Transcriptomic analysis reveals partial epithelial-mesenchymal transition and inflammation as common pathogenic mechanisms in hypertensive nephrosclerosis and Type 2 diabetic nephropathy.

Hypertensive nephrosclerosis (HN) and Type 2 diabetic nephropathy (T2DN) are the leading causes of chronic kidney disease (CKD). To explore shared pathogenetic mechanisms, we analyzed transcriptomes of kidney biopsies from patients with HN or T2DN. Total RNA was extracted from 10 µm whole kidney sections from patients with HN, T2DN, and normal controls (Ctrl) (n = 6 for each group) and processed for RNA sequencing. Differentially expressed (log2 fold change >1, adjusted p < 0.05) genes (DEG) and molecular pathways were analyzed, and selected results were validated by immunohistochemistry (IHC). ELISA on serum samples was performed on a related cohort consisting of patients with biopsy-proven HN (n = 13) and DN (n = 9), and a normal control group (n = 14). Cluster analysis on RNA sequencing data separated diseased and normal tissues. RNA sequencing revealed that 88% (341 out of 384) of DEG in HN were also altered in T2DN, while gene set enrichment analysis (GSEA) showed that over 90% of affected molecular pathways, including those related to inflammation, immune response, and cell-cycle regulation, were similarly impacted in both HN and T2DN samples. The increased expression of genes tied to interleukin signaling and lymphocyte activation was more pronounced in HN, while genes associated with extracellular matrix organization were more evident in T2DN. Both HN and T2DN tissues exhibited significant upregulation of genes connected with inflammatory responses, T-cell activity, and partial epithelial to mesenchymal transition (p-EMT). Immunohistochemistry (IHC) further confirmed T-cell (CD4+ and CD8+ ) infiltration in the diseased tissues. Additionally, IHC revealed heightened AXL protein expression, a key regulator of inflammation and p-EMT, in both HN and T2DN, while serum analysis indicated elevated soluble AXL levels in patients with both conditions. These findings underline the shared molecular mechanisms between HN and T2DN, hinting at the potential for common therapeutic strategies targeting both diseases.

Association of redox and inflammation-related biomarkers with prognosis in IgA nephropathy: A prospective observational study.

BACKGROUND: IgA nephropathy (IGAN) has a variable prognosis. Risk stratification tools are usually based on clinical parameters combined with histologic Oxford-MEST-C score. Circulating redox- and inflammation-related biomarkers may be related to histological changes in IGAN. Therefore, we studied the performance of these biomarkers in predicting the rate of GFR-loss in IGAN. METHODS: This was an observational prospective study. Fifty-seven stable patients with IGAN were examined at baseline and after a mean observational time of 5.9 ± 1.1 years. The main outcome measure was eGFR-loss per year with predefined groups, stable (<1.5 ml/min/1,73 m2/year, intermediate (between 1.5 and 2.5), and progressive (>2.5). RESULTS: Fifteen patients were in the progressive, 11 in the intermediate, and 31 in the stable groups. Positive relationships were detected between eGFR-loss per year and baseline nitrate, oxidized free cysteine, parathyroid hormone, APRIL, TNFR1, CD30, chitinase 3, and LIF-5. The progressive group had elevated concentrations of these markers plus AOPP and osteopontin. Through ROC analysis, it was observed that AOPP, oxidized free cysteine, TNFR1, osteopontin, and LIF-5 had the best ability to identify progressive vs. non-progressive diseases. The combination of urinary albumin/creatinine ratio with AOPP and TNFR1 significantly improved the ability to identify progressive eGFR decline with ROC AUC 95% (adjusted 85%). CONCLUSIONS: We found prognostic biomarkers related to the rate of eGFR-loss in IGAN. These biomarkers may help identify patients at risk of progressive disease. AOPP, oxidized free cysteine, TNFR1, and osteopontin are promising prognostic biomarkers in IGAN, however, further validation studies are needed.

Circulating inflammation-related factors are correlated with systemic redox status in IgA nephropathy; a case-control study.

BACKGROUND: IgA nephropathy (IGAN) is characterized by oxidative stress and inflammation. In the present study, we explored the relationship of redox status vs. that of circulating inflammation-related factors with other biomarkers in patients with IGAN. METHODS: This is a case-control study comparing patients with IGAN (Stage 1-4) to healthy controls. Forty patients and 40 controls were matched for age and sex. Two circulating dynamic redox parameters were analysed: oxidized free cysteine (Cys) and nitrate. Thirty-seven inflammation-related factors were measured in serum. RESULTS: The patients had elevated levels of oxidized free Cys and nitrate, indicating the presence of oxidative stress. Nine circulating inflammation-related factors were higher in the serum of patients than in that of controls. The most important factors were APRIL, MMP-3, osteopontin, TNFR1 and TWEAK. Inflammation-related factors were positively correlated with oxidized free Cys, nitrate, creatinine and parathyroid hormone (PTH) in the patients. The correlation coefficients of Latent Inflammatory Factor vs. oxidized free Cys and nitrate were r = 0.43 (p = 0.007) and r = 0.51 (p = 0.001), respectively. This finding persisted after adjusting for the glomerular filtration rate. CONCLUSIONS: Patients with IGAN had disturbed redox status. Several circulating inflammation-related factors were elevated, suggesting activation of the non-canonical NF-kB pathway. There was a positive relationship between systemic redox status and the level of inflammation-related factors, partially independent of GFR. The present findings raise the question of whether circulating oxidized free Cys and/or nitrate may be employed as prognostic biomarkers for IGAN in the future.

Systemic redox biomarkers and their relationship to prognostic risk markers in autosomal dominant polycystic kidney disease and IgA nephropathy.

BACKGROUND: Oxidative stress is evident from an early stage in chronic kidney disease (CKD). Therefore, we investigated redox biomarkers in polycystic kidney disease (ADPKD) and IgA nephropathy (IGAN). METHODS: This is a case-control study with three groups: ADPKD (n = 54), IGAN (n = 58) and healthy controls (n = 86). The major plasma aminothiols with their redox species were examined: homocysteine (Hcy), cysteinglycine (CG), cysteine (Cys) and glutathione (GSH). The redox ratio was the ratio of reduced free and oxidized aminothiols in plasma. We investigated malonedialdehyde (MDA) and advanced oxidation protein products (AOPP), and ten single nucleotide polymorphisms of antioxidant enzymes. RESULTS: Patients had elevated oxidized free Hcy and Cys with associated low redox ratios - most pronounced in IGAN. Patients with IGAN had elevated AOPP and possibly MDA. Oxidized free Hcy and Cys with redox ratios were correlated to AOPP, MDA and proteinuria. Furthermore, there was an independent relationship to parathyroid hormone (PTH). IGAN had an elevated frequency of Val16Ala SNP rs4880, which influence the function of mitochondrial superoxide dismutase 2 (p = 0.03). CONCLUSIONS: Patients with ADPKD and IGAN have evidence of oxidative stress from stage 1 to 4 - most pronounced in IGAN. In patients, aminothiol redox biomarkers were correlated to AOPP, proteinuria and PTH, which are known prognostic markers in CKD. It may be possible that oxidative stress influences PTH dysregulation in CKD. The association between IGAN and the redox related variant allele rs4880(C) might indicate a new susceptibility locus for IGAN, but this needs verification.

Risk factors for exertional rhabdomyolysis with renal stress.

BACKGROUND: Exercise-induced rhabdomyolysis denotes the exertional damage of myocytes with leakage of sarcoplasmic content into the circulation. The purpose of this study was to determine important risk factors for the development of exertional rhabdomyolysis in a temperate climate and to study the renal effects of myoglobinuria. METHODS: A cluster of eight military recruits was admitted to hospital due to exertional rhabdomyolysis with myoglobinuria. The patients were treated according to current guidelines with isotonic saline and alkalinisation of the urine. The eight patients were compared with a randomly selected control group of 26 healthy fellow recruits. All subjects responded to a standardised questionnaire. RESULTS: There were little differences in baseline characteristics between patients and controls. In the present study, exercise intensity, duration and type were all significant determinants of exertional rhabdomyolysis in univariate models. However, in a multivariate model, high exercise intensity on day -1 was the only significant predictor of rhabdomyolysis (p=0.02). All patients had a stable serum creatinine and cystatin C. There was a significant increase in serum neutrophil gelatinase-associated lipocalin (NGAL) in the patients, suggesting renal stress. CONCLUSIONS: Sustained maximal intensity exercise is a crucial risk factor for rhabdomyolysis with gross pigmenturia. Elevated serum NGAL concentrations indicate the presence of renal stress. It appears to be possible to quantify the risk of rhabdomyolysis by means of a simple questionnaire. In the future, this may be used as a tool to prevent rhabdomyolysis.

Kidney donors and kidney transplants have abnormal aminothiol redox status, and are at increased risk of oxidative stress and reduced redox buffer capacity.

OBJECTIVE: Living kidney donors have been part of a successful kidney transplant programme in Norway for almost 50 years. Glomerular filtration rates (GFRs) have tended to remain stable at about 70% of pre-donation levels. Plasma total homocysteine (Hcy) has an inverse relationship to kidney function, and previous reports indicate elevated levels of Hcy in kidney donors. We wanted to examine the most important plasma aminothiols in kidney donors, i.e. Hcy, cysteine (Cys) and cysteinylglycine (CG) with their redox species. The aminothiol redox-system appears to be an integral part of the extracellular antioxidant defence system in the body. DESIGN AND METHODS: Plasma concentrations of total Hcy were obtained in 82 previous kidney donors, 82 healthy controls and 26 kidney transplants with stable and good kidney function. In a subset of 30 kidney donors, 30 matched controls and 12 kidney transplants plasma samples were analysed for Hcy, Cys, CG and their redox species. There were no differences between groups for B-vitamin status. RESULTS: Kidney donors and kidney transplants had elevated plasma concentrations of total Hcy, Cys and CG. The plasma levels of reduced Hcy species were high - with a high reduced/oxidized ratio. The plasma levels of reduced Cys species were low - with a low reduced/oxidized ratio. CONCLUSIONS: Previous kidney donors have abnormal plasma aminothiol redox status. The present findings indicate that donors may have increased risk of oxidative stress with low redox buffer capacity and disturbed cellular redox-dependent signalling pathways. Similar observations were made in the kidney transplants.

Long-term outcomes after cyclosporine or mycophenolate withdrawal in kidney transplantation - results from an aborted trial.

Long-term triple immunosuppressive therapy with cyclosporine (CsA), mycophenolate mofetil (MMF) and prednisolone may be excessively powerful for many transplant recipients. We compared withdrawal of either MMF or CsA in stable kidney transplants on triple immunosuppression. The study was a prospective, randomized, controlled 12-months trial in stable kidney transplants. The patients who withdrew CsA were given MMF 2 g/d, and CsA troughs were between 75 and 125 ng/mL in MMF withdrawal. Planned inclusion was 298 patients. The study was prematurely aborted after inclusion of 39 patients. Acute rejection rates were 6/20 (30%) in the MMF group compared with 0/19 (0%) in the CsA group (p = 0.02). Time to acute rejections was 4.0-28.7 months after withdrawal. Trough concentrations of mycophenolic acid (MPA) and CsA showed therapeutic levels. The subjects have been observed for eight yr, and of the 28 patients remaining on randomized therapy, the MMF patients preserved graft function better than CsA patients. Death-censored graft survival was 75% and 95% (p = 0.18) and patient survival was 70% and 68% (p = 0.99) in the MMF and CsA groups, respectively, at the end of long-term follow-up. CsA withdrawal was associated with a high rate of acute rejections. Initially, the treatment of acute rejections was successful. However, five of six lost their grafts in the long term.

Serum folate is significantly correlated with plasma cysteine concentrations in healthy industry workers.

BACKGROUND: A low concentration of serum folate is associated with an increased risk of cardiovascular disease. Extracellular cysteine is involved in aging, cancer and cardiovascular disease. The relationship between serum folate and plasma cysteine is poorly understood. Therefore, we investigated this relationship in industry workers, whose health has economic implications. METHODS: The concentration of serum folate was determined by the Access ImmunoAssay System Sanofi Pasteur. Plasma cysteine and homocysteine were measured by an ion-pair HPLC method. The concentrations of serum triglycerides were determined by an enzymatic colorimetric method. RESULTS: We detected a positive correlation between the concentration of serum folate and plasma cysteine, whereas the concentration of serum folate was negatively correlated with plasma homocysteine and serum triglycerides. In a multiple regression analysis with adjustment for age, BMI and smoking, serum folate as the dependent variable exhibited a strong relationship with plasma cysteine, and a negative relationship with plasma homocysteine and serum triglycerides. CONCLUSION: We observed significant correlations between serum folate, plasma cysteine and serum triglyceride concentrations in industry workers, implying that folate may modulate key aspects of the body's cysteine and lipid metabolism.

Drug-induced pertubation of the aminothiol redox-status in patients with epilepsy: improvement by B-vitamins.

OBJECTIVES: Patients with epilepsy have excess morbidity and mortality due to ischemic cardiovascular disease. Many of these patients have elevated concentrations of plasma total homocysteine (Hcy), which is an acknowledged risk factor for cardiovascular disease, venous thromboembolic disease, foetal malformations and dementia. Hyperhomocysteinemia may have negative effects through mechanisms involving oxidative damage. In the present study, we have investigated the aminothiol redox-status in patients on antiepileptic drugs. Thereafter, in a subset of patients with elevated total Hcy, we evaluated the effect of B-vitamin therapy. METHODS: In the first part of the study, 101 patients on antiepileptic drugs were compared with 101 matched healthy controls. The redox-species of Hcy, cysteine and cysteinylglycine, the major aminothiols in plasma, were analyzed by high-performance liquid chromatography (HPLC). Hyperhomocysteinemia was defined as fasting total Hcy above 12 micromol/L and/or post-methionine load concentrations above 38 micromol/L. In the second part of the study, 33 patients identified with hyperhomocysteinemia were supplemented with three B-vitamins for 30 days; folic acid (B9), pyridoxine (B6) and riboflavin (B2). RESULTS: All redox-species of Hcy were significantly elevated in the patients, except the fasting concentrations of reduced Hcy (p=0.09). The reduced/total ratio of cysteine in fasting plasma was lower in the patients than in the controls: 5.20% vs. 6.19%, respectively (p=0.006). After 30 days of B-vitamin supplementation, the plasma concentrations of reduced, oxidized and protein-bound Hcy species were significantly lowered by 17%, 22% and 28%, respectively. The reduced/total ratio of cysteine rose from 4.9% to 7.9% (p=0.007). CONCLUSIONS: Patients on antiepileptic drugs have abnormal aminothiol redox-status associated with hyperhomocysteinemia. This is similar to findings in patients with cardiovascular disease. B-vitamin supplementation partially corrects the abnormal aminothiol redox-status. Possibly, B-vitamin supplementation may be useful in drug-induced hyperhomocysteinemia.

Thyroid function during B-vitamin supplementation of patients on antiepileptic drugs.

OBJECTIVES: Patients on antiepileptic drugs (AEDs) may have low serum concentrations of thyroxine, with or without a compensatory increment in thyroid-stimulating hormone (TSH). Furthermore, patients on AEDs often have hyperhomocysteinemia and low concentrations of vitamins B(6), B(2) and folate. Previously, an inverse relationship between thyroxine and homocysteine concentrations has been observed. In animals, deficiency of vitamin B(6) has been found to impair the hypophyseal release of TRH. We have studied the effect of B-vitamin supplements on thyroid function in patients on AEDs. DESIGN AND METHODS: Thirty-two patients on AEDs were identified with hyperhomocysteinemia and low folate, B(6) and B(2). They were supplemented with pyridoxine, riboflavin and folic acid for 30 days. RESULTS: At baseline, the patients had low serum concentrations of free thyroxin and slightly elevated TSH. On day 30 of the B-vitamin supplements, homocysteine had decreased, however, the thyroid parameters remained unchanged. CONCLUSIONS: Hyperhomocysteinemic patients on AEDs have indications of hypothyroidism, however, supplementation with B-vitamins does not improve their thyroid function.

The effect of B-vitamins on hyperhomocysteinemia in patients on antiepileptic drugs.

Patients on antiepileptic drugs (AEDs) may have elevated levels of plasma total homocysteine (p-tHcy). The aim of this study was to assess the effect of B-vitamin supplementation on the levels of p-tHcy and markers of endothelial activation and lipid peroxidation. A total of 33 adult patients on AEDs were identified with either fasting (Group 1, n=23) or post methionine load (PML) (Group 2, n=10) hyperhomocysteinemia. Subjects were supplemented with B-vitamins for 30 days: folic acid 0.4 mg, pyridoxine 120 mg and riboflavin 75 mg per day. After supplementation, serum folate and pyridoxal phosphate had increased, while fasting and PML p-tHcy had decreased (P<0.0001) by 36 and 26%, respectively. Prior to supplementation, the Group 1 patients had elevated levels of P-selectin and von Willebrand factor (vWF) (P=0.05 and 0.03, respectively). After supplementation, the levels of intercellular cell adhesion molecules had decreased (P=0.01) and E-selectin decreased nonsignificantly (P=0.07). However, the levels of vascular cell adhesion molecules had increased (P<0.0001), while lipid peroxidation were unchanged. In conclusion, the combined supplementation with folic acid, pyridoxine and riboflavin reduced fasting and PML hyperhomocysteinemia in patients on AEDs. Patients with fasting hyperhomocysteinemia had elevated levels of P-selectin and vWF, which may indicate an increased risk of cardiovascular disease. Furthermore, B-vitamin supplementation influenced endothelial activation, although the clinical implication is uncertain.