Kidney injury: The Spleno-renal connection and splenic tyrosine kinase

Abstract

Kidney injury is a major medical burden and one of the most common reasons for hospitalization and poor life quality. Kidney injury can include acute kidney injury, chronic kidney disease, and immune-mediated kidney diseases most of which have no definitive therapy. The spleen is a secondary lymphoid organ in the reticuloendothelial system that plays an important role in protecting the body from various diseases. Notably, spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, is a crucial player that aids in immunity and protection and is highly expressed in the kidney and hematopoietic cells. It has been shown that alterations in Syk function or expression could lead to a wide range of diseases and abnormalities. Over the past decade, the role of spleen and Syk in multiple kidney diseases has emerged. Evidence suggests that modulating the spleno-renal connection through activation of the cholinergic anti-inflammatory pathway can be a promising strategy for protecting against kidney injury. Imitating the protective function of the spleen through interleukin-10-extracellular vesicles can also be of therapeutic value. In addition, evidence showed that inhibition of the Syk leads to amelioration of the kidney injury. However, further exploration and long-term studies are needed to unravel the spleno-renal connection, as well as the efficacy of Syk inhibitors, before they can be used as means for treatment of kidney injury.

Graphical Abstract

Introduction:

The spleen is a small secondary lymphoid organ that performs a crucial immunological and hematopoietic role. Anatomically and functionally the spleen is divided into two parts: white pulp and red pulp. The white pulp has an immunological function by producing white blood cells, antibodies, and cytokines, whereas the red pulp filters the blood from any senescent or abnormal blood cells while also purging it of any harmful infections ^1–3. While the spleen plays a significant role in normal physiology, it is also implicated in a variety of pathological conditions. In recent years, it is becoming evident that the spleen plays a significant role in the pathophysiology of several inflammatory illnesses, autoimmune conditions, and hematologic malignancies ^{4, 5}.

Initially found in the spleen, spleen tyrosine kinase (Syk), a member of the non-receptor tyrosine kinases family, is an essential enzyme expressed by several organs and hematopoietic cells. Syk is highly expressed in the kidney and plays an important role in the regulation and modulation of immunity⁶. Syk is required for the intracellular signaling cascades of active receptors, such as the B-cell and Fc receptors, which engage in a wide range of processes, including the production of antibodies, release of cytokines, phagocytosis, and degranulation ^6–9. Several diseases, including autoimmune disorders, allergy, and some types of cancer, have been associated to dysregulation or aberrant activation of Syk ^10–12.

Studies in recent years have demonstrated the significance of targeting the spleen and Syk in reducing the development and progression of renal diseases. Given that the kidneys are essential for filtering waste materials and extra fluid from the blood, controlling blood pressure, and preserving electrolyte balance, these disorders can have a substantial effect on general health and well-being. These disorders might range from acute conditions that appear haphazardly to chronic diseases that take years to develop, and are often driven by various etiologies, such as diabetes, high blood pressure, infections, hereditary conditions, immune system abnormalities, and certain drugs. Additionally, underlying conditions can contribute to the development of renal diseases.

This review aims to summarize the mechanisms by which the spleen-kidneys interact, as well as the potential benefit of targeting Syk in various kidney diseases.

Spleno-renal connection in AKI:

Acute Kidney Injury (AKI) describes a number of related disorders that cause a loss of kidney function and is marked by an abrupt decrease in glomerular filtration rate (GFR) followed by a rise in serum creatinine concentration or oliguria ¹³. Commonly observed in hospitalized patients ¹⁴, AKI can be instigated by a variety of conditions, such as reduced perfusion or reperfusion, urinary tract obstruction, intrinsic renal factors, sepsis, or certain medications ^15–17. Some studies suggested protective splenic functions in AKI ¹⁸, that might be mediated through multiple pathways in which the spleen and the kidney crosstalk. For instance, during AKI, stimulating the spleen through exposure to ultrasonic waves has been suggested to result in favorable outcomes. In mice that with ischemia-reperfusion injury (IRI) to the kidneys, pre-treatment with ultrasound waves reduced inflammatory infiltration of CD45+ leukocytes, particularly CD11b+Ly6Ghigh (Neutrophils) and CD11b+F4/80high (Dendritic Cells). This led to an improved kidney tissue preservation in the injury model in comparison to the control group. These protective effects are hypothesized to be mediated through the cholinergic anti-inflammatory pathway (CAP) in the spleen^{19, 20}.

The CAP is a neuro-immunomodulatory pathway by which systemic inflammation is ameliorated. It starts by the activation of the efferent vagus nerve and then gets transmitted to the spleen via the splenic nerve. Splenic nerve terminals release norepinephrine, which binds to β-adrenergic receptors on splenic CD4+ T-cells. Acetylcholine is then released and binds to α7nAChR on neighboring splenic macrophages to influence their activity (Figure 1). It is thought that this significantly modulates the systemic immune response ^21–24. To test whether the protective effects of pre-treatment with ultrasonic waves are mediated through the CAP, the mice were treated with an a7nAChR agonist (cytisine), which mimicked the protective effects of ultrasound. Contrarily, an a7nAChR antagonist (α-bungarotoxin) blunted the ultrasonic waves’ protective impact, strongly suggesting that the splenic CAP accounted for the protective benefits of the ultrasonic waves. These ultrasonic waves-mediated protective effects were specifically dependent on CD4+ cells, as Rag1⁻/⁻ mice that lack functional T- and B-lymphocytes were devoid of these protective effects ^{19, 20}. In a similar subsequent study, the CAP was triggered via activation of vagal nuclei in the brainstem, also known as vagal nerve stimulation (VNS). Pre-treatment with VNS 24 hours prior to IRI activates the CAP, leading to the activation of a7nAChR on splenocytes, and finally improvements in renal biomarkers and a shift in the phenotype of macrophages from M1 (Pro-inflammatory) to M2 (Anti-inflammatory)²⁵. In a subsequent study, researchers investigated the entire pathway involved in CAP, and showed that the vagus nerve exerts its protective effects on the kidney via the sympathetic efferent pathway (also known as the vagosympathetic reflex) on one hand, and the vagus afferent pathway on the other hand, both of which lead to protective effects of CAP. This was showcased in that study using a novel mode of VNS: optogenetic stimulation of the neural circuits. The neural circuits responsible for VNS-mediated IRI amelioration start by activating the C1 nucleus, which is situated in the rostral ventrolateral medulla, and in turn activates the vagus nerve activating the splenic nerve to achieve kidney protection. In addition, the spleen also acts as bidirectional signal transducer in AKI. Injured kidneys send a danger signal (such as IL-6) to C1 neurons, possibly via vagal or somatic afferent neurons¹⁸. Taken together, the CAP protective effects on the kidney are mediated by the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis (Figure 2) ²⁶. Recent studies showed that VNS of the auricular branch of vagus nerve led to reduced inflammatory responses in diseases like Rheumatoid Arthritis ²⁷. This makes for a noninvasive and possible prophylactic therapeutic intervention for patients who are at high risk for AKI, such as patients >65 years of age, or those with chronic kidney disease, chronic illnesses, severe infection, or on certain medications. However, targeting this pathway is limited, as it needs to be stimulated 24 hours prior to AKI to be effective, rendering this method effective in prevention rather than treatment of AKI (Table 1).

Figure 1: Ultrasound waves pretreatment in the spleen improves acute kidney injury (AKI) outcome.

In mice exposed to ultrasound waves 24 hours prior to AKI, the cholinergic anti-inflammatory pathway was activated. The splenic nerve then releases norepinephrine to activate the CD4+ T-cells, which in turn release acetylcholine to shift macrophages phenotypes leading to enhanced anti-inflammatory effect. Created with BioRender.com

Figure 2. Vagus nerve stimulation actives spleen neuroimmune circuits to protect the kidney from injury.

The C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis mediates the cholinergic anti-inflammatory pathway. In AKI, injured kidneys send a danger signal (IL-6) to C1 neurons possibly via vagal or somatic afferent neurons. First, the C1 nucleus in the rostral ventrolateral medulla activates the vagus nerve (sympathetic nervous system), which in turn activates the splenic nerve, causing changes in the splenocytes. Lastly, these splenocytes trigger an anti-inflammatory system in the kidney. Created with BioRender.com

Table 1:

Potential therapeutic interventions targeting the spelno-renal connection.

Therapeutic intervention	Therapeutic Target	Principle	Advantages	Limitations	Relevant Study
Ultrasound waves	CAP	Pretreatment with US waves activates CAP promoting anti-inflammatory state and ameliorating kidney injury	1) Non-invasive 2) Effective 3) Cost effective 4) Safe 5) Reproducible 6) Widely available 7) Minimized side effects	1)Ineffective when AKI has set in 2)Lack of long-term studies 3) Lack of Preclinical studies 4) Difficult to predict AKI	19, 20
Auricular branch VNS	CAP	Pretreatment with VNS activates CAP promoting anti-inflammatory state and ameliorating kidney injury	1) Non-invasive 2) Effective 3) Safe 4) Minimized side effects	1)Ineffective when AKI has set in 2)Lack of long-term studies 3) Lack of Preclinical studies 4) Difficult to predict setting of AKI 5) Need for Specialized Equipment and Training 6) Limited evidence in AKI	26, 27
a7nAChR agonist (cytisine, dexmedetomidine)	CAP	Pretreatment with cytisine activates CAP promoting anti-inflammatory state and ameliorating kidney injury	1) Non-invasive 2) Effective 3) Safe	1)Ineffective when AKI has set in. 2)Lack of long-term studies 3) Lack of Preclinical studies 4) Difficult to predict AKI 5) Side effects 6) Narrow therapeutic window	48
IL-10 EVs	Injured Kidneys	IL-10, an anti-inflammatory cytokine, is encapsulated within EVs, which protects it from degradation and ensures its delivery to the injured kidneys.	1)Effective 2)Long half-life 3)Targeted delivery 4) Multi-organ protection 5) Enhanced stability	1) Cost 2) Potential for Off-target Effects 3) Limited Understanding of Mechanisms	30
HIF Hydroxylase inhibitor	HIF1a/mTOR Pathway	Aim to enhance HIF-1α activity by inhibiting prolyl hydroxylase, to reduce inflammation and prevent kidney cell death	1) Enhanced cell survival 2) Effective	1)Lack of knowledge regarding the pathway 2)Limited knowledge of adverse effects 3) Lack of long-term studies 4)Lack of preclinical studies	32, 33

Abbreviations: CAP, cholinergic anti-inflammatory pathway; US, Ultrasound; VNS, vagal nerve stimulation; EV, extracellular vesicles; HIF, hypoxia inducible factor

Yet another notable benefit of activating the spleen-kidney axis is the production of interleukin (IL)-10 in the spleen. In AKI, evolving inflammation can be countered by the production of IL-10 which may position the spleen as a vital element in preventing the deterioration of not only the kidney but also distant organs. For instance, in AKI, the proinflammatory IL-6 produced in the kidneys get buffered by the anti-inflammatory IL-10 secreted by the splenic macrophages, T-cells, and B-cells, protecting the lungs from damage. Consequently, mice with AKI that had undergone splenectomy showed lung inflammation and capillary leakage ^{28, 29}. Intriguingly, IL-10 packed in extracellular vesicles (EV) was found to be beneficial in ischemic AKI, due to improved half-life and thereby enhanced delivery to the target organ compared to serum IL-10. Specifically, IL-10 EVs reduced apoptosis and pro-inflammatory cytokines and shifted macrophages towards a polarized (M2) phenotype (Figure 3). Importantly, renal damage was reduced by IL-10 EV through increased mitophagy which improved the mitochondrial function and diminished the development of chronic kidney disease (CKD) following AKI ³⁰ (Table 1). Hence, IL-10 EVs may represent a promising therapeutic agent for AKI. Further studies are needed to study the in vitro side effects and efficacy in preventing multi-organ failure in the setting of AKI.

Figure 3. The role of IL-10 in spleen-renal immune regulation.

IL10 targeting the kidneys can produce a wide range of benefits during AKI, including reduction in kidney apoptosis, reduction in the inflammatory cytokines, and increased in macrophages of the M2 anti-inflammatory phenotype. IL-10 packed in extracellular vesicles has prolonged half-life and thereby enhanced delivery to the kidneys. Created with BioRender.com

Contrary to studies showing the spleen’s protective effect in AKI, a recent study suggested a role for the spleen in aggravating sepsis-induced AKI. This study demonstrated that CD11b+Gr-1+ cells, myeloid-derived suppressor cells, are essential in the progression of sepsis-induced AKI. Sepsis-induced AKI in mice achieved by cecum ligation and puncture induced an increase in the number of CD11b+Gr-1+ cells in the blood and kidneys and a reduction in the spleen, indicating that these cells were mobilized from the spleen. It has been suggested that these cells suppress the hypoxia inducible factor (HIF)-1a/mTOR pathway, thereby activating caspase-3 and apoptosis (Figure 4)³¹. In the context of sepsis-induced AKI, the spleen releases pro-inflammatory cytokines and immune cells into the bloodstream, which can exacerbate inflammation and lead to further kidney damage. On the other hand, inhibition of HIF hydroxylases leading to the activation HIF-1a resulting in protective effects in the setting of IRI (Table 1) ^{32, 33}. Through targeting HIF hydroxylases, the HIF-1a/mTOR pathway could be utilized in the future as a target for prevention of kidney injuries. However, more research is required to determine the role of the HIF-1a/mTOR pathway in sepsis-induced AKI and its interaction through the spleno-renal connection.

Figure 4. Spleen immune cells aggravate sepsis-induced acute kidney injury.

In sepsis-induced AKI, myeloid-derived suppressor CD11b+Gr-1+ cells migrate from the spleen to the kidneys through the systemic circulation. These cells inhibit the mTor/HIF1a pathway through a decrease in their phosphorylation. These cascades lead to increase in caspase-3 that induces apoptosis, thereby aggravating kidney injury. Created with BioRender.com

Spleno-renal connection in chronic kidney disease:

CKD may be secondary to various etiologies and risk factors, particularly diabetes, hypertension, obesity, and smoking ³⁴. The link between the pathophysiology of CKD and the spleen might be particularly prominent during the association of obesity with CKD. Obesity may be associated with renal damage in the long term with evidence of proteinuria, podocyte hypertrophy, mesangial expansion, glomerular enlargement, focal segmental diabetes, and hypertension ³⁵. Although the precise known molecular mechanisms causing pathological damage to the renal system are still under investigation, studies have shown that obesity is associated with chronic systemic low-grade inflammation due to downregulation of several spleen-produced anti-inflammatory cytokines along with upregulation of pro-inflammatory cytokines like tumor necrosis factor (TNF)-alpha and IL-6 ³⁵. Furthermore IL-10 levels are reduced in the setting of obesity, possibly due to the downregulation of CD20 expression on B-cells, which produce IL-10 ³⁶. A high fat diet leads to multiple morphological changes in the kidneys, including glomerular hypertrophy, decreased nephrin, and increased desmin, that are theorized to be offset by the anti-inflammatory cytokines produced by the spleen, particularly IL-10 ³⁵. This disruption of the spleno-renal crosstalk in the setting of obesity may thus be inducive for progression to CKD in patients with these existing risk factors. Yet additional causative factors for obesity-related CKD remain to be identified.

Role of spleen tyrosine kinase (Syk) in kidney injury:

Initially found in the spleen, Syk is a non-receptor tyrosine kinase highly expressed in multiple organs in the body, namely nerves, kidney, heart, brain, small intestine, and colon ³⁷. It plays a significant role in signal mediation of the adaptive immune system. However, recent studies show that it also is an integral player in other biological functions and molecular mechanisms, such as the innate immune system, osteoclast and platelet function, and vascular function¹⁰. More recently Syk activation has also been implicated in the development of various renal pathologies. When active, Syk phosphorylates targets downstream, raising intracellular Ca2+. It also initiates cascades involving p38 mitogen-activated protein kinase and extracellular signal-regulated kinase and activates transcription factors like NF-κB ³⁸. Due to its function in cell signal mediation, Syk is involved in downstream signaling of immunoreceptors like B-cell and Fc receptors, which participate in other signaling pathways and may lead to an increase in the production of pro-inflammatory cytokines and chemokines as well as trigger cellular proliferation, all mediators in kidney damage ³⁹. For example, in IgA nephropathy, the most common type of primary glomerulonephritis globally ⁴⁰, Syk may impact IgA-producing plasma cells ⁴⁰. Immunohistochemical staining showing increased expression of Syk in renal biopsy specimens from patients with IgA nephropathy which also inversely correlated with renal functions ⁴⁰. By increasing expression of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, and IFN-γ in IgA nephropathy Syk may induce inflammatory damage. During sepsis-induced AKI Syk influences the release of inflammatory cytokines and reactive oxygen species from neutrophils and dendritic cells. In a pertinent experiment lipopolysaccharide (LPS) were injected into mice to induce sepsis-induced AKI and a Syk inhibitor, R406, was administered 1 and 12 hours later. Creatinine and BUN both decreased in treated mice suggesting that Syk inhibition has alleviated sepsis-induced AKI.

Given its influence on the immune system, Syk was further implicated in autoimmune renal diseases. Once a receptor is engaged by various cell signals, Syk becomes phosphorylated and undergoes a conformational change that allows its C-terminal kinase domain to activate other downstream targets such as phospholipase-C gamma, phosphatidylinositol 30-kinase, and mitogen activated protein kinases ⁴¹. Activation of these targets in turn evokes cellular differentiation and proliferation along with increased phagocytosis and cytokine production ⁴¹, all of which play a role in the pathogenesis of autoimmune mediated renal disease, such as diffuse proliferative glomerulonephritis (DPGN). DPGN is often associated with Systemic Lupus Erythematosus ⁴², characterized by decreased C3 complement levels, thickened glomerular capillaries and increased glomerular cellularity ⁴³. While in healthy subjects Syk was expressed only in distal tubular epithelial cells, in the cohort with DPGN the positive staining was also found within in the glomeruli, suggesting it’s the role in the pathogenesis of autoimmune mediated renal diseases ⁴¹. As a result, several pharmacological inhibitors of Syk, such as Fostamatinib, Entospletinib, and Cerdulatinib have been tested, none have been approved specifically for the use in renal diseases (Table 2). Lanraplenib is a recently developed Syk inhibitor that underwent a phase II clinical trial in 2020 to test its efficacy in patients with lupus membranous nephropathy ⁴⁴, but was found to be was poorly tolerated and ineffective ⁴⁴. Further studies are needed in the future to understand the exact molecular pathophysiology by which Syk affects certain renal pathologies, such as IgA nephropathy and diffuse proliferative DPGN. Clinical trials should be considered for Syk inhibitors, such as Fostamatinib, Entospletinib, Cerdulatinib, and Lanraplenib as novel therapies for renal diseases. They should also further explore combination therapies to enhance efficacy and minimize adverse effects and overall improving the outcome in patients with renal pathologies.

Table 2:

Indications for Syk inhibitors.

Drug	Indication	Citation
Fostamatinib	RA, refractory immune thrombocytopenic purpura, leukemia and lymphoma	49
Entospletinib	CLL, non-hodgkin lymphoma	50
Cerdulatinib (dual Syk JAK1/3 inhibitor)	B-cell lymphoma, CLL	50
Lanraplenib	Currently under investigation for lupus membranous nephropathy.	44

Abbreviations: RA, rheumatoid arthritis; CLL, chronic lymphocytic leukemia; Syk, spleen tyrosine kinase

Splenectomy and kidney injury:

Investigations have delved into the potential impact of splenectomy on renal function, particularly in IRI cases and its effects on inflammatory responses. One trial showed that splenectomy in IRI produced encouraging outcomes with notable improvements in histological deterioration and renal function, and a discernible drop in TNF-α levels suggested a reduction in IRI ⁴⁵. This is consistent with the notion that the spleen, as an essential source of lymphocytes, may be involved in IRI injury. This implies that splenectomy may be a feasible strategy to lessen renal injury by lowering lymphocyte-mediated inflammatory cytokines.

Furthermore, the use of splenectomy in conjunction with kidney transplantation has been associated with favorable results regarding IRI. A study on rats revealed that in patients positive for donor-specific antigens, antidonor antibody generation was reduced when splenectomy was combined with kidney transplantation. The outcomes also showed that via lowering blood levels of TNF-α, splenectomy ameliorated renal IRI. The use of splenectomy dramatically reduced the deleterious effects of IRI damage, including markedly raised blood creatinine and BUN levels ⁴⁶.

On the other hand, a study looked at the effects of the α7nAChR agonist dexmedetomidine on LPS-induced kidney damage, apoptosis, and inflammatory protection associated with splenectomy. The findings showed that splenectomy exacerbated inflammation, hindered the removal of apoptotic cells from the injured kidneys, inhibited the protective effects of dexmedetomidine and the activation of monocytes and macrophages. This study highlights the renal protective effect via CAP activation is mediated by the spleen ⁴⁷.

Future directions and research gaps

Current interventions using physical stimulation methods such as ultrasonic or VNS, or IL-10 EVs, have been mostly explored in rodent models of IRI. These studies have shown protective effects when interventions were applied 24 hours before the onset of AKI. The CAP modulates the spleen to release anti-inflammatory cytokines and immune cells. However, there are significant gaps in monitoring spleen-specific biomarkers and inflammatory cytokines.

The number of studies examining the spleen-renal connection remains limited, especially in CKD. Comprehensive strategies combining preclinical validation, treatment approach optimization, and detailed clinical trial designs are necessary to effectively leverage the benefits of the spleen-renal connection in AKI and CKD settings. Further clarification of the molecular pathways involved in the spleen-renal connection and their role in modulating systemic inflammation is essential, and studies using large animal models may help better understand these mechanisms. Refinement of techniques for activating the spleen-kidney axis, such as ultrasonic waves and VNS, is crucial. Additionally, optimizing the administration of IL-10 EVs and HIF hydroxylase inhibitors for better stability and targeting will be important steps forward.

Clinical trials need to identify appropriate patient populations, particularly those at high risk of AKI, establish optimal timing and dosage for interventions, and define clear clinical endpoints and reliable biomarkers such as GFR and creatinine. Long-term studies are required to assess sustained benefits and translate these findings into clinical practice. There is potential in targeting the spleen and Syk for reducing renal disease progression. Clinical trials using Syk inhibitors for autoimmune kidney diseases could also be beneficial.

Combining VNS with other therapeutic strategies, such as anti-inflammatory drugs and cytokine inhibitors, could enhance protective effects against AKI. Consideration of individual variances in response to medications and further examination of the spleen-kidney link across different patient demographics could lead to more effective individualized therapies.

Clinical implications and translation

Recent interest in the pathophysiological connection between the spleen and kidneys, as well as the role of Syk in renal diseases, has increased. Because AKI and CKD are profoundly influenced by the spleen, understanding the spleen-kidney connection and the role of Syk can help identify potential therapeutic targets for these diseases. Various animal studies have shown promise in mitigating kidney disease by leveraging this connection.

Vagus nerve stimulation of the auricular branch has potential as a noninvasive method to prevent AKI in high-risk patients, and IL-10 EVs have shown potential in reducing inflammation in AKI. Clinical trials using Syk inhibitors for autoimmune kidney diseases could also be beneficial. Splenectomy, which modulates kidney responses and enhances outcomes, highlights the spleen’s significant role in this interaction.

Noninvasive interventions like VNS and the production of IL-10 in the spleen show promise in preventing kidney deterioration and protecting other organs. For instance, IL-10 EVs have been beneficial in ischemic AKI by improving delivery and stability compared to serum IL-10, reducing apoptosis, and enhancing macrophage polarization and mitochondrial function.

Despite promising animal studies, translating these findings into clinical practice requires more research. Clinical trials must identify appropriate patient populations, optimal timing and dosage for interventions, and clear clinical endpoints. Long-term studies are necessary to assess the sustained benefits and potential side effects of these therapies.

By addressing these gaps, the translation of spleen-kidney axis findings into clinical practice holds promise for improving outcomes in AKI and CKD patients, reducing the burden of these conditions.

- Data availability statement

We do not have independent data to share for this review.