The Circulating Biomarkers League: Combining miRNAs with Cell-Free DNAs and Proteins.

The potential of liquid biopsy for the prognosis and diagnosis of diseases is unquestionable. Within the evolving landscape of disease diagnostics and personalized medicine, circulating microRNAs (c-miRNAs) stand out among the biomarkers found in blood circulation and other biological fluids due to their stability, specificity, and non-invasive detection in biofluids. However, the complexity of human diseases and the limitations inherent in single-marker diagnostics highlight the need for a more integrative approach. It has been recently suggested that a multi-analyte approach offers advantages over the single-analyte approach in the prognosis and diagnosis of diseases. In this review, we explore the potential of combining three well-studied classes of biomarkers found in blood circulation and other biofluids-miRNAs, DNAs, and proteins-to enhance the accuracy and efficacy of disease detection and monitoring. Initially, we provide an overview of each biomarker class and discuss their main advantages and disadvantages highlighting the superiority of c-miRNAs over the other classes of biomarkers. Additionally, we discuss the challenges and future directions in integrating these biomarkers into clinical practice, emphasizing the need for standardized protocols and further validation studies. This integrated approach has the potential to revolutionize precision medicine by offering insights into disease mechanisms, facilitating early detection, and guiding personalized therapeutic strategies. The collaborative power of c-miRNAs with other biomarkers represents a promising frontier in the comprehensive understanding and management of complex diseases. Nevertheless, several challenges must be addressed before this approach can be translated into clinical practice.

Exploring the Dynamic Relationship between the Gut Microbiome and Body Composition across the Human Lifespan: A Systematic Review.

This systematic review aimed to identify different gut microbiome profiles across the human lifespan and to correlate such profiles with the body composition. PubMed, Scopus, and Cochrane were searched from inception to March 2022. Sixty studies were included in this systematic review. Overall, the gut microbiome composition in overweight participants exhibited decreased α-diversity, decreased levels of the phylum Bacteroidetes and its taxa, and increased levels of the phylum Firmicutes, its taxa, and the Firmicutes/Bacteroidetes ratio, in comparison to normal-weight participants. Other body composition parameters showed similar correlations. Fat mass and waist circumference were found to correlate positively with the Firmicutes taxa and negatively with the Bacteroidetes taxa. In contrast, lean body mass and muscle mass demonstrated a positive correlation with the Bacteroidetes taxa. Notably, these correlations were more pronounced in athletes than in obese and normal-weight individuals. The composition of the gut microbiome is evidently different in overweight individuals or athletes of all age groups, with the former tending towards decreased Bacteroidetes taxa and increased Firmicutes taxa, while a reversed relationship is observed concerning athletes. Further studies are needed to explore the dynamic relationship between energy intake, body composition, and the gut microbiome across the human lifespan.

Exploring the Feasibility of Circulating miRNAs as Diagnostic and Prognostic Biomarkers in Osteoarthritis: Challenges and Opportunities.

Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by progressive cartilage degradation and joint inflammation. As the most common aging-related joint disease, OA is marked by inadequate extracellular matrix synthesis and the breakdown of articular cartilage. However, traditional diagnostic methods for OA, relying on clinical assessments and radiographic imaging, often need to catch up in detecting early-stage disease or i accurately predicting its progression. Consequently, there is a growing interest in identifying reliable biomarkers that can facilitate early diagnosis and prognosis of OA. MicroRNAs (miRNAs) have emerged as potential candidates due to their involvement in various cellular processes, including cartilage homeostasis and inflammation. This review explores the feasibility of circulating miRNAs as diagnostic and prognostic biomarkers in OA, focusing on knee OA while shedding light on the challenges and opportunities associated with their implementation in clinical practice.

Detection of SARS-CoV-2-Specific Antibodies in Human Breast Milk and Their Neutralizing Capacity after COVID-19 Vaccination: A Systematic Review.

SARS-CoV-2 is the virus that causes the infectious disease known as Corona Virus Disease 2019 (COVID-19). The severe impact of the virus on humans is undeniable, which is why effective vaccines were highly anticipated. As of 12 January 2022, nine vaccines have obtained Emergency Use Listing by the World Health Organization (WHO), and four of these are approved or authorized by the Centers for Disease Control and Prevention (CDC) in the United States. The initial clinical trials studying COVID-19 vaccine efficacy excluded pregnant and lactating individuals, meaning that data on the effects of the vaccine on breast milk were lacking. Until today, none of the authorized vaccines have been approved for use in individuals under six months. During the first months of life, babies do not produce their own antibodies; therefore, antibodies contained in their mothers' breastmilk are a critical protective mechanism. Several studies have shown the presence of SARS-CoV-2 antibodies in the breast milk of women who have been vaccinated or had been naturally infected. However, whether these are protective is still unclear. Additionally, research on the BNT162b2 mRNA vaccine developed by Pfizer-BioNTech and the mRNA-1273 vaccine developed by Moderna suggests that these vaccines do not release significant amounts, if any, of mRNA into breast milk. Hence, there is no evidence that vaccination of the mother poses any risk to the breastfed infant, while the antibodies present in breast milk may offer protection against the virus. The primary objective of this systematic review is to summarize the current understanding of the presence of immunoglobulins in human milk that are elicited by SARS-CoV-2 vaccines and to evaluate their ability to neutralize the virus. Additionally, we aim to quantify the side effects experienced by lactating mothers who have been vaccinated, as well as the potential for adverse effects in their infants. This study is critical because it can help inform decision-making by examining the current understanding of antibody secretion in breastmilk. This is particularly important because, although the virus tends to be less severe in younger individuals, infants who contract the disease are at a higher risk of requiring hospitalization compared to older children.

Alterations in Circulating miRNA Levels after Infection with SARS-CoV-2 Could Contribute to the Development of Cardiovascular Diseases: What We Know So Far.

The novel coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and poses significant complications for cardiovascular disease (CVD) patients. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and influence several physiological and pathological processes, including CVD. This critical review aims to expand upon the current literature concerning miRNA deregulation during the SARS-CoV-2 infection, focusing on cardio-specific miRNAs and their association with various CVDs, including cardiac remodeling, arrhythmias, and atherosclerosis after SARS-CoV-2 infection. Despite the scarcity of research in this area, our findings suggest that changes in the expression levels of particular COVID-19-related miRNAs, including miR-146a, miR-27/miR-27a-5p, miR-451, miR-486-5p, miR-21, miR-155, and miR-133a, may be linked to CVDs. While our analysis did not conclusively determine the impact of SARS-CoV-2 infection on the profile and/or expression levels of cardiac-specific miRNAs, we proposed a potential mechanism by which the miRNAs mentioned above may contribute to the development of these two pathologies. Further research on the relationship between SARS-CoV-2, CVDs, and microRNAs will significantly enhance our understanding of this connection and may lead to the use of these miRNAs as biomarkers or therapeutic targets for both pathologies.

Chitosan Nanogel with Mixed Food Plants and Its Relation to Blood Glucose in Type 2 Diabetes: A Systematic and Meta-Analysis Review of Observational Studies.

This systematic review with metanalysis evaluated and analyzed the beneficial effects of certain plants food in type 2 diabetes (T2D) when consumed alone or in combination with chitosan. The main objective of the paper was to examine the relation of chitosan nanogel and mixed food plant (MFP) to control T2D. The databases included Medline, Scopus, PubMed, as well as Cochrane available between the month of January 1990 to January 2021. The eligibility criteria for selecting studies were case-controlled studies that included unripe plantain, bitter yam, okra, and chitosan either used-alone or in combination with non-specified food plants (NSFP). Two-fold autonomous critics retrieved the information required and evaluated the risk of bias of involved studies. Random-effect meta-analyses on blood glucose controls, were performed. Results of 18 studies included: seven that examined unripe plantains, one bitter yam, two okras, and eight chitosan, found regarding the decrease in blood glucose level. Meta-analysis of the results found a large proportion of I2 values for all studies (98%), meaning heterogeneity. As a consequence, the combined effect sizes were not useful. Instead, prediction interval (PI) was used (mean difference 4.4 mg/dL, 95% PI -6.65 to 15.50 and mean difference 3.4 mg/dL, 95% PI -23.65 to 30.50) rather than the estimate of its confidence interval (CI). These studies were at 50% high risk of bias and 50% low risk of bias and there was judged to be an unclear risk of bias due to the insufficient information from the included study protocol (moderately low). The intervention lasted between three and 84 days, indicating potency and effectiveness of the intervention at both short and long durations. Due to the moderately low quality of the studies, the findings were cautiously interpreted. In conclusion, the current evidence available from the study does support the relation of chitosan with mixed unripe plantain, bitter yam and okra for the management of T2D. Further high-quality case-controlled animal studies are required to substantiate if indeed chitosan nanogel should be cross-linked with the specified food plant (SFP) for the management T2D.

Seroprevalence of immunoglobulin G antibodies against SARS-CoV-2 in Cyprus.

Monitoring the levels of IgG antibodies against the SARS-CoV-2 is important during the coronavirus disease 2019 (COVID-19) pandemic, to plan an adequate and evidence-based public health response. After this study we report that the plasma levels of IgG antibodies against SARS-CoV-2 spike protein were higher in individuals with evidence of prior infection who received at least one dose of either an mRNA-based vaccine (Comirnaty BNT162b2/Pfizer-BioNTech or Spikevax mRNA-1273/Moderna) or an adenoviral-based vaccine (Vaxzervia ChAdOx1 nCoV-19 /Oxford-Astra Zeneca) (n = 39) compared to i) unvaccinated individuals with evidence of prior infection with SARS-CoV-2 (n = 109) and ii) individuals without evidence of prior infection with SARS-CoV-2 who received one or two doses of one of the aforementioned vaccines (n = 342). Our analysis also revealed that regardless of the vaccine technology (mRNA-based and adenoviral vector-based) two doses achieved high anti- SARS-CoV-2 IgG responses. Our results indicate that vaccine-induced responses lead to higher levels of IgG antibodies compared to those produced following infection with the virus. Additionally, in agreement with previous studies, our results suggest that among individuals previously infected with SARS-CoV-2, even a single dose of a vaccine is adequate to elicit high levels of antibody response.

Associations between serum biomarkers of cartilage metabolism and serum hyaluronic acid, with risk factors, pain categories, and disease severity in knee osteoarthritis: a pilot study.

BACKGROUND: Specific serum biomarkers of cartilage metabolism such as cartilage oligomeric matrix protein (sCOMP) and procollagen type II C-terminal propeptide (sPIICP) as well as hyaluronan (sHA), a biomarker of synovitis, have been implicated in the pathophysiology of knee osteoarthritis (OA). However, the associations of these biomarkers with the severity of the disease and OA risk factors, including age and obesity remain inconclusive. This analysis examines the associations between these serum biomarkers and the radiographic severity of OA and knee pain, as wells as obesity, the age and gender of the participants, and other OA risk factors. METHODS: From 44 patients with early knee OA and 130 patients with late knee OA we analyzed the radiographic severity of the disease using the Kellgren and Lawrence (KL) grading system. Moreover, 38 overweight healthy individuals were used as a control group. Specific information was collected from all participants during their recruitment. The levels of the three serum biomarkers were quantified using commercially available ELISA kits. Serum biomarkers were analyzed for associations with the average KL scores and pain in both knees, as well as with specific OA risk factors. RESULTS: The levels of sCOMP were elevated in patients with severe late OA and knee pain and correlated weakly with OA severity. A weakly correlation of sHA levels and OA severity OA was observed. We demonstrated that only sPIICP levels were markedly decreased in patients with late knee OA suggesting the alterations of cartilage metabolism in this arthritic disease. Moreover, we found that sPIICP has the strongest correlation with obesity and the severity of OA, as well as with the knee pain at rest and during walking regardless of the severity of the disease. ROC analysis showed that the area under the ROC curve (AUC) was 0.980 (95% CI: 0.945-0.995; p < 0.0001), suggesting high diagnostic accuracy of sPIICP. Interestingly, gender and age had also an effect on the levels of sPIICP. CONCLUSION: This study revealed the potential of serum PIICP to be used as a biomarker to monitor the progression of knee OA, however, further studies are warranted to elucidate its clinical implication.

A bovine miRNA, bta-miR-154c, withstands in vitro human digestion but does not affect cell viability of colorectal human cell lines after transfection.

Colorectal cancer (CRC) is the third most frequent human cancer with over 1.3 million new cases globally. CRC is a complex disease caused by interactions between genetic and environmental factors; in particular, high consumption of red meat, including beef, is considered a risk factor for CRC initiation and progression. Recent data demonstrate that exogenous microRNAs (miRNAs) entering the body via ingestion could pose an effect on the consumer. In this study, we focused on bovine miRNAs that do not share a seed sequence with humans and mice. We identified bta-miR-154c, a bovine miRNA found in edible parts of beef and predicted via cross-species bioinformatic analysis to affect cancer-related pathways in human cells. When bovine tissue was subjected to cooking and a simulation of human digestion, bta-miR-154c was still detected after all procedures, albeit at reduced concentrations. However, lipofection of bta-miR-154c in three different colorectal human cell lines did not affect their viability as evaluated at various time points and concentrations. These data indicate that bta-miR-154c (a) may affect cancer-related pathways in human cells, (b) can withstand digestion and be detected after all stages of an in vitro digestion protocol, but (c) it does not appear to alter epithelial cell viability after entering human enterocytes, even at supraphysiological amounts. Further experiments will elucidate whether bta-miR-154c exerts a different functional effect on the human gut epithelium, which may cause it to contribute to CRC progression through its consumption.

Variability in the levels of exosomal miRNAs among human subjects could be explained by differential interactions of exosomes with the endothelium.

Exosomes are 30-100 nm endosome-derived membrane vesicles, that contain specific RNA transcripts including mRNAs, and microRNAs (miRNAs) and have been implicated in cell-to-cell communication. Exosomal miRNAs in blood circulation have been attracting major interest as potential diagnostic and prognostic biomarkers in a variety of diseases including stroke, cancer, and inflammatory disorders. Despite the progress made in the utilization of circulating exosomal miRNAs as biomarkers for various human diseases and conditions, there are still difficulties in functionally utilizing such methods in the clinic due to the high variability observed among subjects. Attempts to use miRNA signatures have improved but have not eliminated the problem. Additionally, standardized laboratory practices may partially reduce variability but there is still an unknown biological factor that hinders the proper use of miRNAs as biomarkers. We hypothesize that this variability might be partially attributed to a differential interaction among circulating exosomes carrying those miRNAs with endothelial surface molecules that themselves may vary among individuals due to secondary conditions, for example, inflammation status. This differential interaction could potentially add variability to the level of the examined miRNA that is not directly attributed to the primary condition under study.

Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases.

Micro-RNAs (miRNAs) play a pivotal role in the development and physiology of the cardiovascular system while they have been associated with multiple cardiovascular diseases (CVDs). Several cardiac miRNAs are detectable in circulation (circulating miRNAs; c-miRNAs) and are emerging as diagnostic and therapeutic biomarkers for CVDs. c-miRNAs exhibit numerous essential characteristics of biomarkers while they are extremely stable in circulation, their expression is tissue-/disease-specific, and they can be easily detected using sequence-specific amplification methods. These features of c-miRNAs are helpful in the development of non-invasive assays to monitor the progress of CVDs. Despite significant progress in the detection of c-miRNAs in serum and plasma, there are many contradictory publications on the alterations of cardiac c-miRNAs concentration in circulation. The aim of this review is to examine the pre-analytical and analytical factors affecting the quantification of c-miRNAs and provide general guidelines to increase the accuracy of the diagnostic tests in order to improve future research on cardiac c-miRNAs.

Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes.

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation, and is considered to be the earliest podocyte marker. miRNA prediction analysis revealed a full-length target site for the primate-specific miR-548c-5p at a genomic region > 8 kb upstream of FOXC2. We hypothesised that the transcription rates of FOXC2 during podocyte differentiation might be tuned by miR-548c-5p through this target site. Experiments were performed with cultured human podocytes, transfected with luciferase reporter constructs bearing this target site region within an enhancer element of the native plasmid. The results confirmed a seed region-driven targeting potential by the miRNA, with mimics downregulating and inhibitors enhancing luciferase activity. Introducing mutations into the miRNA target seed region abolished the expected response. In cultured podocytes, FOXC2 mRNA and protein levels responded to miR-548c-5p abundance in a coordinated manner before and after induction of differentiation, with high statistical significance. Ago-ChIP experiments revealed occupancy of the miRNA target site by miRNA/RISC in undifferentiated cells and its release when differentiation is initiated, allowing its interaction with the gene's promoter region to amplify FOXC2 expression, as shown by chromosome conformation capture and qRT-PCR. Moreover, the expression pattern of FOXC2 during podocyte differentiation seems to be affected by miR-548c-5p, as removal of either endogenous or mimic miR-548c-5p results in increased FOXC2 protein levels and cells resembling those undergoing differentiation. Collectively, results indicate a well-orchestrated regulatory model of FOXC2 expression by a remote upstream target site for miR-548c-5p.

Catalyzing Transcriptomics Research in Cardiovascular Disease: The CardioRNA COST Action CA17129.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).

MicroRNAs and Aging: Biomarkers or Therapeutic Targets?

Understanding the molecular mechanism of aging is of utmost importance to the scientific communities. To date, various theories have been proposed and many of them were evaluated as potential targets in the battle against aging. MicroRNAs, the universal gene expression regulators, were found to be associated with the aging process as many of them have been linked to biological process associated with cellular deterioration. In this short report, we briefly review the contribution of miRNAs to the aging process and offer an opinion as to how the knowledge of the role of these molecules in aging can be utilized.

Homo sapiens exhibit a distinct pattern of CNV genes regulation: an important role of miRNAs and SNPs in expression plasticity.

Gene expression regulation is a complex and highly organized process involving a variety of genomic factors. It is widely accepted that differences in gene expression can contribute to the phenotypic variability between species, and that their interpretation can aid in the understanding of the physiologic variability. CNVs and miRNAs are two major players in the regulation of expression plasticity and may be responsible for the unique phenotypic characteristics observed in different lineages. We have previously demonstrated that a close interaction between these two genomic elements may have contributed to the regulation of gene expression during evolution. This work presents the molecular interactions between CNV and non CNV genes with miRNAs and other genomic elements in eight different species. A comprehensive analysis of these interactions indicates a unique nature of human CNV genes regulation as compared to other species. By using genes with short 3' UTR that abolish the "canonical" miRNA-dependent regulation, as a model, we demonstrate a distinct and tight regulation of human genes that might explain some of the unique features of human physiology. In addition, comparison of gene expression regulation between species indicated that there is a significant difference between humans and mice possibly questioning the effectiveness of the latest as experimental models of human diseases.

CNVs-microRNAs interactions demonstrate unique characteristics in the human genome. An interspecies in silico analysis.

MicroRNAs (miRNAs) and copy number variations (CNVs) represent two classes of newly discovered genomic elements that were shown to contribute to genome plasticity and evolution. Recent studies demonstrated that miRNAs and CNVs must have co-evolved and interacted in an attempt to maintain the balance of the dosage sensitive genes and at the same time increase the diversity of dosage non-sensitive genes, contributing to species evolution. It has been previously demonstrated that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA binding sites are significantly higher than those of genes found in non-CNV regions. These findings raise the possibility that miRNAs may have been created under evolutionary pressure, as a mechanism for increasing the tolerance to genome plasticity. In the current study, we aimed in exploring the differences of miRNAs-CNV functional interactions between human and seven others species. By performing in silico whole genome analysis in eight different species (human, chimpanzee, macaque, mouse, rat, chicken, dog and cow), we demonstrate that miRNAs targeting genes located within CNV regions in humans have special functional characteristics that provide an insight into the differences between humans and other species.

A miR-1207-5p binding site polymorphism abolishes regulation of HBEGF and is associated with disease severity in CFHR5 nephropathy.

Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3'UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.

Is suppression of cyst growth in PKD enough to preserve renal function?: STAT6 inhibition is a novel promising target.

The autosomal dominant form of polycystic kidney disease (ADPKD) is one of the most frequent monogenic disorders and the most frequent among inherited kidney disorders. In fact it has a prevalence in the population of about 1/1,000 individuals, therefore it does not even satisfy the definition for rare diseases. It is mainly characterized by the formation of multiple cysts filled with fluid that over time develop in number and size leading to the distraction of the structure and function of the kidneys and eventually leading to chronic kidney disease/end stage kidney disease (CKD/ESKD), usually between the 4th and 7th decade of life. There are two known forms of the autosomal dominant type of polycystic kidney disease, type 1 and type 2, caused by mutations in the PKD1 and PKD2 genes, located on chromosomes 16 and 4 respectively. The polycystin 1 protein, encoded by PKD1 and mutated in ~85% of patients, is a huge protein of 4,302 amino acids with multiple transmembrane domains, 200 residues intracytoplasmic part and a huge extracellular part with multiple Ig-like PKD repeats, which probably acts as a receptor to an unknown ligand. Polycystin 1 has been shown to interact with and participate in multiple signal transduction pathways, including the G-protein coupled receptor, cAMP pathway, Wnt, mTOR, MAPK/ERK, AP1 and JAK-STAT pathway, while its intracytoplasmic C-terminal domain has been shown to be cleaved and translocated to the nucleus where it plays a role in gene transcription, in concert with P100 and STAT6.(1.)

Cyst formation in the PKD2 (1-703) transgenic rat precedes deregulation of proliferation-related pathways.

BACKGROUND: Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development. METHODS: In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters. RESULTS: Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Specifically, gene expression analysis demonstrated that at day 0 the RAS system is involved. This is altered at day 6, when Wnt signaling and focal adhesion pathways are affected. However, at and after 24 days, proliferation, apoptosis, altered ECM signaling and many other factors become involved. CONCLUSIONS: Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation.

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2.

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. METHODS: Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. RESULTS: Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. CONCLUSION: Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.