Dynamics of GRK2 in the kidney: a putative mechanism for sepsis-associated 1 kidney injury 2

13 Renal vascular reactivity to vasoconstrictors is preserved in sepsis in opposition to 14 what happens in the systemic circulation. We studied whether this distinct behavior 15 was related to α1 adrenergic receptor density, G protein-coupled receptor kinase 2 16 (GRK2) and the putative role of nitric oxide (NO). Sepsis was induced in female mice by 17 cecal ligation and puncture (CLP). Wild-type mice were treated with prazosin 12 hours 18 after CLP or NOS-2 inhibitor, 30 min before and 6 and 12 hours after CLP. In vivo 19 experiments and biochemistry assays were performed 24 hours after CLP. Sepsis 20 decreased the systemic mean arterial pressure and the vascular reactivity to 21 phenylephrine. Sepsis also reduced basal renal blood flow which was normalized by 22 treatment with prazosin. Sepsis led to a substantial decreased in GRK2 level associated 23 to an increase in α1 adrenergic receptor density in the kidney. The disappearance of 24 renal GRK2 was prevented in NOS-2-KO mice or mice treated with 1400W. Treatment of 25 non-septic mice with a NO donor reduced GRK2 content in the kidney. Therefore, our 26 results show that a NO-dependent reduction in GRK2 level in the kidney leads to the 27 maintenance of a normal α1 adrenergic receptor density, probably. The preservation of 28 the density and/or functionality of this receptor in the kidney together with a higher 29 vasoconstrictor tonus in sepsis lead to vasoconstriction. Thus, the increased 30 concentration of vasoconstrictor mediators together with the preservation (and even 31 increase) of the response to them may help to explain sepsis-induced acute kidney injury.

treatment with prazosin. Sepsis led to a substantial decreased in GRK2 level associated 23 to an increase in α1 adrenergic receptor density in the kidney. The disappearance of 24 renal GRK2 was prevented in NOS-2-KO mice or mice treated with 1400W. Treatment of 25 non-septic mice with a NO donor reduced GRK2 content in the kidney. Therefore, our 26 results show that a NO-dependent reduction in GRK2 level in the kidney leads to the 27 maintenance of a normal α1 adrenergic receptor density, probably. The preservation of 28 the density and/or functionality of this receptor in the kidney together with a higher 29 vasoconstrictor tonus in sepsis lead to vasoconstriction. Thus, the increased 30 concentration of vasoconstrictor mediators together with the preservation (and even 31 increase) of the response to them may help to explain sepsis-induced acute kidney 32 injury.

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Sepsis is defined as a life-threatening organ dysfunction caused by an exaggerated 38 immune response of the host to infection, and it is the leading cause of death in 39 intensive care units worldwide (1,2). Sepsis clinical findings may vary and are 40 dependent on the etiological agent, the initial location of the infection, the previous 41 health condition of the host (1,3). Sepsis high mortality rate and the associated costs 42 remains a challenge in clinical medicine (4,5). Despite many attempts to introduce novel 43 therapeutic molecules, the therapeutic approach to sepsis treatment still consists 44 mainly of antibiotics, fluid, and vasopressors agents (6,7). 45 The cardiovascular dysfunction is one of the most important complication of 46 sepsis and is determinant for both prognosis and outcome. This dysfunction involves 47 myocardial depression, vasodilatation, decrease in vascular reactivity towards 48 vasoconstrictors and increased vascular permeability, all of which may lead to 49 hypovolemia and severe and untreatable hypotension (8,9). Cardiovascular dysfunction 50 directly impacts on sepsis pathophysiology due to the reduction in oxygen delivery to 51 tissues, resulting in anaerobic metabolism and hyperlactatemia, and it is an important 52 contributor to organ failure (10). 53 One of the organs most affected by sepsis is the kidney, which may result in 54 sepsis-associated acute kidney injury (S-AKI) which, in turn, worsens the prognosis and 55 increases sepsis mortality (11,12). S-AKI pathophysiology is not well understood but suggesting that the kidney failure should be more related to a dysfunction than to tissue 62 lesion (14-16). 63       protocol. Next, membranes were blocked with 5% BSA in TBST (Tris-buffered saline,  Alpha-1 adrenergic receptor labeling assay 235 To study α1-adrenoceptor density, we used a fluorescent labeling with an antagonist 236 probe. Tissues were collected and prepared as above. Following fixation, sections were   Renal markers 285 As seen in Figure 2, plasma levels of creatinine ( Figure 2A) and urea ( Figure 2B) were 286 elevated during sepsis. These findings indicated that the kidney is already failing.

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Alpha1 adrenergic receptor density in the kidney and in the heart 288 To explore the α1 adrenergic receptor density in kidney and heart during sepsis, we 289 performed a binding assay with the fluorescent α1-adrenoceptor ligand QAPB. Twenty-290 four hours after sepsis induction, the α1 adrenergic receptor density was increased by 291 three-fold in the kidney when compared to control group (Figures 3A and 3C). Although 292 glomeruli appear to express the receptor, the intensity of the labeling is greater in renal 293 tubules and vessels. In contrast, the α1 adrenoceptor receptor density did not change in 294 septic heart (Figures 3B and D). 296 Since the density of α1 adrenergic receptor was higher in the septic kidney but not in 297 the heart, we sought to study the GRK2 levels in both organs (Figure 4). In a distribution 298 like the α1 adrenergic receptor, GRK2 was present in the glomeruli but its content was 299 greater in tubules and vessels. Sepsis substantially reduced GRK2 content in all 300 structures ( Figures 4A and 4C). As for the heart, GRK2 seems to be evenly distributed in 301 the cardiac tissue and sepsis increased its content (Figures 4B and 4D).

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As shown by Western blot analysis, GRK2 levels were reduced to almost nil in 303 the septic kidney ( Figure 5A) whereas it was even increased in the heart ( Figure 5B).

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To study the role of NO in the renal GRK2 dynamics during sepsis, septic mice 315 were treated with 1400W, a selective inhibitor of NOS-2, 30 min before and 6 and 12 h 316 after sepsis induction. The treatment with 1400W prevented the loss of GRK2 content in 317 septic kidney (Figures 7A and 7B). Confirming that NOS-2 was the main source of NO 318 affecting GRK2 in the septic kidney, NOS-2 KO mice were submitted to sepsis and again 319 the loss in GRK2 content induced by sepsis was prevented (Figures 7C and 7D). An 320 interesting result was obtained when we treated normal, non-septic mice with SNAP, a 321 nitric oxide donor and measured the renal content of GRK2. The treatment with the NO 322 donor reduced GRK2 content in the kidney by 20% (Figures 7E and 7F).  As for the model of sepsis we used, the findings that basal blood pressure of 335 septic mice was lower than that from control animals and that their response to 336 phenylephrine were severely reduced, indicate that mice have developed septic shock.

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When the blood pressure and tissue perfusion fall below certain limits powerful

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The response of the kidney to sepsis is different from most other vascular beds. leading to receptor internalization (33). This mechanism is operative for the heart ß-393 adrenergic receptor during sepsis upon GRK2 phosphorylation, as we have previously 394 shown (28). Therefore, we set out to examine GRK2 profile in the kidney of septic mice. 395 Our results demonstrate that both GRK2 mRNA and protein levels were 396 reduced in the kidney, in sharp contrast with the heart, where GRK2 mRNA was 397 unchanged and protein levels were even increased, probably due to increased mRNA 398 translation to produce the enzyme protein.

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NO has been shown to be a relevant player in sepsis, mainly regarding the 400 cardiovascular dysfunction (reviewed in 23). Our present results confirm that NO is 401 indeed produced in sepsis, as NOS-2 expression in the kidney was elevated and plasma 402 levels of its metabolites nitrate and nitrite were also elevated. We have previously 403 shown that NO has a prominent role in the activation of cardiac GRK during sepsis (28).      Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20210462