Sham 154.85±13.19 ng/g, p<0.05). After RDN, renal NE content in rats of Sham and CHF was very low (<8%) which indicates efficacy of renal denervation procedure (Table-S1).

Expression of SGLT2, MAP17 and PZDK1 proteins in CHF

Western blot analysis demonstrated that the levels of total SGLT2, MAP17 and PDZK1 were significantly higher in the renal cortex of CHF rats compared with the Sham group (CHF 0.21±0.03 vs. Sham 0.06±0.01; CHF 0.52±0.16 vs. Sham 0.03±0.01; CHF 0.114±0.005 vs. Sham 0.089±0.003; n=6, p<0.05, respectively). Membranous SGLT2 and MAP17 were also significantly increased in the renal cortex rats with CHF compared with the Sham rats (CHF 0.025±0.003 vs. Sham 0.011±0.002; CHF 0.67±0.11 vs. Sham 0.28±0.04; p<0.05, respectively). RDN exhibited significant reduction of total SGLT2, MAP17 and PDZK1 as well as membranous levels SGLT2 and MAP17 in the renal cortex of the CHF group, while did not significantly alter in the Sham group (Figure-1).

Figure 1:

Figure 1:

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Representative western blots gel images (A) and densitometry analyses (B) of SGLT2, MAP17 and PDZK1 protein expression within the renal cortex of rats from all groups (Sham, Sham+RDN, CHF, and CHF+RDN). Values are presented as mean±SE (n=6/group). *p<0.05; **p<0.01; ***p<0.001 compared with Sham or CHF.

Colocalization of SGLT2 and MAP17 in the apical membrane of the proximal tubule.

Immunohistochemistry analysis showed enhanced immunofluorescence intensity and apical membrane co-localization of SGLT2 and MAP17 in the renal cortex of rats with CHF compared with the Sham group (CHF 5.77±1.31 vs. Sham 1.49±0.34; CHF 2.65±0.52 vs. Sham 0.53±0.1, p<0.05, respectively). RDN decreased the intensity of immunofluorescence for SGLT2 and MAP17 in the CHF group but did not alter in the Sham group (Figure-2).

Figure 2:

Figure 2:

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Immunofluorescent image (A) and fluorescent intensity (B) from the renal cortex sections stained for immunolocalization of MAP17 (red) and SGLT2 (green) in Sham, Sham+RDN, CHF, and CHF+RDN groups of rats. Scale bar=20µm. Values are presented as mean±SE (n=6/group). *p<0.05; **p<0.01; ***p<0.001 compared with Sham or CHF.

Renal denervation reduced activation of ERK/NF-KB Pathway in the renal cortex of rats with CHF

Western blot analysis revealed significant increase in the phosphorylation of ERK and NF-KB in the renal cortex of CHF group compared with the Sham group (CHF 0.68±0.08 vs. Sham 0.33±0.05; CHF 3.90±1.10 vs. Sham 0.70±0.20, p<0.05, respectively). RDN reduced the phosphorylation of ERK and NF-KB in CHF group but did not significantly change in the Sham group (Supplementary figure-S1).

Expression and membrane localization of SGLT2, MAP17 and PDZK1 after NE treatment in HK2 cells

Western blot analysis revealed that norepinephrine treatment significantly increased the total SGLT2, MAP17 and PDZK1 (NE 0.73±0.06 vs. Control 0.36±0.03; NE 0.042±0.005 vs. Control 0.023±0.002; NE 0.31±0.02 vs. Control 0.22±0.01; n=6, p<0.05, respectively). Also, the levels of SGLT2 and MAP17 is significantly higher in the membrane fraction (NE 0.071±0.014 vs. Control 0.021±0.006; NE 0.27±0.03 vs. Control 0.16±0.02; p<0.05, respectively), whereas cytoplasmic fraction did not show any significant change indicating the trafficking of these proteins to the cell surface (Figure-3). Consistent with these results, significantly enhanced expression and membrane colocalization of SGLT2 and MAP17 was observed using confocal imaging of HK2 cells in vitro after NE treatment (NE 2.29±0.17 vs. Control 0.89±0.10; NE 0.68±0.05 vs. Control 0.29±0.01; p<0.05, respectively) (Figure-4). It further supports the concept of increased trafficking of MAP17 and SGLT2 after the treatment with NE. Simultaneous trafficking of MAP17 and SGLT2 to the cell membrane might be a key step for the activation of SGLT2 to enhance sodium and glucose reabsorption in renal cortex of rats with CHF.

Figure 3:

Figure 3:

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Representative western blots gel images (A) and densitometry analyses (B) of SGLT2, MAP17 and PDZK1 protein expression in HK2 cells (Control, Norepinephrine (NE), NF-KB inhibitor (Bay-11–7082), and NF-KB inhibitor+NE). Values are presented as mean±SE (n=6/group). *p<0.05; **p<0.01 compared with Control or NE.

Figure 4:

Figure 4:

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Immunofluorescent image (A) and fluorescent intensity (B) from the HK2 cells stained for immunolocalization of MAP17 (red) and SGLT2 (green) in Control, Norepinephrine (NE), NF-KB inhibitor (Bay-11–7082), and NF-KB inhibitor+NE. Scale bar=50µm. Values are presented as mean±SE (n=3/group). **p<0.01 compared with Control or NE.

Expression and membrane trafficking of SGLT2 and MAP17 by ERK/NF-KB pathway in HK2 Cells

Significantly increased phosphorylation of ERK and NF-KB was observed after NE treatment (NE 2.71±0.31 vs. Control 1.27±0.25; NE 0.74±0.16 vs. Control 0.17±0.03, p<0.05, respectively) by western blot analysis (Supplementary figure-S2). Pretreatment of ERK inhibitor and NF-KB inhibitor to HK2 cells showed significant reduction of total and membranous MAP17 and SGLT2 which was not enhanced after NE treatment (Figure-3; Supplementary figure-S3), indicating the role of ERK/NF-KB in regulation of expression and membrane trafficking. ERK inhibitor showed dose dependent decrease in the phosphorylation of ERK and NF-KB, exhibiting a key role for ERK in the activation of NF-KB (Supplementary figure-S4). Confocal imaging demonstrated that NF-KB inhibitor pretreatment prevented the expression and membrane colocalization of SGLT2 and MAP17 in vitro, which was also not increased after NE treatment (Figure-4).

Cellular uptake of glucose in HK2 cells by NE

Confocal imaging exhibited significant uptake of 2-NBDG (a fluorescent glucose analog) (NE 6.78±0.42 vs. Control 2.29±0.46, p<0.05) after NE treatment. Prior treatment of NF-KB inhibitor showed reduction of 2-NBDG uptake and this uptake was unaltered after NE treatment supporting the reduction of SGLT2 at the cell surface (Supplementary figure-S5). Similarly, when HK2 cells were treated with SGLT2 inhibitor (Dapagliflozin-DAPA) prior to NE treatment, there was significantly less uptake of 2-NBDG (Figure-5). These results indicate that NE significantly enhanced expression and function of SGLT2, specifically.

Figure 5:

Figure 5:

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Norepinephrine-induced 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) uptake and its inhibition by SGLT2 inhibitor (Dapagliflozin-DAPA) in HK-2 cells. Top panel: Scale bar=100µm. Bottom panel: Scale bar=20µm. Values are presented as mean±SE (n=5/group). ****p<0.0001 compared with Control or NE.

Discussion

This study demonstrates that there is: (1) enhanced expression/membrane localization of SGLT2, MAP17 and PDZK1 in the renal cortex of rats with CHF, (2) increased phosphorylation of ERK/NF-KB in the renal cortex of rats with CHF, (3) RDN reduced the enhanced expression/membrane translocation of SGLT2, MAP17 and PDZK1 as well as phosphorylation of ERK/NF-KB in the renal cortex of rats with CHF; Direct treatment of NE on proximal tubular cells (HK2 cell) in vitro demonstrated; (4) enhanced expression of SGLT2, MAP17 and PDZK1 with simultaneously increased membrane localization of SGLT2 and MAP17, as well as augmented phosphorylation of ERK/NF-KB, (5) increased glucose uptake, specifically by SGLT2 (indexed by 2-NBDG uptake), (6) ERK inhibitor (U0126) and NF-KB inhibitor (Bay-11–7082) mitigated expression/membrane localization of SGLT2 and MAP17 as well as regulated glucose uptake in HK2 cells. Taken together, these findings indicate that the augmented renal sympathetic nerve activation (RSNA) in CHF1,5,11 triggers the expression of SGLT2, MAP17 and PDZK1 as well as trafficking them to the luminal membrane of proximal cell via ERK/NF-KB pathway leading to the increased activity of renal SGLT2 leading to sodium retention in the CHF condition (Figure 6). To our knowledge, this is the first report demonstrating a neurally-elicited expression/membrane trafficking of SGLT2, MAP17 and PDZK1 complex in proximal tubule, and second the utilization of the ERK/NF-KB pathway by NE to enhance expression/membrane trafficking of SGLT2, MAP17 and PDZK1 complex in proximal tubule.

Figure 6:

Figure 6:

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Proposed schematic for the role of enhanced sympathetic nervous system in the regulation of renal SGLT2, MAP17 and PDZK1 expression/membrane trafficking in congestive heart failure (CHF). Activation of renal sympathetic nerves augments the release of norepinephrine (NE) which in turn enhances the expression/membrane trafficking of SGLT2, MAP17 and PDZK1 via activation of ERK/NF-KB pathway in CHF. Renal denervation (RDN) inhibits the release of NE by reducing the renal sympathetic nerve activity which in turn reduces the expression/membrane trafficking of SGLT2, MAP17 and PDZK1. Red arrows represent changes due to CHF and green arrows indicate changes after RDN. SGLT2=Sodium-glucose cotransporter-2; MAP17=Membrane-associated protein-17; PDZK1=PDZ domain containing-1); NHE3=Na+/H+ exchanger isoform 3; AR=Adrenergic Receptor; ERK=Extracellular signal-regulated kinase; NF-KB=Nuclear factor kappa B.

Recently, we reported enhanced expression of renal SGLT2 in rats with CHF10. Altered expression of SGLT2 has been shown in various diseased conditions such as diabetes49–51, nondiabetic heart failure52 and high-fat diet supplementation27. Enhanced RSNA in CHF5 has been known to stimulate proximal tubular reabsorption of sodium and fluid53. Renal sympathetic nerve hyperactivity has also been suggested to contribute to the progression of chronic kidney disease (CKD)54,55. RDN significantly reduced SGLT2 expression in the kidney of rats with CHF 10 and gene expression of SGLT2 in STZ induced diabetic rats 56. Enhanced expression of NHE3, responsible for reabsorption of 70% of all the filtered sodium36,37 in the proximal tubule, has been reported in the renal cortex of rats with CHF1. Activity of NHE3 is regulated by various Na+/H+ exchanger regulatory factors (NHERFs)32,35 and one of them being PDZK1. Interestingly, SGLT2 is structurally associated with NHE3 by two scaffolding proteins namely, MAP17 & PDZK1 and forms a complex SGLT2-MAP17-PDZK1-NHE340. Also, analysis of native mouse kidney via affinity purification with mass spectrometry (MS) showed association of SGLT2 with different sodium and hydrogen transporter–regulating proteins such as MAP17, PDZK1 and NHERF257. Intriguingly, it has been reported that NHE3 knockdown reduced renal SGLT2 expression in Akita diabetic mice58. MAP-17, a key scaffolding protein of a transmembrane complex SGLT2-MAP17-PDZK1-NHE340, interacts with SGLT2 and has been shown to functionally activate SGLT2 at the cell surface29,30,39,59. The present study reports for the first time that MAP17 is significantly increased in the renal cortex of rats with CHF. Intriguingly, enhanced expression of MAP17 is also associated with inflammation, oxidative stress and tumor progression41–43,60,61, thus being subject to possible influence/interaction from these events/factors as well. MAP17 is responsible for the transport of various proteins to the membrane such as PDZK1 and associates with membrane transporter such as NaPi-IIa38. Also, it has been reported that MAP17 enhanced reactive oxygen species (ROS) production44. However, the exact molecular mechanism/trigger for the initiation of the enhanced expression of SGLT2, MAP17 and PDZK1 in renal proximal tubular cells remained undefined.

We present a common relevant link, i.e. ERK/NF-KB pathway, between the enhanced sympathetic nervous system and increased expression/membrane trafficking of SGLT2 and MAP17 in the renal cortex of rats with CHF. The results in the HK2 cells after NE treatment validate a proof of concept for this pathway. In the present study, phosphorylation of ERK and NF-KB was observed in the renal cortex of rats with CHF and similar results were observed in the HK2 cells after NE treatment. Activation of alpha(1a)-AR by NE has been shown to increase the phosphorylated ERK1/2 in a time- and dose-dependent manner62. This relationship is well established in various cell types27,45,46,62. However, this study did not identify specific adrenoceptor/s that mediate sympathetic actions on SGLT2 expression and trafficking. Further studies identifying specific adrenoceptor involved in this process would have therapeutic clinical implications. In addition, NE has also been reported to enhance the activation of NF-KB in rat cultured hepatocytes and pineal gland47,48. There are several reports which indicate that phosphorylation of ERK regulates the activation of NF-KB63–65 and its nuclear translocation for the downstream activity and expression of several proteins. A combination of enhanced expression of SGLT2 and activation of NF-KB was noted after the treatment of high glucose and rhTNF-α in HK2 cells66, similar to our study after NE treatment.

Enhanced activation of transporters at cell membrane is directly proportional to the enhanced expression and/or increase in the membrane translocation of transporters. Activity of transporters depends on the number of transporters and the supportive scaffolding cargo proteins (such as MAP17 for SGLT2) within the plasma membrane as well as the rate of its expression, and/or membrane translocation. In present study, we observed enhanced expression of SGLT2, MAP17 and PDZK1 as well as increased membrane trafficking of SGLT2 and MAP17 after NE treatment indicating augmented availability of SGLT2 for enhanced uptake of glucose and sodium. To confirm the enhanced availability of SGLT2 and MAP17 at plasma membrane, we performed western blot analysis from membrane fraction and immunofluorescence staining, indicating enhanced availability of SGLT2 and MAP17 at the luminal membrane of the proximal tubular cells. Also to confirm increased activity of SGLT2 at the membrane, we evaluated functional activity of SGLT2 for glucose uptake (indexed by 2-NBDG uptake) in HK2 cells after NE treatment. The results demonstrated a significant increase in the uptake of glucose suggesting activation of SGLT2 at cell membrane. To specifically validate that NE-mediated enhanced glucose uptake is due to enhanced function of SGLT2, HK2 cells were treated with SGLT2 inhibitor (Dapagliflozin-DAPA) prior to NE treatment. The results showed the prevention of glucose uptake exhibiting a specific critical role of NE in the regulation of SGLT2. Upregulation of MAP17 has also been linked with chronic inflammation as well as enhanced oxidative stress43,44,60,61, which may lead to altered expression and function of transporters as observed in this study. Low-grade inflammation and enhanced reactive oxygen species (ROS) formation have also been associated with the upregulation of SGLT267. Consistent with our observations, enhanced expression of SGLT2 has been demonstrated in kidney cells after NE treatment10,27 indicating that the expression of SGLT2 is regulated by sympathetic nervous system.

RDN showed significant reduction in the expression and membrane localization of SGLT2 and MAP17 in rats with CHF compared with Sham, further demonstrating the contribution of tonic basal renal sympathetic nerve activation in CHF. Also, RDN significantly reduced activation of ERK/NF-KB. In addition, we also observed that either pretreatment with ERK inhibitor or NF-KB inhibitor prevented NE-induced protein expression and membrane translocation of SGLT2 and MAP17 in HK2 cells indicating involvement of ERK/NF-KB pathway in expression/membrane trafficking of SGLT2 and MAP17. This is corroborated by data showing increased phosphorylation of ERK and NF-KB after 8-Br-cAMP treatment which regulates the expression and membrane localization of SGLTs in renal proximal tubule cells68. Inhibition of NF-KB signaling pathway prevented the increase in expression of SGLT2 after the treatment with palmitic acid and NaCl69 or insulin70 in HK-2 cells, further supporting our results. It has also been reported that inhibition of NF-KB signaling pathway reduced expression of SGLT2 in mice treated with high-fat and high-salt (HF-HS) diet69 as well as in db/db diabetic mice70, thereby improving hypertension and hyperglycemia, respectively. Activation of ERK/NF-KB pathway leads to enhanced expression and activation of various transporters (such as SGLT2 in the present study) in the renal cortex which in turn may cause chronic kidney disease (CKD) in CHF. According to our results, it is evident that enhanced sympathetic nervous system in CHF activates ERK/NF-KB pathway which in turn regulates the expression and membrane trafficking of SGLT2 and MAP17 in the proximal tubular cells (PTCs) (Figure 6). These findings suggest that some of the beneficial effects of SGLT2 inhibitors in patients with heart failure19–21 may relate to the alleviation of the exaggerated sympathetic activation commonly seen in patients with CHF. Specifically, the neural regulation of the SGLT2 complex may be a plausible mechanism by which SGLT2 inhibitors exert benefit beyond glycemic control in patients with heart failure.25,71,72 Perhaps SGLT2 inhibitors and RDN may have some common underlying mechanistic therapeutic actions to benefit patients with heart failure.

The present data demonstrates a unique and novel observation indicating that the enhanced renal sympathetic activity, characteristic of CHF, initiates an increase in expression and membrane translocation of SGLT2 and MAP17 in renal cortex via ERK/NF-KB signaling pathway. Identifying this specific pathway for the regulation of expression and membrane trafficking of scaffolding proteins of a transmembrane complex SGLT2-MAP17-PDZK1-NHE340 contributes fundamentally to the understanding of the molecular mechanisms underlying the neurally mediated regulation of sodium and glucose homeostasis which holds immense potential for the discovery of novel therapeutic strategies in the treatment of various cardiorenal diseases.

Limitations

While our data indicates a significant role for renal nerves in the regulation of SGLT2-MAP17-PDZK1, several limitations warrant consideration and may be addressed in the future work. Although this research was conducted using both, in vivo as well as in vitro models, some experiments could not be performed in vivo because of their severe side effects in the whole organism. In vivo treatments of ERK and NF-KB inhibitors may lead to severe side effects in healthy animals leading to disruption of normal physiological processes, suppressed immune function, and impaired cell survival to name a few. This study did not identify specific adrenoceptor/s that mediate sympathetic actions on SGLT2 expression and trafficking.

Perspectives

This study provides a novel mechanistic insight into how enhanced renal sympathetic nerve activity in heart failure upregulates SGLT2 and associated scaffolding proteins (MAP17 and PDZK1) in the luminal membrane of the proximal tubular cells of the renal cortex. These studies provide a framework of potential targets for therapeutic manipulation of this key regulatory pathway involved in the regulation of glucose and sodium handling by SGLT2-MAP17-PDZK1 complex in heart failure as well as other cardio-renal diseases.

Supplementary Material

Supplemental Publication Material
Full Gels

Novelty and Relevance.

What is New?

Membrane-associated protein-17 (MAP17) and PDZ domain containing-1 (PDZK1) expression is concomitantly increased with SGLT2 in renal proximal tubules of rats with congestive heart failure (CHF). Removal of neural innervation to the kidney normalizes the expression and membrane localization of the SGLT2-MAP17-PDZK1 complex in the luminal membrane of proximal tubules in CHF. ERK/NF-KB pathway regulates the expression and membrane translocation of SGLT2-MAP17-PDZK1 complex in CHF.

What Is Relevant?

Enhanced sympathetic tone with a concomitant sodium and water retention are observed in patients with CHF. Renal denervation potentially reduces sympathetic tone with concomitant sodium and water retention in CHF, however, the underlying molecular mechanisms are unclear. Hence, understanding such mechanisms in CHF may provide opportunity to develop novel therapeutics for the patients with CHF.

Clinical/Pathophysiological Implications?

CHF is characterized by sympathoexcitation and sodium retention by the kidneys. Enhanced renal sympathetic nerve activity in CHF enhances expression and function of SGLT2 in CHF. We show that enhanced RSNA regulates the expression and membrane translocation of SGLT2-MAP17-PDZK1 complex in rats with CHF via ERK/NF-KB pathway. Also, exogenous treatment of norepinephrine to human proximal tubular cells augmented expression and membrane translocation of SGLT2-MAP17-PDZK1 complex and glucose uptake via ERK/NF-KB pathway. The beneficial effects of either SGLT2 inhibition or renal denervation exhibit mutual intracellular pathway for the reduction of sodium and water retention which is commonly observed in CHF.

Sources of Funding

This work was supported by National Institutes of Health (NIH) grants [R01-DK-114663, R01-DK-129311, and endowed McIntyre Professorship fund to K.P.P].

Non-standard Abbreviations and Acronyms

CHF

Congestive heart failure

RDN

Renal denervation

RSNA

Renal sympathetic nerve activity

NE

Norepinephrine

SGLT2

Sodium-glucose cotransporter-2

MAP17

Membrane-associated protein-17

PDZK1

PDZ domain containing-1

NHE3

Na+/H+ exchanger isoform 3

NHERF

Na+/H+ exchanger regulatory factor family

ERK

Extracellular signal-regulated kinase

NF-kB

Nuclear factor-kappa B

References:

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