Risk factors and predictors of urogenous sepsis after percutaneous nephrolithotomy for idiopathic calcium oxalate nephrolithiasis

Introduction

More than 1–5% of people worldwide have a history of urolithiasis (1). Calcium oxalate stones account for more than 80% of kidney stones and usually occur due to a high intake of oxalic acid or calcium forming calcium oxalate stones in the urine, which can be seen on X-ray (2). The most popular method of treatment for kidney stones is surgery. Open surgery is no longer the first option for the surgical removal of kidney stones due to the advancement and popularization of minimally invasive surgery. The preferred method of treating large, complicated upper urinary tract stones or stones that have not responded to retrograde intrarenal surgery or shock wave lithotripsy is percutaneous nephrolithotomy (PCNL) (3), the safety and efficacy of which has been improved through advancements in technology and practice. However, due to bacteria entering the patient’s bloodstream during lithotripsy, postoperative systemic inflammatory response syndrome (SIRS), with a reported incidence of ranging from 9.8% to 37%, continues to be one of the most frequent complications in PCNL (4,5). SIRS is the first step in the sepsis cascade and is closely related to the development of sepsis (6), and 0.3–4.7% of patients can develop severe life-threatening sepsis (7), while the mortality rate can reach 50–66% (8).

According to recent research, Randall’s plaque, the accumulation of calcium phosphate crystals on the renal papilla's surface, is the precursor to stone development (9). Therefore, identifying the key genes and pathways based on Randall calcium plaque is crucial for understanding how idiopathic calcium oxalate kidney stones develop. Bioinformatics analysis based on gene chip data may effectively scan and evaluate critical genes associated with the onset and progression of diseases due to the extensive use of high-throughput genomics research technologies (10). To screen the essential genes and prospective molecular mechanisms in the formation of idiopathic calcium oxalate kidney stones, our investigation first used bioinformatics techniques to explore the kidney stone gene chip data set in the public gene expression database, with the aim to identify new targets and gain insights into treating and preventing idiopathic calcium oxalate kidney stones. Then, to better inform the development of practical settings, we further investigated the association between the key genes we screened and the occurrence of postoperative urogenous sepsis. Then, based on the clinical data of patients with idiopathic calcium oxalate renal stones treated with PCNL in our hospital, we further explore the correlation between the key genes we selected and the occurrence of postoperative urinary sepsis, providing better reference opinions for clinical work. We present this article in accordance with the STREGA reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-23-219/rc).

Methods

Data collection and processing

The National Center for Biotechnology Information's gene expression database, the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) provided the kidney stone-related gene expression data set GSE73680 (11). The data set is based on the GPL17077 platform. It includes 23 cases of renal papilla tissue from individuals who had idiopathic calcium oxalate kidney stones and 6 cases with normal human renal papilla tissue. In combination with R software (The R Foundation for Statistical Computing), the platform annotation data were used to translate the gene names from the gene expression profile probe data.

Differentially expressed gene screening

The R package “limma” (https://www.r-project.org/)was used to find the differentially expressed genes in the processed GSE73680 data set, with P<0.01 and |log₂ fold change (FC)|>1 as the screening conditions. With the “ggplot2” program in R, a volcano map of the differentially expressed genes was constructed.

Examining the Search Tool for the Retrieval of Interacting Genes/Proteins database and the GeneMANIA database for genes that interact with key genes

For physiological and biochemical processes, pathways, and protein–DNA, genetic, and protein-protein interactions (PPI), we used the GeneMANIA database (http://www.genemania.org) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/). Following this, we constructed a PPI network by identifying the linked genes associated with key genes from the phenotype screening, protein domain, and protein expression perspectives.

Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment and Gene Ontology functional annotation analysis

For the differentially expressed genes, analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment and Gene Ontology (GO) functional annotation was carried out through the online biological information annotation library, Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8, with P<0.05 as the screening threshold.

Clinical information

Statistical analysis was conducted on the clinical data of 421 patients who underwent initial PCNL therapy at the Second Affiliated Hospital of Nantong University, between January 2013 and December 2017. Patients with end-stage renal disease, tumors, blood diseases, immunosuppressive disorders, horseshoe kidneys, and preoperative heartbeats of >90 beats per minute were excluded from the study. Only 135 patients experienced SIRS, and the other 21 patients developed urosepsis. As per the 2015 urinary system infection recommendations set by the European Association of Urology (EAU), we defined SIRS as urinary sepsis with severe urinary tract infection that causes pathogenic microorganisms in urine to enter the blood system through blood vessels, causing bacteremia, sepsis, and other adverse conditions (12). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by ethics board of the Second Affiliated Hospital of Nantong University (No. 2021KT158) and informed consent was taken from all the patients.

Preoperative examination and treatment

Preoperative intravenous urography (IVU), CT, ultrasonography, and kidney, ureter, and bladder (KUB) X-ray were all routinely conducted. According to standard procedure, patients with positive urine cultures receive the appropriate antibiotic treatment for 3–7 days based on culture antibiotic test findings, but urine cultures were not be repeated. Then, 30 minutes before surgery, we administered broad-spectrum prophylactic antibiotics.

Recorded metrics

Clinical characteristics including postoperative white blood cell (WBC) count, stone composition, operation time, stone location, stone size, nitrite status, urinary WBC in preoperative urinalysis, gender, and age were examined. The postoperative WBC was assessed immediately following the operation or within 3 hours. Operative time was measured from the start of the renal biopsy to the end of the nephrostomy tube insertion. Meanwhile, the components of the stones were analyzed. However, the composition of many stones can be mixed, and so we recorded the main components. Meanwhile, renal papilla tissue biopsy was performed during the operation, and the expression of the key genes we screened was judged using the immunohistochemical method.

Treatment

Participants were seated in the prone position for all surgeries while under general anesthesia. After establishing the best possible renal access, the opening was expanded with an 8–18 F fascial dilator and a corresponding Amplatz sheath. Then, we applied a pneumatic lithotripter, an 8/9.8 F semirigid ureteroscope, or Hol: YAG laser lithotripsy. Stone tweezers are used to remove larger fragments, and a pulse perfusion pump was used to flush out tiny debris. After the procedure ended, a silicone rubber nephrostomy tube and a double-J stent were inserted.

Statistical analysis

The statistical evaluation was conducted using SPSS software (v.20.0, IBM, Armonk, NY, USA). Continuous variables were compared using the independent samples t test and are presented as the mean and ± SD. The χ² test was used to compare categorical variables. To identify independent characteristics linked with the occurrence of postoperative urosepsis, multivariate logistic regression analysis was employed. The aforesaid online database automatically conducted the statistical analyses of the subsequent investigations. An acceptable P value or log-rank P value of <0.05 was used to determine statistical significance.

Results

The differentially expressed genes between the patients with idiopathic calcium oxalate nephrolithiasis and normal human renal papilla tissue

The GSE73680 data set selected in this study contains 6 cases of normal human renal papilla tissue and 26 cases of patients with calcium oxalate stones. With P<0.01 and |log₂FC|>1 as screening conditions, R software demonstrated that 7,799 genes were differentially expressed in the renal papilla of participants with idiopathic calcium oxalate kidney stones compared to normal people. Moreover, 2,282 genes were upregulated, and 1,930 were downregulated (see the volcano plot, principal component analysis plot, heat map, and sample-normalized box plot in Figure 1A). Compared with those in healthy people, downregulated genes may have more clinical application feasibility and more research value, so we only selected genes with downregulated expression among common differential genes for further analysis. Considering that the pyroptosis mechanism is one of the research hotspots in the current research on the mechanism of kidney stones, we took the intersection of the downregulated differentially expressed genes and all pyroptotic genes to construct a Venn diagram (Figure 1B). From this, we finally obtained 1 differentially expressed gene, nucleotide-binding oligomerization domain-containing protein 2 (NOD2). The definitions of pyroptosis-related genes vary significantly across the different articles, and thus we selected the 33 most widely used genes from the related literature (13-16), the detailed summary of which is shown in Table 1.

Figure 1 Genes that were differently expressed in idiopathic calcium oxalate nephrolithiasis were screened. (A) The volcano map, principal component analysis map, sample-normalized box plot, and the heat map of differentially expressed genes in the GSE73680 data set. Blue points indicate downregulation, red points indicate upregulation, and black points represent |log FC| <1. (B) Venn diagram of the downregulated genes and all pyroptotic genes in the GSE73680 data set. FC, fold change.

Identification of NOD2 interacting genes and proteins

Using GeneMania, we created a gene-gene interaction network of NOD2 and adjusted the neighboring genes (Figure 2A). The network of NOD2 PPIs was established using the STRING database (Figure 2B).

Figure 2 Identification of the NOD2 interacting genes and proteins. (A) Gene-gene interaction network of NOD2 in the GeneMania database. (B) PPI network of NOD2 in the STRING database. NOD2, nucleotide binding oligomerization domain 2; PPI, protein-protein interaction.

KEGG and GO enrichment analysis

Using the DAVID online database, the enrichment analysis of genes interacting with NOD2 in the PPI network diagram was carried out with the background of Homo sapiens, and enrichment information on GO was obtained. The term “biological process” refers to a group of processes that primarily controls the NLRP3 inflammasome complex, necrotic cell death, cytokine-mediated signaling pathways, cytoplasmic pattern recognition receptor signaling pathways, and I-κB kinase/nuclear factor (NF)-κB signaling, among others (Figure 3A and Table 2). For the term “cellular composition”, interacting genes were mainly enriched in the serine/threonine protein kinase complex, plasma membrane receptor complex, CD40 receptor complex, phagophore, inflammasome complex, and ubiquitin junction enzyme complexes, among others (Figure 3B and Table 3). For the term, "molecular function", the interacting genes were mainly linked to the caspase recruitment domain, ubiquitin-binding, cysteine-type endopeptidase, enzyme regulation activity, apoptosis process, protein serine/threonine amino acid kinase activity, cytokine receptor binding, and protein tyrosine kinase activity (Figure 3C and Table 4). The enrichment analysis of KEGG signaling pathways revealed that the interacting genes were mainly related to T helper 1 (Th1) and Th2 cell differentiation, PI3K-Akt signaling pathway, programmed death-ligand 1 (PD-L1) expression in cancer and programmed cell death protein 1 (PD-1) checkpoint pathway, the NOD (Nucleotide Binding Oligomerization Domain Containing)-like receptor signaling pathway, MAPK(mitogen-activated protein kinase) signaling pathway, tumor necrosis factor (TNF) signaling pathway, and T-cell receptor signaling pathway, among others (Figure 3D and Table 5).

Figure 3 The outcomes of the GO and KEGG enrichment analysis. (A) Results of BP enrichment, (B) CC enrichment, (C) MF enrichment, and (D) KEGG signaling pathway enrichment analysis. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cell component; MF, molecular function.

General information about the enrolled patients

This retrospective study ultimately included 156 patients treated for PCNL. Of these, 135 patients only developed SIRS, while 21 developed urosepsis postoperatively. Preoperative urinary nitrite, calculus diameter, operating duration, WBC count, and WBC D values all showed statistically significant differences between the 2 groups (P<0.05; Table 6).

Univariate and multivariate analysis of urogenous sepsis risk variables following PCNL

In renal papillary tissue samples, we assessed the expression of NOD2 using immunohistochemistry. The immunohistochemical data were graded on the basis of the degree of staining in the cells and the percentage of positive cells. They were scored according to staining intensity and the percentage of positively stained cells. Staining intensity was scored as follows: 0 = negative or no staining, 1 = weakly positive, 2 = moderately positive, and 3 = strongly positive. Meanwhile, the percentage of staining was scored as follows: 0= negative, 1=≤25%, 2=25–50%, 3=51–75%, and 4≥75%. The result of multiplying the 2 scores represented the final score for each specimen. The samples with a score lower than 7 were ultimately classified as NOD2 low expression after the arithmetic mean of these scores was computed.

The Cox regression analysis was conducted on the factors that affected the occurrence of urogenous sepsis after PCNL. It was ultimately found that preoperative urinary nitrite, stone diameter, 3-hour postoperative blood WBC count, and NOD2 expression were independent prognostic factors that affected the occurrence of urogenous sepsis. Preoperative positive urinary nitrite, diameter, 3-hour postoperative WBC ≥2.98×10⁹/L, stone diameter >6 cm, and low expression of NOD2 in renal papilla tissue were more likely to cause urogenous idiopathic calcium oxalate kidney stones after PCNL sepsis (Table 7).

Discussion

Kidney stone development is a complicated process. According to the traditional view, kidney stone formation involves several biochemical functions, such as crystal nucleation, urine supersaturation, crystal growth, and aggregation (17). Earlier research presented several models to describe how kidney stones arise, such as vascular calcification and medullary–papillary complex mineralization (18). However, there are certain limitations to these models. The renal papillary Randall calcium spot model is currently a relatively accepted theory. In this model, supersaturated calcium salt crystals are deposited on the top of the renal medulla renal papillae, and the crystals gradually grow and aggregate, eventually forming urinary calculi. With the advancement of laparoscopic technology, research has revealed that Randall's calcium plaques, which frequently have stones attached, are present in the renal papilla of most patients with calcium-containing stones (19). Renal papillary biopsy studies also suggest that Randall's calcium plaques are closely related to urinary calcium levels, calcium oxalate stones, and kidney stones secondary to some metabolic diseases (20). Therefore, bioinformatics methods to study the key genes and signaling pathways of kidney stone formation based on Randall calcium plaques of renal papillae are of great value.

One study showed that urogenous sepsis is a dangerous complication after PCNL (21). The occurrence of urogenous sepsis not only aggravates the primary disease and reduces the effect of PCNL surgery but also prolongs the treatment time and increases the treatment cost and the risk of death, which has become one of the issues of high clinical concern. According to one survey, every 1-hour delay in treating urogenous sepsis increases the risk of death by 10% (22). However, the early clinical symptoms of most patients with urogenous sepsis lack specificity and only manifest as arrhythmia and body temperature changes, making it difficult to diagnose the disease accurately. Therefore, developing an early predictive index with high specificity and sensitivity to diagnose the condition of patients with urosepsis is an urgent clinical need. In order to determine the final factors that influence the development of urogenous sepsis after PCNL and to identify specific clinical values, our study combined the analysis of key genes that promote the occurrence of idiopathic calcium oxalate kidney stones with the clinical data analysis of our hospital.

In this work, the differentially expressed genes for 2,282 upregulated and 1,930 downregulated genes were examined in the renal papilla gene expression matrix of patients with idiopathic calcium oxalate nephrolithiasis, which was retrieved from the GEO database. A variety of factors are known to influence the formation and occurrence of kidney stones: for example, excessive autophagy mediated by endoplasmic reticulum stress effectively protects kidney function and impedes apoptosis and kidney stone formation (23), while protein-mediated renal oxidative damage and inflammation aggravate calcium oxalate stone formation (24); these mechanisms mainly involve inflammation and other factors. Pyroptosis is a newly recognized type of planned cell death in which cells grow until the cell membrane ruptures, spilling the cell's contents and inciting robust inflammatory and immunological responses. In-depth research on pyroptosis, which is closely involved in the development and occurrence of diseases will help us better comprehend its function in the incidence, progression, and outcome of associated diseases and elicit new ideas for clinical treatment and prevention (25,26). Moreover, pyroptosis and inflammation are also complementary in many diseases. Indeed, the mechanism of pyroptosis has emerged as one of the research hotspots in the current research on the mechanism of kidney stones. Recently, there have also been relevant studies, such as miRNA-141-3p mediated by NLRP3 in renal tubular epithelial cell injury induced by calcium oxalate (27); Long chain non coding RNA 00339 (LINC00339) promotes renal tubular epithelial cell apoptosis by regulating the miR-22-3p/NLRP3 axis in calcium oxalate induced renal stones (28). Therefore, we finally constructed a Venn diagram by intersecting the downregulated differentially expressed genes with all known pyroptotic genes and eventually obtained a single differentially expressed gene, NOD2.

Then, we constructed the NOD2-interacting gene network using the GeneMania and STRING databases and performed GO and KEGG analysis on these interacting genes. The results showed that NOD2 might affect inflammation, receptor expression, immune environment, necrosis, apoptosis, and other processes to influence the occurrence of idiopathic calcium oxalate kidney stones.

Next, we retrospectively analyzed 156 patients treated for PCNL in our hospital. Of these, 135 patients only developed SIRS, while 21 developed urosepsis postoperatively. There were statistically significant variations between the 2 groups in terms of preoperative urine WBC count, preoperative urinary nitrite, stone diameter, operation duration, WBC count, and WBC D values. According to Ferry et al. (29), urine sediment microscopy and the WBC nitrite test demonstrated the best diagnostic accuracy for lower urinary tract infections. According to their study, nitrite could improve the diagnostic efficacy for urinary tract infections, which is in agreement with our findings. Preoperative urine nitrite testing revealed that the urosepsis group had a higher positive rate (90.5% vs. 32.6%; P<0.001) than did the SIRS group, and is associated with urinary sepsis as an independent risk factor

Cox regression analysis was conducted on the factors that affect the occurrence of urogenous sepsis after PCNL which ultimately revealed that preoperative urinary nitrite, stone diameter, 3-hour postoperative blood WBC count, and NOD2 expression are the independent prognostic factors that affect the occurrence of urogenous sepsis. WBCs are the primary line of protection against external bacterial infection. In the early stages of urogenic sepsis, WBC will significantly decrease. This is due to the release of endotoxin into the bloodstream and the subsequent production of inflammatory factors that activate the systemic inflammatory response. Typically, this reaction occurs intraoperatively or within the first 2 hours after surgery (30). The WBC count then increases and peaks on the first postoperative day. WBC counts that do not increase are indicative of a poor prognosis. These observations are consistent with our findings, specifically, that urosepsis was independently linked with individuals whose postoperative WBC count was less than 2.98×10⁹/L. A study by Wu et al. (31) reported that a WBC count of 2.85×10⁹/L or below within 2 hours after surgery might predict urinary tract syndrome following endoscopic lithotripsy of the upper urinary tract. Therefore, our research results have certain accuracy and credibility. Although immunohistochemical detection of NOD2 expression is an invasive procedure, this study is more convenient and cost-effective compared with the cost of testing such as single-cell sequencing, so it has good clinical value to help early detection and intervention of urogenic sepsis after PCNL.

Our study does have several limitations, including a limited sample size and its retrospective design. In the future, we will conduct relevant prospective and/or randomized studies to further evaluate the accuracy of these results and provide more valuable information for clinical management.

Conclusions

If the patient has the following preoperative urinary nitrite positivity and blood white blood cells ≥2.98 at 3 hours after surgery × Idiopathic calcium oxalate kidney stones with a stone diameter greater than 6cm and low expression of NOD2 in renal papilla tissue at 10⁹/L are more prone to urinary sepsis after PCNL. The first 6 hours following a medical assessment of urosepsis are regarded as the “golden hour” for interventional therapy (32). Therefore, early judgment and intervention based on the above-mentioned independent factors will improve the effect of treatment for these patients.

Acknowledgments

Funding: This study was supported by the Nantong Science and Education Strong Health project (No. Key 57, Young 68), the Research Project of Nantong Municipal Health Commission (No. MB2021025), the Nantong Science and Technology Project (No. MS22022085), the Nantong Science and Technology Planning Project (No. MSZ2022101), the Jiangsu Provincial Cadre Health Research Project (No. BJ21010), and the Science and Technology Project of Jiangsu Provincial Health Commission (No. LKM2022059).

Footnote

Reporting Checklist: The authors have completed the STREGA reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-23-219/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-219/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-219/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-23-219/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by ethics board of the Second Affiliated Hospital of Nantong University (No. 2021KT158) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)