IgA nephropathy is associated with elevated urinary mitochondrial DNA copy numbers

Mitochondrial injury plays important roles in the pathogenesis of various kidney diseases. However, mitochondrial injury in IgA nephropathy (IgAN) remains largely unexplored. Here, we examined the associations among mitochondrial injury, IgAN, and treatment outcomes. We prospectively enrolled patients with IgAN and age-/sex-matched healthy volunteers (HVs) as controls (n = 31 each). Urinary copy numbers of the mitochondrial DNA (mtDNA) genes cytochrome-c oxidase-3 (COX3) and nicotinamide adenine dinucleotide dehydrogenase subunit-1 (ND1) were measured. Urinary mtDNA levels were elevated in the IgAN group compared with that in HVs (p < 0.001). Urinary ND1 levels were significantly higher in the low proteinuria group than in the high proteinuria group (p = 0.027). Changes in urinary levels of ND1 and COX3 were positively correlated with changes in proteinuria (p = 0.038 and 0.024, respectively) and inversely correlated with changes in the estimated glomerular filtration rate (p = 0.033 and 0.017, respectively) after medical treatment. Mitochondrial injury played important roles in IgAN pathogenesis and may be involved in early-stage glomerular inflammation, prior to pathological changes and increased proteinuria. The correlation between changes in urinary mtDNA and proteinuria suggest that these factors may be promising biomarkers for treatment outcomes in IgAN.

Urinary mtDnA did not correlate with the existing prognostic markers at presentation of igAn. There were no significant relationships between urinary mtDNA copy numbers and traditional prognostic markers, including mean arterial pressure, eGFR, and baseline proteinuria levels, at presentation in the IgAN group (Table 2). Urinary ND1 and COX3 did not correlate with the MEST-C score of the Oxford classification, but tended to be higher in patients with M0 and T0 (Table 3).
Urinary mtDnA did not correlate with renal injury markers. Urinary kidney injury molecule-1 (KIM-1) level were significantly higher in the IgAN group than in HVs (Table 1)  Treatment did not affect the levels of urinary mtDNA in patients with IgAN. Urinary mtDNA in 19 patients in the IgAN group was measured 6 months after medical treatment. In followed patients, urinary ND1 and COX3 were higher in the high proteinuria group than in the low proteinuria group (p = 0.007 and 0.016, respectively). All 19 patients were treated with angiotensin receptor blockers (ARBs), and six were treated with an immunosuppressive agent (five patients were treated with corticosteroids, and one patient was treated with cyclosporin). Medical treatment did not reduce urinary levels of ND1 and COX3 (p = 0.778 and 0.334, respectively). Baseline proteinuria levels were lower (p = 0.029) and urinary ND1 and COX3 levels were higher (p < 0.001, both) in patients treated only with ARBs (only ARB treatment group) than those treated with ARBs and immunosuppressant (concurrent treatment group) in IgAN patients. Urinary levels of ND1 and COX3 were not reduced 6 month after treatment in concurrent treatment group (p = 0.917, both) and only ARBs treatment group (p = 0.701, 0.221, respectively). Changes in proteinuria and eGFR after treatment correlated with changes in urinary mtDNA copy numbers in patients with IgAN. Moreover, changes in urinary levels of ND1 and COX3 showed positive correlations with  www.nature.com/scientificreports www.nature.com/scientificreports/ changes in proteinuria (p = 0.038, 0.024, respectively) at 6 months and were inversely correlated with changes in eGFR at 12 months ( Fig. 3) after medical treatment. There were no significant differences in baseline urinary ND1 (p = 0.968) and COX3 (p = 0.661) levels between the response and no response groups (Fig. 4).
Structural changes in mitochondria were detected in the igAn group. Electron microscopic images showed that the mitochondria in the tubular epithelium and glomerular endothelial cells in kidney donors had many elongated mitochondria with densely stacked cristae membranes that were organized along membrane compartments. However, mitochondria were small and disorganized in patients with IgAN. Cristae membranes from some patients were replaced by a homogenized matrix, and electron-dense particles or droplets were detected in a few mitochondria (Fig. 5). These results suggested that structural changes occurred in the mitochondria of patients with IgAN.

Discussion
In this study, we evaluated changes in urinary mtDNA in patients with IgAN. Our results showed that urinary ND1 and COX3 copy numbers were well correlated with each other and were higher in the IgAN group than in HVs. This result suggested that mitochondrial injury was associated with IgAN. During the follow-up period, changes in urinary mtDNA correlated with changes in eGFR and proteinuria. Because changes in albuminuria are an independent prognostic marker in IgAN 28,29 , urinary mtDNA could be a promising biomarker for IgAN.
We also analyzed correlations between urinary mtDNA and existing clinical and histopathological prognostic markers at presentation in IgAN and found that there were no correlations. These findings are not consistent with previous studies in AKI, HTN, and diabetes mellitus (DM) nephropathy [25][26][27] . Urinary mtDNA levels tended to be inversely correlated with baseline proteinuria levels and were higher in patients who had a mesangial score of less than 0.5 or tubular atrophy/interstitial fibrosis less than or equal to 25% (Oxford classification M0 or T0, respectively). Interestingly, when dividing the IgAN group into two subgroups according to baseline proteinuria, a subgroup of patients with lower proteinuria showed higher urinary mtDNA levels. In the same context, because of more patients with high proteinuria were treated with immunosuppressant, only ARB treatment group showed lower baseline proteinuria levels compared with concurrent treatment group. However, baseline urinary mtDNA levels were higher in only ARB treatment group than concurrent treatment group.
According to our current knowledge, the immunopathogenesis of IgAN involves a multi-hit pathogenic process. The presence of increased amounts of underglycosylated IgA1 induces autoantibodies and the formation of immune complexes, which activate the complement pathway and contribute to mesangial activation and proliferation. Glomerular inflammation results in podocyte injury, proteinuria, tubulointerstitial inflammation, and fibrosis 30,31 . In our patients with IgAN, we found that urinary mtDNA copy numbers were higher in patients with less histological damage and proteinuria. These results may indicate that mitochondrial damage is involved in the early stage of glomerular inflammation and occurs prior to pathological changes. Accordingly, increased proteinuria and reduced renal function and urinary mtDNA may be the earliest signs of kidney injury. However, the duration of our study was relatively short; thus, further studies are needed to confirm these findings as potential prognostic markers in patients with IgAN.
Medical treatment for IgAN did not affect mtDNA copy numbers. Notably, body weight reduction after bariatric surgery did not decrease urinary mtDNA copy numbers in a previous study 11 . In contrast, KIM-1 levels decreased after medical treatment in our IgAN cohort, consistent with previous findings 32 . KIM-1 is a marker of tubule injury in the kidney; however, urinary mtDNA could have been derived from all cells in the nephron and glomerulus. These different sources could explain the lack of changes in mtDNA copy numbers in IgAN. Alternatively, urinary mtDNA copy numbers may reflect overall kidney cell damage and could be used as a unique renal injury marker distinct from existing renal injury markers.
Changes in urinary mtDNA levels were correlated with changes in eGFR and proteinuria levels in our study. Substantial observational data have supported that reduced proteinuria is associated with improved renal www.nature.com/scientificreports www.nature.com/scientificreports/   www.nature.com/scientificreports www.nature.com/scientificreports/ survival 31 . Moreover, a recent meta-analysis also showed that reduced proteinuria is associated with kidney outcomes, including doubling of SCr, ESRD, or death, and could be used as a surrogate endpoint in trials of IgAN 33 . Accordingly, it seems reasonable to conclude that reduced proteinuria is associated with improved kidney outcomes. In our study, percent reduction in urinary mtDNA copy numbers was found to be related to improved eGFR and decreased proteinuria. These findings suggest that urinary mtDNA copy numbers could also be used as a surrogate marker of kidney outcomes in IgAN. Taken together, these results suggest that alleviation of mitochondrial injury through preventive and therapeutic measures may improve clinical outcomes in IgAN, particularly at an early stage.
COX3 and ND1 encode the enzyme responsible for the mitochondrial respiratory chain and the electron transport, respectively. Previous studies showed that ND1 did not differ from COX3 copy numbers in obese African American hypertensive patients 34 and upon chronic renal injury in hypertensive patients 25 . However, urinary COX3 copy numbers from obese patients with glomerular hyperfiltration were not elevated compared with HVs 11 . In this study, urinary ND-1, but not COX3 levels were higher in the low proteinuria group compared with the high proteinuria group. There are various factors that cause mitochondrial injury in kidney disease. We think the causes of mitochondrial injury may be different for each kidney disease, leading to differences in mtDNA injury patterns. Although there was no statistical significance, urinary COX3 levels tended to be higher in the low-vs. high-proteinuria, and given the small sample size, ND-1 is unlikely to be a better mitochondrial injury marker than COX3.
To our knowledge, there has been no study evaluating the urinary mtDNA levels in GN. Since we have evaluated only patients with IgAN, it is not clear whether elevated urinary mtDNA levels is a ubiquitous finding in GN or a unique finding in IgAN. To clarify this, future studies are needed to measure and compare the urinary mtDNA in each GN.
There were some limitations to our study. First, the sample size was small, and the duration of follow-up was short, which may have made the study underpowered for evaluating whether differences in urinary mtDNA levels were useful for predicting treatment responses or prognosis. Attenuated urinary mtDNA levels were correlated with decreased proteinuria and improved eGFR in our study; however, these findings should be confirmed further in additional studies with larger sample sizes and longer follow-up durations. Second, we did not measure serum mtDNA copy numbers. We measured urinary mtDNA levels, because previous studies showed that, unlike circulating mtDNA levels, urinary mtDNA levels are correlated with renal dysfunction and clinical outcomes in various kidney disease including AKI 35,36 , CKD 25 , and obesity-related hyperfiltration 11 . Age-/sex-matched HVs were collected to minimize the effects of other factors that could increase circulating mtDNA levels. However, patients with IgAN tended to be older and have higher body mass index than HVs. An important limitation of this study is that we could not measure circulating mtDNA levels, due to which we cannot exclude the possibility that urinary mtDNA levels were elevated due to increased circulating mtDNA levels by other factors in the urine. In addition, although urinary mtDNA was increased in patients with IgAN compared with HVs, the variation in IgAN group was so high that it was difficult to detect individual differences among patients. This limitation could be compensated by measuring circulating mtDNA. Finally, we could not explain the specific mechanisms through which mitochondrial injury leads to kidney damage or determine in which part of the kidney mitochondrial injury may occur. In early stage of IgAN pathogenesis, galactose-deficient IgA1-containing immune complexes activate mesangial cells that secrete components of extracellular matrix, releasing various mediators including proinflammatory cytokines [37][38][39] and activating renin-angiotensin-alodosterone system [40][41][42] . These events may eventually lead to oxidative stress 43 and mitochondrial dysfunction. Further studies are required to support this hypothesis. In this study, we identified ultrastructural changes in the mitochondria through kidney biopsy specimens of two patients with IgAN. However, we could not quantitatively measure the extent of damage and determine in which part of the kidney mitochondrial www.nature.com/scientificreports www.nature.com/scientificreports/ injury may occur. In future studies, if ultrastructural changes in the mitochondria of kidney biopsy specimen from patients with IgAN is measured quantitatively using electron microscope and compared with urinary mtDNA level, more accurate mitochondrial injury site could be determined.
In conclusion, our findings suggested that mitochondrial injury may play a role in the pathogenesis of IgAN and may be involved in early-stage glomerular inflammation prior to pathological changes and increased proteinuria. Mitochondrial injury may be associated with changes in proteinuria and eGFR and may therefore represent a promising biomarker for treatment outcomes in patients with IgAN.

Methods
Study population. This was a multicenter prospective cohort study. The current study was conducted according to the principles outlined in the Declaration of Helsinki, and clinical data from patients were obtained after approval of the study by the Institutional Review Board (IRB No. 2016-01-002-007) of Soonchunhyang University Hospital. We enrolled patients with biopsy-proven pure IgAN (n = 31) who were collected from the Cohort for Biomarker Inquiry of Renal Aggravation (COBRA) cohort at Soonchunhyang University Seoul, Bucheon, and Cheonan Hospitals from May 2016 to January 2018. Age-and sex-matched HVs were enrolled as controls (n = 31). HVs were included if they had no history of DM, HTN, congestive heart failure, coronary artery disease, and stroke and did not take any medications. All participants provided written informed consent. clinical, laboratory, and pathologic data. We obtained data on demographics and comorbidities, including history of DM, HTN, hyperlipidemia, and hepatitis. We collected body weights, heights, SBP, and diastolic blood pressure at the time of kidney biopsy. We obtained information regarding the type, duration, and total dose of administered medications, including immunosuppressants and ARBs during the follow-up period. We collected laboratory data at every visit during follow-up. We determined the eGFR from SCr values using the CKD Epidemiology Collaboration equation. Proteinuria levels were determined by 24-h urine collection. We reviewed the pathological findings from kidney biopsies and specifically checked the pathological severity in patients with IgAN according to the Oxford classification 44 .
The IgAN group was classified into the low and high proteinuria groups according to baseline proteinuria levels based on median values. We subclassified patients with IgAN into the response group, defined as proteinuria less than 0.3 g/24 h, and the no response group, defined as proteinuria greater than or equal to 0.3 g/24 h or not decreased from the initial value at 6 months after medical treatment 45 . Urinary mtDnA levels. Urinary copy numbers of the mtDNA genes COX3 and ND1 were measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We also measured urinary ND1 and COX3 levels 6 months after medical treatment in IgAN group (n = 19). DNA was isolated from urine samples (1.75 mL) using DNA isolation kits from Norgen Biotek (Thorold, ON, Canada; cat. no. 18100). We analyzed DNA concentrations using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RT-quantitative PCR (qPCR) was performed using the ND1 primers (forward 5′-AGTCACCCTAGCCATCATTCTACT-3′ and reverse 5′-GGAGTAATCAGAGGTGTTCTTGTGT -3 ′ ) a n d C OX 3 p r i m e r s ( f o r w a r d 5 ′ -AG G C AT C AC C C C G C TA A AT C -3 ′ a n d r e v e r s e 5′-GGTGAGCTCAGGTGATTGATACTC-3′), obtained from Thermo Fisher Scientific, and 20 ng template DNA/sample. The PCR conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 60 s. mtDNA copy numbers were corrected to the nuclear control gene RNAse-P (nDNA; cat. no. 4403326; Thermo Fisher Scientific) using human genomic DNA for the standard curve. mtDNA copy numbers were calculated using Copy Caller software (Thermo Fisher Scientific) and expressed as a mtDNA/nDNA ratio 11 .
Renal injury marker. KIM-1 levels were measured using enzyme-linked immunosorbent assays with a commercial kit (Cat. No. MBS264966; MyBioSource, San Diego, CA, USA) according to the manufacturer's protocol and standardized with urine creatinine levels (ng/mg), as previously described 46 . We measured KIM-1 levels in the IgAN group and HVs at the time of kidney biopsy and in the IgAN group at 6 months after medical treatment. A single investigator performed all measurements blinded to the clinical information.
Structural differences in IgAN compared with normal kidneys. We examed structural changes in the mitochondria in two patients with IgAN using transmission electron microscopy. A kidney biopsy specimen from a living kidney donor who had normal kidney function was assessed as a control. To detect mitochondrial ultrastructure morphology, the kidney samples were cut into 1 mm 3 pieces using a scalpel. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a digital electron microscope (JEM-1400; JEOL USA, Peabody, MA, USA). All glomeruli were examined at x3000 and examined at a higher magnification for mitochondria structure 47 .
Statistical analysis. Descriptive characteristics of the study population were reported as means ± standard deviations or medians with interquartile ranges for continuous variables and as frequency counts with percentages for categorical and binary variables. Comparisons of differences between groups were made using Mann-Whitney and Wilcoxon signed rank tests for continuous variables and either χ 2 tests or Fisher's exact tests for categorical variables, as appropriate. Urinary mtDNA copy numbers were log-transformed. Spearman's rank correlation coefficient was used to analyze the relationships between urinary mtDNA copy numbers and clinical and histological variables. All statistical tests were two-sided, and the results were presented with 95% confidence intervals. We considered p values of less than 0.05 to indicate statistical significance. All analyses were performed using SPSS 25 for Windows (SPSS Inc., Chicago, IL, USA).