ABSTRACT
Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. In spite of the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing progression of diabetic kidney disease.
INTRODUCTION
Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States 1–5. The National Diabetes Statistics Report in 2020 estimated that more than 34 million Americans, or 10.5% of the population, had diabetes (https://www.cdc.gov/diabetes/pdfs/data/statistics/national-diabetes-statistics-report.pdf). Further, as many as one in four Americans are expected to become diabetic by the year 2050 4, 6.
In spite of the beneficial interventions implemented in patients with diabetes, including tight glucose control, stringent blood pressure control, angiotensin-converting enzyme inhibition (ACEI), angiotensin II receptor blockade (ARB), mineralocorticoid receptor antagonism, sodium glucose cotransport-2 (SGLT2) inhibition and glucagon-like receptor protein-1 (GLP-1) receptor agonism 7–12, the new therapeutic targets in DKD are emerging based on the further understanding of the mechanisms to cause progression and/or prevention of DKD.
Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases 13, 14. In animal models of DKD, such as db/db mice or KKAy mice, and in diet induced obesity mice, there are increased renal inflammation and oxidative stress 15, 16. Inflammation is closely associated with mitochondria damage 17, 18. In kidneys from DKD, there is a wide variety of mitochondrial dysfunction reported 15, 16, 19, 20. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation 21–25.
In the present studies, we administered a NAD+ booster, nicotinamide riboside (NR) to db/db mice, a model of type 2 diabetes, to evaluate its effects in diabetic nephropathy. We found that long-term NR treatment improved DKD in the db/db mice via preventing the renal inflammation, at least in part by inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. This anti-inflammatory activity was associated with mitochondrial function restoring.
RESULTS
NR treatment improved murine diabetic kidney disease
We treated db/db mice with type 2 diabetes with NR for 20 weeks (Figure 1A). NR treatment did not affect body weight, kidney weight, or blood glucose, but serum cholesterol and triglyceride levels were decreased in NR treated db/db mice (Table 1). We found that NR treated db/db mice had a significant decrease in albuminuria (Figure 1B) and excretion of the urinary kidney injury marker Kim1 (Figure 1C).
NR treatment reduced mesangial expansion in db/db mice, assessed on PAS staining (Figure 1D). Immunofluorescence microscopy showed increased extracellular matrix protein (collagen IV and fibronectin) deposition in the glomeruli of db/db kidneys indicating the presence of glomerulosclerosis, which was improved with NR treatment (Figure 1E-F, Supplementary Figure S1). RNA expression of profibrotic factors, including transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1 and connective tissue growth (CTGF), and α−smooth muscle actin (αSMA), were increased in db/db kidneys. Treatment with NR decreased mRNA abundance of TGFβ1, PAI-1 and αSMA, but not CTGF (Figure 1G).
We next examined podocyte loss with the podocyte nuclear marker p57. Immunohistochemistry analysis of p57 showed decreased podocyte numbers in db/db kidneys. This loss was prevented by NR treatment (Figure 1H).
Ultrastructural examination demonstrated irregular glomerular basement membrane (GBM) thickening in db/db mice, which was ameliorated with NR treatment (Figure 1I). Finally, podocyte foot processes effacement was notable in db/db mice and preserved with NR treatment (Figure 1J).
Taken together, we concluded that NR prevent effects in protecting podocyte and glomerular integrity and preventing tubulointerstitial injury in a model of diabetic kidney disease.
NR treatment improved renal oxidative stress
We examined levels of thiobarbituric acid reactive substances (TBARS), which represent oxidation products of lipid. We found that urinary TBARS level, kidney NADPH oxidase 4 (NOX4) mRNA and kidney 4-hydroxynonenal (4-HNE) protein levels-markers of lipid peroxidation, were all increased in db/db mice. NR treatment decreased urinary TBARS level, and kidney NOX4 mRNA and 4-HNE protein levels in db/db mice (Figures 2A-C).
NR treatment decreased inflammation in diabetic kidneys
We found increased expression of monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF), interleukin (IL)-6, tissue inhibitor of metalloproteinase (TIMP)1 and CD68 mRNA in diabetic kidneys. Expression of toll-like receptor 2 (TLR2), a marker of innate immune pathway triggered by damage-associated molecular patterns, was also increased in db/db kidneys. NR treatment effectively prevented these increases (Figure 3A). Staining of CD45 and CD68, markers of leukocytes and macrophages, respectively, showed glomerular infiltration of immune cells in db/db kidney, which was prevented by NR treatment (Figure 3B and 3C). Thus we demonstrate the role of NR treatment on reducing inflammatory pathways in the db/db model.
NR treatment decreased cGAS-STING activation in diabetic kidneys
To explore how NR treatment exerts anti-inflammatory effects, we analyzed bulk RNAseq and proteomics data. We found that NR treatment downregulated many of the inflammatory genes and proteins upregulated in db/db kidneys (Figure 4 A-C, Supplementary Tables S1 and Figure S2). Interestingly, the expression of interferon-induced transmembrane (IFITM) genes 27, members of the interferon stimulated gene (ISG) family, was upregulated in db/db kidneys and expression of these genes was reduced by NR treatment (Figure 4D). Aside from viral infections, ISG induction can be triggered by activated nucleic acid sensors, such as cGAS/STING, which respond to the leak of nuclear or mitochondrial DNA into the cytoplasm 28–30. In diabetic kidneys, we found marked increases in the cGAS mRNA, STING mRNA and STING protein levels. cGAS and STING levels were significantly decreased by treatment with NR (Figure 4E). Activation of STING activates downstream effectors TBK1 and IRF3, by promoting their phosphorylation. We detected increased phosphorylation of TBK1 (Figure 4F) and IRF3 (Figure 4G) in db/db mice that was significantly reduced by NR treatment, confirming modulation of STING pathway signaling in an NR-dependent manner (Figure 4F-G). We further examined the downstream response and we found that both STAT3 (Figure 4H) and NFκB (Figure 4I) were activated in db/db kidneys. Their activation as determined by the increased levels of phospho-STAT3 and phospho-p65, was reduced by NR treatment.
To further evaluate STING activation in the kidneys, we performed the immunostaining of STING on kidney tissues. In non-diabetic human kidneys, STING expression is primarily limited to endothelial cells (glomerular, peritubular capillaries and larger vessels) and sparse interstitial inflammatory cells. By contrast, in diabetic renal parenchyma, an expanded pattern of STING staining is observed. (1) There is increased endovascular STING staining, enhanced inglomerular segments with prominent endothelium and endocapillary inflammatory cells including lymphocytes and monocyte/macrophages. (2) Prominent parietal epithelial cells showed increased STING expression. (3) By far, the most prominent compartment with STING expression are the increased influx of interstitial inflammatory cells. (4) Also noted is enhanced SITNG staining in atrophic tubule and tubules of the distal nephron (Figure 4J). the pattern of increased STING expression was noted, albeit not as broadly, and included primarily increased STING expression in intraglomerular inflammatory cells (Figure 4K). These data confirm the cellular compartments most notable for STING expression, with conservation of inflammatory cell STING expression in mouse and human.
STING inhibition decreased inflammation in diabetic kidneys
To determine the role of the increased STING activity per se in mediating the inflammation in the diabetic kidney we treated db/m and db/db mice with the STING inhibitor C176 31–33 (Figure 5A). Treatment of db/db mice with C176 prevented the increases in IRF3 and phospho-IRF3 (Figure 5B). C176 also prevented the increases in the inflammatory markers phospho-STAT3 protein and IL-1β mRNA levels (Figure 5C).
To determine a direct role for STING, independent of the potential off-target effects of C176, we performed studies with the STING KO mice (Figure 5D). In these studies, wild type and STING KO mice were made diabetic with the administration of streptozotocin (STZ). In the STZ mice there was a significant increase in STING protein level, which was prevented in the STING KO mice made diabetic with STZ (Figure 5E). STZ induced increases in urinary albumin and urinary KIM-1 which were significantly attenuated in STING KO mice (Figure 5F). In the STZ mice there was increased protein abundance of phospho-Stat3 which was also significantly decreased in STING KO mice (Figure 5G).
Inflammation was associated with NAD+ reduction and mitochondrial dysfunction
To evaluate the injury mediated by inflammation per se, we used a LPS-induced acute kidney injury model. While LPS activated innate immune response, a cascade of downstream signaling triggered increased STING expression and decreased mitochondrial complex I activity. Other factors involved in the mitochondrial homeostasis were also found to be decreased in LPS kidneys, such as NAD+ level, and expression of PGC-1a, estrogen-receptor related protein-α (ERRα) and sirtuin (SIRT) 3 (Supplementary Figure S3).
NR treatment increased NAD+ level and increased SIRT3 expression and activity
The complexity of interconnecting crosstalk between inflammatory response and mitochondria dysfunction prompted us to further assess the effects of NR treatment in the kidneys. We measured NAD+ levels in db/m and db/db kidneys. Although there was no change in baseline NAD+ levels between db/m and db/db kidneys, NR significantly increased NAD+ levels in both db/m and db/db kidneys (Figure 6A).
There were increases in acetylated lysine levels in whole kidney lysate (Figure 6B) and in isolated mitochondrial fractions (Figure 6C) in db/db mice, an indication of decreased deacetylase activity. Treatment with NR restored acetylated lysine proteins to levels seen in db/m mice, suggesting the improved deacetylase activity in the mitochondria. SIRT3 is the main mitochondrial deacetylase and is involved in regulating mitochondrial functions, including fatty acid oxidation (FAO). To explore whether SIRT3 was involved in the NR effect, we examined SIRT3 expression and activity. SIRT3 protein abundance (Figure 6D) and activity (Figure 6E) were reduced in the diabetic kidney. NR treatment increased SIRT3 protein abundance (Figure 6D) and SIRT3 activity (Figure 6E). In human diabetic kidneys, SIRT3 expression (Figure 6F, Supplementary Table S2) was also decreased. Another NAD+-dependent deacetylase SIRT1 expression did not change in human diabetic kidneys when compared with the non-diabetic controls (Figure 6G, Supplementary Table S2).
To further assess a role for SIRT3 in regulating acetylated lysine protein in the kidney, we studied SIRT3 KO mice 34, 35. In SIRT3 KO kidneys, total protein acetylation level was increased, as well as the acetylation of SOD2 and IDH2 (Figure 6H), two proteins previously identified as SIRT3 targets 36, 37. In diabetic kidneys, acetylated SOD2 and IDH2 protein levels are increased, and NR treatment decreased acetylated SOD2 and IDH2 protein levels (Figure 6I and 6J). Acetylation of K68 and K122 residues of SOD2 has been reported to regulate SOD2 activity 37.
NR treatment enhanced mitochondrial biogenesis in diabetic kidneys
NR treatment increased the mitochondrial DNA/nuclear DNA ratio in db/db kidneys, indicative of increased mitochondrial biogenesis (Figure 7A). NR treatment also increased PGC1α mRNA and protein abundance (Figure 7B). PGC1α is a master mitochondrial biogenesis regulator 38. As expected, the direct targets of PGC1α, Nrf1 and the mitochondrial transcription factor Tfam1, expression were increased in the diabetic kidney following the NR treatment (Figure 7C).
Increased mitochondrial biogenesis was accompanied by increased expression of genes related to the mitochondrial ETC complexes, including complex I subunit Ndufa4, complex III subunit Uqcrc2 and complex IV subunit Cox6a2 (Figure 7D). Complex I activity was decreased in the diabetic kidneys and treatment with NR restored complex I activity. NR treatment also restored complex IV activity (Figure 7E).
In db/db mice, mitochondria within proximal tubule epithelial cells exhibited severe polymorphic structural changes. Based on the type of mitochondrial restructuring, 4 categories were assigned (I-IV), as previously described 39. Type I mitochondria are oval-shaped, with longitudinally oriented and tightly packed cristae. Type II mitochondria are structurally abnormal, with indistinct shape and/or non-uniform size together with hypoplasia. The cristae are swollen and/or have signs of homogenization, irregular or whirling, which usually have lost the longitudinal orientation, tightness, regular spacing, and electron-lucent matrix. Type III mitochondria manifest hypoplasia and degenerative changes. The shape and size of these mitochondria vary, often with a discontinuous outer membrane. The focal disruption of the inner membrane leads to an uneven increase in the crista thickness, homogenization and fragmentation, with a swollen electron-lucent matrix. Type IV mitochondria exhibit disrupted and discontinuous outer membranes, deficiency in cristae and “myelin-like” cristae transformations (Figure 7F). In db/db mice, up to 20% of the total mitochondria corresponded to type III and up to 5% were found to have type IV structural damage. These manifestations were absent in all db/m mice. NR treated db/db mice exhibited a significant decrease in severely damaged mitochondria compared to untreated db/db mice (Figure 7F).
Morphometric analysis revealed that in db/m control mice the mitochondrial area ranged from 0.2 - 0.9 μm2 and mitochondria < 0.2 μm2 constituted up to 24% of all mitochondria. In diabetic db/db mice, mitochondria < 0.2μm2 were found up to 58% which was significantly higher than in control mice (Figure 7F). The increase in mitochondria < 0.2μm2 size indicates the enhancement of mitochondria fission. NR treated diabetic mice showed a significant reduction in number of mitochondria < μm2 size, and this may suggest the improvement of mitochondrial functional. In addition, renal expression of mitochondrial enzymes that mediate mitochondrial fatty acid β-oxidation, including Cpt1a mRNA, Lcad mRNA, and Mcad mRNA and MCAD protein, were all upregulated by NR treatment of db/db mice (Figure 7G), suggesting that NR treatment promotes mitochondrial fatty acid β-oxidation. Consistent with these effects, NR treatment prevented triglyceride accumulation in the kidney (Figure 7H).
Finally, we found increased mtDNA damage in the db/db kidneys and NR treatment protected the kidney from mtDNA damage (Figure 7I). mtDNA damage triggers the leakage of mtDNA into cytosol, activating the nucleic acid sensors such as cGAS and STING.
DISCUSSION
Inflammation and mitochondrial dysfunction have been proposed to play an important role in the progression of diabetic kidney disease 40–43. It is not clear whether and how these two processes are linked with each other. We have found that treating diabetic mice with nicotinamide riboside (NR), an NAD+ precursor, improved inflammation as well as mitochondrial function.
The increases in the interferon-induced transmembrane proteins 1,2, and 3 (IFITMs) from RNAseq prompted us to find the regulation of cGAS/STING signaling in diabetic kidneys. A role for increased STING expression and activity per se in mediating inflammation is demonstrated in studies where we inhibited STING with a well-established inhibitor C-176 and also in STING knockout mice made diabetic with streptozotocin. In both studies we found that STING inhibition prevents inflammation. In addition, and importantly we determined that STING inhibition prevents diabetic kidney injury as demonstrated by preventing increases in urine albumin and urine KIM1 excretion.
While we failed to document a working phospho-STING antibody for the mouse tissue with STING knockout mice, total STING antibody has been found working in the staining of kidney tissue. We found that STING expression is also increased in the kidneys of human subjects with diabetes. STING staining of control kidney biopsies show endothelial staining in glomeruli, peritubular capillaries and larger vessels with sparse staining of interstitial inflammatory cells. In diabetic renal parenchyma, an expanded pattern of staining is observed. There is increased endovascular staining in glomeruli correlating with segments with prominent endothelium and endocapillary inflammatory cells including lymphocytes and monocyte/macrophages. Prominent parietal epithelial cells show increased expression. By far, the most prominent compartment with STING expression are interstitial inflammatory cells. Also noted is enhanced staining in two tubular elements: atrophic tubule and distal nephron. Although STING has been previously reported in other kidney injury models 31, 33, 51, this is the first time that the localization of STING activation in the kidney has been examined. In contrast to previous reports, the proximal tubules did not seem to be a major site for STING activation in diabetic kidneys. The infiltrated immune cells express most of the STING found in the kidney. The expression of STING in other cell types such as podocytes, in diabetic kidneys, may well be a result of co-purifying immune cells. This raises the question of how the mitochondrial DNA damage in the tubules or podocytes relates to STING activation in the immune cells, which warrants further investigation.
From STING inhibition studies we found that this inhibition did not achieve what NR treatment showed in db/db kidneys. This could be due to other players of nucleic acid sensors that we also found to be activated in diabetic kidneys, and blocked by NR, such as AIM2 and NLRP3. This indicates that NR can target to a mechanism upstream of other nucleic acid sensors besides cGAS/STING, making itself a better treatment than STING inhibition alone.
NR treatment improved several parameters of mitochondrial dysfunction including restoration of mitochondrial fatty acid β-oxidation. This may be mediated by increasing the mitochondrial sirtuin 3 activity. The increase in sirtuin 3 activity was associated with decreasing the acetylation of proteins important for mitochondrial function including SOD2 and IDH2. The increase in the mitochondrial antioxidant SOD2 is reflected by the ability of NR to decrease renal oxidative stress. NR treatment also induced an increase in mitochondrial DNA/nuclear DNA ratio, indicative of an increase in mitochondrial biogenesis, as well as increases in Tfam, Nrf1, and PGC-1α, complex I and complex IV activities, and enzymes that mediate mitochondrial fatty acid β-oxidation (FAO). Other NAD+ precursors have also been reported beneficial renal effects 44, 45. However, in this report, we further connected the NAD+ effects in diabetic kidney disease model to its direct target SIRT3 by showing the increase in SIRT3 deacetylase activity and the decrease of acetylation level in the mitochondrial targets of SIRT3. SIRT3 relies on the availability of NAD+ level to improve the mitochondrial functions including FAO 46–48. Restoring FAO is a critical step to reverse the kidney injury. Recently, increasing the FAO enzyme Cpt1a in renal tubules was shown to protect against kidney fibrosis 49.
How these mitochondrial functional changes relate to regulation of inflammation is not fully known, however in diabetes there is evidence for increased mitochondrial DNA damage which is prevented by NR treatment. The increase in mitochondrial DNA damage may be mediated by increased oxidative stress and also by decreases in Tfam and Nrf1 50. The increase in mitochondrial DNA damage on the other hand can result in activation of nucleic acid sensor signaling, which are major mediators of inflammation via inducing the NFκB and STAT3 regulated inflammatory pathways. Treatment with NR prevents the decreases in Tfam and Nrf1, the increase in mitochondrial damage, and the increases in the interferon-induced transmembrane proteins 1,2, and 3, NFκB and STAT3. Overall, our data showed that NR supplementation boosted the NAD metabolism to modulate inflammation and mitochondrial function and prevent progression of diabetic kidney disease.
METHODS
Animal studies
All mouse experiments were conducted according with the Guide for Care and Use of Laboratory Animals, National Institute of Health, Bethesda, MD and were approved by Institutional Animal Care and Use Committee of Georgetown University, Washington, D.C.
10-week-old male db/m (non-diabetic controls) (catalog # 00662) and db/db (diabetic) (catalog # 00642) mice were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were housed in animal care facility with 12/12-hour light-dark cycle and fed for 20 weeks on regular chow diet (TD 190694, Envigo, Madison, WI) or chow diet supplemented with 500 mg/kg body weight nicotinamide riboside (NR) (ChromaDex, Irvine, CA) (TD 190695, Envigo, Madison, WI). One week prior to the end of the study, the mice were placed in metabolic cages for a 24-hour urine collection. At the end of the study, heparinized plasma and kidneys were harvested for further processing.
To determine the role of SIRT3, whole body SIRT3 knockout mice on C57BL/6 background were obtained from Matthew Hirschey (Duke University) 35 to compare their kidneys with the age, gender matched wild-type control kidneys.
To determine the role of STING inhibition in the diabetic kidneys, 20-week-old male db/m and db/db mice were treated with C-176 32 (Focus Biomolecules, Plymouth Meeting, PA) at 1.2mg/kg body weight dose with daily i.p. injection for 4 weeks.
To further determine the role of STING in the diabetic kidneys, 12-week-old male wild type and whole-body STING knockout mice on C57BL/6J background (JAX catalog #025805) were treated with streptozotocin at 50mg/kg body weight as 5-day daily consecutive i.p. injection to induce diabetes. These mice were sacrificed after 12 weeks on diabetes.
Blood and urine biochemical analysis
Blood glucose levels were assessed with a glucometer (Elite XL, Bayer, Tarrytown, NY). Blood urea nitrogen (BUN) was measured with colorimetric QuantiChrom assay kit (Bioassay systems, Hayward, CA). Total cholesterol and triglycerides levels in plasma were determined with calorimetric assay kit provided by Pointe Scientific (Canton, MI, USA). Urine assays followed the instructions from the kits listed in Supplementary Table S1.
NAD+ measurement
20 mg of kidney tissue was homogenized in extraction buffer provided by the kit (E2ND-100, Bioassay systems) and NAD+ was determined immediately according to manufacturer’s protocol.
Immunoblotting
Total protein was quantified using the BCA protein assay kit (Thermo Scientific, Rockford, IL). Western blotting was done as previously described 16. Antibody information can be found in Supplementary Table S1.
Quantitative Real-Time PCR
Total RNA from kidneys were isolated according to manufacturer’s protocol with Qiagen RNeasy mini kit (Qiagen, Gaithersburg, MD) and cDNA was made using reverse transcript reagents from Thermo Scientific (Catalog # 4374967). qRT–PCR was performed using Quant Studio Real-Time PCR machine (Thermo Fisher Scientific). Expression levels of target genes were normalized to 18S level. Primer sequences are listed in Supplementary Table S1.
Mitochondrial enzymatic complex activity assay and sirtuin 3 activity assay
Mitochondrial fraction was isolated according to previously described protocol 16. Isolated mitochondria were assayed for the complex I (Catalog # ab109729), complex IV (Catalog # ab109911) and sirtuin 3 activity (Catalog # ab156067) with the kits purchased from Abcam, Boston, MA.
Analysis of mitochondrial DNA damage
Total DNA from kidney tissue was isolated using Genomic Tip (Qiagen, Valencia, CA), and its concentration was measured by PicoGreen dye (Invitrogen, Carlsbad, CA). 15Lng DNA was used to amplify a long 10kb mitochondria DNA target followed by real-time PCR based quantification to determine mitochondria DNA lesions, as previously described 52.
Immunohistochemical (IHC) and PAS staining
Immunohistochemical staining was performed on formalin fixed and paraffin embedded 5 µm kidney sections. Following deparaffinization and rehydration, the slides were subjected to heat mediated antigen retrieval in citrate buffer pH 6 and blocked with 3% BSA. The sections were probed with Sirt1 (Abcam catalogue # ab110304), Sirt3 (Sigma catalogue # S4072) or STING (Cell Signaling, catalogue # 13647S) antibody and incubated in room temperature for 1.5 hours. Mouse/Rabbit PolyDetector reagent (Bio SB, Catalog No. BSB 0269) or UnoVue HRP secondary antibody detection reagent (Diagnostic BioSystems, Pleasanton, CA) was applied followed by DAB chromogen. The Periodic Acid-Schiff (PAS) staining was performed with a PAS stain kit (Thermo Scientific, Catalog No. 87007). Imaging was done with Nanozoomer (Hamamatsu Photonics, Japan) and Motic Digital Slide Scanner (Richmond, BC, Canada).
Quantification of Morphology
Glomeruli were extracted from images of PAS staining and the PAS components of each glomerulus were segmented as described before 53. To quantify mesangial expansion, the ratio of PAS positive pixels to detected glomerular pixels was used.
Immunofluorescence microscopy
The kidney tissue was snap frozen by embedding in to optimum cutting temperature (OCT) medium (Thermo Scientific, CA). The tissues were sectioned at 5-µm in thickness and transferred over the superfrost slides. Immunofluorescence staining performed as descried previously 16, 54. Antibody information can be found in Supplementary Table S1.
Electron Microscopy
Renal cortex tissues were fixed in the 2.5% glutaraldehyde/2% paraformaldehyde/ 0.05M cacodylate solution, post-fixed with 1% osmium tetroxide, and embedded in EmBed812. For imaging acquisition, ultrathin sections (70 nm) were post-stained with uranyl acetate and lead citrate and examined in the Talos F200X FEG transmission electron microscope (FEI, Hillsboro, OR) at 80 kV located at the George Washington University Nanofabrication and Imaging Center. Digital electron micrographs were recorded with the TIA software (FEI). Ultrathin sections (120 nm) were mounted in silicon wafers and observed with a Teneo LV FEG scanning electron microscope (FEI, ThermoFischer Scientific). For optimal results, we used the optiplan mode (high-resolution) equipped with an in-lens T1 detector (Segmented A+B, working distance of 8 mm). Low-magnification images (600×) were first taken for the observation then we performed high magnification tile images of our regions of interest (35,000) using 2LkV and 0.4 current landing voltage.
Morphometric analysis was performed under blinded conditions by systematic uniform random sampling with the Fiji Software using 20 randomly selected images. In EM images, volume fraction of mitochondria was determined using the morphometric technique with a dot grid. The size of each individual mitochondria was calculated by using the FIJI ImageJ software. Mitochondrial types were determined using the point counting method 55.
Bulk RNA-seq
One microgram of total RNA samples was sent to Novogene (Sacramento, CA) for mRNA sequencing. RNA-seq fastQ files were filtered and trimmed from adaptors using Trimmomatic algorithm 56. The reads were aligned to Mus musculus genome assembly and annotation file GRCM38:mm10 using STAR algorithm 57. Gene expression was estimated in FPKM counts using RSEM algorithm 58. Differential expression was quantified with DeSeq2 algorithm 59. Absolute fold change of 1.5 and Bonferroni adjusted p-value of less than 0.05 was considered as significant change. All bioinformatics analysis was performed on T-BioInfo Platform (http://tauber-data2.haifa.ac.il:3000/). DAVID Bioinformatics 60, 61 and PANTHER Classification System (http://PANTHERdb.org/) were used to classify the differentially expressed genes into functional groups. To identify proteins that are localized to mitochondria, we used a curated database of mitochondrial localized proteins – MitoCarta3 database 62.
Proteomic analysis
Frozen mouse kidney samples were lysed in 8 M urea and 50 mM triethylammonium bicarbonate (TEAB). Proteins were then reduced and alkylated followed by digestion with LysC (Fujifilm Wako Pure Chemical, Osaka, Japan) in the ratio of 1:100 (enzyme-to-protein, w/w) at 37°C for 3 hours. Subsequently, the proteins were further digested with trypsin (Promega, Fitchburg, WI) in the ratio of 1:50 (enzyme-to-protein, w/w) at 37°C overnight after diluting the urea concentration from 8 M to 2 M. Proteins were then acidified, desalted, and lyophilized sequentially. The dried peptides were labeled with 16-plex TMT reagents (Thermo Scientific). The labeled peptides was fractionated using basic pH RPLC for total proteome analysis as described previously 63. The LC-MS/MS was analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled with an Ultimate3000 RSLCnano nano-flow liquid chromatography system (Thermo Scientific). The resulting spectra were analyzed by Proteome Discoverer (version 2.4.1.15 software package, Thermo Scientific) following standard procedures. Downstream analysis was performed with Perseus 64 using log2 normalized intensities of protein abundance. Absolute fold change of 1.5 and Bonferroni adjusted p-value of less than 0.05 was considered as significant change.
Lipidomics
Kidney samples were pulverized in liquid nitrogen and dissolved in 300 μL of isopropanol extraction buffer containing internal standard for lipid classes. The samples were vortexed for 30 seconds and homogenized for 1-2 min on ice and incubated on ice for 20 min followed by incubation at -20 ℃ for 20 min. Samples were centrifuged at 13,000 rpm for 20 min at 4 ℃. The supernatant was transferred to MS vial for LC-MS analysis using QTRAP® 5500 LC-MS/MS System (Sciex, Framingham, MA).
Statistical Analysis
All the resulted data sets were calculated and presented as meanL±LSEM. One-way ANOVA fallowed by Student-Newman-Keuls post hoc analysis were used to analyze the variance among multiple groups and between two groups. The statistically significant differences were designated as a P values of <0.05. GraphPad prism 8.1.2 software package was used for statistical analysis (www.graphpad.com).
Data and Resource Availability
All reagents and data from this article are available from the corresponding author upon request.
Acknowledgements
Funding. This study was funded by NIH R01 Grants DK116567 (ML), DK127830 (ML), F30 Fellowship DK129003 (BAJ), AHA Postdoctoral Fellowship 19POST34381041 (KM), and National Center for Advancing Translational Sciences of NIH under Award Number TL1TR001431 (BAJ). We also thank Chromadex (Los Angeles, CA) for supplying us NR.
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. ML and XXW conceived and designed research; KM, XXW performed most experiments; BAJ, MDH, XY, AZR, BG and PS performed the histology work; LB, YJ, CHN, YQ, XZ, UG, PL, CW, JM, AC, and JP conducted the –omics work and analysis. KM and XXW analyzed data and interpreted results of experiments; KM, XXW and JP prepared figures; XXW, KM, JP and ML wrote the manuscript with input from all the co-authors; ML is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Footnotes
More data have been added.