Podocyte-Mediated Protection from Kidney Injury

The tripartite glomerular filtration barrier is composed of the fenestrated endothelial cells, the glomerular basement membrane (GBM), and the podocytes. Damage to any of these glomerular components may lead to the leakage of albumin and larger proteins into urine. In addition to systemic factors, the glomerular cells themselves control the functionality of the filtration barrier by actively communicating in an autocrine or paracrine manner through the secretion of soluble and other factors.¹ In the context of kidney disease, these signals may either enhance or protect from the disease progression.

Inflammation is a driving force in the development and progression of various kidney diseases, including the many different forms of GN. The injury in GN can be mediated through several mechanisms. A physical and functional alteration to the GBM may affect communication between endothelial cells and podocytes. Circulating anti-GBM antibodies lead to the formation of immune complexes (composed of antibodies bound to antigens) that can deposit in the glomeruli. They trigger complement activation and attract inflammatory cells, leading to glomerular injury.² This can further lead to proteinuria, hematuria, edema, and hypertension, which often require careful management to prevent progression to CKD or kidney failure.²

In this issue of JASN, Truong et al. turned their attention to a new, dynamic aspect by studying the putative protective role of acetylcholine produced locally in the kidney by choline acetyltransferase (ChAT).³ For this, they used a transgenic mouse line expressing enhanced green fluorescent protein under the control of the endogenous transcriptional regulatory elements of ChAT (ChAT transgenic mice). Anti-GBM antibodies were derived from rabbits immunized with rat GBM preparations. ChAT transgenic mice were preimmunized with rabbit IgG and thereafter given passively the rabbit anti-rat GBM antibodies,³ generating GN with features of both an anti-GBM and immune complex GN. In humans, this model would resemble anti-GBM disease, various forms of membranoproliferative or systemic lupus erythematosus-type GN, or membranous nephropathy. Because these types of diseases are often seen in nephrology clinics and pose therapeutic challenges, any new means to alleviate the symptoms and promote healing would be welcome.

Acetylcholine is a neurotransmitter produced as a result of the stimulation of the vagus nerve.⁴ In addition to its many neuronal cholinergic effects, acetylcholine reduces inflammation by affecting multiple types of immune cells.⁴ ChAT is widely expressed in human cells and tissues.⁵ Truong et al. observed that ChAT was mainly expressed by podocytes in the ChAT transgenic mouse kidneys.³ In vitro studies revealed that podocytes, but not endothelial or mesangial cells, can be induced to produce acetylcholine.³ Also the muscarinic and nicotinic acetylcholine receptors are expressed by numerous human cells,⁵ including podocytes and glomerular endothelial cells.⁶ Stimulation of these receptors enables acetylcholine to regulate various cellular activities, such as immune mediator release, in these cells.

Acetylcholine, being a vagal (parasympathetic) neurotransmitter, is well known for counteracting the effects of NE and epinephrine (adrenaline), which are involved in the sympathetic nervous system and released during stress. The latter can cause vasoconstriction of kidney blood vessels to reduce kidney blood flow and GFR. Previous work revealed that acetylcholine lowered the proinflammatory cytokine levels systemically, by reducing their production by macrophages in the spleen, and thereby protected kidneys from ischemia-reperfusion injury.⁷ Truong et al. observed that the expression of ChAT was induced mainly in podocytes after induction of anti-GBM GN, enhancing acetylcholine synthesis locally in the kidney (Figure 1).³ The mice with anti-GBM GN show proteinuria, inflammatory cell infiltration, glomerular necrosis, and crescents,⁸ features similar to the respective human diseases. Although not mentioned by Truong et al., the disease development involves complement activation. Marked tubular and interstitial injury along with fibrosis starts to develop 1 week after anti-GBM antibody injection.⁸ The key observation of the study of Truong et al. was that the ChAT transgenic mice with GN were protected from glomerular and tubular injury showing lower serum creatinine levels and less severe albuminuria than the wild-type mice with GN.³ Nephrotic ChAT transgenic mice also showed amelioration of kidney inflammation (Figure 1). However, ChAT was not increased in the spleen, ruling out a systemic effect.³ Thus, increased local expression of ChAT and synthesis of acetylcholine appear to protect the kidney against inflammatory injury. Truong et al. confirmed the role of ChAT in kidney protection by inducing anti-GBM GN in mice lacking ChAT specifically in podocytes, and, indeed, the knockout mice showed more severe kidney injury.³

Figure 1.

Acetylcholine produced by podocytes leads to anti-inflammatory and angiogenic effects in GN and protects from kidney injury. Induction of anti-GBM GN in ChAT transgenic mice increases the expression of ChAT and synthesis of acetylcholine in podocytes. This reduces inflammation, increases the expression of eNOS, and attenuates the reduction of angiogenic factors caused by GN. Glomerular and tubular damage become alleviated, and the kidneys are protected from a functional decline. ChAT inducer rolipram (acts by inhibiting phosphodiesterase 4) and acetylcholinesterase inhibitor donepezil (through a vagal positive feedback effect) promote the expression of ChAT and synthesis of acetylcholine in ChAT transgenic mice. It remains to be tested whether these treatments protect kidneys from injury in anti-GBM GN. Further studies are also needed to define whether acetylcholine affects podocytes in an autocrine manner. ChAT, choline acetyltransferase; E, fenestrated endothelial cell; eNOS, endothelial nitric oxide synthase; GBM, glomerular basement membrane; P, podocyte.

Alleviation of tissue injury by locally produced acetylcholine in the model used offers potentially novel possibilities for the treatment of glomerular immunoinflammatory diseases, especially GN. The current treatments targeting the immune background of GN are nonspecific and come with a wide spectra of side effects. Controlling the BP and reducing proteinuria are standard therapies in kidney diseases. Depending on the type of GN, corticosteroids or immunosuppressants like cyclophosphamide, mycophenolate mofetil, or azathioprine, or, in the case of complement-mediated diseases, complement inhibitors are being used. Because the effects of these drugs are usually wider than needed and they may also have side effects, more targeted or gentle therapies are needed.

As the stimulation of acetylcholine synthesis remarkably reduced inflammation and tissue damage, means to convert this effect to therapeutic use could prove useful. The beneficial effects of acetylcholine include regulation of angiogenesis and induction of nitric oxide synthesis by endothelial cells (Figure 1).³ This emphasizes the cross-talk between podocytes and endothelial cells and the benefits of locally produced acetylcholine—in circulation, acetylcholine becomes rapidly inactivated by acetylcholinesterase. In addition, the potential of acetylcholine to protect podocytes in an autocrine manner in podocytopathies, as proposed by the authors, is worth further investigation. Altogether, for any therapeutic application in glomerular disease, the challenge is that the stimulation of acetylcholine synthesis needs to be locally targeted.

The highlights in the study by Truong et al. were the analyses defining whether ChAT and acetylcholine production can be induced in mice using available medications. Indeed, phosphodiesterase 4 inhibitor rolipram and acetylcholinesterase inhibitor donepezil administered to ChAT transgenic mice increased ChAT expression in podocytes, visualized by increased enhanced green fluorescent protein signal, and enhanced acetylcholine production (Figure 1).³ Donepezil increased acetylcholine levels also in the wild-type mouse kidneys.³ As a proof-of-concept for these treatments, it would be interesting to treat mice with anti-GBM GN with these drugs to define whether this reduces glomerular and tubular damage and albuminuria. To achieve the first step in translating the data to humans, one could define whether the drugs induce acetylcholine synthesis and lower inflammatory cytokine production in human-induced pluripotent stem cell–derived organoids or ex vivo human glomerular outgrowths cultured under inflammatory conditions.⁹ Support for the beneficial effects of blocking acetylcholinesterase in kidney disease comes from a study on people with Alzheimer disease treated with donepezil, revealing that use of donepezil was associated with lower risk of the progression of CKD.¹⁰

The study by Truong et al. demonstrates an interesting link between a neurotransmitter effect and immunoinflammatory mechanisms. In general, the effects of neurotransmitters on the broad range of diseases affecting kidney glomeruli and immunoinflammatory syndromes provide interesting new study areas. In addition, coming from an unexpected angle, they may also offer new possibilities for treatment.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/E986.

Funding

S. Lehtonen: Sigrid Juséliuksen Säätiö and Novo Nordisk Fonden (NNF22OC0078449). S. Meri: Sigrid Juséliuksen Säätiö and Helsingin ja Uudenmaan Sairaanhoitopiiri (TYH2019311 and TYH2022315).

Author Contributions

Conceptualization: Sanna Lehtonen, Seppo Meri.

Visualization: Sanna Lehtonen.

Writing – original draft: Sanna Lehtonen, Seppo Meri.

Writing – review & editing: Sanna Lehtonen, Seppo Meri.