Renal serotonin signaling in ischemic kidney insufficiency

Renal ischemia–reperfusion (IR) injury is a major cause of acute kidney injury (AKI), a syndrome associated with high morbidity, mortality, and long-term risk of chronic kidney disease. IR injury occurs when renal blood flow is transiently interrupted (ischemia) and subsequently restored (reperfusion). Although reperfusion is necessary to prevent irreversible tissue damage, it paradoxically exacerbates renal injury through mechanisms involving inflammation, oxidative stress, and microvascular dysfunction. Despite restoration of perfusion, IR injury produces persistent renal hemodynamic abnormalities characterized by increased renal vascular resistance (RVR), reduced renal blood flow (RBF), decreased glomerular filtration rate (GFR), diminished urine output, and accumulation of nitrogenous waste products in the circulation. These alterations in renal hemodynamics are central to the pathophysiology of ischemic AKI. Although several vasoactive mediators have been implicated in the development of post-ischemic microvascular dysfunction, the molecular and cellular mechanisms responsible for these hemodynamic disturbances remain incompletely understood. Peripheral serotonin (5-hydroxytryptamine, 5-HT) is a potent vasoactive mediator that strongly influences renal vascular tone. Unlike central serotonin, which is synthesized by tryptophan hydroxylase-2 (Tph2) in neurons, peripheral serotonin is generated primarily through the rate-limiting enzyme tryptophan hydroxylase-1 (Tph1), which is predominantly expressed in non-neuronal tissues. In the kidney, Tph1 expression has been localized largely to proximal tubule epithelial cells, indicating that the kidney possesses the machinery for local serotonin biosynthesis. Previous studies have shown that administration of exogenous serotonin or its precursor 5-hydroxytryptophan (5-HTP) causes both functional and structural kidney injury. Serotonin exerts many of these effects through activation of 5-HT₂ receptors expressed in the renal vasculature. However, the upstream mechanisms regulating intrarenal serotonin production remain poorly understood. In particular, whether renal IR injury stimulates local serotonin synthesis in the kidney and contributes to post-ischemic renal hemodynamic dysfunction has not been previously established. Hypoxia, a hallmark of ischemia, has been shown to upregulate Tph1 expression and serotonin biosynthesis by activating the transcription factor cAMP response element-binding protein (CREB). CREB is a key regulator of gene transcription during cellular stress and may represent a mechanistic link between ischemic signaling pathways and serotonin production. Based on this premise, we hypothesized that renal IR injury stimulates CREB-dependent Tph1 expression and serotonin production in the kidney, thereby contributing to renal vasoconstriction and the development of ischemic AKI. To test this hypothesis, we employed complementary genetic, cellular, and pharmacological approaches. These included global Tph1 hypomorphic rats generated through CRISPR/Cas9-mediated frameshift mutation, renal proximal tubule-specific Tph1 conditional knockout mice using the Cre-Lox system under the γ-glutamyl transferase (GGT) promoter, in vitro proximal tubule cell models of chemical ischemia, and pharmacologic inhibitors targeting serotonin biosynthesis and CREB signaling. Our findings demonstrate that renal IR injury significantly increases Tph1 expression and serotonin levels in the kidney, is associated with marked increases in RVR and reductions in RBF and GFR, and is accompanied by marked increases in Tph1 expression and serotonin levels in the kidney. Pharmacological inhibition of serotonin synthesis or receptor signaling significantly attenuated these hemodynamic disturbances and reduced the severity of AKI. Notably, CRISPR/Cas9-mediated Tph1 hypomorphism did not alter baseline arterial pressure, heart rate, renal blood flow, glomerular filtration rate, or kidney injury markers, indicating that reduced serotonin synthesis does not disrupt basal cardiovascular or renal function. However, global Tph1 hypomorphic rats exhibited marked protection against IR-induced renal dysfunction. Similarly, proximal tubule-specific deletion of Tph1 preserved renal perfusion, reduced renal vascular resistance, and improved renal functional outcomes following IR injury, without affecting baseline renal physiology. Mechanistic studies further revealed that proximal tubule cells subjected to chemical ischemia displayed increased CREB phosphorylation, enhanced CREB transcriptional activity, and subsequent upregulation of Tph1 expression, leading to increased serotonin production. Pharmacologic inhibition of CREB binding activity prevented ischemia-induced Tph1 expression and serotonin biosynthesis, thereby identifying a previously unrecognized CREB-dependent regulatory pathway controlling intrarenal serotonin production during ischemic stress. Collectively, these findings demonstrate that renal IR injury induces CREB-dependent upregulation of Tph1 and local serotonin production in the kidney, which contributes to renal vasoconstriction, impaired renal perfusion, and the development of ischemic AKI. These results identify intrarenal serotonin biosynthesis as a previously unrecognized driver of post-ischemic renal hemodynamic dysfunction. Targeting serotonin synthesis or signaling pathways may therefore represent a novel therapeutic strategy to mitigate renal injury and preserve kidney function following IR.