Quantitative Reduction of Gut Microbiota-Derived Short-Chain Fatty Acids in Stool and Serum in Diabetic Kidney Disease

Objectives Previous studies found the dysbiosis of intestinal microbiota in individuals with diabetic kidney disease (DKD),especially the decreased SCFA-producing bacteria. We aimed to investigate stool and serum short-chain fatty acids (SCFAs), gut microbiota-derived metabolites, in individuals with DKD and the correlations. Methods A total of 30 participants with DKD, 30 participants with type 2 diabetes mellitus (DM) and 30 normal controls (NC) in HwaMei Hospital were recruited from 1/1/2018 to 12/31/2019. Participants with DKD were divided into low estimated glomerular ltration rate (eGFR) (eGFR<60ml/min, n=14) and high eGFR (eGFR ≥ 60ml/min, n=16) subgroups. Stool and serum were measured for SCFAs with gas chromatograph-mass spectrometry.


Introduction
Diabetic kidney disease (DKD) is the most serious complication of diabetic mellitus (DM) and the leading cause of chronic kidney disease (CKD) in the world. A recent study indicated that the prevalence of DM in China was 11.2% (95% con dence interval 10.5% to 11.9%), especially in Han ethnicity [1]. About 35% of patients with type 2 DM (T2DM) would eventually develop DKD, with an increased mortality [2], but the etiology of diabetic kidney disease is yet still unclear.
Recent studies highlighted the involvement of gut microbiome-kidney axis in nephropathy [3,4]. Tao et al. demonstrated that gut microbiota was associated with the development of DKD, and the individuals with DKD could be accurately screened out by testing g_Escherichia-Shigella and g_ Prevotella_9 among the individuals with diabetes [5]. Another study showed that fecal microbiota transplantation could reverse intestinal microbiota dysbiosis and improve renal function in rats with DKD [6]. These suggested that gut microbiota dysbiosis may play an important role in the pathogenesis of DKD.
Besides, studies also indicated that gut microbiota and kidney were interacted via microbiome-kidney axis, which also participated in kidney injury process. Being one of the major metabolites of microbiota-mediated ber fermentation process in the gut, short-chain fatty acids (SCFAs) have attracted considerable interest. SCFAs are a subset of fatty acids that contain 6 or less carbon molecules and have showed bene cial effects on kidney [4,7]. SCFAs played a role in biological modulation by attenuating the in ammatory response and reduce mean arterial pressure, via inhibiting histone deacetylases (HDACs) and activating G protein receptor 41(GPR41), GPR43, GPR109a and Olfr78 [8,9]. However, SCFAs presented markedly varied concentrations in different diseases [10,11]. The change of fecal and serum SCFAs levels in DKD remains unclear.
In this study, all 90 participants were included from HwaMei Hospital. Fecal and serum samples were measured for SCFAs with gas chromatograph-mass spectrometry (GC-MS). We reported the substantial variations in the levels of fecal and serum SCFAs among normal controls, participants with diabetes and participants with DKD. SCFAs levels in participants with diabetic kidney disease were further analyzed within subgroups by renal function.

Participants
There were 30 participants with DKD, 30 participants with type 2 diabetes, and 30 normal controls included in HwaMei Hospital, University of Chinese Academy of Science from 1 January 2018 to 31 December 2019. The diagnosis of T2DM were de ned by the criterion issued by American Diabetes Association (ADA) in 2017 [12]. Diabetic kidney disease can be diagnosed when patients with type 2 diabetes meet any of the following situation: 1)microalbuminuria with an ACR between 30-300mg/g; 2)macroalbuminuria with an ACR>300mg/g. 3) 2 of 3 samples should fall within the microalbuminuric or macroalbuminuric range to con rm clsaai cation in the absence of urinary tract infection with 2 additional rst-void specimens collected over the next 3 to 6 months. [13]. All participants underwent a medical history screening, a physical examination and body mass index (BMI) was calculated. Lab tests were complete blood count and metabolic panel including albumin, fasting glucose, lipid pro le, renal function and urinary albumin creatinine ratio (UACR) . Estimated glomerular ltration rate (eGFR) was calculated with the CKD-EPI Scr formula.
Participants in NC group from physical examination center were given tests including metabolic panel, urinalysis, stool test, HBsAg (Hepatitis B surface antigen ) and anti-HCV( Hepatitis C antibody ). Exclusions include: receiving antibiotics or probiotics within 2 months, gastro-intestinal or systemic diseases known to affect gut bacterial composition, primary or other secondary kidney disease, obesity, liver cirrhosis with/without complications, nonalcoholic fatty liver disease, HBsAg or anti-HCV positive. The clinical parameters are shown in Table 1 Fecal and serum sample collection Fresh fecal samples were collected and a portion of 200mg was utilized for each test. Blood samples were collected in the fasting status and serum was obtained by centrifugation at 3,500rpm for 5min at 4℃. These samples were then stored at -80°C until usage. One fecal sample and one serum sample in DKD group were later found not usable and were excluded in the study. 30 serum samples in NC group were not collected from physical examination center. Hence, 30 fecal samples in NC group, 30 fecal and serum samples in the diabetes group, 29 fecal and serum samples in the group with DKD were used for data determination.
Fecal and serum sample processing Each fecal sample of 200mg was mixed with 0.8mL of ultrapure water, crushed with a tissue grinder and then centrifuged at 12,000 rpm for 20 min at 4℃. Each 0.4ml supernatant was mixed with 0.1mL of 50% sulfuric acid (ultrapure water diluted), 0.5ml of ether(containing 50ug /mL of internal standard dimethylvaleric acid) for 1 min, centrifuged at 12,000 rpm for 20 min at 4℃, and then stood for 30 min at 4℃. The supernatant ether layer was ltered through anhydrous sodium sulfate for GC-MS analysis.
Each serum sample (100μL) was mixed with 50μL of 50% sulfuric acid (ultrapure water diluted), 200μL of ether (containing standard dimethylvaleric acid) for 1 min, centrifuged at 12,000 rpm for 20 min at 4℃, and then stood for 30 min at 4℃. The supernatant ether layer was ltered through anhydrous sodium sulfate and the solution later transferred to a glass vial for GC-MS analysis.
Determination of SCFAs using gas chromatograph-mass spectrometry (GC-MS) The analysis was performed using the GC-MS 7890A-5975C (Agilent Technology, USA

The correlations between SCFAs and the biochemical indicators
Correlations between the fecal SCFAs and clinical indicators were estimated by Spearman's correlation analysis ( Figure 3). As expected, an inverse relationship was observed between blood urea nitrogen and fecal acetate, propionate and butyrate levels(r=-0. 22 We further investigated the correlations between serum SCFAs and biochemical indicators ( Figure 4). Unexpectedly, no statistical correlations were found between renal function markers and serum SCFAs, except for a negative correlation between age and acetate level (r=-0. 25 and BMI between the two groups were matched with no statistical difference (P>0.05). UACR, serum creatinine and blood urea nitrogen were statistical higher (P<0.05) in the low GFR subgroup compared with high GFR subgroup.
There were no differences in fecal SCFAs between the two subgroups (P>0.05). As shown in Supplementary table 2, serum acetate and total SCFAs were lower and with borderline signi cant in low GFR subgroup versus high GFR subgroup (P=0.055, P=0.050, respectively). However, other SCFAs had no difference between these two subgroups (P>0.05).

Discussion
We are the rst to investigate fecal and serum SCFAs simultaneously in individuals with DKD. In this study, fecal acetate, propionate, butyrate and total SCFAs were markedly lower in DKD group. Serum acetate and total SCFAs were also found lower in low eGFR subgroup. Furthermore, fecal and serum acetate seem to be respectively correlated with eGFR in DKD patients. Besides, serum total SCFAs seems to be an independent factor for renal function.
SCFAs are end products of bacterial carbohydrate fermentation, and function as an important energy source and signaling molecules [14]. The concentration of SCFAs varies among different diseases. In DKD mice, there was a signi cantly decreasing in propionic acid and butyric acid contents in DKD progression [15]. The study conducted by Wang et al showed that fecal SCFAs declined in CKD patients, and negatively correlated with the renal function [16]. It was consistent with our study that SCFAs, mainly acetate, propionate and butyrate levels were evidently lower in DKD patients compared to DM and NC groups.
The gut microbiota, yielding SCFAs as the major products, was also believed to involve with DKD. Studies have clearly outlined the changes in microbiota in DKD patients [5,17], that the richness of gut microbiota and the variation of bacteria population were found different in DKD compared to DM [5] and SCFAs-producing bacteria Prevotella declining in DKD patients [5]. We speculated that this reduction of SCFAs-producing bacteria was accompanied by the decrease of yielding SCFAs. Maybe this was the result of the lowest fecal SCFAs levels in DKD.Besides, it may be related with diet, as patients with kidney diseases are required to avoid the intake of fruits, vegetables, and high-ber diets [16]. So it is warranted to analysis the composition and construction of gut microbiota in newly diagnosed DKD patients in future.
Despite the nding of fecal SCFAs changes, there has not been a de ned study on the subsequent serum SCFAs in DKD patients. Our study revealed that the serum acetate was lower in low GFR subgroup than high GFR subgroup with signi cant difference. This change is postulated to be caused by changes in diet, medication, gastrointestinal microecology and host physiology and pathology. However, we noticed that the main types of SCFAs, including acetate, propionate, butyrate, and valerate did not change signi cantly in DKD patients versus DM patients, which was unexpected given recent literature identifying a signi cant decline in SCFA-producing bacteria with advancing kidney disease [17]. Wang  investigate the types and concentrations of SCFAs in a larger group of DKD patients. Notably, we identi ed that a signi cant decline of the level of serum caproate in DKD patients than DM patients in our study. It in in line with with the study that serum caproate concentration decreased in CKD 3 patients compared to non-CKD participants conducted by Wu et al [11].
SCFAs diffuse through the intestinal mucosa and enter the bloodstream via the portal vein [19,20]. Samuel et al found that the intestinal absorption of SCFA seems to be in uenced by the G protein-coupled receptor (GPCR), which are broadly distributed in mammalian organisms [21]. However, serum SCFAs were not in parallel with fecal SCFAs changes in DM and DKD patients in our study. It is assumed that SCFAs measured in circulation may not be utilized in fecal SCFAs excretion, therefore fecal SCFA may be more accurate in revealing SCFAs absorption or production [22]. Several in vitro and in vivo studies have con rmed signi cant disruption of the colonic, ileal, jejunal and gastric epithelial tight junction in different models of CKD in rats and in cultured human colonocytes exposed to uremic human plasma [23,24]. Meanwhile, several observation have provided indirect evidence of increased intestinal permeability in the CKD patients and animals [25,26]. A human study showed that the participants with lower fecal acetate tended to have higher acetate absorption [22]. However, the transit time of SCFAs in the large intestine does not indicate speci c phases of a certain disease. Also, the level of serum SCFAs is in uenced by diet manipulations.
Herein, we agree that serum SCFAs are effected by many factors and it was necessary to assess both fecal and circulating SCFAs in certain disease to achieve a better understanding of the microbiota change.
Gut microbiota participates in the progression of metabolic diseases via its metabolites. Several studies have demonstrated that SCFAs play a protective role in kidney disease. Yang et al. revealed that dietary ber supplement signi cantly reversed kidney injuries in CKD mice due to increased SCFAs production from microbial fermentation [27].
Andrade-Oliveira et al. demonstrated that intraperitoneal injection with SCFAs improved acute kidney injury (AKI) by decreasing in ammatory cytokines and chemokines locally and systemically via suppressing NF-κB signaling pathway [28]. In the recent studies, SCFAs played an important effect on multipe aspects of renal physiology, inhibiting in ammation, immunity, and brosis, decreasing blood pressure, and adjusting energy metabolism [29].
Protective effects of SCFAs on DKD have also been reported, via activation of GPCRs and the inhibition of HDAC activity. Administration of sodium butyrate (NaBu), the major members of SCFAs, ameliorates mesangial matrix expansion, brosis and in ammation in the kidneys of STZ-induced diabetic rats [30,31]. In vitro study, NaBu acted as an antioxidant in HG-induced NRK-52E cells and suppressed HG-induced apoptosis of NRK-52E cells through inhibiting HDAC2 [32]. In vivo study, dietary ber protects against DKD through modulation of the gut microbiota, enrichment of SCFA-producing bacteria, and increased SCFA production, so that it reduced expression of genes encoding in ammatory cytokines, chemokines, and brosis-promoting proteins in diabetic kidneys via GPR43 and GPR109A [33].Recent studies found GPR41 and GPR43 protein expressed in the distal renal tubules and collecting tubules, and found SCFAs lowered TNF-α induced MCP-1 expression by reducing phosphorylation of p38 and JNK in a GPR41/43-dependent manner in human renal cortical epithelial cells(HRCEs) [34]. Iso-butyrate, valerate and isovalerate, have not been studied as extensively as other SCFAs, and details of the physiological effects are sparse.
Previous work has identi ed these as ligands for GPCR [35], which in uence a variety of metabolic, immune and vascular processes [36].
There are some limitations in our cross-section study, consequently we could not demonstrate the causal relationship between fecal, serum SCFAs and the presence of DKD. This monocentric study included a small number of patients in China and dietary assessment was not included in the study design . Prudence need to be taken when trying to extrapolate our data to other populations. Besides, the composition and construction of gut microbiota in participants were not analyzed, therefor the relationship between fecal and serum SCFAs and gut microbiota was not identi ed.
In conclusion, this study provides evidence for quantitative reduction of gut microbial product -SCFAs fecal acetate, propionate and butyrate in particular in DKD patients, demonstrating the association of SCFAs with worse renal function in DKD.