Proposal for pathogenesis-based treatment options to reduce calcium oxalate stone recurrence.

Objective: Prevalence of kidney stone disease continues to increase globally with recurrence rates between 30% and 50% despite technological and scientific advances. Reduction in recurrence would improve patient outcomes and reduce cost and stone morbidities. Our objective was to review results of experimental studies performed to determine the efficacy of readily available compounds that can be used to prevent recurrence. Methods: All relevant literature up to October 2020, listed in PubMed is reviewed. Results: Clinical guidelines endorse the use of evidence-based medications, such as alkaline agents and thiazides, to reduce urinary mineral supersaturation and recurrence. However, there may be additional steps during stone pathogenesis where medications could moderate stone risk. Idiopathic calcium oxalate stones grow attached to Randall's plaques or plugs. Results of clinical and experimental studies suggest involvement of reactive oxygen species and oxidative stress in the formation of both the plaques and plugs. The renin-angiotensin-aldosterone system (RAAS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mitochondria, and NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome have all been implicated at specific steps during stone pathogenesis in animal models. Conclusion: In addition to supersaturation-reducing therapies, the use of anti-oxidants, free radical scavengers, and inhibitors of NADPH oxidase, NLRP3 inflammasome, and RAAS may prove beneficial for stone prevention. Compounds such as statins and angiotensin converting enzyme inhibitors are already in use as therapeutics for hypertension and cardio-vascular disease and have previously shown to reduce calcium oxalate nephrolithiasis in rats. Although clinical evidence for their use in stone prevention in humans is limited, experimental data support they be considered along with standard evidence-based medications and clinical expertise when patients are being counselled for stone prevention.

Antiurolithic effects of medicinal plants: results of in vivo studies in rat models of calcium oxalate nephrolithiasis-a systematic review.

Urolithiasis is one of the oldest diseases affecting humans, while plants are one of our oldest companions providing food, shelter, and medicine. In spite of substantial progress in understanding the pathophysiological mechanisms, treatment options are still limited, often expensive for common people in most parts of the world. As a result, there is a great interest in herbal remedies for the treatment of urinary stone disease as an alternative or adjunct therapy. Numerous in vivo and in vitro studies have been carried out to understand the efficacy of herbs in reducing stone formation. We adopted PRISMA guidelines and systematically reviewed PubMed/Medline for the literature, reporting results of various herbal products on in vivo models of nephrolithiasis/urolithiasis. The Medical Subject Heading Terms (Mesh term) "Urolithiasis" was used with Boolean operator "AND" and other related Mesh Unique terms to search all the available records (July 2019). A total of 163 original articles on in vivo experiments were retrieved from PubMed indexed with the (MeshTerm) "Urolithiasis" AND "Complementary Therapies/Alternative Medicine, "Urolithiasis" AND "Plant Extracts" and "Urolithiasis" AND "Traditional Medicine". Most of the studies used ethylene glycol (EG) to induce hyperoxaluria and nephrolithiasis in rats. A variety of extraction methods including aqueous, alcoholic, hydro-alcoholic of various plant parts ranging from root bark to fruits and seeds, or a combination thereof, were utilized. All the investigations did not study all aspects of nephrolithiasis making it difficult to compare the efficacy of various treatments. Changes in the lithogenic factors and a reduction in calcium oxalate (CaOx) crystal deposition in the kidneys were, however, considered favorable outcomes of the various treatments. Less than 10% of the studies examined antioxidant and diuretic activities of the herbal treatments and concluded that their antiurolithic activities were a result of antioxidant, anti-inflammatory, and/or diuretic effects of the treatments.

Randall's plaque and calcium oxalate stone formation: role for immunity and inflammation.

Idiopathic calcium oxalate (CaOx) stones often develop attached to Randall's plaque present on kidney papillary surfaces. Similar to the plaques formed during vascular calcification, Randall's plaques consist of calcium phosphate crystals mixed with an organic matrix that is rich in proteins, such as inter-α-trypsin inhibitor, as well as lipids, and includes membrane-bound vesicles or exosomes, collagen fibres and other components of the extracellular matrix. Kidney tissue surrounding Randall's plaques is associated with the presence of classically activated, pro-inflammatory macrophages (also termed M1) and downregulation of alternatively activated, anti-inflammatory macrophages (also termed M2). In animal models, crystal deposition in the kidneys has been associated with the production of reactive oxygen species, inflammasome activation and increased expression of molecules implicated in the inflammatory cascade, including osteopontin, matrix Gla protein and fetuin A (also known as α2-HS-glycoprotein). Many of these molecules, including osteopontin and matrix Gla protein, are well known inhibitors of vascular calcification. We propose that conditions of urine supersaturation promote kidney damage by inducing the production of reactive oxygen species and oxidative stress, and that the ensuing inflammatory immune response promotes Randall's plaque initiation and calcium stone formation.

Immunotherapy for stone disease.

PURPOSE OF REVIEW: In addition to traditional risk factors such as low urine volume or hypercalciuria, emerging data suggest that calcium oxalate (CaOx), one of the most common mineral complexes in the urine, elicits a strong immunologic response. This review highlights those studies and projects how future therapies may be directed for kidney stone prevention. RECENT FINDINGS: Over the last 2 years, several groups have studied the response of the immune system to CaOx crystals using cell culture and animal models. Dominguez et al. found that CaOx crystals were recognized by monocytes through an lipopolysaccharide-mediated mechanism, leading to M1 'inflammatory' macrophage phenotype. Patel et al. proposed excessive oxalate-mediated reactive oxygen species within macrophage mitochondria may impair their ability to properly clear stones. Two other groups developed mouse models (an androgen receptor knock-out and an overexpression of Sirtuin 3 protein) and demonstrated increased renal anti-inflammatory macrophage differentiation and decreased CaOx deposition in experimental compared with controls. Anders et al. fed hyperoxaluric mice 1,3-butanediol, which blocks an inflammatory form of cell death called NLRP3 inflammasome and found less intrarenal oxidative damage and higher anti-inflammatory renal infiltrates in experimentals. Finally, monocytes exposed to CaOx crystals followed by hydroxyapatite had reduced inflammatory cytokine and chemokine production compared with those without hydroxyapatite, suggesting that Randall's plaque may play a role in dampening M1-mediatiated CaOx inflammation. SUMMARY: By modulating the immune response, immunotherapy could provide the means to prevent stone recurrences in certain individuals. The promotion of M2 over M1 macrophages and inhibition of inflammation could prevent the cascade that leads to CaOx nucleation. Future therapies may target the ability of macrophages to degrade CaOx crystals to prevent stones.

Opportunities for future therapeutic interventions for hyperoxaluria: targeting oxidative stress.

INTRODUCTION: Oxalate is a toxic byproduct of metabolism and is normally produced in quantities easily removed from the body. However, under specific circumstances oxalate production is increased resulting in deposition of calcium oxalate (CaOx) crystals in the kidneys as well as other organs causing inflammation and injury. Excessive buildup of crystal deposits in the kidneys causes eventual loss of renal function requiring renal transplantation. Areas covered: Cellular exposure to CaOx crystals induces the production of reactive oxygen species (ROS) with the involvement of renin-angiotensin aldosterone system (RAAS), mitochondria, and NADPH oxidase. Inflammasomes are activated and pro-inflammatory cytokines, such as IL-1ß and IL-18 are produced. We reviewed results of experimental and clinical studies of crystal renal epithelial cell interactions with emphasis on cellular injury and ROS production. Expert opinion: Treatment should depend upon the level of hyperoxaluria and whether it is associated with CaOx crystal deposition. Persistent low grade or intermittent hyperoxaluria can be treated with antioxidants, free radical scavengers. Hyperoxaluria associated with CaOx crystal deposition will require administration of angiotensin II receptor blockers, and NADPH oxidase or NLRP3 inflammasome inhibitors. DASH-style diet will be beneficial in both cases.

Development of a two-stage in vitro model system to investigate the mineralization mechanisms involved in idiopathic stone formation: stage 1-biomimetic Randall's plaque using decellularized porcine kidneys.

Idiopathic calcium oxalate (CaOx) stone formers form stones that are commonly attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane, where they exhibit a morphology of concentrically laminated apatitic spherules, while in the interstitial regions, the collagen fibrils and vesicles become mineralized. We hypothesize that these minerals might form by non-classical crystallization mechanisms, such as via amorphous precursors, some of which might originate from a polymer-induced liquid-precursor (PILP) process. Thus, our goal is to identify mineralogical 'signatures' of various stone formation mechanisms. To do this for idiopathic CaOx stones, we are developing a two-stage model system of CaP-CaOx composite stones, consisting of stage (1) CaP mineralized plaque, followed by stage (2) CaOx overgrowth into a stone. For the studies presented here, decellularized porcine kidneys were mineralized with CaP using polyaspartic acid or the protein osteopontin (OPN) to induce the PILP process and create biomimetic RP. Analysis of the PILP-mineralized tissues shows features that resemble the native plaques, including mineral spherules and collagen with intrafibrillar mineral. In contrast, the classical crystallization produced large apatitic spherulites, which is a very different morphology, but one which is also found in some stones. An alternative hypothesis regarding Randall's plaque, and if or when it becomes pathological, is discussed.

Development of a two-stage model system to investigate the mineralization mechanisms involved in idiopathic stone formation: stage 2 in vivo studies of stone growth on biomimetic Randall's plaque.

Idiopathic stone formers often form calcium oxalate (CaOx) stones that are attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane and spread into the interstitial regions where collagen fibrils and vesicles become mineralized; if the epithelium is breached, the RP becomes overgrown with CaOx upon exposure to urine. We have developed a two-stage model system of CaP-CaOx composite stones, consisting of Stage (1) CaP mineralized plaque, followed by Stage (2) CaOx overgrowth into a stone. In our first paper in this series (Stage 1), osteopontin (and polyaspartate) were found to induce a non-classical mineralization of porcine kidney tissues, producing features that resemble RP. For the Stage 2 studies presented here, biomimetic RPs from Stage 1 were implanted into the bladders of rats. Hyperoxaluria was induced with ethylene glycol for comparison to controls (water). After 4 weeks, rats were sacrificed and the implants were analyzed using electron microscopy and X-ray microanalyses. Differences in crystal phase and morphologies based upon the macromolecules present in the biomimetic plaques suggest that the plaques have the capacity to modulate the crystallization reactions. As expected, mineral overgrowths on the implants switched from CaP (water) to CaOx (hyperoxaluric). The CaOx crystals were aggregated and mixed with organic material from the biomimetic RP, along with some amorphous and spherulitic CaOx near the "stone" surfaces, which seemed to have become compact and organized towards the periphery. This system was successful at inducing "stones" more similar to human idiopathic kidney stones than other published models.

Calcium Oxalate Differentiates Human Monocytes Into Inflammatory M1 Macrophages.

Purpose: A number of hyperoxaluric states have been associated with calcium oxalate (CaOx) deposits in the kidneys. In animal models of stone disease, these crystals interact with circulating monocytes that have migrated into the kidney as part of innate immunity. Similarly, macrophages surround CaOx crystals in kidneys of patients excreting high levels of oxalate. We investigate the effect of this exposure and subsequent human immunological response in vitro. Materials and methods: Primary human monocytes were collected from healthy donors and exposed to CaOx, potassium oxalate, and zinc oxalate (ZnOx). Cytokine production was measured with a multiplex ELISA. Quantitative reverse transcription-polymerase chain reaction was done to validate the mRNA profile expression. M1 macrophage phenotype was confirmed with immunofluorescence microscopy. Results: Both primary monocytes and THP-1 cells, a human monocytic cell line, respond strongly to CaOx crystals in a dose-dependent manner producing TNF-α, IL-1ß, IL-8, and IL-10 transcripts. Exposure to CaOx followed by 1 h with LPS had an additive effect for cytokine production compared to LPS alone, however, LPS followed by CaOx led to significant decrease in cytokine production. Supernatants taken from monocytes were previously exposed to CaOx crystals enhance M2 macrophage crystal phagocytosis. CaOx, but not potassium or ZnOx, promotes monocyte differentiation into inflammatory M1-like macrophages. Conclusion: In our in vitro experiment, human monocytes were activated by CaOx and produced inflammatory cytokines. Monocytes recognized CaOx crystals through a specific mechanism that can enhance or decrease the innate immune response to LPS. CaOx promoted M1 macrophage development. These results suggest that monocytes have an important role promoting CaOx-induced inflammation.

Development of a personalized diagnostic model for kidney stone disease tailored to acute care by integrating large clinical, demographics and laboratory data: the diagnostic acute care algorithm - kidney stones (DACA-KS).

BACKGROUND: Kidney stone (KS) disease has high, increasing prevalence in the United States and poses a massive economic burden. Diagnostics algorithms of KS only use a few variables with a limited sensitivity and specificity. In this study, we tested a big data approach to infer and validate a 'multi-domain' personalized diagnostic acute care algorithm for KS (DACA-KS), merging demographic, vital signs, clinical, and laboratory information. METHODS: We utilized a large, single-center database of patients admitted to acute care units in a large tertiary care hospital. Patients diagnosed with KS were compared to groups of patients with acute abdominal/flank/groin pain, genitourinary diseases, and other conditions. We analyzed multiple information domains (several thousands of variables) using a collection of statistical and machine learning models with feature selectors. We compared sensitivity, specificity and area under the receiver operating characteristic (AUROC) of our approach with the STONE score, using cross-validation. RESULTS: Thirty eight thousand five hundred and ninety-seven distinct adult patients were admitted to critical care between 2001 and 2012, of which 217 were diagnosed with KS, and 7446 with acute pain (non-KS). The multi-domain approach using logistic regression yielded an AUROC of 0.86 and a sensitivity/specificity of 0.81/0.82 in cross-validation. Increase in performance was obtained by fitting a super-learner, at the price of lower interpretability. We discussed in detail comorbidity and lab marker variables independently associated with KS (e.g. blood chloride, candidiasis, sleep disorders). CONCLUSIONS: Although external validation is warranted, DACA-KS could be integrated into electronic health systems; the algorithm has the potential used as an effective tool to help nurses and healthcare personnel during triage or clinicians making a diagnosis, streamlining patients' management in acute care.

Modulation of calcium oxalate dihydrate growth by phosphorylated osteopontin peptides.

Osteopontin (OPN) is a significant component of kidney stone matrix and a key modulator of stone formation. Here, we investigated the effects of different phosphorylated states of a synthesized peptide of OPN (the ASARM peptide; acidic, serine- and aspartate-rich motif) on calcium oxalate dihydrate (COD) crystals, a major mineral phase of kidney stones. In vitro, phosphorylated OPN-ASARM peptides strongly inhibited COD crystal growth in solution as compared to the nonphosphorylated state, with increasing inhibitory potency correlating with the degree of peptide phosphorylation. Scanning electron microscopy revealed that the inhibition from the phosphopeptides resulted in distinctive, rosette-like crystal aggregates called spherulites. The OPN-ASARM peptides preferentially bound and specifically inhibited the {1 1 0} crystallographic faces of COD, as identified by combining atomic force microscopy and computational simulation approaches. These {1 1 0} surfaces of COD have high lattice calcium occupancy (exposure), providing preferential binding sites for the highly acidic peptides; binding and inhibition by OPN-ASARM peptides at the {1 1 0} faces led to crystal aggregation and intergrowth. The crystal spherulite formations obtained in vitro when using the most phosphorylated form of OPN-ASARM peptide at a high concentration, resembled crystal morphologies observed in vivo in a rat model of urolithiasis, in which crystal deposits in the kidney contain abundant OPN as revealed by immunogold labeling. A mechanistic model for spherulite formation is proposed based on the symmetry and crystallographic structure of COD, where the phosphate groups of OPN-ASARM bind to calcium atoms at [1 1 1] step risers on the COD {1 1 0} surface, inducing the periodic emergence of new COD crystals to form spherulites.

Transcriptional study of hyperoxaluria and calcium oxalate nephrolithiasis in male rats: Inflammatory changes are mainly associated with crystal deposition.

Hyperoxaluria associated with renal deposition of calcium oxalate (CaOx) crystals causes renal injury and inflammation leading to number of diseases including chronic kidney disease (CKD). It is however, not been possible to separate the renal consequences of hyperoxaluria from that of CaOx crystal deposition. We decided to utilize ethylene glycol (EG) model where hyperoxaluria and CaOx crystal deposition can be separated in time. To test our hypothesis, male rats were made hyperoxaluric by administering EG, rats were euthanized and kidneys were extracted on day 14, when occasional crystal is seen in the kidneys and day 28, when all animals have developed renal CaOx crystal deposits. Total RNA was extracted for microarray analysis and genome wide analysis of differentially expressed genes was performed to investigate differences between hyperoxaluria and crystal induced alterations in the kidneys. Immunohistochemical and Hematoxylin and Eosin (H&E) staining was also done for macromolecules with significant role in stone formation. All EG fed rats became hyperoxaluric by day 7, showed a few crystal deposits on day 14, and had heavy crystal deposition by day 28. There were significant changes in the expression of genes encoding for NADPH Oxidases; macromolecular crystallization modulators; genes involved in inflammasome activation; and osteogenic marker genes. Results demonstrate major differences between hyperoxaluria and CaOx crystal induced changes in the kidneys. Injury and inflammation are mainly associated with crystal deposition indicating significant role played by crystal retention.

Pathogenesis of calcium oxalate urinary stone disease: species comparison of humans, dogs, and cats.

Idiopathic calcium oxalate nephrolithiasis is a highly recurrent disease that is increasing in prevalence. Decades of research have not identified effective methods to consistently prevent the formation of nephroliths or induce medical dissolution. Idiopathic calcium oxalate nephroliths form in association with renal papillary subepithelial calcium phosphate deposits called Randall's plaques (RPs). Rodent models are commonly used to experimentally induce calcium oxalate crystal and stone formation, but a rodent model that conclusively forms RPs has not been identified. Both dogs and cats form calcium oxalate uroliths that can be recurrent, but the etiopathologic mechanisms of stone formation, especially renal pathologic findings, are a relatively unexploited area of study. A large animal model that shares a similar environment to humans, along with a shorter lifespan and thus shorter time to recurrence, might provide an excellent means to study preventative and therapeutic measures, along with enhancing the concepts of the one health initiative. This review article summarizes and compares important known features of idiopathic calcium oxalate stone disease in humans, dogs, and cats, and emphasizes important knowledge gaps and areas for future study in the quest to discover a naturally occurring animal model of idiopathic calcium oxalate stone disease.

How do stones form? Is unification of theories on stone formation possible?

There are two basic pathways for formation of calcium based kidney stones. Most idiopathic calcium oxalate (CaOx) stones are formed in association with sub-epithelial plaques of calcium phosphate (CaP), known as Randall's plaques, on renal papillary surfaces. Crystal formation and retention within the terminal collecting ducts, the ducts of Bellini, leading to the formation of Randall's plugs, is the other pathway. Both pathways require supersaturation leading to crystallization, regulated by various crystallization modulators produced in response to changing urinary conditions. High supersaturation, as a result of a variety of genetic and environmental factors, leads to crystallization in the terminal collecting ducts, eventually plugging their openings into the renal pelvis. Stasis behind the plugs may lead to the formation of attached or unattached stones in the tubular lumen. Deposition of crystals on the plug surface facing the pelvic or tubular urine may result in stone formation on the Randall's plugs. Kidneys of idiopathic stone formers may be subjected to oxidative stress as a result of increased urinary excretion of calcium/oxalate/phosphate and/or decrease in the production of functional crystallization inhibitors or in relation to co-morbidities such as hypertension, atherosclerosis, or acute kidney injury. We have proposed that production of reactive oxygen species (ROS) causes dedifferentiation of epithelial/endothelial cells into osteoblast type cells and deposition of CaP in the basement membrane of renal tubules or vessels. Growth, aggregation and melding of CaP crystals leads to the formation of plaque which grows by further calcification of interstitial collagen and membranous vesicles. Plaque becomes exposed to pelvic urine once the covering papillary epithelium is breached. Surface layers of CaP are replaced by CaOx through direct transformation or demineralization of CaP and mineralization of CaOx. Alternatively, or in addition, CaOx crystals nucleate directly on the plaque surface. Stone growth may also depend upon supersaturation in the pelvic urine, triggering further nucleation, growth and aggregation.

Histological aspects of the "fixed-particle" model of stone formation: animal studies.

Crystallization by itself is not harmful as long as the crystals are not retained in the kidneys and are allowed to pass freely down the renal tubules to be excreted in the urine. A number of theories have been proposed, and studies performed, to determine the mechanisms involved in crystal retention within the kidneys. It has been suggested that urinary transit through the nephron is too fast for crystals to grow large enough to be retained. Thus, free particle mechanism alone cannot lead to stone formation, and there must be a mechanism for crystal fixation within the kidneys. Animal model studies suggest that crystal retention is possible through both the free- and fixed-particle mechanisms. Crystal-cell interaction leads to pathological changes which promote crystal attachment to either epithelial cells or their basement membrane. Alternatively, crystals aggregate and produce large enough particles to block the tubules particularly at sites, where urinary flow is affected because of changes in the luminal diameter of the tubule. Crystal deposits plugging the openings of the ducts of Bellini may be the result of such a phenomenon. Intratubular crystals translocating to renal interstitium may produce osteogenic changes in the epithelial or endothelial cells resulting in the formation of the Randall's plaques. Thus, fixation appears to be either through the formation of Randall's plugs, crystal plugs clogging the openings of the ducts of Bellini or sub-epithelial crystal deposits, and the Randall's plaques.

Kidney stones.

Kidney stones are mineral deposits in the renal calyces and pelvis that are found free or attached to the renal papillae. They contain crystalline and organic components and are formed when the urine becomes supersaturated with respect to a mineral. Calcium oxalate is the main constituent of most stones, many of which form on a foundation of calcium phosphate called Randall's plaques, which are present on the renal papillary surface. Stone formation is highly prevalent, with rates of up to 14.8% and increasing, and a recurrence rate of up to 50% within the first 5 years of the initial stone episode. Obesity, diabetes, hypertension and metabolic syndrome are considered risk factors for stone formation, which, in turn, can lead to hypertension, chronic kidney disease and end-stage renal disease. Management of symptomatic kidney stones has evolved from open surgical lithotomy to minimally invasive endourological treatments leading to a reduction in patient morbidity, improved stone-free rates and better quality of life. Prevention of recurrence requires behavioural and nutritional interventions, as well as pharmacological treatments that are specific for the type of stone. There is a great need for recurrence prevention that requires a better understanding of the mechanisms involved in stone formation to facilitate the development of more-effective drugs.