Urinary C3 levels associated with sepsis and acute kidney injury-A pilot study.

Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 μg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine.

Introduction

The complement system with the central component C3 represents a member of the innate immune system. Three pathways of complement cascade activation have been characterized that converge at factor C3 and it is involved in immunological reactions in the kidney [1]. A most interesting example is its involvement in reperfusion injury [2], a serious form of acute kidney injury (AKI), through activation of the cascade and by influencing the regenerative potential through autophagy [3].

The most frequent cause of AKI in humans is infections such as sepsis [4, 5]. On a cellular level, the proximal tubular epithelium [6] represents the most vulnerable part of the nephron. Epithelial cells undergo damage at the brush border, loose cell polarity and are driven towards apoptosis [6, 7]. This occurs together with cast formation and tubular proteinuria. However, various cell types at the nephron possess compensatory mechanisms to counteract damage [8]. Thereby the tubular epithelial cells alter their gene transcription leading to changes in the regulation of autophagy [9]. This is of importance in order to balance and counteract stressing factors such as reactive oxygen species [10] which could finally result in AKI. Such regulations occur within various functional pathways [11] such as energy expenditure, inflammation, cell cycle [12, 13], lipid modification, metabolisms and autophagy [3]. As the result of such an ongoing process it has to be assumed that upregulated gene products, of which some represent secreted [13] or cleaved protein fragments [14, 15], might be detectable in urine and can serve as biomarkers [13, 16, 17]. In this line it is of note that at the kidney glomerular mesangial cells [18], glomerular epithelial cells [19] and tubular epithelia the complement factor C3 gene is induced for transcription upon pathogenic stimuli [2, 20, 21] given by interferon gamma (INFγ), immunocomplexes [22] and interleukin (IL)-1⍺ [23]. C3 induction is also seen in kidneys of deceased organ donors [21]. It has been shown that this locally synthesized C3 is of relevance for complement-mediated injury of ischemic kidney allografts [2]. But this is just one side of the coin, the other side is that C3 has been shown to be involved in the regulation of autophagy in pancreatic beta cells which contributes to cell survival in inflammatory states [24] in pre-diabetic and diabetic conditions. This functional, cytoprotective mechanism of C3 has also been found to be of importance in limiting damage in renal epithelial cells upon exposure to toxic compounds such as cisplatin [25].

Motivated by the demonstration of the increasing importance of C3 in renal disease we have generated a monoclonal antibody recognizing a conformational structure of the anterior part of the C3a/C3 molecule [26]. We have selected a clone among others specifically recognizing native C3a/C3. Using this antibody, we followed patients admitted to the intensive care unit (ICU) because of serious infections for the secretion and appearance of C3a/C3 in urine. In addition, we analyzed the urinary sediment to visualize C3a/C3 distribution in various cellular structures. Concomitantly we sought to evaluate whether C3a/C3 is involved in urinary tract infections (UTI) often associated with such disease conditions.

Materials and methods

Sample collection

The study was approved by the ethics committee of the Medical University of Vienna under the EC number (721/2007). Eighty-five patients admitted to the ICU or intermediate care unit between 2009 and 2012 were included into this study after giving oral and written informed consent for participation. Only patients fulfilling at least 4 of 10 sepsis criteria [27] were selected and urine was collected from an indwelling urinary catheter every morning over the course of 6 days. Twenty one out of the 85 subjects were categorized as non-AKI patients. The interval between first and second time point of urine collection varied between 10 to 24 hours. Collection intervals 2, 3, 4, and 5 each represent a span of 24 hours.

The data on serum creatinine and other kidney function parameters as well as C-reactive protein (CRP) levels and leukocyte counts were extracted from the hospital data files. C3a/C3 urine analysis was performed in retrospect.

Antibody generation

A C3a/C3 specific mouse monoclonal antibody (mAb 3F7E2) was produced and characterized as described earlier [26]. In brief, following three immunizations in two-week intervals, splenocytes were fused with the HAT sensitive murine myeloma cell line 536 using the polyethylene-glycol method as described earlier [28]. Outgrowing clones were screened by a multi-slot immunoblotting device (Merck Millipore, Bedford, MA, USA). Clones recognizing SDS treated C3 under non-reducing conditions were kept and further used for experiments.

Dot blot

Thirty μl of urine were applied into an S&S Minifold I (Schleicher&Schüll, Germany) dot blotting device and filtered onto nitrocellulose by vacuum. The filter was then briefly dried and exposed to blocking solution for 30 mins. Following this the mAb 3F7E2 (tissue culture supernatant) was incubated over night at 4°C and next day the goat anti mouse HRP conjugated detection antibody (Dako, P0447) was incubated for 60 mins after two washing steps with TPBS. The filter was then developed by using a chemiluminescence reagent (Roche; BM Chemiluminescence Blotting Substrate, 11500694001) and recorded on an imaging device (Fusion Fx Vilber Lourmat) using Fusion software for signal detection. Pictures were further processed using Adobe Photoshop 6. Dot blot densitometric evaluation was performed using FusionCapt Advance Solo 4 16.06.

Immunoblot

Human urine or human serum/plasma was loaded onto a 10% SDS-PAGE gel and run under non-reducing conditions, transferred onto nitrocellulose and incubated with tissue culture supernatant of mAb 3F7E2. The site of antibody binding was visualized such as described for the dot blot above.

Urine C3a/C3 ELISA

Goat anti mouse pre-coated plates (Pierce) were washed once with PBS and the mAb 3F7E2 was bound onto the plate by incubating hybridoma cell supernatant for 2 hours. After one wash with TPBS, urine samples (100μl) were loaded together with a C3 standard series. Following an incubation period of 2 hours the ELISA plate was washed three times with TPBS (Tween20 PBS, 0,1%) using an ELISA washing machine. The rabbit anti human C3 Ab (Abcam, ab48342), conjugated with biotin, was incubated using Ray Bio-assay diluent (item E2, 1:2500) for 1.5 hours under constant shaking. Following three washes with TPBS, the streptavidin HRP (Dako, Po397), diluted 1:2500 in Ray Bio-assay diluent, was bound to the biotin for 30 mins at room temperature under constant shaking. Following another three washes with TPBS, the two-component TMB peroxidase substrate chromogen solution (KPL, 50-65-00 and 50-76-01) was added and kept in the dark for 10 mins. The reaction was stopped with 1M HCl and read with the ELISA reader. Each value was calculated according to a standard curve.

Urine C3a ELISA

The human C3a ELISA (Invitrogen, BMS2089) was carried out as indicated in the test manual. Urine samples were thawed under airflow. The diluted (1:4 or 1:2 in sample diluent) urine samples (100μl) were pipetted into the prewashed test wells and incubated for two hours under constant shaking together with the provided standard series which was prepared in the sample diluent. Following washing on an automated ELISA washer (three times) the biotin-conjugate prepared in the assay buffer (100μl) was pipetted into each test well and incubated for one hour at RT under constant shaking. Following a second washing step again three times, the streptavidin-HRP prepared in the assay buffer provided with the test kit (100μl) was added into each well and incubated for one hour under constant shaking at RT. After a final washing step, the TMB substrate solution (100μl) was pipetted into each well and reacted at RT for 20 minutes under light protection. The reaction was then stopped by adding 100μl stop solution. The test signal was read at 450nm at an ELISA reader and the sample concentrations were calculated according to the standard curve using the Gen5 version 2.03 program.

Urinary sediment analysis

Seven ml of urine were centrifuged at 3000 RPM for 5 mins and the resultant sediment was re-suspended in a tissue culture medium (RPMI 1640 containing 10% fetal bovine serum). Cyto-slides were prepared using a Shandon cytocentrifuge and air dried for at least 2 hours. These were either wrapped in aluminum foil and frozen at -20°C or analyzed immediately. In brief, slides were fixed in acetone for 5 mins and the cell containing area was marked by drawing a cycle around the cells. Tissue culture supernatant of mAb 3F7E2 was then applied onto the slide and incubated over night at 4°C in a moist chamber. The next day the slides were washed in PBS and incubated in Alexa fluor 488 labelled donkey anti mouse secondary antibody for 1 hour at room temperature. Following two washing steps in PBS the slides were mounted in Vectashield (VECTOR, Z0619) mounting media containing propidium iodide for staining DNA and nuclei. Slides were viewed and recorded at a Zeiss confocal microscope or a Leica Aristoplan microscope. Pictures were further processed using Adobe Photoshop 6.

Statistical analyses

Adherence to a Gaussian distribution was determined using the Kolmogorov-Smirnov test. Normally distributed data were described as means±SDs. The paired samples t-test was utilized to compare continuous variables within the same group. Qualitative variables were described with counts and percentages. The strength of association between CRP, leukocyte count, age, peak creatinine and urinary peak C3a/C3 was measured with the Pearson correlation coefficient. Data were analysed with SAS (version 9.2 for Windows) and SPSS (version 26 for Macintosh). All p-values result from 2-sided tests, with significance inferred at p<0.05.

Results

Acute phase proteins in the serum are represented by CRP, fibrinogen, ferritin, complement factor C3 and others. These have their origin mainly in the liver. However, for the kidney such an acute phase reaction has not been described, but C3 production and secretion from various kidney epithelial cells has been demonstrated before [18, 19, 23, 29].

Dot blot initial screens

In order to look for kidney born urinary proteins, which might be of diagnostic value in critical illness, previous literature indicated that some complement factors representing secreted proteins are induced by pathogenic stimuli such as infections. This motivated us to perform pilot testing using a dot blot analysis and the C3a/C3-specific monoclonal antibody 3F7E2. Using a 96-well dot blotting device a transient elevation of C3 secretion in urine was found in patients suffering from serious infection (). Most patients exhibited a decline in urinary C3a/C3 secretion before transfer to the open ward (left panel of ), however, an increase of urinary C3a/C3 on days 5 and 6 could also be found and was in some individuals associated with worsening disease (right panel in ) and a fatal outcome (patient o in , ).

C3a/C3 dot blot.

Urine samples obtained at consecutive time points from ICU patients suffering from serious infections. Patient (o) did not survive this episode due to multiorgan failure the same day as urine sample #5 was collected. Shown is a representative experiment out of four. Left panel of the figure represents patients with decreasing urinary C3a/C3 levels. Right panel shows patients with increasing urinary C3a/C3 levels towards 5th and 6th day of ICU treatment.

ELISA testing

As a next step, we investigated if urinary C3a/C3 secretion correlated with other markers of systemic inflammation or stages of AKI. Therefore, urine from 85 sepsis patients () undergoing ICU treatment was screened for C3a/C3 levels over a period of 6 days. As depicted in the left panel of the highest mean overall level of C3a/C3 was found on the second day of measurement, which represented the second day of treatment (all p<0.05). Selectively measured C3a was detectable in urine over all 6 days ( right panel). Out of 230 samples 178 exhibited detectable levels of C3a as shown in ( right panel). Thirty-seven patients had AKI stage 1, ten patients had AKI stage 2, and 17 had AKI stage 3. Twenty-one ICU patients with sepsis showed no signs of AKI (). The measured urinary C3a/C3 level did not correlate with AKI stage () and did not allow any conclusion regarding disease outcome.

ELISA-measurement of urinary complement factor C3a/C3 and C3a in 85 ICU patients.

Data are presented in columned scatter graphs; the horizontal lines mark the mean values of the respective C3a/C3 (left) and C3a (right) levels. Day 1: n = 78; day 2: n = 78; day 3: n = 72; day 4: n = 66; day 5: n = 56; day 6: n = 48.

Peak urinary C3a/C3 level in patients categorized for stage of AKI.

Peak levels of urinary C3a/C3 did not correlate with AKI stage. AKI 0 (n = 21), AKI 1 (n = 37), AKI 2 (n = 10) AKI 3 (n = 17).

There were 29 patients with a pre-existent chronic renal disease, out of whom 28 underwent an additional AKI during the infection period.

Most of the 85 patients were undergoing a remarkable acute phase reaction with a mean peak level of CRP of 18.00 mg/dl (±11.24) and a mean peak leukocyte count of 12.613 G/l (±6.746). Sixteen patients did not recover and died at the ICU.

Urinary C3a/C3 levels correlated negatively with serum CRP (p<0.0001). There was no correlation with peak serum creatinine, peak leukocyte count and age ().

In order to evaluate of what biological relevance this urinary C3a/C3 secretion might be, we sought to discover whether C3a/C3 is present in its entire molecular structure or only in fragments. For this reason, we loaded high C3a/C3 containing urine in SDS loading buffer onto SDS-PAGE gels. The blotted samples were developed with mAb 3F7E2. As demonstrated in the entire 190 kDa C3 molecule was detected in the urine samples. For comparison the peripheral blood derived C3 is shown aside the immunoblot result from urine (, middle panel). In addition, urine was concentrated up to 20 times using protein concentrators and the resultant urine concentrate was similarly immunoblotted as described above. C3 fragmentation and the C3a fragment were detected in lane 3 of .

C3 serum und urine immunoblot using mAb 3F7E2.

Ten μl of human urine (patient#1 and #2) was loaded onto a 10% SDS-PAGE transferred to nitrocellulose and incubated with mAb 3F7E2 (right panel). 0.4μl of human serum (patient#1 and #2) was loaded onto a 10% SDS-PAGE transferred to nitrocellulose and incubated with mAb 3F7E2 (middle panel). Molecular weight marker is shown at the left panel. Shown is a representative experiment out of two. The entire 190 kDa C3 molecule was detected and exhibited some extent of degradation. C3a could not be delineated.

C3a/C3 immunoblot developed with 3F7E2 mAb.

Twenty times (20x) concentrated urine from a patient with high C3a/C3 level in urine ELISA (lane 1); ten times (10x) concentrated urine from a patient with medium range C3a/C3 level in urine ELISA (lane 2); three times (3x) concentrated urine from patient with C3a fragment (shown by arrow) in urine (lane 3); three times (3x) concentrated urine from patient with undetectable C3a/C3 level in urine ELISA (lane 4); non-concentrated (native urine) urine (lane 5).

Immunofluorescence

As a next step it was considered important to evaluate the origin of the urinary C3a/C3. Therefore, we stained urinary sediment for C3a/C3 expression obtained from 12 patients with serious infections. As depicted in intracellular granules of C3a/C3 could be visualized in cells with epithelial morphology. The staining pattern was highly variable, with large granular and vesicular staining indicating an autophagic granular expression (90% of positive cells, ), by contrast, reticular cytoplasmic distribution indicated endoplasmatic reticulum production . This mode of positive staining was mainly restricted to epithelial cells. In addition, bacteria were found enveloped in a C3a/C3 detectable coat (). A negative control from a patient with non-inflammatory condition is included in .

Immunofluorescence staining of urinary sediment using mAb 3F7E2.

Tubular epithelial cells contain intracellular granules with C3 (A, B). Bacteria present in the urinary sediment are veiled in C3a/C3 to some extent (C). The scale bar represents 10 μm. Immunofluorescence staining of a urinary sediment of a patient without systemic inflammation and AKI served as control (D). The scale bar represents 10 μm.

In addition to the urinary sediment, human renal tissue from a patient with tumor nephrectomy showed areas of tubular epithelial staining as demonstrated in .

Immunofluorescence staining of cryosections of a kidney removed because of a renal cell carcinoma using rabbit anti human C3-specific antibody.

Tubular epithelial cells contain intracellular C3 (left panel). Merging C3 staining with DAPI nuclear staining (right panel).

Discussion

In this study, we sought to investigate a urinary secreted factor indicative for stressing renal conditions. In this respect urine of 85 ICU patients, all admitted because of a serious infection and critical illness, was analysed. Sixty percent of these patients showed high secretion of C3a/C3 (>20μg/ml peak level) mostly about 12–24 hours following admission. About 40% did not present with such high urinary C3a/C3 levels, which did not appear to be associated with a milder course of disease compared to those presenting with high C3a/C3 levels. In addition, urinary C3a/C3 levels were not associated with AKI stage. Furthermore, we recorded peak CRP levels during the urine collection period. Interestingly, peak CRP levels correlated inversely with peak urinary C3a/C3. This unexpected observation might be due to a discordant gene regulation regarding C3 between the liver and the kidney tissue, mechanistically caused by different transcription enhancers and repressors in these functionally very different tissue types. A second explanation might be that complement activation and consumption in urine leads to lower C3 levels in some individuals. A third and interesting option is higher endophagocytosis (autophagy) in patients with higher CRP levels. Such results have already been demonstrated in beta cells of diabetic individuals [24]. Similarly to CRP, the peak leukocyte count was not associated with urinary C3a/C3. These data seem to indicate that C3 induction at the kidney is caused by stimuli other than those inducing CRP levels and leucocytosis in the peripheral blood during some forms of infection and sepsis.

Acute phase proteins are rapidly increased in transcription and translation upon inflammatory stimuli. The increase of these proteins in the serum is mainly orchestrated by the liver. However, it is of high interest whether other organs such as the kidney are also capable of producing following a pathogenic stimulus. For complement factors extrahepatic sites of synthesis have been researched for several years and their history is reviewed [29]. Interleukins such as INFγ, IL-1 and others have been effective in stimulating C3 production in in vitro studies although to a much lower extent when compared with that in the liver. The level of urinary C3 is much lower (in ng-μg/ml) than in serum (mg/ml) even under stress conditions. However, it is well demonstrated by earlier authors that proximal tubular cells can produce both C3 and factor B following IL-1α stimulation in a time and concentration dependent manner [23]. Local synthesis of C3 has already been described in the past. Sack S and colleagues were able to describe that glomerular mesangial cells are capable of producing C3 and C4, with an increase in C4 expression after stimulation with INFγ, whereas C3 expression remains unaffected under INFγ stimulation [18]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed, and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with INFγ lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by INFγ [19]. In addition, increased local C3 synthesis has been described in human diseases, such as postischemic acute renal failure and immune-mediated nephritis [2, 22].

The role of the complement cascade in AKI has been extensively reviewed by McCullough [30]. It is of particular note that the C3a receptor (C3aR) [31] and C5a receptor (C5aR) [32] are expressed at the tubular cell surface which is involved in upregulation of pro-inflammatory factors and chemokines to initiate granulocyte infiltration. In this respect, our article demonstrates for the first time to our knowledge, that tubular epithelial cells can secrete C3 upon infectious stimuli. Whether this C3 secretion in urine is of specific biological relevance or represents a by-product only must be left open by this study. However, as demonstrated in some bacteria are covered by C3a/C3 products. Therefore, it appears that C3 might also be involved in antibacterial defence and thereby relevant for opsonization and chemotaxis to attract macrophages and granulocytes to the site of infection.

C3 is secreted from tubular epithelium and might represent a determinant of tubular epithelial stress. When this stress is elevated the transcription and secretion is increasing. We strongly suggest that urinary C3 of critically ill patients suffering from serious infections is the result of synthesis and secretion from renal epithelial cells. For this reason, we have excluded patients suffering from nephrotic syndrome to circumvent the chance of detecting blood born C3. Furthermore, we stained urinary born cells fixed on cytopreparation slides for presence of C3a/C3 in tubular epithelial cells. This demonstrated the various extent and morphologic distribution of intracellular C3 in renal tubular epithelial cells. Two modes of secretion are known in biology. It has to be assumed that C3 is not constitutively secreted into urine, but that there must be a regulated mode of secretion from nephronic epithelial cells. A specific stimulus secretion coupling appears to exist with one of them occurring in infection and sepsis. The exact triggering molecule has not been characterized yet but must differ from that in the liver. Transcription factor and repressor proteins are usually expressed in a tissue- and cell subtype-specific manner. Thereby, renal epithelial cells express different transcription factors and repressor proteins than hepatocytes. The function of these proteins is to interact with specific responsive elements on the DNA of genes. In recent years, miRNAs have been characterized as similar players at the mRNA level and thereby can regulate translation and mRNA turnover. As a result, different tissues are able to respond differently to the same stimulus such as interleukins, toxic compounds, drugs, and nutrients. This response is achieved by regulating gene expression and protein production. With regard to our study, we must hypothesize that the C3 gene in the kidney is regulated differently than the CRP gene in the liver. Moreover, the dynamics of CRP in serum is much brisker than that of C3.

At this point the question should be elaborated, why urinary C3a/C3 levels correlate negatively with serum CRP (p<0.0001) in our study. The consumption and activation of C3 is a well-known marker for determining the activity of inflammatory diseases, such as rheumatic diseases or atypical hemolytic uremic syndrome. In this context, a decrease in C3 represents increased inflammation. Consequently, C3 can be said to represent an inverse acute phase parameter. In relation to our study, it is important to mention that mAb 3F7E2-based ELISA detects both C3 and C3a. To clarify the activation of C3 at the site of renal secretion, a commercially available C3a ELISA was performed that showed the presence of C3a in urine on all 6 days of measurement. The hypothesis that this measured C3a originates from the blood, because it is a small molecule and, unlike C3, could pass through the glomerular filter, must be rejected because C3a has a very short half-life and is rapidly degraded in the blood by proteases.

Overall, the question arises as to the purpose of this urinary C3 provided by the kidney. In this respect much earlier work by other authors has demonstrated that C3 transcription and protein production can be induced in a time and dose dependent fashion in proximal tubular epithelial cells by IL-1α [23]. In addition, other cytokines such as TNF-α, IL-6, IL-8, and MCP-1 are produced in response to IL-2 stimulation. It represents a matter of speculation if invading leukocytes or stressed epithelial cells themselves are the source of IL-1 and thereby induce C3 transcription and protein production. This then can act in both directions causing cell injury but at the same time preventing cell death through autophagy.

The shortcoming of this study is that C3a/C3 excretion could not be measured for the complete 6 days in all patients, as some died or were discharged before.

C3a/C3 is detected in urine upon infection-associated stimuli but declines rapidly following recovery from disease. It appears to represent an indicator of an acute phase reaction and stress causing condition, which in part might be involved in the regeneration of AKI-associated damage to epithelial cells, despite AKI-stages not being associated with urinary C3a/C3 levels in the measured timely interval.

(PDF)

Click here for additional data file.

29 Mar 2021

PONE-D-21-05396

Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease? – A pilot study

PLOS ONE

Dear Dr. Gerges,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscripts was reviewed by two experts and they are critical of the experimental design of the experiments. I am giving you an option for revision to address those technical issues.

Please submit your revised manuscript by May 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Please compile all raw blot and gel images in a single PDF file titled S1_raw_images, and upload this file as Supporting Information or provide it via a publicly available data repository and include the dataset identifier (DOI or equivalent) in the Data Availability Statement. We ask that you ensure that every image in the file is clearly labeled to annotate the loading order, identity of experimental samples, method used to capture the image, and which figure panel was generated from that original image. Molecular weight markers should be included or indicated on each blot/gel image, and any lanes not included in the final figure should be marked with an “X” above the lane label on the original blot/gel image. All labeling and annotations should be performed without obscuring any data or background bands. Please note, there is a 20 MB maximum file size for Supporting Information files. If your PDF size is larger, please use a suitable repository or discuss with the journal staff.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available.

PLOS policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

Furthermore, please specify in your ethics statement: 1) whether the ethics committee approved the verbal/oral consent procedure, 2) why written consent could not be obtained, and 3) how verbal/oral consent was recorded.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The current study “Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease? – A pilot study” attempts to study the relationship of urinary C3 and acute phase renal reaction. However, there are many major problems in the experimental design.

1. The authors aim for the C3 molecules derived from the kidney. However, there is no indication the C3 molecules detected are from the kidney, instead of liver. In kidney injury, especially septic injury as discussed in the current work, it is very likely complement molecules leak through the blood vessels into the urine.

2. The authors used sepsis patients and analyzed the kidney injury and C3. However, C3 and kidney injury are both too much related to sepsis and any correlation shown in this study may be the result of their respective correlation with sepsis. This is a major statistical error in correlation study.

3. The authors found no correlation between AKI stage and C3 concentration. This means the study gives a negative result. However, the authors claims C3 molecules a good indicator. The conclusion contradicts with the data.

4. Urine concentration of a specific protein is very variable. So many factors interfere with the urine amount. Unless the authors provide a practical and standardized method, the study and its clinical relevance are not valid.

Reviewer #2: In the present study “Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease?-A pilot study” the authors try to discover the C3a/C3 as the adjunctive diagnostic marker for Acute kidney injury. The article might be helpful to understand urine C3a/C3 in the development of AKI. However, several concerns must be addressed for better quality. Here are some major concerns.

1. This study mainly rely on mAb 3F7E2 (C3a/C3 specific mouse monoclonal antibody) to detect C3a/C3. Why the specific Abs each of C3 and C3a were not used? Using of mAb 3F7E2 made it hard to distinguish whether the production of C3 or the activation of C3 is altered during the study.

2. Table 1 shows majority of patients had Pneumonia. Is there the possibility C3 from respiratory system transfer to Urinary system and affect the urine C3 level?

3. For Table 2, the finding from this study show Peak-uC3a/C3 is negatively correlated with CRP. Based on relevant literature, activation of complement factors are associated with increase inflammatory reaction. C3a levels or C3a to C3 ratio should be check to support authors’ idea.

4. For Fig1A, the authors claim that Dot blot initial screens test show decline in urinary C3a/C3 on Day 5 and 6. Please consider developing numeric scoring according to dot color and its C3a/C3 levels.

5. For Fig1B, degradation of C3 is mentioned in results. Though, C3a band seems not clear. Please indicate C3 and C3a location.

6. For Fig2, the indication should be changed to urinary C3a/C3. Some patients couldn’t make it until Day 5-6 with worsened condition. If C3/C3a is related with disease severity, it may affect decreased C3a/C3 level on Day 5-6.

7. For Fig3, please change the graph to dot form for the consistency.

8. Fig4 shows intracellular deposit of C3a/Ca in epithelial cells from urine sediment. Please include IF from control group without systemic inflammation or AKI.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 May 2021

Dear Editors in Chief,

Please find enclosed a revised version of our systematic review entitled Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease? – A pilot study for possible publication in PLOS ONE.

We thank the reviewers for their comments. Please find a point-by-point response letter to the reviewers’ comments below.

Thank you for considering our manuscript!

Yours sincerely,

Daniela Gerges, MD, MSc

Reviewer comments: black font

Response: blue font

Modifications in the revised version of the manuscript: red font

Major concerns:

Reviewer #1:

(C1) The authors aim for the C3 molecules derived from the kidney. However, there is no indication the C3 molecules detected are from the kidney, instead of liver. In kidney injury, especially septic injury as discussed in the current work, it is very likely complement molecules leak through the blood vessels into the urine.

(R1A): Thank you for this valuable and important comment as this point apparently needs further clarification in our work. Local synthesis of C3 has already been shown in the past by Sacks S et al. Sack S and colleagues were able to describe that glomerular mesangial cells are capable of producing C3 and C4, with an increase in C4 expression after stimulation with interferon-gamma, whereas C3 expression remains unaffected under interferon-gamma stimulation [1]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with interferon-gamma lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by interferon-gamma [2]. In a further study using human samples of patients with postischemic acute renal failure, local synthesis of C3 in the tubule was described and the authors were able to distinguish tubular C3 from C3 of hepatic origin [3]. In addition, another study described enhanced local C3 production in immune-mediated nephritis [4].

Regarding our study, immunofluorescence staining clearly exhibits tubular epithelial cells containing intracellular C3 (Fig 7 in the manuscript). It is therefore hard to imagine how hepatic C3 could enter a tubular cell. It must be emphasized that the molecule size of 185 to 190 kDa is not suitable for passing through the glomerular filter. Although the slit diaphragm may be vulnerable in sepsis, such large molecules should not enter the urine in kidneys of sepsis patients.

What appears very likely to occur, is that C3 is locally synthesized in tubular cells as has already been described in the literature. We included a statement regarding the origin of C3 in the discussion section of our manuscript.

Discussion, paragraph 2, page 13:

Local synthesis of C3 has already been described in the past. Sack S and colleagues were able to describe that glomerular mesangial cells are capable of producing C3 and C4, with an increase in C4 expression after stimulation with interferon-gamma (INF-gamma), whereas C3 expression remains unaffected under interferon-gamma stimulation [1]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed, and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with INF-gamma lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by INF-gamma [2]. In addition, increased local C3 synthesis has been described in human diseases, such as postischemic acute renal failure and immune-mediated nephritis [3, 4].

Figure 7 - manuscript

Fig 7. Immunofluorescence staining of cryosections of a kidney removed because of a renal cell carcinoma using rabbit anti human C3-specific antibody. Tubular epithelial cells contain intracellular C3 (left panel). Merging C3 staining with DAPI nuclear staining (right panel).

(R1B): To further substantiate and affirm our data, we performed a C3-specific RT-qPCR transcriptome analysis from the urinary sediment of AKI patients with and without kidney transplantation. This investigation was initiated in response to the comments of the reviewers.

These data were performed solely for the purpose of review and were not included into our manuscript as they provide data from a partially different cohort. This is because urine sediment was only available from a minor number of study participants:

Methods: 125 urine samples of a total of 64 patients were utilized. Twenty-four patients were kidney transplant recipients, one patient had received bone marrow transplantation and all patients experienced AKI. Urine sediment was palleted and RNA was extracted out of the sediment by mixing with Trizol. The Trizol lysate was then mixed with chlorophorm for precipitating the total RNA using isopropanol, as described in the Trizol test manual. Purified RNA was dissolved in RNAse free water and mixed with dNTPs random hexamer primers and reverse transcriptase using superscript enzyme. The resulting cDNA was diluted 1:4 with H2O and amplified using C3 specific probes from TaqMan® (Hs01100881_m1, Thermo Fisher Scientific) and 2x TaqMan® Universal Master Mix in a StepOnePlus qPCR machine (Applied Biosystems®). Data recording was performed over 46 cycles. Individual C3 expression levels in terms of cycle threshold (Ct) were normalized using GAPDH as house-keeping gene resulting in a ΔCt value, and further calculated using the ΔΔCt method [5]. Results: Forty-six samples showed no expression of C3 until 44 cycles and were therefore considered negative. Seventy-nine samples exhibited C3 transcripts in urine sediment cells or cell fragments, indicative for C3 synthesis.

Relative C3 expression normalized to GAPDH is given below in Reviewer material I.

Reviewer material I. Relative C3 expression in urinary sediments of AKI patients normalized to GAPDH. 125 urine samples of 64 patients were analyzed: 24 patients were kidney transplant recipients; one patient had received bone marrow transplantation and all patients experienced AKI. Each bar represents one patient. Values are given as relative expression of C3 normalized to GAPDH in urinary sediments.

(C2) The authors used sepsis patients and analyzed the kidney injury and C3. However, C3 and kidney injury are both too much related to sepsis and any correlation shown in this study may be the result of their respective correlation with sepsis. This is a major statistical error in correlation study.

(R2): As mentioned in R1, several other conditions are known, which lead to renal synthesis of C3, such as postischemic injury and immune mediated nephritis [3, 4]. However, we have included an analysis for the reviewer to validate our data and exhibit that also in contrast nephropathy C3/C3a excretion in urine is elevated one day after administration of contrast agent. It might be assumed that C3/C3a is upregulated in response to the adverse effect caused by the contrast agent or might exhibit cell-protective effects, such as autophagy. However, this is the content of work only performed for affirming our data and will not be included in the present study but are elucidated for the reviewer.

Reviewer material II. Urinary C3/C3a levels increase one day after intravenous CT contrast agent application. 45 patients were included in this work. Day 1 represents urinary C3/C3a levels prior to contrast agent application. Urinary C3/C3a levels increased on day 2, after application of contrast-CT and went down on day 3. Before contrast application all patients were checked for being negative for any inflammatory process.

(C3) The authors found no correlation between AKI stage and C3 concentration. This means the study gives a negative result. However, the authors claims C3 molecules a good indicator. The conclusion contradicts with the data.

(R3): Thank you for this valuable comment. Our study stated that C3a/C3 level correlated negatively with serum CRP, however, did not correlate with peak serum creatinine, peak leukocyte count and age. The individual patients’ inflammatory levels (i.e. CRP and urinary C3a/C3 levels) were independent of urinary output and glomerular filtration rate. We do not claim in this study, that urinary C3a/C3 levels are a good indicator for renal function (which would be of little to no clinical use), but rather an indicator for an acute phase reaction and cellular stress condition to the kidneys. We acknowledge that our statement can be misleading and therefore included in the Discussion section as follows:

Discussion, last paragraph , page 15:

C3a/C3 is detected in urine upon infection-associated stimuli but declines rapidly following recovery from disease. It appears to represent an indicator of an acute phase reaction and stress causing condition, which in part might be involved in the regeneration of AKI-associated damage to epithelial cells, despite AKI-stages not being associated with urinary C3a/C3 levels in the measured timely interval.

(C4) Urine concentration of a specific protein is very variable. So many factors interfere with the urine amount. Unless the authors provide a practical and standardized method, the study and its clinical relevance are not valid.

(R4): Thank you for this comment. The study was conducted as pilot study as mentioned in the title and does not claim to provide a novel, practical or standardized method for the measurement of urinary inflammatory parameters but rather give a new viewpoint into the understanding of inflammatory or stressing mechanisms involving the kidneys. Standardizing urinary C3a/C3 expression to urinary output or GFR would unfortunately miss the point of this study as inflammation is not necessarily associated with urinary output or renal function. Due to individual patients’ genetics and comorbidities standardizing urinary outputs to C3a/C3 levels would probably falsify the data.

Reviewer #2:

(C1) This study mainly rely on mAb 3F7E2 (C3a/C3 specific mouse monoclonal antibody) to detect C3a/C3. Why the specific Abs each of C3 and C3a were not used? Using of mAb 3F7E2 made it hard to distinguish whether the production of C3 or the activation of C3 is altered during the study.

(R1): Thank you for this important comment. To estimate activation of C3 a C3a-ELISA was performed and methods and results included into this revision.

Methods and materials, Page 7, paragraph 2:

Urine C3a ELISA

The human C3a ELISA (Invitrogen, BMS2089) was carried out as indicated in the test manual. Urine samples were thawed under airflow. The diluted (1:4 or 1:2 in sample diluent) urine samples (100µl) were pipetted into the prewashed test wells and incubated for two hours under constant shaking together with the provided standard series which was prepared in the sample diluent. Following washing on an automated ELISA washer (three times) the biotin-conjugate prepared in the assay buffer (100µl) was pipetted into each test well and incubated for one hour at RT under constant shaking. Following a second washing step again three times, the streptavidin-HRP prepared in the assay buffer provided with the test kit (100µl) was added into each well and incubated for one hour under constant shaking at RT. After a final washing step, the TMB substrate solution (100µl) was pipetted into each well and reacted at RT for 20 minutes under light protection. The reaction was then stopped by adding 100µl stop solution. The test signal was read at 450nm at an ELISA reader and the sample concentrations were calculated according to the standard curve using the Gen5 version 2.03 program.

Results, Page 9-10, subheading ELISA testing:

As depicted in the left panel of Fig 2 the highest mean overall level of C3a/C3 was found on the second day of measurement, which represented the second day of treatment (all p<0.05). Selectively measured C3a was detectable in urine over all 6 days (Fig 2 right panel). Out of 230 samples 178 exhibited detectable levels of C3a as shown in (Fig 2 right panel).

Page 14-15, last and first paragraph:

For that reason, we performed a C3a specific ELISA measurement, but C3a represents a small molecule capable of passing the glomerular filter.

Figure 2 manuscript:

Fig 2. ELISA-measurement of urinary complement factor C3a/C3 and C3a in 85 ICU patients. Data are presented in columned scatter graphs; the horizontal lines mark the mean values of the respective C3a/C3 (left) and C3a (right) levels. Day 1: n=78; day 2: n=78; day 3: n=72; day 4: n=66; day 5: n=56; day 6: n=48.

(C2) Table 1 shows majority of patients had Pneumonia. Is there the possibility C3 from respiratory system transfer to Urinary system and affect the urine C3 level?

(R2): Thank you for this important comment. It is very unlikely that C3 from the respiratory system was transferred to the urinary tract. In fact, local synthesis of C3 in renal cells has been described in the past. We therefore added a statement to the discussion, giving already published data of local C3 synthesis by former authors.

Discussion, paragraph 2, page 13:

Local synthesis of C3 has already been described in the past. Sack S and colleagues were able to describe that glomerular mesangial cells are capable of producing C3 and C4, with an increase in C4 expression after stimulation with interferon-gamma (INF-gamma), whereas C3 expression remains unaffected under interferon-gamma stimulation [1]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with INF-gamma lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by INF-gamma [2]. In addition, increased local C3 synthesis has been described in human diseases, such as postischemic acute renal failure and immune-mediated nephritis [3, 4].

(C3) For Table 2, the finding from this study show Peak-uC3a/C3 is negatively correlated with CRP. Based on relevant literature, activation of complement factors are associated with increase inflammatory reaction. C3a levels or C3a to C3 ratio should be check to support authors’ idea.

(R3): Due to the fact that the mAb 3F7E2 based ELISA detects both C3a and C3 no ratio of C3a and C3 could be calculated. However, we performed following the suggestion of the reviewer a commercially available C3a specific ELISA and provide urine concentration levels of C3a levels in an additional graph (Fig. 2 right panel).

(C4) For Fig1, the authors claim that Dot blot initial screens test show decline in urinary C3a/C3 on Day 5 and 6. Please consider developing numeric scoring according to dot color and its C3a/C3 levels.

(R4): Thank you for this important comment. Values were assigned and the following table was added to the manuscript.

Methods section, subheading Dot plot, page 6:

Dot blot densitometric evaluation was performed using FusionCapt Advance Solo 4 16.06.

Table 1, page 17:

Table 1. Densitometric values of C3/C3a dot blot analysis of 20 AKI patients at the ICU. Urine samples were obtained at consecutive time points over 6 days.

ID Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

a 307 260 355 411 100 91

b 56 98 165 79 97 38

c 167 154 223 52 61 na

d 36 44 222 74 38 na

e 105 97 74 41 39 58

f 34 50 158 169 109 na

g 40 78 93 50 184 na

h 294 na 239 95 na na

i 164 70 130 107 36 47

j 248 239 370 198 85 na

k 48 207 78 63 76 na

l 104 138 115 68 90 77

m 372 327 162 168 249 na

n 161 238 123 69 na na

o 112 113 85 297 724 na

p 108 49 52 59 113 200

q 51 134 140 45 97 144

r na na 54 40 141 267

s na 302 197 116 686 490

t 186 150 398 327 436 438

(C5) For Fig 4, degradation of C3 is mentioned in results. Though, C3a band seems not clear. Please indicate C3 and C3a location.

(R5): Thank you for this important comment. Fig 4 does show C3 only, C3a is not shown. This was clarified in the Figure caption. Also, the sentence regarding the degradation was corrected.

Figure 4 - manuscript:

Fig 4. C3 serum und urine immunoblot using mAb 3F7E2. Ten microlitres of human urine (patient#1 and #2) was loaded onto a 10% SDS-PAGE transferred to nitrocellulose and incubated with mAb 3F7E2 (right panel). 0.4µl of human serum (patient#1 and #2) was loaded onto a 10% SDS-PAGE transferred to nitrocellulose and incubated with mAb 3F7E2 (middle panel). Molecular weight marker is shown at the left panel. Shown is a representative experiment out of two. The entire 190 kDa C3 molecule was detected. C3a could not be delineated.

(C6) For Fig2, the indication should be changed to urinary C3a/C3. Some patients couldn’t make it until Day 5-6 with worsened condition. If C3/C3a is related with disease severity, it may affect decreased C3a/C3 level on Day 5-6.

(R6): Thank you for this important comment. The indication was changed accordingly (see C1 – Figure 2 manuscript).

(C7) For Fig3, please change the graph to dot form for the consistency.

(R7): Thank you for this valuable comment. We have changed the graph.

Figure 3 manuscript:

Fig 3. Peak urinary C3a/C3 level in patients categorized for stage of AKI. Peak levels of urinary C3a/C3 did not correlate with AKI stage. AKI 0 (n=21), AKI 1 (n=37), AKI 2 (n=10) AKI 3 (n=17).

(C8) Fig 6 shows intracellular deposit of C3a/C3 in epithelial cells from urine sediment. Please include IF from control group without systemic inflammation or AKI.

(R8): Thank you for this valuable comment. We included a figure with IF of a control patient without systemic inflammation or AKI and included it into Figure 6.

Results, subheading Immunofluorescence, page 11:

A negative control from a patient with non-inflammatory condition is included in Fig 6D.

Figure 6:

Fig 6. Immunofluorescence staining of urinary sediment using mAb 3F7E2. Tubular epithelial cells contain intracellular granules with C3 (A, B). Bacteria present in the urinary sediment are veiled in C3a/C3 to some extent (C). The scale bar represents 10 µm. Immunofluorescence staining of a urinary sediment of a patient without systemic inflammation and AKI served as control (D). The scale bar represents 10 µm.

References

1. Sacks S, Zhou W, Campbell RD, Martin J. C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells. Clin Exp Immunol. 1993;93(3):411-7. Epub 1993/09/01. PubMed PMID: 8370168; PubMed Central PMCID: PMC1554924.

2. Sacks SH, Zhou W, Pani A, Campbell RD, Martin J. Complement C3 gene expression and regulation in human glomerular epithelial cells. Immunology. 1993;79(3):348-54. Epub 1993/07/01. PubMed PMID: 8406564; PubMed Central PMCID: PMC1421987.

3. Farrar CA, Zhou W, Lin T, Sacks SH. Local extravascular pool of C3 is a determinant of postischemic acute renal failure. Faseb J. 2006;20(2):217-26. Epub 2006/02/02. doi: 10.1096/fj.05-4747com. PubMed PMID: 16449793.

4. Sacks SH, Zhou W, Andrews PA, Hartley B. Endogenous complement C3 synthesis in immune complex nephritis. Lancet. 1993;342(8882):1273-4. Epub 1993/11/20. PubMed PMID: 7901586.

5. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. Epub 2002/02/16. doi: 10.1006/meth.2001.1262. PubMed PMID: 11846609.

NOTE: Please note, that Figure numbers were updated, as they appear in the latest version of the manuscript.

18 Jun 2021

PONE-D-21-05396R1

Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease? – A pilot study

PLOS ONE

Dear Dr. Gerges,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

Specifically:

Your revised manuscript did not address main technical concerns and the data is sufficient to support the conclusions. However, the manuscript is interesting and  you may submit it as "new submission" after addressing all major technical aspects.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The current version of “Are urinary C3 levels associated with the renal acute phase reaction in acute kidney disease? – A pilot study” did not answer the following critical concerns. The results are not sufficient to support the conclusion of the study.

1. The authors cannot trace the source of urine C3. Even though other studies and the authors found an expression of C3 from kidney cells, it is still very likely that most of the C3 found in the urine is from the liver. The authors proposed that the C3 is too high in molecular weight so that it cannot pass the filtration barrier. However, immunoglobulins are frequently found to pass through the same barrier in kidney conditions and they have similar molecular weight. These claims are thus not convincing.

2. The authors did not exclude the possibility that the C3 production and the kidney damage are both independently correlated to the sepsis, while C3 production/leakage and kidney damage are not linked in the current study.

3. The authors claimed that the urinary C3 an “indicator for an acute phase reaction and cellular stress condition to the kidneys”. However, according to the experimental method, the authors did not trace the course of disease to define an acute phase. Refining an acute phase of sepsis is very difficult to achieve in practice. Data show that C3 and CRP concentration are negatively correlated, which is the opposite of the conclusion, as CRP is one of the best indicators of acute phase reaction. The authors did not examine cellular stress of the kidney either. These conclusions are not supported by the data.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

- - - - -

For journal use only: PONEDEC3

12 Jul 2021

(C1) The authors cannot trace the source of urine C3. Even though other studies and the authors found an expression of C3 from kidney cells, it is still very likely that most of the C3 found in the urine is from the liver. The authors proposed that the C3 is too high in molecular weight so that it cannot pass the filtration barrier. However, immunoglobulins are frequently found to pass through the same barrier in kidney conditions and they have similar molecular weight. These claims are thus not convincing.

(R1A): We hereby attempt to defend and clarify our experience and position, although we understand and have included the reviewer's statements as strong arguments: As mentioned before, local synthesis of C3 has already been shown in the past by Sacks S et al. Sack S et al described the capability of glomerular mesangial cells to produce C3 and C4, with an increase in C4 expression after stimulation with interferon-gamma, whereas C3 expression remains unaffected under interferon-gamma stimulation [1]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed, and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with interferon-gamma lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by interferon-gamma [2]. In a further study using human samples of patients with postischemic acute renal failure, local synthesis of C3 in the tubule was described and the authors were able to distinguish tubular C3 from C3 of hepatic origin [3]. In addition, another study described enhanced local C3 production in immune-mediated nephritis [4].

IgG is ascribed a size of 150 kDa, whereas C3 is estimated to be significantly larger at 190 kDa; moreover, the molecular structure of IgG is quite different from that of C3. This argues against the reviewer's hypothesis that IgG excretion can be taken as a comparative molecule to C3 in its amount of excretion through the slit diaphragm of the glomerulus in sepsis.

In addition, in all individuals included in the Human Protein Atlas, which is open access, C3 is expressed to variable extent such as shown below. The extracted figure below represents the individual CAB004209, Female, age 56 and was stained for C3 by a rabbit monospecific antibody.

Reviewer material I. This figure has not been produced by our research group. It represents one extract from the human protein atlas: https://www.proteinatlas.org/ENSG00000125730-C3/tissue/kidney#img. Further images can be seen obtained each from another individual even in children age 7, but all express C3.

Concerning our study, immunofluorescence staining clearly demonstrates tubular epithelial cells containing intracellular C3 (Fig. 7 in the manuscript).

Figure 7 from main manuscript

Fig 7. Immunofluorescence staining of cryosections of a kidney removed because of a renal cell carcinoma using rabbit anti human C3-specific antibody. Tubular epithelial cells contain intracellular C3 (left panel). Merging C3 staining with DAPI nuclear staining (right panel).

Two modes of secretion are known in biology. It has to be assumed that C3 is not constitutively secreted into urine. There must be a regulated mode of secretion from nephronic epithelial cells. A specific stimulus secretion coupling has to exist. One of them might occur in infection and sepsis. The exact triggering molecule is not characterized but it must differ from that in the liver.

To further support and confirm our data, we performed C3-specific RT-qPCR transcriptome analysis from urine sediments of AKI patients with and without kidney transplantation.

This study was initiated in response to reviewer comments. These data were performed for review purposes only and were not included in our manuscript because they provide data from a partially different cohort. This is because urine sediment was only available from a small number of study participants. In this analysis, forty-six samples showed no expression of C3 by cycle 44 and were therefore considered negative. Seventy-nine samples had C3 transcripts in the cells or cell fragments of the urine sediment, indicating C3 synthesis.

Methods: 125 urine samples of a total of 64 patients were utilized. Twenty-four patients were kidney transplant recipients, one patient had received bone marrow transplantation and all patients experienced AKI. Urine sediment was palleted and RNA was extracted out of the sediment by mixing with Trizol. The Trizol lysate was then mixed with chlorophorm for precipitating the total RNA using isopropanol, as described in the Trizol test manual. Purified RNA was dissolved in RNAse free water and mixed with dNTPs random hexamer primers and reverse transcriptase using superscript enzyme. The resulting cDNA was diluted 1:4 with H2O and amplified using C3 specific probes from TaqMan® (Hs01100881_m1, Thermo Fisher Scientific) and 2x TaqMan® Universal Master Mix in a StepOnePlus qPCR machine (Applied Biosystems®). Data recording was performed over 46 cycles. Individual C3 expression levels in terms of cycle threshold (Ct) were normalized using GAPDH as house-keeping gene resulting in a ΔCt value, and further calculated using the ΔΔCt method [5].

Results: Forty-six samples showed no expression of C3 until 44 cycles and were therefore considered negative. Seventy-nine samples exhibited C3 transcripts in urine sediment cells or cell fragments, indicative for C3 synthesis. Relative C3 expression normalized to GAPDH is given below in Reviewer material II.

Reviewer material II. Relative C3 expression in urinary sediments of AKI patients normalized to GAPDH. 125 urine samples of 64 patients were analyzed: 24 patients were kidney transplant recipients; one patient had received bone marrow transplantation and all patients experienced AKI. Each bar represents one patient. Values are given as relative expression of C3 normalized to GAPDH in urinary sediments.

Furthermore, we have again included a further analysis only performed for the purpose of review, which shall serve to validate our data. It exhibits that also in contrast nephropathy excretion of C3/C3a in urine is increased one day after administration of contrast agent. Therefore, it might be assumed that C3/C3a is upregulated or the secretion out of the tubular cells is stimulated in response to the adverse effect caused by the contrast agent or might exhibit cell-protective effects, such as autophagy. At this point it must be mentioned that none of these patients exhibited signs of infection and did not show elevated CRP levels. These data are nevertheless content of another study and are shown here for the sole purpose of review to again substantiate our data and will not be included in the present study but are elucidated for the reviewer.

Reviewer material III. Urinary C3/C3a levels increase one day after intravenous CT contrast agent application. 45 patients were included in this work. Day 1 represents urinary C3/C3a levels prior to contrast agent application. Urinary C3/C3a levels increased on day 2, after application of contrast-CT and went down on day 3. Before contrast application all patients were checked for being negative for any inflammatory process.

What seems very probable is that C3 is synthesized locally in tubular cells, as has been described in prior literature. We have included a statement about the origin of C3 with the available literature to our discussion section within the previous revision.

Page 13, paragraph 1, Discussion,

Local synthesis of C3 has already been described in the past. Sack S and colleagues were able to describe that glomerular mesangial cells are capable of producing C3 and C4, with an increase in C4 expression after stimulation with INF𝛾, whereas C3 expression remains unaffected under interferon-gamma stimulation [1]. These data are substantiated by a further study, which demonstrates that C3 is synthesized, processed, and secreted by glomerular epithelial cells under basal conditions, with the C3 alpha and beta polypeptide chains having identical electrophoretic mobilities with those of hepatic C3. In contrast to the study mentioned above, stimulation with INF𝛾 lead to an increase in C3 gene expression, indicating that C3 expression in glomerular epithelial cells is regulated by INF𝛾 [2]. In addition, increased local C3 synthesis has been described in human diseases, such as postischemic acute renal failure and immune-mediated nephritis [3, 4].

(C2) The authors did not exclude the possibility that the C3 production and the kidney damage are both independently correlated to the sepsis, while C3 production/leakage and kidney damage are not linked in the current study.

(R2) Transcription factor and repressor proteins are expressed in a tissue- and cell subtype-specific manner. For example, renal epithelial cells express different transcription factors and repressor proteins than hepatocytes. The function of these proteins is to interact with specific responsive elements on the DNA of genes. In recent years, miRNAs have been characterized as similar players at the mRNA level and thereby can regulate translation and mRNA turnover. As a result, different tissues are able to respond differently to the same stimulus such as interleukins, toxic compounds, drugs, and nutrients. This response is achieved by regulating gene expression and protein production. With regard to our study, we must hypothesize that the C3 gene in the kidney is regulated differently than the CRP gene in the liver. Moreover, the dynamics of CRP in serum is much more brisk than that of C3.

(C3) The authors claimed that the urinary C3 an “indicator for an acute phase reaction and cellular stress condition to the kidneys”. However, according to the experimental method, the authors did not trace the course of disease to define an acute phase. Refining an acute phase of sepsis is very difficult to achieve in practice. Data show that C3 and CRP concentration are negatively correlated, which is the opposite of the conclusion, as CRP is one of the best indicators of acute phase reaction. The authors did not examine cellular stress of the kidney either. These conclusions are not supported by the data.

(R3): As described in the Methods section under the subheading “sample collection” only patients admitted to the intensive care unit (ICU) fulfilling at least 4 of 10 sepsis criteria were included into this study [6]. The sepsis criteria were established to define an acute phase of sepsis and are therefore the adequate tool to substantiate the fact that our patients were acutely ill. It is true, that CRP and urinary C3a/C3 levels correlated negatively with serum CRP (p<0.0001), however the mAb 3F7E2 based ELISA detects both, C3 and C3a. Consumption and activation of C3 is a known marker for determining the activity of inflammatory diseases, such as rheumatic diseases or atypical hemolytic uremic syndrome. Thereby, a decrease of C3 stands for increased inflammation. Therefore, it can be said, that for example like serum albumin, C3 represents an inverse acute phase parameter.

It is true that the CRP level is the best indicator of an acute-phase response, which is orchestrated by the liver and represents the response to a pathological stimulus. However, we disagree with the reviewer's assumption that the CRP level may be one of the best indicators of an acute-phase response in the kidney. The site in the kidney directly affected by sepsis is the endothelium and signaling to the tubular epithelial cells and podocytes occurs with a time delay. The tubular epithelial cells undergo specific response processes involving IFN signaling and STAT1. The kidney has different modalities to respond, often delayed, such as upregulation of alpha-2-macroglobulin, while CRP is not found elevated or not elevated in the blood in such disease [7].

An attempt to follow the reviewer's suggestion to better represent the acute phase reaction in the kidney, we wanted to elucidate the activation of C3 at the site of renal secretion, a commercially available C3a ELISA was performed in response to an acute phase reaction, and the data have already been included in the previous review. Again, the reviewer might say that this is from blood, since C3a is a small molecule. This is contradicted by the assumption that C3a has a very short half-life and is rapidly degraded by proteases in the blood.

Methods, page 7, paragraph 2

Urine C3a ELISA

The human C3a ELISA (Invitrogen, BMS2089) was carried out as indicated in the test manual. Urine samples were thawed under airflow. The diluted (1:4 or 1:2 in sample diluent) urine samples (100µl) were pipetted into the prewashed test wells and incubated for two hours under constant shaking together with the provided standard series which was prepared in the sample diluent. Following washing on an automated ELISA washer (three times) the biotin-conjugate prepared in the assay buffer (100µl) was pipetted into each test well and incubated for one hour at RT under constant shaking. Following a second washing step again three times, the streptavidin-HRP prepared in the assay buffer provided with the test kit (100µl) was added into each well and incubated for one hour under constant shaking at RT. After a final washing step, the TMB substrate solution (100µl) was pipetted into each well and reacted at RT for 20 minutes under light protection. The reaction was then stopped by adding 100µl stop solution. The test signal was read at 450nm at an ELISA reader and the sample concentrations were calculated according to the standard curve using the Gen5 version 2.03 program.

Results, page 9-10, paragraph 3

As depicted in the left panel of Fig 2 the highest mean overall level of C3a/C3 was found on the second day of measurement, which represented the second day of treatment (all p<0.05). Selectively measured C3a was detectable in urine over all 6 days (Fig 2 right panel). Out of 230 samples 178 exhibited detectable levels of C3a as shown in (Fig 2 right panel).

Fig 2. ELISA-measurement of urinary complement factor C3a/C3 and C3a in 85 ICU patients. Data are presented in columned scatter graphs; the horizontal lines mark the mean values of the respective C3a/C3 (left) and C3a (right) levels. Day 1: n=78; day 2: n=78; day 3: n=72; day 4: n=66; day 5: n=56; day 6: n=48.

References

1. Sacks S, Zhou W, Campbell RD, Martin J. C3 and C4 gene expression and interferon-gamma-mediated regulation in human glomerular mesangial cells. Clin Exp Immunol. 1993;93(3):411-7. Epub 1993/09/01. PubMed PMID: 8370168; PubMed Central PMCID: PMC1554924.

2. Sacks SH, Zhou W, Pani A, Campbell RD, Martin J. Complement C3 gene expression and regulation in human glomerular epithelial cells. Immunology. 1993;79(3):348-54. Epub 1993/07/01. PubMed PMID: 8406564; PubMed Central PMCID: PMC1421987.

3. Farrar CA, Zhou W, Lin T, Sacks SH. Local extravascular pool of C3 is a determinant of postischemic acute renal failure. Faseb J. 2006;20(2):217-26. Epub 2006/02/02. doi: 10.1096/fj.05-4747com. PubMed PMID: 16449793.

4. Sacks SH, Zhou W, Andrews PA, Hartley B. Endogenous complement C3 synthesis in immune complex nephritis. Lancet. 1993;342(8882):1273-4. Epub 1993/11/20. PubMed PMID: 7901586.

5. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. Epub 2002/02/16. doi: 10.1006/meth.2001.1262. PubMed PMID: 11846609.

6. Funk D, Sebat F, Kumar A. A systems approach to the early recognition and rapid administration of best practice therapy in sepsis and septic shock. Curr Opin Crit Care. 2009;15(4):301-7. Epub 2009/06/30. doi: 10.1097/MCC.0b013e32832e3825. PubMed PMID: 19561493.

7. Menon R, Otto EA, Hoover P, Eddy S, Mariani L, Godfrey B, et al. Single cell transcriptomics identifies focal segmental glomerulosclerosis remission endothelial biomarker. JCI Insight. 2020;5(6). Epub 2020/02/29. doi: 10.1172/jci.insight.133267. PubMed PMID: 32107344; PubMed Central PMCID: PMCPMC7213795.

Submitted filename: point-to-point response PlosONEdocx.docx

Click here for additional data file.

27 Oct 2021

Urinary C3 levels associated with sepsis and acute kidney injury – A pilot study

PONE-D-21-05396R2

Dear Dr. Gerges,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

29 Oct 2021

PONE-D-21-05396R2

Urinary C3 levels associated with sepsis and acute kidney injury – A pilot study

Dear Dr. Gerges:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Partha Mukhopadhyay

Section Editor

PLOS ONE

Table 1

Densitometric values of C3/C3a dot blot analysis of 20 AKI patients at the ICU.

Urine samples were obtained at consecutive time points over 6 days.

ID	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
a	307	260	355	411	100	91
b	56	98	165	79	97	38
c	167	154	223	52	61	na
d	36	44	222	74	38	na
e	105	97	74	41	39	58
f	34	50	158	169	109	na
g	40	78	93	50	184	na
h	294	na	239	95	na	na
i	164	70	130	107	36	47
j	248	239	370	198	85	na
k	48	207	78	63	76	na
l	104	138	115	68	90	77
m	372	327	162	168	249	na
n	161	238	123	69	na	na
o	112	113	85	297	724	na
p	108	49	52	59	113	200
q	51	134	140	45	97	144
r	na	na	54	40	141	267
s	na	302	197	116	686	490
t	186	150	398	327	436	438

na = not applicable.

Table 2

Demographics and disease status.

	ICU (n = 85)
Age in y (mean ± SD)	58.88 ± 15.02
Age in y (median; min, max)	62 (15, 86)
Male/Female	49/36
Pneumonia n (%)	49 (57.6%)
UTI n (%)	6 (7.1%)
CPR n (%)	16 (18.8%)
MCI n (%)	8 (9.4%)
TX n (%)	10 (11.8%)
sCr (mean ± SD; mg/dL)	1.87 ± 1.66
CRP (mean ± SD; mg/dL)	18.00 ± 11.24
no AKI n (%)	21 (24.7%)
AKI stage 1 n (%)	37 (43.5%)
AKI stage 2 n (%)	10 (11.8%)
AKI stage 3 n (%)	17 (20.0%)
AKI on CKD n (%)	28 (32.9%)
CKD without AKI (%)	1 (1.2%)

AKI–acute kidney injury; CKD–chronic kidney disease; CPR–cardiopulmonary resuscitation; CRP–C-reactive protein; MCI–myocardial infarction; sCr–serum creatinine; SD-standard deviation; TX–history of organ transplantation; UTI–urinary tract infection.

Table 3

Correlation of peak urinary C3a/C3 with age, CRP, WBC, peak sCr.

	CRP	WBC	age	Peak sCR	Peak-uC3a/C3
CRP	1.00	0.20	-0.04	-0.11	-0.41
	N/A	0.061	0.74	0.33	<0.0001
WBC	0.20	1.00	-0.11	0.02	-0.08
	0.06	N/A	0.30	0.87	0.46
Age	-0.04	-0.11	1.00	0.16	-0.02
	0.74	0.30	N/A	0.15	0.87
Peak sCr	-0.11	0.02	0.16	1.00	0.09
	0.33	0.87	0.15	N/A	0.39
Peak-uC3a/C3	-0.41	-0.08	-0.02	0.09	1.00
	<0.0001	0.46	0.87	0.39	N/A

Pearson correlation of CRP, WBC, log10 of peak serum creatinine during the 6 observation days (peak sCr) and log10 of peak urinary C3a/C3 (peak-uC3a/C3). The upper white line represents the correlation coefficient, the lower line in gray represents the p-value. A p-value of <0.05 was considered as statistically significant (bold). CRP–C-reactive protein; sCr–serum creatinine; WBC–white blood count.