Renal Effects of Chronic D-Serine Administration in a Parkinson’s Disease Rat Model: Evidence for a Threshold Nephrotoxic Dose

Renal Effects of Chronic D-Serine Administration in a Parkinson’s Disease Rat Model: Evidence for a Threshold Nephrotoxic Dose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Renal Effects of Chronic D-Serine Administration in a Parkinson’s Disease Rat Model: Evidence for a Threshold Nephrotoxic Dose Giorgia Ferniani, Camilla Lucotti, Silvia Fanni, Jessica Bratzu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9551872/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract D-serine, an endogenous co-agonist at the glycine binding site of NMDA receptor NR1 subunits, is increasingly studied in Parkinson’s disease, where altered levels have been detected in both patients and animal models. Using a rat model based on α-synuclein overexpression, we evaluated renal effects after eight weeks of daily D-serine administration, alone or in combination with L-DOPA. Given the known acute nephrotoxicity of D-serine in rats, this study addresses the lack of data on chronic exposure. Our results show that a dose of 200 mg/kg induces biological alterations - reflected in growth patterns and water consumption - and peritubular inflammatory infiltrates comparable to the nephrotoxic effect reported after acute treatment. These findings suggest that 200 mg/kg could represent a threshold dose for chronic D-serine administration in rats, providing a useful reference for future studies involving prolonged treatment. D-serine nephrotoxicity rat Parkinson’s disease L-DOPA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction D-amino acids are emerging as interesting molecules in the context of neurodegenerative and psychiatric disorders, either as therapeutic agents or as biomarkers (Madeira et al. 2015 ; Seckler and Lewis 2020 ; Nuzzo et al. 2020 ; Piubelli et al. 2021 ; Taniguchi et al. 2022 ; Souza et al. 2023 ). In this context, D-serine, an endogenous co-agonist at the glycine binding site of NMDA receptor NR1 subunits, has attracted particular attention as its levels have been found altered in animal models of Parkinson’s disease (PD) as well as in patients (Di Maio et al. 2023 ; Serra et al. 2023 ). Intense research is currently ongoing to understand whether these alterations, found at both central and peripheral levels, play a role in the neurodegenerative process and/or represent a specific signature of the disease (Imarisio et al. 2024 ; Yahyavi et al. 2025 ). To this aim, animal models of PD are instrumental as they provide the unique opportunity to investigate, in controlled conditions, the consequence of the neurodegenerative event affecting the nigrostriatal dopaminergic system on peripheral and central D-serine levels, as well as the impact of its exogenous administration. Among different models, the rat model based on overexpression of human α-synuclein (α-syn) via an adeno-associated viral vector represents a validated and extensively used tool in this field of research. Indeed, this model reproduces cardinal features of the disease, such as the formation of cellular and axonal α-syn rich inclusions, the slowly progressive neurodegenerative process, and the appearance of related motor defects (Decressac et al. 2012 ). Based on this rationale, we designed a study in which animals overexpressing α-syn received daily D-serine treatment, alone or in combination with L-DOPA, the most used antiparkinsonian drug, for eight weeks, before being sacrificed for ex-vivo analyses. Whereas the primary objective of the experimental design was to investigate the central effects of these treatments, which are the subjects of a separate study, kidneys were also collected to assess potential nephrotoxic effects associated with prolonged D-serine administration, which are reported here. In fact, previous studies have highlighted an acute nephrotoxic effect of D-serine, specifically in rats, but not in mice. Thus, while mice have been treated with doses up to 1 g/kg, with no adverse effects on renal function, histological and biochemical changes have emerged in rats after a single administration of D-serine at a dose of 200 mg/kg or higher (Ganote et al. 1974 ; Williams et al. 2003 ; Maekawa et al. 2005 ; Orozco-Ibarra et al. 2007 ; Hasegawa et al. 2019 ; Meftah et al. 2021 ). The potential risk of D-serine-induced nephrotoxicity in rats has been described since the 1940s (Morehead R et al. 1945 ). In particular, acute pathological events at kidney level induced by D-serine are tubular structural changes, serum creatinine increase, glycosuria, proteinuria, and presence of urinary N-acetyl-β-D-glucosaminidase (NAG) activity, indicating tubular damage (Ganote et al. 1974 ; Williams et al. 2003 ; Delraso et al. 2009 ; Hasegawa et al. 2019 ; Meftah et al. 2021 ). The most marked perturbations are observed between 8–24 h postdosing (Williams et al. 2003 ), and, despite these acute pathological events, D-serine-induced nephrotoxicity appears to be fully reversible (Ganote et al. 1974 ; Meftah et al. 2021 ). Specifically, after acute kidney damage, urine values of proteins, glucose, and amino acids begin to normalize 24–48 h after the last dose of D-serine and by 120 h post-dose largely return to normal. Pathological changes also completely resolve within this timeframe, with complete regrowth of new epithelium in tubules and renal tubular basophilia (Williams et al. 2003 ). Despite this evidence, no single study has focused on the consequences of chronic daily administration of D-serine. This report is therefore meant to cover this gap and guide the choice of the D-serine dose in future rat studies where this D-amino acid is administered chronically, both in physiological and pathological conditions. Results Experimental design Sprague-Dawley male rats were subjected to stereotaxic surgery for the nigral delivery of an adeno-associated viral vector encoding the human α-syn gene. Three days after surgery, animals were allocated into different subgroups in order to receive D-serine, either alone or in combination with L-DOPA, L-DOPA alone, or vehicle, as a control group. Treatments were carried out daily for eight weeks. At the end of the treatment period, one-spot urine samples were collected from the animals. Thereafter, animals were sacrificed by transcardiac perfusion with paraformaldehyde (PFA) to collect central and peripheral tissues, including the kidneys (see Fig. 1 for the experimental timeline). A first study (n = 40) was conducted using a D-serine dose of 200 mg/kg (study 1). In light of the results, a subsequent study (n = 60) was performed employing two D-serine doses, 100 and 200 mg/kg (study 2). Water consumption and body weight, study 1 Water consumption was estimated weekly based on the scale of the drinking bottles, and body weight was measured at the same time points. In the first study, D-serine treatment, with or without L-DOPA, produced a marked increase in water intake during the first week, with D-serine-treated rats consuming approximately twice as much as control animals (Fig. 2 A). Moreover, a moderate reduction in body weight was observed in the same period (on average 30 g, corresponding to about 10% of the initial body weight; Fig. 3 ). Notably, water consumption normalized by the second week of treatment (Fig. 2 B), and animals began to gain weight similarly to controls during the same week. Nevertheless, a slight but persistent difference in body weight between D-serine-treated animals and the control group remained throughout the study. Specifically, animals receiving D-serine weighed, on average, approximately 10 g less than controls from week 2 until week 8, before being sacrificed. Histological observations and urinalysis, study 1 Perfused kidneys were fixed in formalin and subsequently sampled, processed, and embedded in paraffin for histopathological evaluation. Morphological alterations of the renal parenchyma, including tubular epithelial attenuation, cytoplasmic hypereosinophilia, cytoplasmic blebbing, the presence of cellular debris, fibrin, and/or necrosis, chronic peritubular inflammatory infiltrates, tubular thyroidization, and fibrotic changes, were systematically assessed. The main histopathological alteration found was a mild to moderate chronic peritubular inflammatory infiltrate, only in the rat groups treated with D-serine, both in the presence and in the absence of L-DOPA. No significant damage to the glomeruli or renal tubules was observed (Fig. 4 A-D). Inflammatory cell count revealed a significant increase in the inflammatory cells only in rats treated with D-serine, both in the presence and in the absence of L-DOPA (Fig. 4 E). These kinds of tubular injury overlap with those observed for other nephrotoxins (Kim and Moon 2012 ; Meftah et al. 2021 ; Hall et al. 2022 ). As mentioned in the introduction, D-serine nephrotoxicity is associated with proteinuria and urinary NAG activity. For this reason, we also evaluated these markers of kidney injury in rats’ urines. As shown in Fig. 5 , no significant differences emerged among the analyzed groups for both proteinuria (Fig. 5 A) and NAG activity (Fig. 5 B). The overall results obtained from this experimental set led us to hypothesize that 200 mg/kg could represent a threshold dose for chronic D-serine administration in rats. Indeed, both the observed biological alterations (i.e., growth evaluation and water consumption) and the presence of peritubular inflammatory infiltrate suggest that the chronic administration of D-serine induces effects comparable to those reported after acute administration (Ganote et al. 1974 ; Delraso et al. 2009 ; Hasegawa et al. 2019 ). Water consumption and body weight, study 2 Considering these results, a second study was performed using two different D-serine doses, 100 and 200 mg/kg, administered alone or in combination with L-DOPA. Rats were split into two subgroups: in group A, rats meant to receive the higher D-serine dose, received 200 mg/kg from the first administration; in group B, rats meant to receive the 200 mg/kg dose, were initially given 100 mg/kg for the first 7 days; thereafter, the dose was increased to 200 mg/kg. Administration of 200 mg/kg, but not 100 mg/kg, of D-serine led to increased water consumption during the first week in group A (Fig. 6 A). By contrast, starting the treatment with the lower dose, as done in group B, did not result in increased water consumption upon dose escalation (Fig. 6 B). Nevertheless, a reduced body weight was observed both in group A and B for all D-serine-treated rats also throughout study 2. Compared with controls, D-serine-treated animals weighed, on average, approximately 30 g less (Fig. 7 ) from week 2 until sacrifice at week 8. This represents a more pronounced difference than what was observed for study 1; the smaller size of the rats subjected to D-serine treatment in this second study compared to the first one may account for this discrepancy. Histological observations and urinalysis, study 2 Perfused kidneys were processed as for study 1. Microscopic examination showed that 200 mg/kg of D-serine, both alone and in combination with L-DOPA, induced morphological alterations of the renal parenchyma, confirming what was seen in the first group of animals. Also here, the main histopathological alteration was a mild to moderate chronic peritubular inflammatory infiltrate, without significant damage to the glomeruli or renal tubules, in D-serine 200 mg/kg treated rats (Figs. 8 D and E) compared to the vehicle group (Fig. 8 A) in group A. Importantly, no detrimental effects were observed with D-serine 100 mg/kg (Figs. 8 B and C). Inflammatory cell evaluation fully overlapped with the histopathological results (Fig. 8 G). Interestingly enough, when adopting the escalation design, as for group B, the inflammation was almost undetectable (Fig. 9 ), and no significant differences in the inflammatory cell count were seen (Fig. 9 G). From the biochemical point of view, urine analysis showed again no significant differences among the experimental groups, for the analyzed parameters, suggesting that the mild morphological alterations had no functional impact (Fig. 10 ). Discussion In the present study, we investigated the long-term effects of chronic D-serine administration, either alone or in combination with L-DOPA, on renal morphology and function in a PD rat model. Our findings demonstrate that daily administration of D-serine for eight weeks at a dose of 200 mg/kg, but not 100 mg/kg, induced early signs of nephrotoxicity. These alterations were mainly characterized by changes in the renal parenchyma, including a clear chronic peritubular inflammatory infiltrate, in the absence of glomerular structural damage and without detectable alterations in conventional urinary biomarkers of kidney injury. Quantitative analysis of inflammatory cells confirmed a significant increase exclusively in the D-serine 200 mg/kg–treated groups, indicating that D-serine is the primary driver of the observed inflammatory response. Importantly, these effects were independent of L-DOPA co-administration, as similar outcomes were seen whether D-serine was administered alone or in combination with L-DOPA, suggesting that L-DOPA does not affect D-serine–induced renal effects under the conditions tested. This study was originally designed to investigate the central effects of D-serine as an add-on therapy to L-DOPA, which is the most effective drug in treating motor symptoms in PD. Thus, this design allowed us to also demonstrate that L-DOPA chronic administration, at a dose commonly used in animal models, is devoid of significant detrimental effects on kidneys. An additional relevant observation was the transient increase in water consumption observed at the beginning of treatment with the higher D-serine dose (200 mg/kg), and the slightly reduced body weight at both doses, which, conversely, persisted throughout the study. These abnormalities prompted us to collect the kidneys at the end of the experiments and perform the present investigations. In fact, increased water intake is often an indirect marker of impaired renal function and may reflect alterations in urine concentrating ability, hormonal regulation, or waste dilution mechanisms (Hopp et al. 2015 ; Choi et al. 2015 ). Interestingly, the abnormal water consumption normalized after the first week of treatment and was not observed when D-serine was initiated at the lower dose and subsequently escalated to the higher one after the first week (study 2, group B). Notably, this experimental design also prevented inflammation, seen, by contrast, when the 200 mg/kg D-serine dose was administered from the first treatment (study 1 and study 2, group A). This observation may suggest the existence of some adaptive mechanisms to the exogenous D-serine administration in our rat model, which appear to take place after a few drug injections. Although the molecular basis of this event remains unknown, potential mechanisms include the upregulation of enzymes involved in D-serine metabolism, such as D-amino acid oxidase (DAAO), or the downregulation of renal NMDA receptors. Previous studies have demonstrated that acute D-serine nephrotoxicity is characterized by proximal tubular damage accompanied by functional alterations, including proteinuria and increased urinary NAG activity (Delraso et al. 2009 ; Hasegawa et al. 2019 ). By contrast, in the present chronic model, neither proteinuria nor NAG activity differed significantly among experimental groups. Differences in exposure duration may also explain this discrepancy, as chronic administration may allow the induction of adaptive or compensatory mechanisms that limit overt tubular dysfunction despite the presence of slight histological alterations. The mechanisms underlying D-serine nephrotoxicity in rats are not fully understood but appear to involve species-specific differences in renal handling and metabolism of D-serine. Rats exhibit a higher degree of renal reabsorption of D-serine compared with other species, as reflected by low urinary D-serine levels, despite serum concentrations comparable to those observed in humans and dogs (Huang et al. 1998 ). Increased tubular reabsorption leads to elevated local concentrations of D-serine, which is metabolized by DAAO; DAAO activity is associated with the generation of reactive oxygen species (ROS) that, in turn, may drive nephrotoxic events (Maekawa et al. 2005 ). Additionally, it has been proposed that rats possess a greater capacity for utilizing D-amino acids and that renal NMDA receptors may be directly involved in mediating D-serine–induced nephrotoxicity (Tseng et al. 2021 ). Regardless of the specific mechanisms involved, our data suggest that chronic D-serine administration in rats does not exacerbate renal injury over time, even when particularly prolonged. It is worth noting that, compared with rats, humans show lower renal tubular reabsorption and higher urinary excretion of D-serine, resulting in reduced intrarenal accumulation (Huang et al. 1998 ). In fact, across all published human studies on D-serine, only one subject has been reported to exhibit abnormal renal values related to D-serine treatment (Kantrowitz et al. 2010 ). Despite human D-serine administration appears safe, the plasma levels are increased in chronic kidney disease, especially at advanced stages, making D-serine a possible “sensor” of the kidney healthy status as well as a possible uremic toxin (Okada et al. 2017 ; Hesaka et al. 2019 ; Kimura et al. 2023 ). A reduced body weight was observed upon D-serine treatment throughout the study and should also be considered when designing future rat studies. However, this effect is unlikely to be related to nephrotoxicity, as it was also present at the lower dose, which was not associated with any morphological or functional renal alterations in our analyses. Whereas this issue goes beyond the aim of this study, it is worth mentioning that previous studies have shown that chronic D-serine administration can reduce body weight also in mice, possibly by reducing food consumption and/or altering insulin secretion (Sasaki et al. 2015 ; Suwandhi et al. 2018 ). To our knowledge, this study represents the first systematic evaluation of the renal effects of chronic daily D-serine administration in rats. While mice are preferentially used for the apparent lack of D-serine nephrotoxicity, even at doses up to 1 g/kg, rats remain a valuable experimental model in certain pathological contexts, including PD, where the role of D-serine is increasingly recognized in both preclinical and clinical studies (Gelfin et al. 2012 ; Di Maio et al. 2023 ; Serra et al. 2023 ). Therefore, this study is relevant to better define its long-term toxicity in this species. Our data suggest that daily doses lower than 200 mg/kg are advisable for chronic D-serine administration and that a dose-escalation approach could be used to prevent adverse events, such as abnormal water consumption and early signs of morphological alterations. Although this study was conducted in a rat model of PD, it is important to note that the viral vector encoding human α-syn was injected unilaterally into the substantia nigra, resulting in a model of hemi-parkinsonism characterized by moderate dopaminergic degeneration and minimal motor impairment. Therefore, no significant peripheral alterations are expected to take place in consequence of this procedure and alter the response to D-serine in the kidneys. For this reason, we believe that the findings of this study, although obtained in a pathological model, can extend to physiological conditions and be a reference for any rat study concerning chronic D-serine administration. Importantly, our parallel investigation on the central effects of this D-amino acid shows that these doses are capable of inducing measurable effects in the CNS, such as increased synaptic plasticity (manuscript in preparation). On the other hand, a previous rat NMR study showed that a D-serine dose as low as 50 mg/kg can activate specific brain regions, further suggesting that even relatively low doses are effective in rats (Panizzutti et al. 2005 ). In conclusion, by defining a dose range devoid of nephrotoxicity, this study covers a gap regarding the effect of chronic D-serine administration in rats, thus providing a useful reference for future studies investigating the central or peripheral effects of this D-amino acid. Materials and Methods Animals Male Sprague-Dawley rats (275–300 g; Envigo, Italy) were housed in groups of 3–4 per cage under a 12 h light/dark cycle in a temperature- and humidity-controlled environment, with ad libitum access to food and water. All animal procedures were conducted in accordance with European Directive 2010/63/EU and Italian Legislative Decree no. 26/2014. The present study was approved by the local Ethical Committee (OPBA) and the Italian Ministry of Health (authorization no. 779/2024). The authors complied with the ARRIVE guidelines. Animal model Animals underwent stereotaxic surgery for the unilateral nigral infusion of the adeno-associated virus (AAV) vector expressing a human wild-type α-syn, as previously described (Decressac et al. 2012 ). All surgical procedures were performed under general anesthesia induced by intraperitoneal injection of a 20:1 mixture of fentanyl (Fentadon, Dechra) and medetomidine hydrochloride (Domitor, Orion Pharma) at a dose of 6 mL/kg. Rats were allocated in a stereotaxic frame (2Biological Instruments), and vector solution was delivered using a 10 µl Hamilton syringe fitted with a glass capillary to minimize mechanical tissue damage. Each animal received a total volume of 4 µL of AAV-α-syn solution, split into 2 different sites and infused at a rate of 0.5 µL/min. The capillary was kept in place for an additional 3 min per site before being slowly retracted. The AAV solution was injected into the right Substantia Nigra pars compacta at the following coordinates (flat skull position): 1) antero-posterior (AP): −5.3 mm (from bregma), medio-lateral (ML): −1.6 mm, dorso-ventral (DV): −7.2 mm (from dura surface); 2) AP: −5.3 mm, ML: −2.6 mm, DV: −6.7 mm, according to the stereotaxic atlas of Paxinos and Watson ( 1986 ) (Björklund et al. 2022 ). Anesthesia awakening was promoted by subcutaneous administration of atipamezole hydrochloride (Revertor, Virbac). After the surgery, animals were placed in clean cages and monitored daily until full recovery. Drugs formulations and administration L-DOPA as the methyl ester hydrochloride and D-serine (both from Biosynth s.r.o., Bratislava, SK) were dissolved in 0,9% NaCl before use and administered once daily. L-DOPA (12 mg/kg), in association with benserazide (6 mg/kg), was injected subcutaneously (s.c.), whereas D-serine (100 or 200 mg/kg, depending on experimental group) was administered intraperitoneally (i.p.). Control animals received 0,9% NaCl (vehicle) via both subcutaneous and intraperitoneal routes. Animals were randomly assigned to the different treatment groups. Study 1 consisted of 4 groups (10 rats/group), daily treated with: 1) vehicle s.c. + vehicle i.p., 2) L-DOPA s.c. + vehicle i.p., 3) vehicle s.c. + D-serine (200 mg/kg) i.p., 4) L-DOPA s.c. + D-serine (200 mg/kg) i.p. Study 2 consisted of 6 groups (10 rats/group): 1–4) as in study 1, 5) vehicle s.c. + D-serine (100 mg/kg) i.p., 6) L-DOPA s.c. + D-serine (100 mg/kg) i.p. Animals in study 2 were split into two subgroups: group A, in which rats meant to receive the higher D-serine dose, received 200 mg/kg from the first administration; group B, where rats meant to receive the 200 mg/kg dose, were initially given 100 mg/kg for the first 7 days before escalating to 200 mg/kg. All treatments were administered starting from 3 days after surgery and continued daily for 8 weeks until sacrifice. Urine collection For urine collection, animals were individually housed in single cages during the eighth week of treatment. The last treatment was administered 24 h prior to urine sampling. One-spot urine samples were collected using a micropipette, transferred into Eppendorf tubes, and stored at -80°C until biochemical analysis. Sacrifice Under anesthesia, animals were sacrificed 24 h after the last D-serine administration by transcardiac perfusion with saline (0.9% NaCl), followed by ice-cold 4% PFA. After perfusion, kidneys were collected into Falcon tubes containing 4% PFA and stored at 4°C until histopathological analyses. Histopathology For light microscopy, kidney tissue samples were fixed by immersion in buffered formalin (pH 7.4) and embedded in paraffin. For histological analysis, 5-µm thickness sections were produced from paraffin blocks and stained with hematoxylin and eosin. Slides were observed under an Olympus optical microscope equipped with a digital imaging camera. Histological analyses were performed by investigators blinded to treatment allocation. Inflammatory Cells Counting Cell counts were performed for quantitative assessment of inflammatory intensity. Representative slides were photographed using an Olympus optical microscope equipped with a digital imaging camera. Ten photographs/slide were taken at 20 × magnification. Digital analysis of the images was performed using the public domain ImageJ 1.44p software (National Institutes of Health, USA; http://imagej.nih.gov/ij ). The manual cell counting tool, “cell counter”, was chosen (Pinheiro et al. 2024 ). Biochemical Measurements Urine samples were centrifuged at 10,000 rpm for 3 minutes, and supernatant was used for the subsequent analysis. Urinary proteins were quantified by using Rat Urinary Protein Assay Kit # 9040 (Condrex, Inc.), following manufactures instructions. Briefly, urine was diluted 1:2 with the provided dilution buffer, and the turbidity assay was conducted by adding 3% Sulfosalicylic acid. Absorbance was recorded at 450 nm. Each sample was analyzed in duplicate and subtracted from the respective blank (sample treated with 0.1N HCl). The protein level in samples was calculated using regression analysis. The obtained concentration was normalized with creatinuria (Creatinine assay kit – MAK 475, Sigma Aldrich). N-acetyl-β-D-glucosaminidase (NAG) activity (E-BC-K064-M - Elabscience®) was evaluated by following manufactures instructions. The NAG activity expressed as Unit/Liter (U/L) was calculated as following specified: NAG activity (U/L) = (∆A 400 - b) ÷ a ÷ T × f × 1000 (U/L), where ΔA 400 : The change OD value of sample well. (ΔA 400 = A sample - A control ). T: The time of reaction, 10 min. f: Dilution factor of sample before test. 1000: 1 mmol/L = 1000 µmol/L. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 10). Data normality was assessed using standard normality tests. Since normality was not satisfied for the evaluated parameters, differences among treatment groups were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Statistical significance was set at p < 0.05. Declarations Conflicts of Interest The authors declare no competing interests. Data Availability Data will be made available on reasonable request. Author Contributions M.C. and A.U. conceived the study. G.F., C.L., S.F., J.B., S.L., and M.B. curated the data. G.F. and M.B. performed the formal analysis. M.C. acquired funding. G.F., C.L., S.F., J.B., M.B., S.L., and R.F. conducted the investigations. M.C. and G.F. developed the methodology. M.C. administered the project and supervised the research. M.B. and M.C. validated the results. G.F., M.B., and M.C. prepared the visualizations. G.F., M.B., and M.C. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript. Funding The research was supported and funded by: #NEXTGENERATIONEU (NGEU); the Ministry of University and Research (MUR); the National Recovery and Resilience Plan (NRRP); project MNESYS (PE0000006) – A Multiscale integrated approach to the study of the nervous system in health and disease (DN. 1553 11.10.2022). Acknowledgments The authors gratefully acknowledge the MNESYS consortium, Spoke 2, for their support. 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Toxicology 229:123–135. https://doi.org/10.1016/j.tox.2006.10.008 Panizzutti R, Rausch M, Zurbrügg S et al (2005) The pharmacological stimulation of NMDA receptors via co-agonist site: An fMRI study in the rat brain. Neurosci Lett 380:111–115. https://doi.org/10.1016/j.neulet.2005.01.062 Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates Pinheiro BC, Pinheiro TN, Leite MGM et al (2024) Effect of ELVAX polymer subgingival implants with echistatin on extracted and reimplanted rats’ teeth. Odontology 112:112–124. https://doi.org/10.1007/s10266-023-00814-z Piubelli L, Pollegioni L, Rabattoni V et al (2021) Serum d-serine levels are altered in early phases of Alzheimer’s disease: towards a precocious biomarker. Transl Psychiatry 11. https://doi.org/10.1038/s41398-021-01202-3 Sasaki T, Kinoshita Y, Matsui S et al (2015) N-methyl-D-aspartate receptor coagonist D-serine suppresses intake of high-preference food. Am J Physiol Regul Integr Comp Physiol 309:561–575. https://doi.org/10.1152/ajpregu.00083.2015 Seckler JM, Lewis SJ (2020) Advances in D-amino acids in neurological research. Int J Mol Sci 21. https://doi.org/10.3390/ijms21197325 Serra M, Di Maio A, Bassareo V et al (2023) Perturbation of serine enantiomers homeostasis in the striatum of MPTP-lesioned monkeys and mice reflects the extent of dopaminergic midbrain degeneration. https://doi.org/10.1016/j.nbd.2023.106226 . Neurobiol Dis 184: Souza IN, de O, Roychaudhuri R, de Belleroche J, Mothet JP (2023) D-Amino acids: new clinical pathways for brain diseases. Trends Mol Med 29:1014–1028. https://doi.org/10.1016/j.molmed.2023.09.001 Suwandhi L, Hausmann S, Braun A et al (2018) Chronic D-serine supplementation impairs insulin secretion. Mol Metab 16:191–202. https://doi.org/10.1016/j.molmet.2018.07.002 Taniguchi K, Sawamura H, Ikeda Y et al (2022) D-Amino Acids as a Biomarker in Schizophrenia. https://doi.org/10.3390/diseases10010009 . Diseases 10 Tseng YS, Liao CH, Wu W, Bin, Ma MC (2021) N-methyl-D-aspartate receptor hyperfunction contributes to D-serine-mediated renal insufficiency. Am J Physiol Ren Physiol 320:F799–F813. https://doi.org/10.1152/ajprenal.00461.2020 Williams RE, Jacobsen M, Lock EA (2003) 1H NMR Pattern Recognition and 31P NMR Studies with D-Serine in Rat Urine and Kidney, Time- and Dose-Related Metabolic Effects. Chem Res Toxicol 16:1207–1216. https://doi.org/10.1021/tx030019q Yahyavi I, Carrillo F, Nuzzo T et al (2025) Differences in sex and genetic status affect the disruption of NMDAR-related amino acid homeostasis in Parkinson’s disease. medRxiv Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 03 May, 2026 Reviewers invited by journal 03 May, 2026 Editor assigned by journal 01 May, 2026 Submission checks completed at journal 29 Apr, 2026 First submitted to journal 28 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9551872","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":635991787,"identity":"71db6fa7-d921-4aa7-9d34-69bca263e9e4","order_by":0,"name":"Giorgia Ferniani","email":"","orcid":"","institution":"University of Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Giorgia","middleName":"","lastName":"Ferniani","suffix":""},{"id":635991790,"identity":"2e52952a-ebe0-4322-8c10-09c4cbc0bbf6","order_by":1,"name":"Camilla Lucotti","email":"","orcid":"","institution":"University of Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Camilla","middleName":"","lastName":"Lucotti","suffix":""},{"id":635991793,"identity":"69aa9fa1-a718-4cdc-be8b-1afaf820c45b","order_by":2,"name":"Silvia Fanni","email":"","orcid":"","institution":"University of Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Fanni","suffix":""},{"id":635991796,"identity":"38ac47b5-ad7f-40e4-81e4-412d23a35db8","order_by":3,"name":"Jessica Bratzu","email":"","orcid":"","institution":"University of Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Bratzu","suffix":""},{"id":635991799,"identity":"56a382ac-8860-43dd-a61b-a5482b25c359","order_by":4,"name":"Stefano Lucà","email":"","orcid":"","institution":"University of Campania \"Luigi Vanvitelli\"","correspondingAuthor":false,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Lucà","suffix":""},{"id":635991802,"identity":"d039b4be-4112-4e24-a495-ee03e1fec699","order_by":5,"name":"Renato Franco","email":"","orcid":"","institution":"University of Campania \"Luigi Vanvitelli\"","correspondingAuthor":false,"prefix":"","firstName":"Renato","middleName":"","lastName":"Franco","suffix":""},{"id":635991806,"identity":"736b484f-9f4a-42b0-a7f4-80d8a00309a5","order_by":6,"name":"Alessandro Usiello","email":"","orcid":"","institution":"Ceinge Biotecnologie Avanzate (Italy)","correspondingAuthor":false,"prefix":"","firstName":"Alessandro","middleName":"","lastName":"Usiello","suffix":""},{"id":635991811,"identity":"f700f7fd-1890-4902-8d37-2c34c7175649","order_by":7,"name":"Manolo Carta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYLCCBxCKEUhLgOgGwloSQAQbA7MBVEsjYT1QLWwSUD5+a3Tbzx58kFBhJ28+v/lZNe8OC3n+Bub2B/i0mJ3JSzZIOJNsOOcYm9lt3jMShjMOEHCY2YEcM4nEtgOMM9gYgFraJBIYCGo5/wasxX4GG/u3YpAWeYJabkBsSZzBxmPGDNJiQFjLG2OQX5JnsOUUS85tkzDceJixcQZ+h+UYPvhQYWc7g/n4xg9v2+rk5Y63P/iATwsWwEyi+lEwCkbBKBgFmAAA31BIKrMDz9YAAAAASUVORK5CYII=","orcid":"","institution":"University of Cagliari","correspondingAuthor":true,"prefix":"","firstName":"Manolo","middleName":"","lastName":"Carta","suffix":""},{"id":635991820,"identity":"7ae693a8-4347-48a0-b573-d06e17d58574","order_by":8,"name":"Margherita Borriello","email":"","orcid":"","institution":"University of Campania \"Luigi Vanvitelli\"","correspondingAuthor":false,"prefix":"","firstName":"Margherita","middleName":"","lastName":"Borriello","suffix":""}],"badges":[],"createdAt":"2026-04-28 09:38:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9551872/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9551872/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109076651,"identity":"0b39a396-484a-4a7d-9731-0b480abf633f","added_by":"auto","created_at":"2026-05-12 11:03:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51192,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental timeline.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/905e124ef992ed6f28758cc0.png"},{"id":109076653,"identity":"1a33aae8-3c6a-4180-b8dc-43d78b5b1657","added_by":"auto","created_at":"2026-05-12 11:04:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45118,"visible":true,"origin":"","legend":"\u003cp\u003eWater consumption of rats in study 1. Mean daily water intake (ml/day/rat) was estimated during the first week of treatment (A) and the second week of treatment (B). Animals received vehicle (Veh; saline); D-serine (200 mg/kg; D-ser), L-DOPA (LD), or their combination (D-ser + LD).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/43f614a60bd0dcd481789906.png"},{"id":109076660,"identity":"75fcd44a-d489-4f7e-8e28-5898be987b04","added_by":"auto","created_at":"2026-05-12 11:04:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":77150,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curve of rats in study 1. Body weight (g) was measured weekly. Data are presented as mean ± SEM for each treatment group at each time point. Veh: vehicle (saline); D-ser: D-serine; LD: L-DOPA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/37017d7f27a89393f4f70886.png"},{"id":109076966,"identity":"826e3b2a-8f40-4890-8148-34283ffcbe63","added_by":"auto","created_at":"2026-05-12 11:05:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1037892,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin-stained kidney sections from rats treated with: A) Vehicle; B) D-Serine; C) D-Serine + L-DOPA; D) L-DOPA (study 1). E) Inflammatory cell count performed by using ImageJ software. Data are presented as median (IQR). ****p \u0026lt; 0.0001. See Materials and Methods section for further details.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/e0c9d9135d963855fbe51511.png"},{"id":109076658,"identity":"6b63c944-93fc-4f74-a48f-0e7aa0a58570","added_by":"auto","created_at":"2026-05-12 11:04:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69799,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical urinary evaluation after chronic D-serine administration (study 1). A) Proteinuria/creatinuria ratio. B) N-acetyl-β-D-glucosaminidase (NAG) activity. Data are presented as median (IQR). No statistically significant differences were observed among groups. See Materials and Methods section for further details.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/722c7257e6118cb95e2aeb3c.png"},{"id":109076657,"identity":"fba615fa-c4bf-4f91-b0b4-6e8968f59d8d","added_by":"auto","created_at":"2026-05-12 11:04:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46547,"visible":true,"origin":"","legend":"\u003cp\u003eWater consumption of rats in study 2 during the first week of treatment. (A) Group A, in which animals received the D-serine dose assigned to their experimental group starting from the first administration. (B) Group B, in which animals were treated with the lower dose of D-serine during the first week before transitioning to the dose corresponding to each group. Data are expressed as mean daily water intake (ml/day/rat). Veh: vehicle (saline); D-ser: D-serine; LD: L-DOPA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/8f0786c94b8620da6ca0eaa6.png"},{"id":109076908,"identity":"d8926dc4-026b-4663-9e29-2600512e13ff","added_by":"auto","created_at":"2026-05-12 11:05:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":164072,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curves of rats in study 2. (A) Group A, in which rats received the D-serine dose assigned to their group starting from the first administration. (B) Group B, in which D-serine treatment started with the lower dose for the first week before transitioning to the dose corresponding to each group. Body weight (g) was measured weekly. Data are presented as mean ± SEM for each treatment group at each time point. Veh: vehicle (saline); D-ser: D-serine; LD: L-DOPA.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/dfed8247bfac248b82af08ac.png"},{"id":109076627,"identity":"ede5f86d-70c9-4fee-bd3b-940c2103e126","added_by":"auto","created_at":"2026-05-12 11:03:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1008920,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin-stained kidney sections from rats of group A treated with: A) Vehicle; B) D-serine 100 mg/kg; C) D-serine 100 mg/kg + L-DOPA; D) D-serine 200 mg/kg; E) D-serine 200 mg/kg + L-DOPA; F) L-DOPA, starting from the first administration (study 2). G) Inflammatory cell count performed using ImageJ software. Data are presented as median (IQR). **p \u0026lt; 0.01. See Materials and Methods section for further details.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/876829247f17d8efbb3256cd.png"},{"id":109076998,"identity":"0d7486e6-3b8f-44bb-ae15-db4107304419","added_by":"auto","created_at":"2026-05-12 11:06:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":986202,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative hematoxylin and eosin-stained kidney sections from rats of group B treated with: A) Vehicle; B) D-serine 100 mg/kg; C) D-serine 100 mg/kg + L-DOPA; D) D-serine 200 mg/kg; E) D-serine 200 mg/kg + L-DOPA; F) L-DOPA, starting with the lower dose of D-serine for the first week before transitioning to the dose corresponding to each group (study 2). G) Inflammatory cell count performed using ImageJ software. Data are presented as median (IQR). No statistically significant differences were observed among groups. See Materials and Methods section for further details.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/b93e92c768cd69367604391f.png"},{"id":109076647,"identity":"c4a2c35e-31ea-403a-872b-5d83a5dd40d9","added_by":"auto","created_at":"2026-05-12 11:03:59","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":108965,"visible":true,"origin":"","legend":"\u003cp\u003eBiochemical urinary evaluation after chronic D-serine administration. A) Proteinuria/creatinuria ratio – group A. B) NAG activity – group A. C) Proteinuria/creatinuria ratio – group B. D) NAG activity – group B. Data are presented as median (IQR). No statistically significant differences were observed among groups. See Materials and Methods section for further details.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/bf3e32e3687a3e128b9cd2eb.png"},{"id":109081446,"identity":"3e31ff71-8ce1-48ca-aef3-7da6cda6d5d5","added_by":"auto","created_at":"2026-05-12 12:18:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3899625,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9551872/v1/d616e2d3-aa35-4974-8ab9-97aebf6c8d33.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Renal Effects of Chronic D-Serine Administration in a Parkinson’s Disease Rat Model: Evidence for a Threshold Nephrotoxic Dose","fulltext":[{"header":"Introduction","content":"\u003cp\u003eD-amino acids are emerging as interesting molecules in the context of neurodegenerative and psychiatric disorders, either as therapeutic agents or as biomarkers (Madeira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Seckler and Lewis \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nuzzo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Piubelli et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Taniguchi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this context, D-serine, an endogenous co-agonist at the glycine binding site of NMDA receptor NR1 subunits, has attracted particular attention as its levels have been found altered in animal models of Parkinson\u0026rsquo;s disease (PD) as well as in patients (Di Maio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Serra et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Intense research is currently ongoing to understand whether these alterations, found at both central and peripheral levels, play a role in the neurodegenerative process and/or represent a specific signature of the disease (Imarisio et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Yahyavi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To this aim, animal models of PD are instrumental as they provide the unique opportunity to investigate, in controlled conditions, the consequence of the neurodegenerative event affecting the nigrostriatal dopaminergic system on peripheral and central D-serine levels, as well as the impact of its exogenous administration. Among different models, the rat model based on overexpression of human α-synuclein (α-syn) via an adeno-associated viral vector represents a validated and extensively used tool in this field of research. Indeed, this model reproduces cardinal features of the disease, such as the formation of cellular and axonal α-syn rich inclusions, the slowly progressive neurodegenerative process, and the appearance of related motor defects (Decressac et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Based on this rationale, we designed a study in which animals overexpressing α-syn received daily D-serine treatment, alone or in combination with L-DOPA, the most used antiparkinsonian drug, for eight weeks, before being sacrificed for \u003cem\u003eex-vivo\u003c/em\u003e analyses. Whereas the primary objective of the experimental design was to investigate the central effects of these treatments, which are the subjects of a separate study, kidneys were also collected to assess potential nephrotoxic effects associated with prolonged D-serine administration, which are reported here. In fact, previous studies have highlighted an acute nephrotoxic effect of D-serine, specifically in rats, but not in mice. Thus, while mice have been treated with doses up to 1 g/kg, with no adverse effects on renal function, histological and biochemical changes have emerged in rats after a single administration of D-serine at a dose of 200 mg/kg or higher (Ganote et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Maekawa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Orozco-Ibarra et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hasegawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meftah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The potential risk of D-serine-induced nephrotoxicity in rats has been described since the 1940s (Morehead R et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1945\u003c/span\u003e). In particular, acute pathological events at kidney level induced by D-serine are tubular structural changes, serum creatinine increase, glycosuria, proteinuria, and presence of urinary N-acetyl-β-D-glucosaminidase (NAG) activity, indicating tubular damage (Ganote et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Delraso et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hasegawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meftah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The most marked perturbations are observed between 8\u0026ndash;24 h postdosing (Williams et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and, despite these acute pathological events, D-serine-induced nephrotoxicity appears to be fully reversible (Ganote et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Meftah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Specifically, after acute kidney damage, urine values of proteins, glucose, and amino acids begin to normalize 24\u0026ndash;48 h after the last dose of D-serine and by 120 h post-dose largely return to normal. Pathological changes also completely resolve within this timeframe, with complete regrowth of new epithelium in tubules and renal tubular basophilia (Williams et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite this evidence, no single study has focused on the consequences of chronic daily administration of D-serine. This report is therefore meant to cover this gap and guide the choice of the D-serine dose in future rat studies where this D-amino acid is administered chronically, both in physiological and pathological conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eSprague-Dawley male rats were subjected to stereotaxic surgery for the nigral delivery of an adeno-associated viral vector encoding the human α-syn gene. Three days after surgery, animals were allocated into different subgroups in order to receive D-serine, either alone or in combination with L-DOPA, L-DOPA alone, or vehicle, as a control group. Treatments were carried out daily for eight weeks. At the end of the treatment period, one-spot urine samples were collected from the animals. Thereafter, animals were sacrificed by transcardiac perfusion with paraformaldehyde (PFA) to collect central and peripheral tissues, including the kidneys (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for the experimental timeline).\u003c/p\u003e \u003cp\u003eA first study (n\u0026thinsp;=\u0026thinsp;40) was conducted using a D-serine dose of 200 mg/kg (study 1). In light of the results, a subsequent study (n\u0026thinsp;=\u0026thinsp;60) was performed employing two D-serine doses, 100 and\u003c/p\u003e \u003cp\u003e200 mg/kg (study 2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWater consumption and body weight, study 1\u003c/h3\u003e\n\u003cp\u003eWater consumption was estimated weekly based on the scale of the drinking bottles, and body weight was measured at the same time points. In the first study, D-serine treatment, with or without L-DOPA, produced a marked increase in water intake during the first week, with D-serine-treated rats consuming approximately twice as much as control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, a moderate reduction in body weight was observed in the same period (on average 30 g, corresponding to about 10% of the initial body weight; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, water consumption normalized by the second week of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and animals began to gain weight similarly to controls during the same week. Nevertheless, a slight but persistent difference in body weight between D-serine-treated animals and the control group remained throughout the study. Specifically, animals receiving D-serine weighed, on average, approximately 10 g less than controls from week 2 until week 8, before being sacrificed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHistological observations and urinalysis, study 1\u003c/h3\u003e\n\u003cp\u003ePerfused kidneys were fixed in formalin and subsequently sampled, processed, and embedded in paraffin for histopathological evaluation. Morphological alterations of the renal parenchyma, including tubular epithelial attenuation, cytoplasmic hypereosinophilia, cytoplasmic blebbing, the presence of cellular debris, fibrin, and/or necrosis, chronic peritubular inflammatory infiltrates, tubular thyroidization, and fibrotic changes, were systematically assessed. The main histopathological alteration found was a mild to moderate chronic peritubular inflammatory infiltrate, only in the rat groups treated with D-serine, both in the presence and in the absence of L-DOPA. No significant damage to the glomeruli or renal tubules was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D).\u003c/p\u003e \u003cp\u003eInflammatory cell count revealed a significant increase in the inflammatory cells only in rats treated with D-serine, both in the presence and in the absence of L-DOPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThese kinds of tubular injury overlap with those observed for other nephrotoxins (Kim and Moon \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Meftah et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hall et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As mentioned in the introduction, D-serine nephrotoxicity is associated with proteinuria and urinary NAG activity. For this reason, we also evaluated these markers of kidney injury in rats\u0026rsquo; urines. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, no significant differences emerged among the analyzed groups for both proteinuria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and NAG activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe overall results obtained from this experimental set led us to hypothesize that 200 mg/kg could represent a threshold dose for chronic D-serine administration in rats. Indeed, both the observed biological alterations (i.e., growth evaluation and water consumption) and the presence of peritubular inflammatory infiltrate suggest that the chronic administration of D-serine induces effects comparable to those reported after acute administration (Ganote et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Delraso et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hasegawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eWater consumption and body weight, study 2\u003c/h3\u003e\n\u003cp\u003eConsidering these results, a second study was performed using two different D-serine doses, 100 and 200 mg/kg, administered alone or in combination with L-DOPA.\u003c/p\u003e \u003cp\u003eRats were split into two subgroups: in group A, rats meant to receive the higher D-serine dose, received 200 mg/kg from the first administration; in group B, rats meant to receive the 200 mg/kg dose, were initially given 100 mg/kg for the first 7 days; thereafter, the dose was increased to 200 mg/kg. Administration of 200 mg/kg, but not 100 mg/kg, of D-serine led to increased water consumption during the first week in group A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). By contrast, starting the treatment with the lower dose, as done in group B, did not result in increased water consumption upon dose escalation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Nevertheless, a reduced body weight was observed both in group A and B for all D-serine-treated rats also throughout study 2. Compared with controls, D-serine-treated animals weighed, on average, approximately 30 g less (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) from week 2 until sacrifice at week 8. This represents a more pronounced difference than what was observed for study 1; the smaller size of the rats subjected to D-serine treatment in this second study compared to the first one may account for this discrepancy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHistological observations and urinalysis, study 2\u003c/h3\u003e\n\u003cp\u003ePerfused kidneys were processed as for study 1. Microscopic examination showed that 200 mg/kg of D-serine, both alone and in combination with L-DOPA, induced morphological alterations of the renal parenchyma, confirming what was seen in the first group of animals. Also here, the main histopathological alteration was a mild to moderate chronic peritubular inflammatory infiltrate, without significant damage to the glomeruli or renal tubules, in D-serine 200 mg/kg treated rats (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD and E) compared to the vehicle group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) in group A. Importantly, no detrimental effects were observed with D-serine 100 mg/kg (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and C). Inflammatory cell evaluation fully overlapped with the histopathological results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eInterestingly enough, when adopting the escalation design, as for group B, the inflammation was almost undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e), and no significant differences in the inflammatory cell count were seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eFrom the biochemical point of view, urine analysis showed again no significant differences among the experimental groups, for the analyzed parameters, suggesting that the mild morphological alterations had no functional impact (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we investigated the long-term effects of chronic D-serine administration, either alone or in combination with L-DOPA, on renal morphology and function in a PD rat model. Our findings demonstrate that daily administration of D-serine for eight weeks at a dose of 200 mg/kg, but not 100 mg/kg, induced early signs of nephrotoxicity. These alterations were mainly characterized by changes in the renal parenchyma, including a clear chronic peritubular inflammatory infiltrate, in the absence of glomerular structural damage and without detectable alterations in conventional urinary biomarkers of kidney injury.\u003c/p\u003e \u003cp\u003eQuantitative analysis of inflammatory cells confirmed a significant increase exclusively in the D-serine 200 mg/kg\u0026ndash;treated groups, indicating that D-serine is the primary driver of the observed inflammatory response. Importantly, these effects were independent of L-DOPA co-administration, as similar outcomes were seen whether D-serine was administered alone or in combination with L-DOPA, suggesting that L-DOPA does not affect D-serine\u0026ndash;induced renal effects under the conditions tested.\u003c/p\u003e \u003cp\u003eThis study was originally designed to investigate the central effects of D-serine as an add-on therapy to L-DOPA, which is the most effective drug in treating motor symptoms in PD. Thus, this design allowed us to also demonstrate that L-DOPA chronic administration, at a dose commonly used in animal models, is devoid of significant detrimental effects on kidneys.\u003c/p\u003e \u003cp\u003eAn additional relevant observation was the transient increase in water consumption observed at the beginning of treatment with the higher D-serine dose (200 mg/kg), and the slightly reduced body weight at both doses, which, conversely, persisted throughout the study. These abnormalities prompted us to collect the kidneys at the end of the experiments and perform the present investigations. In fact, increased water intake is often an indirect marker of impaired renal function and may reflect alterations in urine concentrating ability, hormonal regulation, or waste dilution mechanisms (Hopp et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Choi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Interestingly, the abnormal water consumption normalized after the first week of treatment and was not observed when D-serine was initiated at the lower dose and subsequently escalated to the higher one after the first week (study 2, group B). Notably, this experimental design also prevented inflammation, seen, by contrast, when the 200 mg/kg D-serine dose was administered from the first treatment (study 1 and study 2, group A). This observation may suggest the existence of some adaptive mechanisms to the exogenous D-serine administration in our rat model, which appear to take place after a few drug injections. Although the molecular basis of this event remains unknown, potential mechanisms include the upregulation of enzymes involved in D-serine metabolism, such as D-amino acid oxidase (DAAO), or the downregulation of renal NMDA receptors.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that acute D-serine nephrotoxicity is characterized by proximal tubular damage accompanied by functional alterations, including proteinuria and increased urinary NAG activity (Delraso et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hasegawa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). By contrast, in the present chronic model, neither proteinuria nor NAG activity differed significantly among experimental groups. Differences in exposure duration may also explain this discrepancy, as chronic administration may allow the induction of adaptive or compensatory mechanisms that limit overt tubular dysfunction despite the presence of slight histological alterations.\u003c/p\u003e \u003cp\u003eThe mechanisms underlying D-serine nephrotoxicity in rats are not fully understood but appear to involve species-specific differences in renal handling and metabolism of D-serine. Rats exhibit a higher degree of renal reabsorption of D-serine compared with other species, as reflected by low urinary D-serine levels, despite serum concentrations comparable to those observed in humans and dogs (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Increased tubular reabsorption leads to elevated local concentrations of D-serine, which is metabolized by DAAO; DAAO activity is associated with the generation of reactive oxygen species (ROS) that, in turn, may drive nephrotoxic events (Maekawa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Additionally, it has been proposed that rats possess a greater capacity for utilizing D-amino acids and that renal NMDA receptors may be directly involved in mediating D-serine\u0026ndash;induced nephrotoxicity (Tseng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegardless of the specific mechanisms involved, our data suggest that chronic D-serine administration in rats does not exacerbate renal injury over time, even when particularly prolonged.\u003c/p\u003e \u003cp\u003eIt is worth noting that, compared with rats, humans show lower renal tubular reabsorption and higher urinary excretion of D-serine, resulting in reduced intrarenal accumulation (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In fact, across all published human studies on D-serine, only one subject has been reported to exhibit abnormal renal values related to D-serine treatment (Kantrowitz et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Despite human D-serine administration appears safe, the plasma levels are increased in chronic kidney disease, especially at advanced stages, making D-serine a possible \u0026ldquo;sensor\u0026rdquo; of the kidney healthy status as well as a possible uremic toxin (Okada et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hesaka et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kimura et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA reduced body weight was observed upon D-serine treatment throughout the study and should also be considered when designing future rat studies. However, this effect is unlikely to be related to nephrotoxicity, as it was also present at the lower dose, which was not associated with any morphological or functional renal alterations in our analyses. Whereas this issue goes beyond the aim of this study, it is worth mentioning that previous studies have shown that chronic D-serine administration can reduce body weight also in mice, possibly by reducing food consumption and/or altering insulin secretion (Sasaki et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Suwandhi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo our knowledge, this study represents the first systematic evaluation of the renal effects of chronic daily D-serine administration in rats. While mice are preferentially used for the apparent lack of D-serine nephrotoxicity, even at doses up to 1 g/kg, rats remain a valuable experimental model in certain pathological contexts, including PD, where the role of D-serine is increasingly recognized in both preclinical and clinical studies (Gelfin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Di Maio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Serra et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, this study is relevant to better define its long-term toxicity in this species. Our data suggest that daily doses lower than 200 mg/kg are advisable for chronic D-serine administration and that a dose-escalation approach could be used to prevent adverse events, such as abnormal water consumption and early signs of morphological alterations.\u003c/p\u003e \u003cp\u003eAlthough this study was conducted in a rat model of PD, it is important to note that the viral vector encoding human α-syn was injected unilaterally into the substantia nigra, resulting in a model of hemi-parkinsonism characterized by moderate dopaminergic degeneration and minimal motor impairment. Therefore, no significant peripheral alterations are expected to take place in consequence of this procedure and alter the response to D-serine in the kidneys. For this reason, we believe that the findings of this study, although obtained in a pathological model, can extend to physiological conditions and be a reference for any rat study concerning chronic D-serine administration.\u003c/p\u003e \u003cp\u003eImportantly, our parallel investigation on the central effects of this D-amino acid shows that these doses are capable of inducing measurable effects in the CNS, such as increased synaptic plasticity (manuscript in preparation). On the other hand, a previous rat NMR study showed that a D-serine dose as low as 50 mg/kg can activate specific brain regions, further suggesting that even relatively low doses are effective in rats (Panizzutti et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, by defining a dose range devoid of nephrotoxicity, this study covers a gap regarding the effect of chronic D-serine administration in rats, thus providing a useful reference for future studies investigating the central or peripheral effects of this D-amino acid.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale Sprague-Dawley rats (275\u0026ndash;300 g; Envigo, Italy) were housed in groups of 3\u0026ndash;4 per cage under a 12 h light/dark cycle in a temperature- and humidity-controlled environment, with \u003cem\u003ead libitum\u003c/em\u003e access to food and water.\u003c/p\u003e \u003cp\u003e All animal procedures were conducted in accordance with European Directive 2010/63/EU and Italian Legislative Decree no. 26/2014. The present study was approved by the local Ethical Committee (OPBA) and the Italian Ministry of Health (authorization no. 779/2024). The authors complied with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimal model\u003c/h2\u003e \u003cp\u003eAnimals underwent stereotaxic surgery for the unilateral nigral infusion of the adeno-associated virus (AAV) vector expressing a human wild-type α-syn, as previously described (Decressac et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll surgical procedures were performed under general anesthesia induced by intraperitoneal injection of a 20:1 mixture of fentanyl (Fentadon, Dechra) and medetomidine hydrochloride (Domitor, Orion Pharma) at a dose of 6 mL/kg. Rats were allocated in a stereotaxic frame (2Biological Instruments), and vector solution was delivered using a 10 \u0026micro;l Hamilton syringe fitted with a glass capillary to minimize mechanical tissue damage.\u003c/p\u003e \u003cp\u003eEach animal received a total volume of 4 \u0026micro;L of AAV-α-syn solution, split into 2 different sites and infused at a rate of 0.5 \u0026micro;L/min. The capillary was kept in place for an additional 3 min per site before being slowly retracted.\u003c/p\u003e \u003cp\u003eThe AAV solution was injected into the right Substantia Nigra \u003cem\u003epars compacta\u003c/em\u003e at the following coordinates (flat skull position): 1) antero-posterior (AP): \u0026minus;5.3 mm (from bregma), medio-lateral (ML): \u0026minus;1.6 mm, dorso-ventral (DV): \u0026minus;7.2 mm (from dura surface); 2) AP: \u0026minus;5.3 mm, ML: \u0026minus;2.6 mm, DV: \u0026minus;6.7 mm, according to the stereotaxic atlas of Paxinos and Watson (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) (Bj\u0026ouml;rklund et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnesthesia awakening was promoted by subcutaneous administration of atipamezole hydrochloride (Revertor, Virbac). After the surgery, animals were placed in clean cages and monitored daily until full recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDrugs formulations and administration\u003c/h2\u003e \u003cp\u003eL-DOPA as the methyl ester hydrochloride and D-serine (both from Biosynth s.r.o., Bratislava, SK) were dissolved in 0,9% NaCl before use and administered once daily. L-DOPA (12 mg/kg), in association with benserazide (6 mg/kg), was injected subcutaneously (s.c.), whereas D-serine (100 or 200 mg/kg, depending on experimental group) was administered intraperitoneally (i.p.). Control animals received 0,9% NaCl (vehicle) via both subcutaneous and intraperitoneal routes.\u003c/p\u003e \u003cp\u003eAnimals were randomly assigned to the different treatment groups. Study 1 consisted of 4 groups (10 rats/group), daily treated with: 1) vehicle s.c. + vehicle i.p., 2) L-DOPA s.c. + vehicle i.p., 3) vehicle s.c. + D-serine (200 mg/kg) i.p., 4) L-DOPA s.c. + D-serine (200 mg/kg) i.p.\u003c/p\u003e \u003cp\u003eStudy 2 consisted of 6 groups (10 rats/group): 1\u0026ndash;4) as in study 1, 5) vehicle s.c. + D-serine (100 mg/kg) i.p., 6) L-DOPA s.c. + D-serine (100 mg/kg) i.p.\u003c/p\u003e \u003cp\u003eAnimals in study 2 were split into two subgroups: group A, in which rats meant to receive the higher D-serine dose, received 200 mg/kg from the first administration; group B, where rats meant to receive the 200 mg/kg dose, were initially given 100 mg/kg for the first 7 days before escalating to 200 mg/kg.\u003c/p\u003e \u003cp\u003eAll treatments were administered starting from 3 days after surgery and continued daily for 8 weeks until sacrifice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eUrine collection\u003c/h2\u003e \u003cp\u003eFor urine collection, animals were individually housed in single cages during the eighth week of treatment. The last treatment was administered 24 h prior to urine sampling. One-spot urine samples were collected using a micropipette, transferred into Eppendorf tubes, and stored at -80\u0026deg;C until biochemical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSacrifice\u003c/h2\u003e \u003cp\u003eUnder anesthesia, animals were sacrificed 24 h after the last D-serine administration by transcardiac perfusion with saline (0.9% NaCl), followed by ice-cold 4% PFA. After perfusion, kidneys were collected into Falcon tubes containing 4% PFA and stored at 4\u0026deg;C until histopathological analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eFor light microscopy, kidney tissue samples were fixed by immersion in buffered formalin (pH 7.4) and embedded in paraffin. For histological analysis, 5-\u0026micro;m thickness sections were produced from paraffin blocks and stained with hematoxylin and eosin. Slides were observed under an Olympus optical microscope equipped with a digital imaging camera.\u003c/p\u003e \u003cp\u003eHistological analyses were performed by investigators blinded to treatment allocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory Cells Counting\u003c/h2\u003e \u003cp\u003eCell counts were performed for quantitative assessment of inflammatory intensity. Representative slides were photographed using an Olympus optical microscope equipped with a digital imaging camera. Ten photographs/slide were taken at 20 \u0026times; magnification. Digital analysis of the images was performed using the public domain ImageJ 1.44p software (National Institutes of Health, USA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://imagej.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://imagej.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The manual cell counting tool, \u0026ldquo;cell counter\u0026rdquo;, was chosen (Pinheiro et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Measurements\u003c/h2\u003e \u003cp\u003eUrine samples were centrifuged at 10,000 rpm for 3 minutes, and supernatant was used for the subsequent analysis. Urinary proteins were quantified by using Rat Urinary Protein Assay Kit # 9040 (Condrex, Inc.), following manufactures instructions. Briefly, urine was diluted 1:2 with the provided dilution buffer, and the turbidity assay was conducted by adding 3% Sulfosalicylic acid. Absorbance was recorded at 450 nm. Each sample was analyzed in duplicate and subtracted from the respective blank (sample treated with 0.1N HCl). The protein level in samples was calculated using regression analysis. The obtained concentration was normalized with creatinuria (Creatinine assay kit \u0026ndash; MAK 475, Sigma Aldrich). N-acetyl-β-D-glucosaminidase (NAG) activity (E-BC-K064-M - Elabscience\u0026reg;) was evaluated by following manufactures instructions. The NAG activity expressed as Unit/Liter (U/L) was calculated as following specified: NAG activity (U/L) = (∆A\u003csub\u003e400\u003c/sub\u003e - b)\u0026thinsp;\u0026divide;\u0026thinsp;a\u0026thinsp;\u0026divide;\u0026thinsp;T \u0026times; f \u0026times; 1000 (U/L), where\u003c/p\u003e \u003cp\u003eΔA\u003csub\u003e400\u003c/sub\u003e: The change OD value of sample well. (ΔA\u003csub\u003e400\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;A\u003csub\u003esample\u003c/sub\u003e - A\u003csub\u003econtrol\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eT: The time of reaction, 10 min.\u003c/p\u003e \u003cp\u003ef: Dilution factor of sample before test.\u003c/p\u003e \u003cp\u003e1000: 1 mmol/L\u0026thinsp;=\u0026thinsp;1000 \u0026micro;mol/L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 10). Data normality was assessed using standard normality tests. Since normality was not satisfied for the evaluated parameters, differences among treatment groups were analyzed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparisons test. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.C. and A.U. conceived the study. G.F., C.L., S.F., J.B., S.L., and M.B. curated the data. G.F. and M.B. performed the formal analysis. M.C. acquired funding. G.F., C.L., S.F., J.B., M.B., S.L., and R.F. conducted the investigations. M.C. and G.F. developed the methodology. M.C. administered the project and supervised the research. M.B. and M.C. validated the results. G.F., M.B., and M.C. prepared the visualizations. G.F., M.B., and M.C. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was\u0026nbsp;supported and funded by: #NEXTGENERATIONEU (NGEU); the Ministry of University and Research (MUR); the National Recovery and Resilience Plan\u003cbr\u003e(NRRP); project MNESYS (PE0000006) \u0026ndash; A Multiscale integrated\u003cbr\u003eapproach to the study of the nervous system in health and disease (DN.\u003cbr\u003e1553 11.10.2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the MNESYS consortium, Spoke 2, for their support.\u003c/p\u003e\n\u003cp\u003eThe authors also thank all personnel of CeSASt (\u003cem\u003eCentro Servizi di Ateneo per gli Stabulari\u003c/em\u003e) for their skillful assistance in animal care.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBj\u0026ouml;rklund A, Nilsson F, Mattsson B et al (2022) A Combined α-Synuclein/Fibril (SynFib) Model of Parkinson-Like Synucleinopathy Targeting the Nigrostriatal Dopamine System. 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Using a rat model based on α-synuclein overexpression, we evaluated renal effects after eight weeks of daily D-serine administration, alone or in combination with L-DOPA. Given the known acute nephrotoxicity of D-serine in rats, this study addresses the lack of data on chronic exposure. Our results show that a dose of 200 mg/kg induces biological alterations - reflected in growth patterns and water consumption - and peritubular inflammatory infiltrates comparable to the nephrotoxic effect reported after acute treatment. These findings suggest that 200 mg/kg could represent a threshold dose for chronic D-serine administration in rats, providing a useful reference for future studies involving prolonged treatment.\u003c/p\u003e","manuscriptTitle":"Renal Effects of Chronic D-Serine Administration in a Parkinson’s Disease Rat Model: Evidence for a Threshold Nephrotoxic Dose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-12 10:46:32","doi":"10.21203/rs.3.rs-9551872/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-10T16:10:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334912765165328575847768645963802565617","date":"2026-05-07T07:42:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132514187279860725899137850563085132854","date":"2026-05-03T20:40:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-03T18:33:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T17:53:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T17:44:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Amino Acids","date":"2026-04-28T09:20:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amino-acids","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amac","sideBox":"Learn more about [Amino Acids](http://link.springer.com/journal/726)","snPcode":"726","submissionUrl":"https://submission.nature.com/new-submission/726/3","title":"Amino Acids","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b7f570cc-f025-4d96-8d3a-bb84f69dee77","owner":[],"postedDate":"May 12th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-10T16:10:47+00:00","index":10,"fulltext":""},{"type":"reviewerAgreed","content":"334912765165328575847768645963802565617","date":"2026-05-07T07:42:55+00:00","index":9,"fulltext":""},{"type":"reviewerAgreed","content":"132514187279860725899137850563085132854","date":"2026-05-03T20:40:10+00:00","index":7,"fulltext":""},{"type":"reviewersInvited","content":"3","date":"2026-05-03T18:33:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-01T17:53:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T17:44:15+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T10:46:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-12 10:46:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9551872","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9551872","identity":"rs-9551872","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}