Unveiling Dynamic Changes and Regulatory Mechanisms of T Cell Subsets in Sepsis Pathogenesis.

Purpose: The pathogenesis of T cell subsets in sepsis during the body's resistance to infection is currently unknown. We aimed to investigate the dynamics and molecular mechanisms of T cells during the development of sepsis. Patients and Methods: Perform single-cell data analysis on peripheral blood mononuclear cells (PBMCs) specimen samples from seven healthy controls, five early-stage sepsis patients, and four late sepsis patients, and the atlas were mapped and analyzed using reference mapping to identify the T cell subpopulations specific to early sepsis. Expression network upstream to investigate the changes of regulatory transcription factors and pathways by pySCENIC. Results: Twenty-two CD4+ T-cell subpopulations and 10 CD8+ T-cell subpopulations were identified by mapping analysis. At the early stage of sepsis, we observed altered ratios of multiple immune cells in PBMCs. Three cell types CD4 Tn cells, CD8 (GZMK+ early Tem), and CD8 (ZNF683+CXCR6- Tm) showed an upward trend (p < 0.05) in the early stages of sepsis compared to normal and returned to normal levels after two weeks. In addition, we found the presence of four sepsis-specific transcription factors (MXI1, CHD1, ARID5A, KLF9) in these three types of cells, which were validated in two external datasets. The differentially expressed genes in CD4 Tn cells, CD8 (GZMK+ early Tem), and CD8 (ZNF683+CXCR6- Tm) cells between the healthy group and the early-stage sepsis group are commonly enriched in the allograft rejection pathway. In addition, it was found that CD8 cells exhibit a trend towards differentiation into CD8 Temra cells in sepsis. Conclusion: We successfully depicted the dynamic changes of T cell subsets during sepsis onset and progression, which provides important clues for an in-depth understanding of T cells' function and regulatory mechanisms during sepsis pathogenesis.

Parameters and Morphological Changes of Erythrocytes and Platelets of COVID-19 Subjects: A Longitudinal Cohort Study.

Purpose: Information about dynamic changes occurring in the parameters and morphology of erythrocytes and platelets during the coronavirus disease 2019 (COVID-19) infection and convalescence is scarce. To explore potential associations between dynamic erythrocyte and platelet parameters, morphological changes, and the course or severity of the disease is essential. Patients and Methods: From January 17th, 2020, to February 20th, 2022, we followed up on 35 patients with non-severe and 11 patients with severe COVID-19 following their discharge. We collected clinical features, dynamic complete blood count (CBC), and peripheral blood smears (PBS) and analyzed parameter and morphological changes of erythrocytes and platelets depending on the course or severity of the disease. The course of the disease included four periods, namely onset (T1), discharge (T2), 1-year follow-up (T3), and 2-year follow-up (T4). Results: Red blood cell (RBC) counts and hemoglobin were the lowest in T2, followed by T1, and lower in T1 and T2 than in T3 and T4. Inversely, the red blood cell distribution width (RDW) was the highest in T2, followed by T1, and higher than in T3 and T4. Compared to non-severe patients, the platelet of severe patients was lower in T1 and T2. In contrast, the mean platelet volume (MPV) and platelet distribution width (PDW) tended to be higher in severe patients. Similarly, anisocytosis was more common in peripheral blood smears at early stages and in severe patients. Finally, large platelets were more common in severe patients. Conclusion: Anisocytosis of erythrocytes and large platelets are found in patients with severe COVID-19, these changes may help primary hospitals to identify patients with a high risk of severe COVID-19 at an early stage.

Proteomics Investigation of Diverse Serological Patterns in COVID-19.

Serum antibodies IgM and IgG are elevated during Coronavirus Disease 2019 (COVID-19) to defend against viral attacks. Atypical results such as negative and abnormally high antibody expression were frequently observed whereas the underlying molecular mechanisms are elusive. In our cohort of 144 COVID-19 patients, 3.5% were both IgM and IgG negative, whereas 29.2% remained only IgM negative. The remaining patients exhibited positive IgM and IgG expression, with 9.3% of them exhibiting over 20-fold higher titers of IgM than the others at their plateau. IgG titers in all of them were significantly boosted after vaccination in the second year. To investigate the underlying molecular mechanisms, we classed the patients into four groups with diverse serological patterns and analyzed their 2-year clinical indicators. Additionally, we collected 111 serum samples for TMTpro-based longitudinal proteomic profiling and characterized 1494 proteins in total. We found that the continuously negative IgM and IgG expression during COVID-19 were associated with mild inflammatory reactions and high T cell responses. Low levels of serum IgD, inferior complement 1 activation of complement cascades, and insufficient cellular immune responses might collectively lead to compensatory serological responses, causing overexpression of IgM. Serum CD163 was positively correlated with antibody titers during seroconversion. This study suggests that patients with negative serology still developed cellular immunity for viral defense and that high titers of IgM might not be favorable to COVID-19 recovery.

Physical condition, psychological status, and posttraumatic stress disorder among recovered COVID-19 subjects: A mediation analysis.

The physical condition of individuals who contracted COVID-19 had a profound influence on mitigating the physical and psychological impact of the disease and the symptoms of posttraumatic stress disorder (PTSD). Little attention has been focused on the influence of physical condition on PTSD among recovered COVID-19 subjects. This study explored the relationship between physical and psychological status and PTSD and the potential mechanisms. Questionnaires were completed by 73 (50.7%, 73/144) COVID-19 recovered subjects who were diagnosed in Taizhou, Zhejiang, China. We conducted a face-to-face survey from January 17 to March 10, 2020. The mediation analysis approach was applied in this research. Our data show that recovered COVID-19 subjects who were in better physical condition exhibited fewer psychological problems [B (95%CI), (-1.65 -3.04, -0.26)] and lower PTSD [B (95%CI), -6.13 (-9.43, -2.83)]. In addition, the worse the psychological status of recovered COVID-19 subjects was, the stronger the PTSD (B [95%CI], 0.58 [0.02, 1.14]). Moreover, psychological status could significantly mediate the impact of physical condition on PTSD (ß1θ2 = -0.87). Together, COVID-19 recovered subjects who have better physical condition could decrease their PTSD, and the worse the physical condition of COVID-19 recovered subjects would increase their psychological problems. Our finding about psychological status could significantly mediate the impact of the physical condition on PTSD might be useful for medical institutions and the government seeking to help with the follow-up rehabilitation training of recovered COVID-19 subjects.

The evaluation of the immune status of COVID-19 recovered subjects with persistent abnormal lung CT after one year: A longitudinal cohort study.

OBJECTIVES: COVID-19 is an immune-related disease caused by novel Coronavirus SARS-COV-2. Lung lesions persist in some recovered patients, making long-term follow-up monitoring of their health necessary. The mechanism of these abnormalities is still unclear. In this study, the immune status was observed to explore the immune mechanism of persistent lung CT abnormalities in one-year COVID-19 recovered subjects. METHODS: One-year follow-up of 73 recovered patients from COVID-19 confirmed in Taizhou City, Zhejiang Province, was conducted to collect laboratory indicators such as blood immune cells, cytokines, complement series, immunoglobulin, and lung imaging; According to the results of lung CT, 60 patients were divided into normal CT group (n = 40) and abnormal CT group (n = 20). We compared the dynamic changes of immune indexes at three timepoints namely onset (T1), discharge (T2), and 1-year follow-up (T3), and studied the relationship between immune indexes and pulmonary sequelae. RESULTS: Compared with the healthy control, there was no significant difference in immune-related indexes, and immune levels had recovered. Patients with elder age, high BMI, severe patients, and those with underlying diseases (hypertension or diabetes) had a higher CT abnormal rate after recovery. Longitudinal observation showed that immunoglobulin increased first and then decreased, immune cell TBNK decreased in the onset period and increased in the recovery period, cytokine level increased significantly in the onset period and decreased to the normal level in the recovery period, and complement series C1q, C3 and C4 increased at the onset and decreased during the one-year follow-up. Complement C3 remained at a high level in the CT abnormal group (CT normal group vs CT abnormal group; P = 0.036). Correlation analysis showed that C3 negatively correlated restrictive ventilation index (TLC-He (ratio) (r = -0.302, P = 0.017). The above results suggest that complement C3 is a negative factor correlating abnormal pulmonary function 1 year after the recovery. CONCLUSION: After one year recovering from COVID-19, the subjects were with stable immune indicators. High levels of complement C3 were associated with persistent lung abnormalities in COVID-19 recovered subjects.

Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19.

The utility of the urinary proteome in infectious diseases remains unclear. Here, we analyzed the proteome and metabolome of urine and serum samples from patients with COVID-19 and healthy controls. Our data show that urinary proteins effectively classify COVID-19 by severity. We detect 197 cytokines and their receptors in urine, but only 124 in serum using TMT-based proteomics. The decrease in urinary ESCRT complex proteins correlates with active SARS-CoV-2 replication. The downregulation of urinary CXCL14 in severe COVID-19 cases positively correlates with blood lymphocyte counts. Integrative multiomics analysis suggests that innate immune activation and inflammation triggered renal injuries in patients with COVID-19. COVID-19-associated modulation of the urinary proteome offers unique insights into the pathogenesis of this disease. This study demonstrates the added value of including the urinary proteome in a suite of multiomics analytes in evaluating the immune pathobiology and clinical course of COVID-19 and, potentially, other infectious diseases.

Potential Use of Serum Proteomics for Monitoring COVID-19 Progression to Complement RT-PCR Detection.

RT-PCR is the primary method to diagnose COVID-19 and is also used to monitor the disease course. This approach, however, suffers from false negatives due to RNA instability and poses a high risk to medical practitioners. Here, we investigated the potential of using serum proteomics to predict viral nucleic acid positivity during COVID-19. We analyzed the proteome of 275 inactivated serum samples from 54 out of 144 COVID-19 patients and shortlisted 42 regulated proteins in the severe group and 12 in the non-severe group. Using these regulated proteins and several key clinical indexes, including days after symptoms onset, platelet counts, and magnesium, we developed two machine learning models to predict nucleic acid positivity, with an AUC of 0.94 in severe cases and 0.89 in non-severe cases, respectively. Our data suggest the potential of using a serum protein-based machine learning model to monitor COVID-19 progression, thus complementing swab RT-PCR tests. More efforts are required to promote this approach into clinical practice since mass spectrometry-based protein measurement is not currently widely accessible in clinic.

Longitudinal observation of viral load in patients infected with Omicron variant and its relationship with clinical symptoms.

Objective: In 2022, a new coronavirus variant (Omicron) infection epidemic broke out in Shanghai, China. However, it is unclear whether the duration of this omicron variant is different from that of the prototype strain. Methods: We retrospectively analyzed 157 cases of Omicron variant infection in Taizhou Public Health Center from March 29, 2022, to April 18, 2022, and observed the dynamics of nucleic acid Ct values during the admission and discharge of patients. Clinical and laboratory indicators of these patients were also obtained. Results: Compared to the prototype strain, the Omicron variant showed a broad population susceptibility in infected individuals (regardless of age and presence of underlying disease) and had slight damage to the immune system and renal function; the viral loads peaked was 2-3 days from disease onset; the median duration of omicron variant was 15-18 days; the nucleic acid Ct value of nasopharyngeal swabs of infected patients is lower than that of throat swabs, and the Ct value of oropharyngeal swabs is unstable during the recovery period. Conclusion: Therefore, we found that the time to peak viral load of this Omicron variant was 2-3 days after the onset of the disease, and the duration was 15-18 days; symptomatic patients had higher viral load and longer hospitalization time. This finding will provide a basis for understanding omicron variants and formulating the national prevention and control strategy.

UPLC-MS/MS-based plasma lipidomics reveal a distinctive signature in systemic lupus erythematosus patients.

Global lipidomics is of considerable utility for exploring altered lipid profiles and unique diagnostic biomarkers in diseases. We aim to apply ultra-performance liquid chromatography-tandem mass spectrometry to characterize the lipidomics profile in systemic lupus erythematosus (SLE) patients and explore the underlying pathogenic pathways using the lipidomics approach. Plasma samples from 18 SLE patients, 20 rheumatoid arthritis (RA) patients, and 20 healthy controls (HC) were collected. A total of 467 lipids molecular features were annotated from each sample. Orthogonal partial least square-discriminant analysis, K-mean clustering analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated disrupted lipid metabolism in SLE patients, especially in phospholipid, glycerol, and sphingolipid metabolism. The area under curve (AUC) of lipid metabolism biomarkers was better than SLE inflammation markers that ordinarily used in the clinic. Proposed model of monoglyceride (MG) (16:0), MG (18:0), phosphatidylethanolamine (PE) (18:3-16:0), PE (16:0-20:4), and phosphatidylcholine (PC) (O-16:2-18:3) yielded AUC 1.000 (95% CI, 1.000-1.000), specificity 100% and sensitivity 100% in the diagnosis of SLE from HC. A panel of three lipids molecular PC (18:3-18:1), PE (20:3-18:0), PE (16:0-20:4) permitted to accurately diagnosis of SLE from RA, with AUC 0.921 (95% CI, 0.828-1.000), 70% specificity, and 100% sensitivity. The plasma lipidomics signatures could serve as an efficient and accurate tool for early diagnosis and provide unprecedented insight into the pathogenesis of SLE.

Eleven routine clinical features predict COVID-19 severity uncovered by machine learning of longitudinal measurements.

Severity prediction of COVID-19 remains one of the major clinical challenges for the ongoing pandemic. Here, we have recruited a 144 COVID-19 patient cohort, resulting in a data matrix containing 3,065 readings for 124 types of measurements over 52 days. A machine learning model was established to predict the disease progression based on the cohort consisting of training, validation, and internal test sets. A panel of eleven routine clinical factors constructed a classifier for COVID-19 severity prediction, achieving accuracy of over 98% in the discovery set. Validation of the model in an independent cohort containing 25 patients achieved accuracy of 80%. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 0.70, 0.99, 0.93, and 0.93, respectively. Our model captured predictive dynamics of lactate dehydrogenase (LDH) and creatine kinase (CK) while their levels were in the normal range. This model is accessible at https://www.guomics.com/covidAI/ for research purpose.

Proteomic and metabolomic investigation of serum lactate dehydrogenase elevation in COVID-19 patients.

Serum lactate dehydrogenase (LDH) has been established as a prognostic indicator given its differential expression in COVID-19 patients. However, the molecular mechanisms underneath remain poorly understood. In this study, 144 COVID-19 patients were enrolled to monitor the clinical and laboratory parameters over 3 weeks. Serum LDH was shown elevated in the COVID-19 patients on admission and declined throughout disease course, and its ability to classify patient severity outperformed other biochemical indicators. A threshold of 247 U/L serum LDH on admission was determined for severity prognosis. Next, we classified a subset of 14 patients into high- and low-risk groups based on serum LDH expression and compared their quantitative serum proteomic and metabolomic differences. The results showed that COVID-19 patients with high serum LDH exhibited differentially expressed blood coagulation and immune responses including acute inflammatory responses, platelet degranulation, complement cascade, as well as multiple different metabolic responses including lipid metabolism, protein ubiquitination and pyruvate fermentation. Specifically, activation of hypoxia responses was highlighted in patients with high LDH expressions. Taken together, our data showed that serum LDH levels are associated with COVID-19 severity, and that elevated serum LDH might be consequences of hypoxia and tissue injuries induced by inflammation.

Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-Cov-2 IgM/IgG.

BACKGROUND: Since December 2019, there had been an outbreak of COVID-19 in Wuhan, China. At present, diagnosis COVID-19 were based on real-time RT-PCR, which have to be performed in biosafe laboratory and is unsatisfactory for suspect case screening. Therefore, there is an urgent need for rapid diagnostic test for COVID-19. OBJECTIVE: To evaluate the diagnostic performance and clinical utility of the colloidal gold immunochromatography assay for SARS-Cov-2 specific IgM/IgG anti-body detection in suspected COVID-19 cases. METHODS: In the prospective cohort, 150 patients with fever or respiratory symptoms were enrolled in Taizhou Public Health Medical Center, Taizhou Hospital, Zhejiang province, China, between January 20 to February 2, 2020. All patients were tested by the colloidal gold immunochromatography assay for COVID-19. At least two samples of each patient were collected for RT-PCR assay analysis, and the PCR results were performed as the reference standard of diagnosis. Meanwhile 26 heathy blood donor were recruited. The sensitivity and specificity of the immunochromatography assay test were evaluated. Subgroup analysis were performed with respect to age, sex, period from symptom onset and clinical severity. RESULTS: The immunochromatography assay test had 69 positive result in the 97 PCR-positive cases, achieving sensitivity 71.1% [95% CI 0.609-0.797], and had 2 positive result in the 53 PCR-negative cases, achieving specificity 96.2% [95% CI 0.859-0.993]. In 26 healthy donor blood samples, the immunochromatography assay had 0 positive result. In subgroup analysis, the sensitivity was significantly higher in patients with symptoms more than 14 days 95.2% [95% CI 0.741-0.998] and patients with severe clinical condition 86.0% [95% CI 0.640-0.970]. CONCLUSIONS: The colloidal gold immunochromatography assay for SARS-Cov-2 specific IgM/IgG anti-body had 71.1% sensitivity and 96.2% specificity in this population, showing the potential for a useful rapid diagnosis test for COVID-19. Further investigations should be done to evaluate this assay in variety of clinical settings and populations.

Renal and cerebral RAS interaction contributes to diabetic kidney disease.

The diabetes mellitus has posed a grave threat on human health, and is bound to result in renal trauma by uncertain mechanisms. Increasing evidences indicated that the activation of the renin-angiotensin system plays a pivotal role during the progression of diabetic kidney disease. In streptozotocin (STZ)-induced type 1 diabetic rat model, the losartan (a selective angiotensin II type 1 (AT1) receptor antagonist) and tempol (4-Hydroxy-TEMPO, reactive oxygen species scavenger) were administrated through intracerebroventricular injection or intragastric gavage. Intracerebroventricular administration of clonidine or renal denervation was carried out to block sympathetic nerve traffic. Compared with non-diabetic rats, the reno-cerebral axis was over-activated, including activity of renin-angiotensin system (RAS), oxidative stress, and sympathetic activity in diabetic rats. Central blockade of RAS inhibited the central oxidative stress and sympathetic activity, which led to decrease of intrarenal RAS activity and oxidative stress. Meanwhile, central administration of tempol reduced brain RAS, thus downregulated renal RAS activity and oxidative stress. Importantly, oral administration by intragastric gavage of high dose of losartan and tempol achieved the same effect. The results suggested that there is a cross-talk between renal and cerebral RAS/reactive oxygen species, contributing to the progression of diabetic kidney disease. The subfornical organ, paraventricular nucleus, and supraoptic nucleus in the forebrain also play a key role in development and progression of renal trauma through reno-cerebral reflex axis.

A rare case of an HIV-seronegative AIDS patient with Pneumocystis jirovecii pneumonia.

BACKGROUND: As technology progresses, several highly sensitive human immunodeficiency virus (HIV) screening kits are being researched and developed to quickly and efficiently identify serum HIV antibodies within the non-window period. In individuals who are HIV-seronegative, HIV infections that are not within a window period are rare. In such cases, all antibody detection methods will fail, and misdiagnosing these patients will have catastrophic consequences. CASE PRESENTATION: A 22-year-old male Chinese patient with diffuse exudative lesions in both lungs and initial symptoms of cough and dyspnoea was diagnosed with Pneumocystis jirovecii pneumonia (PJP) by aetiological examination, and the patient's plasma CD4+ T-cell count was extremely low. In China, PJP is prevalent in HIV-infected individuals. Pneumocystis jirovecii (P. jirovecii) has a high colonisation rate in patients with HIV infections. This patient was naturally suspected of being an HIV patient; however, serum HIV antibody tests were negative using both an enzyme-linked immunosorbent assay (ELISA) and a latex agglutination assay, and HIV was not detected by western blotting. Subsequently, the plasma HIV viral load was found to be extremely high on two repeated plasma HIV RNA tests, thus confirming HIV-seronegative acquired immunodeficiency syndrome (AIDS) in this patient. With administration of effective anti-P. jirovecii treatment and highly active antiretroviral therapy (HAART) after diagnosis, the patient's disease condition was rapidly controlled. CONCLUSION: This is the second reported case in China of an HIV-seronegative AIDS patient. Such cases are also rare worldwide. Although HIV-seronegative HIV infections are rare, AIDS should be considered in immunodeficient patients with opportunistic infections, even if the test results are HIV-seronegative. Plasma HIV RNA testing is important for such patients.

Increased Apoptosis in the Paraventricular Nucleus Mediated by AT1R/Ras/ERK1/2 Signaling Results in Sympathetic Hyperactivity and Renovascular Hypertension in Rats after Kidney Injury.

Background: The central nervous system plays a vital role in the development of hypertension, but the molecular regulatory mechanisms are not fully understood. This study aimed to explore signaling in the paraventricular nucleus (PVN) which might contribute to renal hypertension. Methods: Renal hypertension model was established by five-sixth nephrectomy operation (5/6Nx) in male Sprague Dawley rats. Ten weeks afterwards, they were random assigned to no treatment, or intracerebroventricular injection (ICV) with artificial cerebrospinal fluid, losartan [angiotensin II receptor type 1 (AT1R) antagonist], farnesylthiosalicylic acid (Ras inhibitor), PD98059 (MEK inhibitor), or SB203580 (p38 inhibitor) and Z-DEVD-FMK (caspase-3 inhibitor). Before and after treatment, physiological and biochemical indices were measured. Immunohistochemistry, western blot and RT-PCR were applied to quantify key components of renin-angiotensin system, apoptosis-related proteins, Ras-GTP, and MAPKs in the PVN samples. TUNEL assay was used to measure the situ apoptosis in PVN. Results: The 5/6Nx rats showed significantly elevated systolic blood pressure, urinary protein excretion, serum creatinine, and plasma norepinephrine (p < 0.05) compared to sham rats. The expression of angiotensinogen, Ang II, AT1R, p-ERK1/2, or apoptosis-promoting protein Bax were 1.08-, 2.10-, 0.74-, 0.82-, 0.83-fold higher in the PVN of 5/6Nx rats, than that of sham rats, as indicated by immunohistochemistry. Western blot confirmed the increased levels of AT1R, p-ERK1/2 and Bax; meanwhile, Ras-GTP and p-p38 were also found higher in the PVN of 5/6Nx rats, as well as the apoptosis marker cleaved caspase-3 and TUNEL staining. In 5/6Nx rats, ICV infusion of AT1R antagonist, Ras inhibitor, MEK inhibitor or caspase-3 inhibitor could lower systolic blood pressure (20.8-, 20.8-, 18.9-, 14.3%-fold) together with plasma norepinephrine (53.9-, 57.8-,63.3-, 52.3%-fold). Western blot revealed that blocking the signaling of AT1R, Ras, or MEK/ERK1/2 would significantly reduce PVN apoptosis as indicated by changes of apoptosis-related proteins (p < 0.05). AT1R inhibition would cause reduction in Ras-GTP and p-ERK1/2, but not vice versa; such intervention with corresponding inhibitors also suggested the unidirectional regulation of Ras to ERK1/2. Conclusion: These findings demonstrated that the activation of renin-angiotensin system in PVN could induce apoptosis through Ras/ERK1/2 pathway, which then led to increased sympathetic nerve activity and renal hypertension in 5/6Nx rats.

Proteomic and phosphoproteomic analysis of renal cortex in a salt-load rat model of advanced kidney damage.

Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex. We found 318 proteins differentially expressed in 5/6Nx group relative to sham group, and 310 proteins significantly changed in response to salt load in 5/6Nx animals. Totally, 1810 unique phosphopeptides corresponding to 550 phosphoproteins were identified. We identified 113 upregulated and 84 downregulated phosphopeptides in 5/6Nx animals relative to sham animals. Salt load induced 78 upregulated and 91 downregulated phosphopeptides in 5/6Nx rats. The differentially expressed phospholproteins are important transporters, structural molecules, and receptors. Protein-protein interaction analysis revealed that the differentially phosphorylated proteins in 5/6Nx group, Polr2a, Srrm1, Gsta2 and Pxn were the most linked. Salt-induced differential phosphoproteins, Myh6, Lmna and Des were the most linked. Altered phosphorylation levels of lamin A and phospholamban were validated. This study will provide new insight into pathogenetic mechanisms of chronic kidney disease and salt sensitivity.

Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model) or sham operation were treated for 2 weeks with a normal-(0.4% NaCl), or high-salt (4% NaCl) diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54%) phosphopeptides upregulated and 76 (46%) phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49%) phosphopeptides and downregulation of 88 (51%) phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.