Mitochondrial Dysfunction and Kidney Stone Disease

Gao A., Jiang J., Xie F., Chen L.

The present review is a summary of the recent literature concerning Bnip3 expression, function, and regulation, along with its implications in mitochondrial dysfunction, disorders of mitophagy homeostasis, and development of diseases of secondary mitochondrial dysfunction. As a member of the Bcl-2 family of cell death-regulating factors, Bnip3 mediates mPTP opening, mitochondrial potential, oxidative stress, calcium overload, mitochondrial respiratory collapse, and ATP shortage of mitochondria from multiple cells. Recent studies have discovered that Bnip3 regulates mitochondrial dysfunction, mitochondrial fragmentation, mitophagy, cell apoptosis, and the development of lipid disorder diseases via numerous cellular signaling pathways. In addition, Bnip3 promotes the development of cardiac hypertrophy by mediating inflammatory response or the related signaling pathways of cardiomyocytes and is also responsible for raising abnormal mitophagy and apoptosis progression through multiple molecular signaling pathways, inducing the pathogenesis and progress of hepatocellular carcinoma (HCC). Different molecules regulate Bnip3 expression at both the transcriptional and post-transcriptional level, leading to mitochondrial dysfunction and unbalance of mitophagy in hepatocytes, which promotes the development of non-alcoholic fatty liver disease (NAFLD). Thus, Bnip3 plays an important role in mitochondrial dysfunction and mitophagy homeostasis and has emerged as a promising therapeutic target for diseases of secondary mitochondrial dysfunction.

Missiroli S., Genovese I., Perrone M., Vezzani B., Vitto V.A., Giorgi C.

The main features that are commonly attributed to mitochondria consist of the regulation of cell proliferation, ATP generation, cell death and metabolism. However, recent scientific advances reveal that the intrinsic dynamicity of the mitochondrial compartment also plays a central role in proinflammatory signaling, identifying these organelles as a central platform for the control of innate immunity and the inflammatory response. Thus, mitochondrial dysfunctions have been related to severe chronic inflammatory disorders. Strategies aimed at reestablishing normal mitochondrial physiology could represent both preventive and therapeutic interventions for various pathologies related to exacerbated inflammation. Here, we explore the current understanding of the intricate interplay between mitochondria and the innate immune response in specific inflammatory diseases, such as neurological disorders and cancer.

Lambert J.P., Murray E.K., Elrod J.W.

Geto Z., Molla M.D., Challa F., Belay Y., Getahun T.

Schöttl T., Pachl F., Giesbertz P., Daniel H., Kuster B., Fromme T., Klingenspor M.

Previous studies have revealed decreased mitochondrial respiration in adipocytes of obese mice. This study aimed to identify the molecular underpinnings of altered mitochondrial metabolism in adipocytes.Untargeted proteomics of mitochondria isolated from adipocytes and metabolite profiling of adipose tissues were conducted in diet-induced obese (DIO) and lean mice. Subcutaneous and intra-abdominal adipose tissues were studied to depict depot-specific alterations.In subcutaneous adipocytes of DIO mice, changes in proteins related to mitochondrial structure and function were observed. Mitochondrial proteins of the inner and outer membrane were strongly reduced, whereas proteins of key matrix metabolic pathways were increased in the obese versus lean state, as further substantiated by metabolite profiling. A pronounced decrease in the oxidative phosphorylation (OXPHOS) enzymatic equipment and cristae density of the inner membrane was identified. In intra-abdominal adipocytes, similar systematic downregulation of the OXPHOS machinery in obesity occurred, but there was no regulation of outer membrane or matrix proteins.Protein components of the OXPHOS machinery are systematically downregulated in adipose tissues of DIO mice compared with lean mice. Loss of the mitochondrial OXPHOS capacity in adipocytes may aggravate the development of metabolic disease.

Abate M., Festa A., Falco M., Lombardi A., Luce A., Grimaldi A., Zappavigna S., Sperlongano P., Irace C., Caraglia M., Misso G.

Mitochondria are the key energy-producing organelles and cellular source of reactive species. They are responsible for managing cell life and death by a balanced homeostasis passing through a network of structures, regulated principally via fission and fusion. Herein we discuss about the most advanced findings considering mitochondria as dynamic biophysical systems playing compelling roles in the regulation of energy metabolism in both physiologic and pathologic processes controlling cell death and survival. Precisely, we focus on the mitochondrial commitment to the onset, maintenance and counteraction of apoptosis, autophagy and senescence in the bioenergetic reprogramming of cancer cells. In this context, looking for a pharmacological manipulation of cell death processes as a successful route for future targeted therapies, there is major biotechnological challenge in underlining the location, function and molecular mechanism of mitochondrial proteins. Based on the critical role of mitochondrial functions for cellular health, a better knowledge of the main molecular players in mitochondria disfunction could be decisive for the therapeutical control of degenerative diseases, including cancer.

Dominguez-Gutierrez P.R., Kwenda E.P., Khan S.R., Canales B.K.

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.

Ducasa G.M., Mitrofanova A., Fornoni A.

The goal of this review is to review the role that renal parenchymal lipid accumulation plays in contributing to diabetic kidney disease (DKD), specifically contributing to the mitochondrial dysfunction observed in glomerular renal cells in the context of DKD development and progression. Mitochondrial dysfunction has been observed in experimental and clinical DKD. Recently, Ayanga et al. demonstrate that podocyte-specific deletion of a protein involved in mitochondrial dynamics protects from DKD progression. Furthermore, our group has recently shown that ATP-binding cassette A1 (a protein involved in cholesterol and phospholipid efflux) is significantly reduced in clinical and experimental DKD and that genetic or pharmacological induction of ABCA1 is sufficient to protect from DKD. ABCA1 deficiency in podocytes leads to mitochondrial dysfunction observed with alterations of mitochondrial lipids, in particular, cardiolipin (a mitochondrial-specific phospholipid). However, through pharmacological reduction of cardiolipin peroxidation DKD progression is reverted. Lipid metabolism is significantly altered in the diabetic kidney and renders cellular components, such as the podocyte, susceptible to injury leading to worsened DKD progression. Dysfunction of the lipid metabolism pathway can also lead to mitochondrial dysfunction and mitochondrial lipid alteration. Future research aimed at targeting mitochondrial lipids content and function could prove to be beneficial for the treatment of DKD.

Ralto K.M., Rhee E.P., Parikh S.M.

The mammalian kidney relies on abundant mitochondria in the renal tubule to generate sufficient ATP to provide the energy required for constant reclamation of solutes from crude blood filtrate. The highly metabolically active cells of the renal tubule also pair their energetic needs to the regulation of diverse cellular processes, including energy generation, antioxidant responses, autophagy and mitochondrial quality control. Nicotinamide adenine dinucleotide (NAD+) is essential not only for the harvesting of energy from substrates but also for an array of regulatory reactions that determine cellular health. In acute kidney injury (AKI), substantial decreases in the levels of NAD+ impair energy generation and, ultimately, the core kidney function of selective solute transport. Conversely, augmentation of NAD+ may protect the kidney tubule against diverse acute stressors. For example, NAD+ augmentation can ameliorate experimental AKI triggered by ischaemia–reperfusion, toxic injury and systemic inflammation. NAD+-dependent maintenance of renal tubular metabolic health may also attenuate long-term profibrotic responses that could lead to chronic kidney disease. Further understanding of the genetic, environmental and nutritional factors that influence NAD+ biosynthesis and renal resilience may lead to novel approaches for the prevention and treatment of kidney disease. Here, the authors discuss evidence for a role of NAD+ imbalance in the pathogenesis of acute kidney injury (AKI) and chronic kidney disease (CKD). They suggest that disruption of NAD+ metabolism may contribute to mechanistic links among AKI, CKD and ageing.

Wei P.Z., Szeto C.C.

Although diabetic kidney disease (DKD) is the most common cause of end-stage kidney disease worldwide, the pathogenic mechanisms are poorly understood. There is increasing evidence that mitochondrial dysfunction contributes to the development and progression of DKD. Because the kidney is the organ with the second highest oxygen consumption in our body, it is distinctly sensitive to mitochondrial dysfunction. Mitochondrial dysfunction contributes to the progression of chronic kidney disease irrespective of underlying cause. More importantly, high plasma glucose directly damages renal tubular cells, resulting in a wide range of metabolic and cellular dysfunction. Overproduction of reactive oxygen species (ROS), activation of apoptotic pathway, and defective mitophagy are interlinked mechanisms that play pivotal roles in the progression of DKD. Although renal tubular cells have the highest mitochondrial content, podocytes, mesangial cells, and glomerular endothelial cells may all be affected by diabetes-induced mitochondrial injury. Urinary mitochondrial DNA (mtDNA) is readily detectable and may serve as a marker of mitochondrial damage in DKD. Unfortunately, pharmacologic modulation of mitochondrial dysfunction for the treatment of DKD is still in its infancy. Nonetheless, understanding the pathobiology of mitochondrial dysfunction in DKD would facilitate the development of novel therapeutic strategies.

Annesley S.J., Fisher P.R.

Mitochondria are best known as the sites for production of respiratory ATP and are essential for eukaryotic life. They have their own genome but the great majority of the mitochondrial proteins are encoded by the nuclear genome and are imported into the mitochondria. The mitochondria participate in critical central metabolic pathways and they are fully integrated into the intracellular signalling networks that regulate diverse cellular functions. It is not surprising then that mitochondrial defects or dysregulation have emerged as having key roles in ageing and in the cytopathological mechanisms underlying cancer, neurodegenerative and other diseases. This special issue contains 12 publications—nine review articles and three original research articles. They cover diverse areas of mitochondrial biology and function and how defects in these areas can lead to disease. In addition, the articles in this issue highlight how model organisms have contributed to our understanding of these processes.

Sun X., Zhang H., Liu J., Ouyang J.

Porphyra yezoensis polysaccharide repaired oxalate-injured renal epithelial cells and decreased COM crystal adhesion on the cell surface.

Neagu M., Constantin C., Popescu I.D., Zipeto D., Tzanakakis G., Nikitovic D., Fenga C., Stratakis C.A., Spandidos D.A., Tsatsakis A.M.

Cancer metabolism is an essential aspect of tumorigenesis, as cancer cells have increased energy requirements in comparison to normal cells. Thus, an enhanced metabolism is needed in order to accommodate tumor the cells’ accelerated biological functions, including increased proliferation, vigorous migration during metastasis and adaptation to tissues different from the primary invasion site. In this context, the assessment of tumor cell metabolic pathways generates crucial data pertaining to the mechanisms through which tumor cells survive and grow in a milieu of host defense mechanisms. Indeed, various studies have demonstrated that the metabolic signature of tumors is heterogeneous. Furthermore, these metabolic changes induce the exacerbated production of several molecules, which result in alterations that aid an inflammatory milieu. The therapeutic armentarium for oncology should thus include metabolic and inflammation regulators. Our expanding knowledge of the metabolic behavior of tumor cells whether from solid tumors or hematologic malignancies, may provide the basis for the development of tailor-made cancer therapies.

Bajic V.P., Van Neste C., Obradovic M., Zafirovic S., Radak D., Bajic V.B., Essack M., Isenovic E.R.

More people die from cardiovascular diseases (CVD) than from any other cause. Cardiovascular complications are thought to arise from enhanced levels of free radicals causing impaired “redox homeostasis,” which represents the interplay between oxidative stress (OS) and reductive stress (RS). In this review, we compile several experimental research findings that show sustained shifts towards OS will alter the homeostatic redox mechanism to cause cardiovascular complications, as well as findings that show a prolonged antioxidant state or RS can similarly lead to such cardiovascular complications. This experimental evidence is specifically focused on the role of glutathione, the most abundant antioxidant in the heart, in a redox homeostatic mechanism that has been shifted towards OS or RS. This may lead to impairment of cellular signaling mechanisms and elevated pools of proteotoxicity associated with cardiac dysfunction.

Ham J., Lim W., Park S., Bae H., You S., Song G.

Propyl gallate (propyl 3,4,5-trihydroxybenzoate, PG) is a phenolic antioxidant that has been used for oil-containing foods to prevent acidification. Owing to its antioxidant properties, PG has been applied to various fields and active research is currently underway to prove PG as an anticancer agent. However, there are still concerns about PG as a possible reproductive toxicant. Therefore, we determined whether PG induced male infertility. Our results indicated that PG induced testicular dysfunction in both Leydig and Sertoli cells via suppression of cell viability and steroidogenesis. These normal testis functions were destroyed by PG-induced mitochondrial dysfunction and calcium homeostasis dysregulation. In addition, PG disrupted the expression of several genes associated with the testis function and induced endoplasmic reticulum stress. Furthermore, we verified PG-induced mRNA expression changes in steroidogenesis enzymes and hormone receptors in vitro and in vivo. From the results of the qPCR analysis, we further confirmed the PG-mediated reduction in the mRNA expression of genes related to testis functions by in situ hybridization. Finally, we demonstrated that PG induced testicular toxicity via the disruption of mitochondrial or ER function and the inhibition of testicular development-related genes in mice.

Tang K., Ye T., He Y., Ba X., Xia D., Peng E., Chen Z., Ye Z., Yang X.

Finch R.H., Vitry G., Siew K., Walsh S.B., Behesti A., Hardiman G., da Silveira W.A.

Abstract Incidence of kidney stones in astronauts is a major risk factor associated with long-term missions, caused by increased blood calcium levels due to bone demineralisation triggered by microgravity and space radiation. Transcriptomic changes have been observed in tissues during spaceflight, including the kidney. We analysed kidney transcriptome patterns in two different strains of mice flown on the International Space Station, C57BL/6J and BALB/c. Here we show a link between spaceflight and transcriptome patterns associated with dysregulation of lipid and extracellular matrix metabolism and altered transforming growth factor-beta signalling. A stronger response was seen in C57BL/6J mice than BALB/c. Genetic differences in hyaluronan metabolism between strains may confer protection against extracellular matrix remodelling through the downregulation of epithelial-mesenchymal transition. We intend for our findings to contribute to the development of new countermeasures against kidney disease in astronauts and people here on Earth.

Peerapen P., Rattananinsruang P., Putpeerawit P., Boonmark W., Thongboonkerd V.

Zhang L., Song Z., Mao X., Yang Y., Hou B., Chen Y., Hao Z.

Wu J.

The evidence regarding the relationship between dietary niacin intake and kidney stones is limited. This study aims to investigate the association of dietary niacin intake with the prevalence of kidney stones among adults in the United States. The present cross-sectional study used data from the National Health and Nutrition Examination Survey (NHANES) 2007–2018. Dietary niacin intake was obtained from the first 24-h dietary recalls. The history of kidney stones was assessed by a questionnaire. Weighted logistic regression, restricted cubic splines (RCS) and stratified analysis were used to evaluate the relationship between dietary niacin intake and the risk of kidney stones. A total of 28,508 participants were included. In the full adjusted model, an inverse relationship between dietary niacin intake and kidney stones was observed. Compared to individuals in the lowest dietary niacin intake group, those in the highest group had lower odds of developing kidney stones (OR = 0.78, 95% CI = 0.63–0.97). RCS analysis showed a L-shaped relationship between niacin intake and kidney stones (P for non-linearity = 0.047). Stratified analysis indicated that this inverse associations appeared to be stronger among participants aged blew 60 years (P for interaction < 0.05). This cross-sectional study indicated that dietary niacin intake was associated with a decreased risk of kidney stones in US adults, especially in individuals under 60 years of age. Our findings suggest that appropriate doses of niacin intake may reduce the risk of kidney stones. Further research is required to confirm our findings and clarify the casual relationship.

Suttapitugsakul S., Sassanarakkit S., Peerapen P., Thongboonkerd V.

Nephrolithiasis is a multifactorial disease associated with urinary and matrix proteins that become a focal point of research for diagnostic and preventative strategies. The functional relevance of these proteins in lithogenesis, along with their origins and impacts, remains a major subject of ongoing lithogenic research. Here, an integrated analysis was done on multiple proteome datasets compiled from various studies of normal urine (NU), urine from calcium oxalate stone formers (SFU), and calcium oxalate stone matrix (SM). Functional annotation and network analysis revealed the profound enrichment of proteins associated with oxidative stress and inflammation only in the stone-related samples (both “SFU but not NU” and “SM but not NU” cohorts). The oxidative stress and inflammation-related proteins were most abundant in the “SM but not NU” cohort and had higher proportions in the “SFU but not NU” cohort than the “NU only” cohort. KEGG pathway analysis corroborated such observation and highlighted the inclusion of proteins in the complement and coagulation pathways, particularly in SM. The findings of this study inform some mechanistic insights into the roles of calcium oxalate stone-related proteins and may help develop effective prevention and treatment strategies for nephrolithiasis.

Song W., Li S., Wang G., Gao S.

Background This study investigates the relationship between sagittal abdominal diameter (SAD), a measure of abdominal obesity, and kidney stone disease (KSD) in the U.S. population. Additionally, it explores potential underlying mechanisms and evaluates the clinical utility of a predictive model. Methods Data were collected from 11,671 participants, including 1,136 cases of KSD. Univariate and multivariate logistic regression analyses, dose-response curves, and mediation effect assessments were employed to examine the association between SAD and KSD. A predictive model was developed and validated using calibration curves, receiver operating characteristic (ROC) curves, and clinical decision curves. Additionally, hematological indicators were analyzed to identify potential mediating factors. Results SAD showed a strong and positive association with KSD, even after adjusting for confounders such as gender, age, and education. The predictive model demonstrated moderate accuracy (AUC =  0.661) and clinical utility. Hematological analyses indicated that granulocyte count (GRAN) significantly mediated the relationship between SAD and KSD (P <  0.001). Conclusions SAD is a significant risk factor for KSD, underscoring the role of abdominal obesity in kidney stone formation. The predictive model demonstrates potential clinical applications for early risk assessment and management of KSD.

Huang H., Han Y., Zhang Y., Zeng J., He X., Cheng J., Wang S., Xiong Y., Yin H., Yuan Q., Huang L., Xie Y., Meng J., Tao L., Peng Z.

AbstractRenal fibrosis is a common pathway involved in the progression of various chronic kidney diseases to end‐stage renal disease. Recent studies show that mitochondrial injury of renal tubular epithelial cells (RTECs) is a crucial pathological foundation for renal fibrosis. However, the underlying regulatory mechanisms remain unclear. Pyruvate carboxylase (PC) is a catalytic enzyme located within the mitochondria that is intricately linked with mitochondrial damage and metabolism. In the present study, the downregulation of PC in various fibrotic animal and human kidney samples is demonstrated. Renal proximal tubule–specific Pcx gene knockout mice (PcxcKO) has significant interstitial fibrosis compared to control mice, with heightened expression of extracellular matrix molecules. This is further demonstrated in a stable PC knock‐out RTEC line. Mechanistically, PC deficiency reduces its interaction with sulfide:quinone oxidoreductase (SQOR), increasing the ubiquitination and degradation of SQOR. This leads to mitochondrial morphological and functional disruption, increased mtDNA release, activation of the cGAS‐STING pathway, and elevated glycolysis levels, and ultimately, promotes renal fibrosis. This study investigates the molecular mechanisms through which PC deficiency induces mitochondrial injury and metabolic reprogramming in RTECs. This study provides a novel theoretical foundation and potential therapeutic targets for the pathogenesis and treatment of renal fibrosis.

Zhang J., Wang R., Xie L., Ren H., Luo D., Yang Y., Xie H., Shang Z., Liu C.

Peerapen P., Thongboonkerd V.

Wu K., Lee J., Lee Y., Shen J., Wang H., Tsao Y., Wu Y., Huang S., Chen S., Jhan J., Geng J.

Background Menopause is associated with an increased risk of kidney stone disease (KSD). However, for postmenopausal women, how to avoid KSD has rarely been studied. The aim of this study was to explore whether drinking tea is associated with a reduction in the prevalence of KSD in postmenopausal women. Methods We collected 11,484 postmenopausal women from the Taiwan Biobank, and used questionnaires to obtain information on tea drinking, KSD, and comorbidities. The participants were divided into two groups according to habitual tea consumption: tea-drinking and non-tea-drinking groups. The association between habitual tea consumption and KSD was examined by logistic regression analysis. Results There were 2,035 postmenopausal women in the tea-drinking group and 9,449 postmenopausal women in the non-tea-drinking group. The mean age of all participants was 61 years. Compared to the non-tea-drinking group, the tea-drinking group had a significantly lower prevalence of KSD (7% vs. 5%). The odds ratio (OR) of KSD was lower in those who habitually drank tea than in those who did not (OR = 0.78; 95% confidence interval [CI] [0.63 to 0.96]) after adjusting for confounders. Moreover, postmenopausal women with a daily intake of two cups of tea or more had a 30% reduced risk of KSD compared to those who did not habitually drink tea (OR = 0.71, 95% CI [0.56 to 0.90]). Conclusions Our results suggest that habitual tea drinking may be associated with a reduction in the prevalence of KSD in postmenopausal women. Further studies are warranted to investigate the protective effect of tea on the development of KSD.

Duan Y., Wang Q., Chen X., Deng G., Huang K., Sun F., Zhu J., Jiang K.

Wang Q., Yang Z., Chen X., Yang Y., Jiang K.

Nephrolithiasis is one of the most common diseases in urology, characterized by notable incidence and recurrence rates, leading to significant morbidity and financial burden. Despite its prevalence, the precise mechanisms underlying stone formation remain incompletely understood, thus hindering significant advancements in kidney stone management over the past three decades. Investigating the pivotal biological molecules that govern stone formation has consistently been a challenging and high-priority task. A significant portion of mammalian genomes are transcribed into noncoding RNAs (ncRNAs), which have the ability to modulate gene expression and disease progression. They are thus emerging as a novel target class for diagnostics and pharmaceutical exploration. In recent years, the role of ncRNAs in stone formation has attracted burgeoning attention. They have been found to influence stone formation by regulating ion transportation, oxidative stress injury, inflammation, osteoblastic transformation, autophagy, and pyroptosis. These findings contributes new perspectives on the pathogenesis of nephrolithiasis. To enhance our understanding of the diagnostic and therapeutic potential of nephrolithiasis-associated ncRNAs, we summarized the expression profiles, biological functions, and clinical significance of these ncRNAs in the current review.

Hanstock S., Chew B., Lange D.

Microbiome dysbiosis is closely related to the etiology of kidney stone disease (KSD) and influences a multitude of pathways. Due to our knowledge gaps on this topic, it is still unclear if microbiome interventions can be translated to demonstrate clinical efficacy. Current evidence suggests that the enhancement of butyrate-producing pathways should be the next step for KSD research. While we are not yet at a point where we can make clinical recommendations for KSD, there are many simple dietary or supplement-based approaches that could be applied in the future for prophylaxis or treatment of KSD.

Yoodee S., Peerapen P., Thongboonkerd V.

We have recently reported a set of urinary proteins that inhibited calcium oxalate (CaOx) stone development. However, physicochemical properties that determine their inhibitory activities remained unknown. Herein, human urinary proteins were chromatographically fractionated into 15 fractions and subjected to various CaOx crystal assays and identification by nanoLC-ESI-Qq-TOF MS/MS. Their physicochemical properties and crystal inhibitory activities were subjected to Pearson correlation analysis. The data showed that almost all urinary protein fractions had crystal inhibitory activities. Up to 128 proteins were identified from each fraction. Crystallization inhibitory activity correlated with percentages of Ca