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Urolithiasis is an increasingly common clinical problem worldwide. The formation of stones is a combination of metabolic status, environmental factors, family history and many other aspects. It is important to find new ways to quickly detect and assess urolithiasis because it causes sudden, severe pain and often comes back.
- urolithiasis
- protein biomarkers
- biomarkers
1. Introduction
Urolithiasis (i.e., kidney stone disease) remains a global public health problem with increasing prevalence [1,2]. This disease is spreading in developed countries and affecting both adults and children, becoming a problem for society.
Women have a higher risk of early onset lithiasis, with a tendency to have recurrences. Late stone formation occurs more frequently in those with a BMI > 30 [3].
Urolithiasis is a disease in which deposits are formed in different parts of the urinary tract: the pyelocalyceal system, the ureter or even the bladder. The pathogenesis of the disease is complex and multifactorial. An imbalance between promoter (like calcium, oxalic acid, uric acid, cystine, cell fragments) and inhibitor (like magnesium, citrates, zinc, glycosaminoglycans, uromodulin, osteopontin, osteocalcin, bicunin) concentrations, along with specific physicochemical conditions, increases the risk of lithiasis. A very important aspect is the pH of the urine, which, depending on the type of deposits, can determine their precipitation. In the case of uric acid stones, an acidic environment stimulates their precipitation. The formation of stones is also influenced by antibiotic therapy and the reduction in intestinal colonization with the bacterium Oxalobacter formigenes. Indeed, this organism uses oxalic acid as an energy source, reducing its absorption in the gastrointestinal tract [4]. Also, a sedentary lifestyle, a diet high in animal-based protein with a high intake of salt and a low intake of water and the presence of obesity can increase the risk of stone crystallization. The chance of urolithiasis is 2–16 times higher in patients with a family history of stone disease [5]. The most common type, accounting for 70–80% of cases, is calcium oxalate (CaOx). Uric acid urolithiasis is caused by excessive excretion of uric acid as a metabolite of purine metabolism. Cystine is associated with cystinuria. Magnesium ammonium phosphate (struvite) accompanies urinary tract infection. Other less common types include calcium phosphate, xanthine and 2,8-dihydroxyadenine [6]. On suspicion of urolithiasis, a comprehensive metabolic diagnosis is required to identify risk factors. The most common include hypercalciuria, hypocitraturia, hyperuricosuria and hyperoxaluria [5,7]. Most cases of deposits in the urinary tract are diagnosed during a renal colic episode or incidentally during an abdominal ultrasound performed because of non-specific symptoms, such as abdominal pain. Ultrasonography is the most important imaging study for diagnosing urolithiasis. It is widely available and sensitive and does not involve radiation doses. In the case of diagnostic problems, such as difficult localization or size of the deposit, spiral computed tomography without contrast should be performed [8,9]. Metabolic assessment is important after stone expulsion and should be repeated multiple times. It includes blood tests, urine samples and a 24 h urine collection to test for crystallization promoters and inhibitors. Analysis of the composition of the expelled stone is also an extremely important aspect. There are currently have three methods: infrared spectroscopy, polarization microscopy and X-ray diffraction [8,10]. Based on the collected results, mainly from the metabolic assessment, targeted prophylaxis and conservative treatment can be implemented. Up to 80% of the deposits are expelled spontaneously, mainly those with a small diameter of up to 5 mm. The treatment of urolithiasis can be divided into conservative and invasive. Medical expulsion therapy (MET) relies on analgesics (NSAIDs) and agents that facilitate expulsion: alpha-blockers (doxazosin, tamsulosin, alfuzosin), calcium channel blockers (nifedipine) and corticosteroids. When conservative therapy is unsuccessful, procedures that are as minimally invasive as possible should be implemented. These include extracorporeal shock wave lithotripsy (ESWL), lithotripsy during ureteroscopy (URSL), percutaneous nephrolithotripsy (PCNL) and retrograde intrarenal surgery (RIRS). Because of the efficacy and prevalence of the above methods, classical surgical treatment is a rare choice [8,9].
2. Protein Biomarkers in Urolithiasis
2.1. Biomarkers of Tubular Injury
There is a strong link between urolithiasis and renal tubular injury. On the one hand, there is evidence that oxalate and CaOx crystals injure tubular epithelium.
Research using animals found that having CaOx crystals in the kidney can cause tubular cell damage and result in enzymes and debris in urine [37,38,39].
On the other hand, there are studies suggesting that tubular cell injury may stimulate crystallization. The report of Wiessner J. H. et al. revealed that both individual cells and total tubular monolayer injury exposed cell surfaces resulting in increased affinity for crystal adhesion and their further retention in the collecting duct [40].
Bigger stones, during passage down the ureter, may obstruct renal outflow, causing acute or chronic kidney injury. In patients with renal colic because of urolithiasis, elevation in creatinine serum concentration is often observed. Nevertheless, this classical kidney function indicator lacks specificity in detecting the underlying cause of renal damage and rises late in the process of acute injury. There are various biomarkers proposed to identify and monitor the process of kidney injury. A few of them may apply to urolithiasis as well.
2.2. Cystatin C
Cystatin C has definitely gained importance in diagnosing AKI and CKD. It is a low molecule (13.3 KD) removed from the bloodstream by the kidneys, and its serum levels are a more precise test of kidney function than serum creatinine levels [41]. Most studies prove that cystatin C levels are less dependent on age, gender, ethnicity, diet or muscle mass when compared to creatinine [42]. Cystatin C is a good kidney biomarker in a range of different conditions, including diabetic patients, CKD and after kidney transplantation [43].
2.3. Neutrophil Gelatinase-Associated Lipocalin (NGAL)
Neutrophil gelatinase-associated lipocalin NGAL is a 25 kDa protein bound to gelatinase from neutrophils. Its expression was shown in the proximal and distant tubular cells of the kidney [44]. NGAL is upregulated during the inflammatory process because it meditates cellular proliferation and differentiation and has a bacteriostatic effect [45]. It is a marker of great interest in acute tubular damage, as the expression is upregulated 2–4 h post nephrotoxic and ischemic kidney injury [46,47,48]. Evidence from several studies points out that NGAL is useful in the detection or monitoring of kidney disorders where tubules are affected [49]. In the report of Bolgeri M. et al., not only patients with obstructive uropathy but also those who had urolithiasis without blockade in urine flow had higher sNGAL and uNGAL when compared to healthy peers [50]. Some reports give evidence that the highest uNGAL levels are observed in patients with urolithiasis combined with urinary tract infection [51], which seems to be understood as this marker rises in inflammatory conditions. In the more recent study of Tasdemir M. et al. who compared, among others, uNGAL levels in patients with nephrolithiasis, it was shown that only those who had hydronephrosis (HN) had also elevation in uNGAL [15]. This observation may propose a very careful hypothesis that in patients with nephrolithiasis without HN, markers of tubular injury are not increased because urine flow is not interrupted and tubular injury is not present in contrast with those where dilation of the renal pelvis was observed together with a rise in uNGAL/cr and uNAG/cr. The biggest limitation of this observation is the small number of patients with HD that were included. Other recent findings on NGAL in stone formers are presented in the table.
2.4. Kidney Injury Molecule 1
Kidney Injury Molecule 1 (KIM-1) is a transmembrane protein produced by proximal tubules and is present in plasma and urine after renal injury [52]. It was detected 12–24 h post-AKI, and higher urinary values were observed in patients with ischemic acute tubular necrosis than in other conditions, including CKD, diabetic nephropathy or steroid-resistant nephrotic syndrome [53]. Several studies assessed KIM-1 in nephrolithiasis, giving conflicting results. Some researchers found that uKIM-1 was increased in patients with obstructive nephropathy [54,55]. Similarly, in the study of Fahmy et al., the elevation of uKIM-1 was clear in patients who underwent retrograde intrarenal surgery and shock wave lithotripsy because of kidney stones compared to healthy controls [56]. These findings are in contrast to Urbschat A. et al. who found no difference in uKIM-1 between participants with obstructive nephropathy and controls [57].
2.5. Carbohydrate Antigen 19-9
Carbohydrate Antigen 19-9 (CA 19-9) is a 36-kD glycoprotein normally expressed in different tissues starting from the gastrointestinal tract, through the bronchi or endometrium and ending in the prostate. It is best known as a marker of pancreatic and other gastrointestinal cancers [58,59,60]. Nevertheless, some investigators revealed its higher urinary levels in urinary tract obstruction [61,62,63]. Suzuki K. and Kajbafzadeh A.M. found that CA 19-9 could be a marker for kidney injury related to urinary obstruction in their research on patients with hydronephrosis [61,62]. Amini E. et al. conducted a study on people with urolithiasis and HN before and after a procedure called transurethral lithotripsy [13]. The affected group had a significant elevation before the operation, which decreased in the following measurements.
2.6. N-Acetyl-B-D-Glucosaminidase
N-acetyl-B-D-glucosaminidase (NAG) is also one marker of tubular damage; however, in vivo studies on urolithiasis and its urinary levels were not elevated in stone formers [64]. Nevertheless, in the recent study of Xiaohong F. et al., it was found that bilateral stone formers were more endangered with CKD and had, among others, an increased urine NAG/creatinine ratio (OR 1.95; 95% CI 1.21–3.16) when compared with healthy peers [32].
2.7. Myeloperoxidase
Myeloperoxidase (MPO) is involved in the generation of oxygen radicals by neutrophils in inflammatory conditions [65]. It may rise in the kidney formers; however, it was not proved in an in vivo study. Hughes S. et al. compared pre- and post-URS MPO values in stone-forming participants, and no differences were observed between these two groups [23].
2.8. Markers of Inflammation
2.8.1. Interleukins
Interleukins are cytokines involved mainly in inflammatory response. Most studied in the kidney disorders are listed below. Different tissues like macrophages, osteoblasts and smooth muscles in vessels produce IL-6, which can cause inflammation. It is also a myokine released by muscles in response to excessive contractions, and, in this role, it has mainly an anti-inflammatory effect by inhibition of TNF-alpha [65]. Its importance was shown in many diseases, including different cancers, obesity and severe COVID-19 infection [66]. In sepsis with AKI, an elevation of IL-6 was also noticed [67,68]. IL-8 is another potent cytokine that accelerates inflammation. It induces chemotaxis in target cells (neutrophils and other granulocytes) to make them migrate to the site of infection and then stimulates phagocytosis [66]. As a marker of inflammation, a urinary IL-8 increase was noticed in the course of pyelonephritis [69]. IL-18 can modulate innate and adaptive immunity, and dysregulation of its distribution can lead to autoimmune or inflammatory diseases. The primary site of IL-18 production is macrophages in various organs. It was found in the proximal tubular cells of the kidney as well. IL-18 seems to be the most involved cytokine in kidney disorders. It was even proposed to be the marker of early AKI as it increases 6–24 h post-starting factor. In the kidney, IL-18 is also associated with excessive urinary protein excretion and can be a marker of the progression of diabetic nephropathy [70,71]. Research on inflammatory cytokines in urolithiasis is not consistent. In the study of Memmos D. et al., who compared the effect of standard percutaneous nephrolithotomy (sPCNL) with miniaturized PCNL (mPCNL) and retrograde intrarenal surgery (RIRS) as a nephrolithiasis treatment and measured, among others, uIL-18/cr ratios at baseline and 2, 6, 24 and 48 h postoperatively in the above patients, no significant differences in its level were shown. Similarly, no between-group changes were observed for urinary IL-18/cr at 2 h and later time points postoperatively. Within particular groups, increases for IL-18/cr from baseline were noted at 2 h and progressively lower rises from time zero in all participants at 6, 24 and 48 h post-procedure. No significant difference in this marker level was noticed in AKI or other complications [35].
2.8.2. Tumor Necrosis Factor—α
Tumor necrosis factor—α (TNF-α) is both an adipokine and cytokine. As a cytokine, it is used for cell signaling. Macrophages detecting an infection release TNF to alert other immune system cells and start an inflammatory response. TNF-α regulates cell proliferation, differentiation and apoptotic death and may be used in the detection of various renal disorders [72].
2.8.3. Monocyte Chemoattractant Protein 1
Monocyte chemoattractant protein 1 (MCP-1) is an inflammatory chemokine produced by mononuclear and intrinsic cells in the kidney to activate and recruit monocytes [73]. Its upregulation is the response to various damaging factors. Studies on several kidney disorders demonstrated its potential as a biomarker. Lupus nephritis severity correlated with urinary MCP-1 (uMCP-1) levels in pediatric patients. Similarly, MCP-1 was higher in patients with chronic kidney disease (CKD) or autosomal recessive polycystic kidney disease (ARPKD) when compared to healthy controls [74]. In urolithiasis, MCP-1 may find its place as well.
2.8.4. C-Reactive Protein, Procalcitonin
CRP was first identified by Tillet and Francis in 1930. They found that it can make streptococcus pneumoniae C-polysaccharide precipitate. CRP is synthesized in the liver as a fast response to inflammation and decreases rapidly after its resolution [76]. Similarly, procalcitonin (PCT) concentration rises as a reaction to a pro-inflammatory stimulus, especially of bacterial origin. Both CRP and PCT are commercially used in blood laboratory tests to detect severe inflammatory diseases. In several studies on urolithiasis, it was shown that elevation in CRP and PCT occurs, especially when stones cause obstruction leading to inflammation of the surrounding tissue [77]. Choosing the best treatment option for ureter obstruction depends on different factors, including the size of a stone and its location. According to The European Association of Urology, medical expulsive therapy (MET) with the use of an alpha blocker should be started in patients with renal colic and distal ureteric stones less than 5 mm, whose symptoms are controlled [78]. Classical inflammatory markers can also support the decision-making process.
2.9. Macrophage Inflammatory Protein 1beta
Macrophage Inflammatory Protein 1beta (MIP1β) is a chemokine starting recruitment of the immune cells responsible for innate and adaptive immune activity. It is involved in the process of inflammatory response during infection [82]. As another inflammatory molecule, it may be present in stone-induced kidney injury. In the study of Kusumi K. et al., urinary MIP1β levels were significantly elevated in stone-forming adolescents compared to healthy controls [21].
2.10. Other Urinary Proteins
2.10.1. Osteopontin
Osteopontin (OPN) is a phosphorylated protein that plays an essential role in bone mineralization [83]. Most probably, it is also involved in the process of inflammation, cell survival and leukocyte recruitment [84]. It has a wide tissue distribution associated with abnormal calcification including an organic matrix of the kidney stones. OPN is produced in the kidney and found in human urine. It probably acts as one of the urolithiasis inhibitors by preventing the formation of CaOx and further adhesion of crystals to renal epithelial cells [85,86]. Nevertheless, some research on animals gives exactly the opposite conclusions [87,88].
2.10.2. Nephrocalcin
Nephrocalcin (NC) is one of the most studied molecules, taking part in the process of stone formation as an inhibitor of calcium oxalate (CaOx) crystallization. It was first described in 1978 by Nakagawa et al. as an unidentified acidic polypeptide [89]. During the next ten years, several studies showed its inhibitory effect on CaOx crystallization, and in 1987, it was isolated from the urine and named nephrocalcin [89,90,91,92,93]. This glycoprotein is in the proximal tubule and thick ascending limb of the Henle’s loop [94]. It has many polymeric forms and at least four isoforms: NC-A, NC-B, NC-C and NC-D. The risk of CaOx crystallization and nephrolithiasis depends on the proportion of the isoforms in the urine [95]. Those who are more likely to develop kidney stones excrete greater proportions of NC-C and -D than NC-A and -B [96].
2.11. Bikunin
Another protein that slows CaOx crystallization is bikunin. It is a small chondroitin sulfate proteoglycan joined with a single glycosaminoglycan chain localized in the proximal tubule and the thin descending part of the Henle loop [98]. It is a potent inhibitor of CaOx crystal nucleation and aggregation mostly in healthy humans, whereas in the presence of urolithiasis, its preventing role is limited [99,100]. The existing literature on bikunin’s role gives conflicting results. Higher levels of bikunin were found in children with urolithiasis while in another study on adults, those affected by kidney stones had 50% lower urinary concentration when compared to healthy peers [101,102].
2.12. Calgranulins
Calgranulins, otherwise S100 proteins, are a family of calcium-binding molecules present in the cytosol. Some of them, including S100A8 (calgranulin A) and S100A9 (calgranulin B) have been classified as danger-associated molecular patterns of endogenous origin—alarmins, a group of molecules released as inflammatory signal mediators after cell death [103]. Normally, calgranulins A and B are produced mainly by neutrophils and monocytes, as well as dendritic cells, while in other cell types, they appear after the activating signal [104,105].
2.13. Matrix Gla Protein
Matrix Gla protein (MGP) was identified in the bone matrix and then in other tissues, including vascular [108]. Although it is suspected to have an inhibitory effect on calcification, the recent study on stone formers does not confirm this hypothesis [24,108].
2.14. Tamm–Horsfall Protein
Tamm–Horsfall protein (THP), known as uromodulin, is one of the most extensively investigated macromolecules in nephrolithiasis. It is found in the urine of all placental invertebrates as a polymer with a molecular weight of up to several million Da [109,110]. It is involved in the pathogenesis of nephrolithiasis and tubule interstitial nephritis [111]. The studies on the particular role of THP in stone formation give conflicting answers. Some point out that it is an inhibitor of crystallization [112,113,114,115], whereas others point out its promoting role [112,113,114,115,116,117,118]. Perhaps its action depends on the concentration of this protein and other solutes [119].
2.15. Urinary Prothrombin Fragment—1
This entry is adapted from the peer-reviewed paper 10.3390/jcm12227135
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