Part Ⅱ：Validation Of Candidate Phospholipid Biomarkers Of Chronic Kidney Disease in Hyperglycemic Individuals And Their Organ-Specific Exploration in Leptin Receptor-Deficient Db/db Mouse
Apr 20, 2023
Discussion
Based on the natural history of diabetic nephropathy, the early phase presents with normal renal function (normal GFR) and is not clinically suspicious. This is followed by a brief period of glomerular hyperfiltration (elevated GFR), followed by a return to normal and a slow decline to a sharp decline in GFR at a relatively late stage. Our preliminary findings in a longitudinal human cohort suggest that elevated serum SM C18:1 and PC aa C38:0 levels are predictive of CKD development in hyperglycemic individuals with normal renal function at baseline. The finding of this animal and cross-sectional human study is that these metabolites are associated with further stages of hyperglycemia-related CKD evolution, including (i) early changes characterized by glomerular hyperfiltration (8-week-old db/db mice) and (ii) later changes characterized by reduced eGFR (KORA FF4 study).
This cross-sectional KORA FF4 study showed a significant association between serum SM C18:1 and PC aaC38:0 levels and reduced eGFR in patients with prediabetes or T2D. Their association with renal function was independent of systolic blood pressure, lipids, HbA1C and UACR, suggesting that these two candidate phospholipid biomarkers are independent risk factors for CKD. Both metabolites SM C18:1 and PC aa C38:0 are phospholipids known to regulate inflammation and fibrosis, and their alterations in diabetes and metabolic syndrome occur in multiple body systems. In addition to hyperglycemia-associated CKD, metabolomics studies have shown that plasma PC aa C38:0 is positively associated with mortality from coronary artery disease, and alterations in systemic SM levels predict T1D, T2D, and myocardial infarction. As these findings are risk factors or subsequent outcomes for hyperglycemia-associated CKD, it is necessary to further investigate the disease specificity of the emerging phospholipid biomarkers before applying them to clinical diagnosis. Since not all diabetic patients develop CKD and not all CKD patients follow the same disease trajectory, exploring their mechanisms of action is also important for better patient stratification and accelerating targeted screening programs.
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Glomerular hyperfiltration is a hallmark of diabetic renal insufficiency. The blood flow-related effects of altered glomerular and tubular changes caused by the mechanical stress associated with glomerular hyperfiltration play an important role in the pathogenesis of the glomerular disease, and reducing hyperfiltration is a key therapeutic target for diabetes-induced CKD. In young diabetic mice (6-10 weeks), elevated GFR and increased creatinine clearance have been reported. In our 8-week-old db/db mice, we observed a potential effect of glomerular hyperfiltration with reduced plasma and urinary creatinine levels. Creatinine is a toxic by-product of phosphocreatine metabolism and is excreted through glomerular filtration and proximal tubular secretion with little reabsorption. In our db/db mice, in addition to plasma and urine, lower creatinine concentrations were found in the liver and lungs, which may be due to reduced creatine biosynthesis and/or phosphocreatine energy metabolism in skeletal muscle and other organs. Factors known to affect serum creatinine values (age, sex, race, muscle mass, protein diet, and drug intake) had little effect, as these factors were controlled for in our mouse studies. Diabetic mice already exhibit reduced bone mass at 5 weeks of age and before the onset of T2D, whereas low blood creatinine in T2D patients indicates muscle loss and predicts T2D independently of glomerular filtration. in summary, creatinine measurements in 8-week-old db/db mice suggest not only altered renal function, such as glomerular hyperfiltration but also high-energy phosphate metabolism.
Our db/db mice had significantly higher levels of the lung metabolites SM C18:1 and PC aa C38:0 than WT mice. This may suggest pulmonary dysfunction, as pc and SMs are key components of lung surface-active substances and their dysregulation is associated with respiratory failure. db/db mice are prone to pulmonary edema and asthma-related symptoms such as airway hyperresponsiveness. Sphingomyelin synthase 2 (SMS2) deficiency attenuates inflammation and improves recovery after lung injury in mice. Pulmonary dysfunction is a common comorbidity in patients with CKD but is less commonly managed clinically. Despite some early and controversial evidence of better adult respiratory distress syndrome (ARDS) survival in T2D patients, studies on pulmonary dysfunction in T2D patients have been urged.
Adipose tissue of the epididymis of db/db mice showed lower concentrations of SM C18:1 and PC aa C38:0 (Figure 3). In agreement with our findings, reduced adipose tissue levels of certain SMs and PCs were also detected in 30-week-old db/db mice [38]. Phospholipid metabolism in white adipose tissue and macrophages was largely disrupted in obese animals. We speculate that the lower adipose levels in SM C18:1 and PC aa C38:0 may be due to increased efflux of SM or PC lipid-containing proteins by the upregulated ATP-binding cassette transporter ABCG1 in obese mice.
In db /db mice, elevated SM C18:1 levels in the liver may be the result of the upregulation of fatty liver-associated SMS2 activity, which determines liver and plasma sm values. SMS2 activity promotes fatty acid uptake and hepatic steatosis, whereas SMS2 deficiency prevents HFD-induced hepatic steatosis (44) and increases insulin sensitivity. The liver is a central hub for the synthesis and recirculation of phospholipids via lipoprotein particles (e.g., LDL/VLDL).
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We observed that higher concentrations of PC aa C38:0 in the adrenal glands may be associated with reduced polyunsaturated fatty acid biosynthesis in the adrenal glands of db / db mice. These mice also exhibit increased adrenal steroid synthesis, which can stimulate pulmonary PC synthesis ((Figure 3)).
Biological fluids such as blood and urine provide insight into inter-organ metabolic crosstalk and renal activity, respectively. Similar to creatinine, lower levels of SMC18:1 and PC aa C38:0 in the urine of db/db mice may reflect altered glomerular filtration and accumulation of phospholipids in renal tissue, as shown in HFD-fed db/db mice. accumulation of SMs in the glomeruli of diabetic and HFD-fed mice may promote CKD. Diabetes in db/db mice Nephropathy manifests around 8 weeks of age as albuminuria and increased glomerular surface area, similar to the early stages of human diabetic nephropathy, followed by a progressive increase in thylakoid matrix and hypertrophy. The kidney regulates HDL metabolism, and its early dysfunction may impair reverse cholesterol transport and further lead to reduced urinary concentrations of both phospholipids (Figure 3). In conclusion, a detailed assessment of two biological fluids and six tissues in a mouse model of diabetic nephropathy showed altered levels of SM C18:1 and PC aa C38:0 in the liver, lung, adrenal adipose tissue, and urine. Of these, the lung appears particularly interesting due to the association of phospholipids with various lung diseases and injuries. At this stage of knowledge, it is not clear but possible (based on the literature) that these organs may also contribute to the circulatory regulation of SM C18:1 and PC aa C38:0.
The present study has several limitations and strengths. The limited availability of mouse data prevented us from analyzing kidney tissue and validating metabolite profiles by histological analysis. Differences in the genetic background of db/db mice that cause hyperglycemia and diabetic nephropathy compared to humans may confound metabolite profiles. Therefore, the multi-organ contribution of SM C18:1 and PC aa C38:0 in systemic dysregulation and their potential functional significance in renal function (by feeding experiments in diabetic mouse models) need to be further investigated. One of the strengths of our study is the validation of two candidate CKD biomarkers, not only in cross-sectional human studies but also in multi-organ mouse models suffering from hyperglycemia and obesity. Our study reveals for the first time a multi-stage association of CKD, characterized by glomerular hyperfiltration in the early phase (8-week-old db/db mice) and reduced eGFR in the late phase (KORA FF4 study), as well as a potential multi-organ contribution of two phospholipid metabolites to the circulatory regulation of CKD.
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Conclusions
This study provides biological insight into our recent discovery of SM C18:1 and PC aa C38:0 as predictive metabolites for the development of CKD in hyperglycemic individuals. Cross-sectional analysis revealed that the negative association of both phospholipids with glomerular filtration was independent of systolic blood pressure, cholesterol, triglycerides, HbA1C, and UACR in hyperglycemic individuals. Multi-organ analysis of mouse models of early diabetic nephropathy revealed possible contributions of the lung, liver, adipose tissue, and adrenal glands in their systemic regulation and CKD progression. As a remarkable example of interdisciplinary collaboration, this human and animal study confirms our initial findings and provides insights into the relationship with potential effects on renal function and other organs. This study contributes to the human validation of SM C18:1 and PC aa C38:0 as novel biomarkers for the early identification of diabetic (pre-) patients at increased risk of CKD and is a step forward in risk stratification and improved targeted screening programs for CKD. These new metabolites are necessary to predict an in-depth molecular phenotype of CKD.
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Jialing Huang 1,2,3, Marcela Covic 1,2,3, Cornelia Huth 2, Martina Rommel 1,2, Jonathan Adam 1,2, Sven Zukunft 4,5 , Cornelia Prehn 6, Li Wang 1,2,7, Jana Nano 2,3, Markus F. Scheerer 8,9, Susanne Neschen 8,10, Gabi Kastenmüller 11, Christian Gieger 1,2,3, Michael Laxy 12, Freimut Schliess 13 , Jerzy Adamski 4,14,15, Karsten Suhre 16 , Martin Hrabe de Angelis 3,8,15, Annette Peters 2,3 and Rui Wang-Sattler 1,2,3.
1 Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; jialing.huang@helmholtz-muenchen.de (J.H.); marcela.covic@helmholtz-muenchen.de (M.C.); martina.troll@helmholtz-muenchen.de (M.R.); jonathan.adam@helmholtz-muenchen.de (J.A.); wlrst@126.com (L.W.); christian.gieger@helmholtz-muenchen.de (C.G.)
2 Institute of Epidemiology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; cod.huth@gmail.com (C.H.); jana.nano@helmholtz-muenchen.de (J.N.); peters@helmholtz-muenchen.de (A.P.)
3 German Center for Diabetes Research (DZD), 85764 München-Neuherberg, Germany; hrabe@helmholtz-muenchen.de
4 Research Unit of Molecular Endocrinology and Metabolism, Helmholtz Zentrum München, 85764 Neuherberg, Germany; zukunft@vrc.uni-frankfurt.de (S.Z.); adamski@helmholtz-muenchen.de (J.A.)
5 Centre for Molecular Medicine, Institute for Vascular Signaling, Goethe University, 60323 Frankfurt am Main, Germany
6 Metabolomics and Proteomics Core Facility, Helmholtz Zentrum München, 85764 Neuherberg, Germany; prehn@helmholtz-muenchen.de
7 Liaocheng People’s Hospital—Department of Scientifific Research, Shandong University Postdoctoral Work Station, Liaocheng 252000, China
8 Institute of Experimental Genetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; markus@scheerer-home.de (M.F.S.); susanne.neschen@mail.com (S.N.)
9 Bayer AG, Medical Affairs & Pharmacovigilance, 13353 Berlin, Germany
10 Sanofifi Aventis Deutschland GmbH, Industriepark Hoechst, 65929 Frankfurt am Main, Germany
11 Institute of Computational Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; g.kastenmueller@helmholtz-muenchen.de
12 Institute of Health Economics and Health Care Management, Helmholtz Zentrum München, 85764 Neuherberg, Germany; michael.laxy@helmholtz-muenchen.de
13 Profifil, 41460 Neuss, Germany; Freimut.Schliess@profifil.com
14 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore
15 Chair of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, 85353 Freising, Germany
16 Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar (WCMC-Q), Education City, Qatar Foundation, Doha P.O. Box 24144, Qatar; karsten@suhre.fr
