Dilated Cardiomyopathy in an Adult Renal Transplant Recipient: Recovery Upon Tacrolimus to Sirolimus Switch: A Case Report.

The objective of immunosuppressive drugs used in solid organ transplantation is to achieve acceptable rejection rates, minimize infections, and prolong graft and patient survival. Cardiovascular disease is a major cause of death in kidney transplant recipients. The drugs commonly used to prevent rejection (calcineurin inhibitors [CNIs] and steroids) contribute to cardiac disease seen in transplant patients by increasing the risk of hypertension and diabetes. Direct cardiac toxicity of chemotherapeutic drugs such as doxorubicin is well-known but potential direct effect of CNIs on myocardium is less explored and understood. Cardiac toxicity a rare but serious adverse effect of tacrolimus, has been observed in patients receiving solid organ transplants such as liver, bowel and kidney. In this report, we describe a case of new onset severe dilated cardiomyopathy after kidney transplantation. Reversal of heart failure occurred after tacrolimus discontinuation and the switch to a mammalian target of rapamycin (mTOR) inhibitor: sirolimus

High glucose-induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model.

BACKGROUND: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. METHODS: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. RESULTS: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition. CONCLUSIONS: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

A Prokaryotic S1P Lyase Degrades Extracellular S1P In Vitro and In Vivo: Implication for Treating Hyperproliferative Disorders

Sphingosine-1-phosphate (S1P) regulates a broad spectrum of fundamental cellular processes like proliferation, death, migration and cytokine production. Therefore, elevated levels of S1P may be causal to various pathologic conditions including cancer, fibrosis, inflammation, autoimmune diseases and aberrant angiogenesis. Here we report that S1P lyase from the prokaryote Symbiobacterium thermophilum (StSPL) degrades extracellular S1P in vitro and in blood. Moreover, we investigated its effect on cellular responses typical of fibrosis, cancer and aberrant angiogenesis using renal mesangial cells, endothelial cells, breast (MCF-7) and colon (HCT 116) carcinoma cells as disease models. In all cell types, wild-type StSPL, but not an inactive mutant, disrupted MAPK phosphorylation stimulated by exogenous S1P. Functionally, disruption of S1P receptor signaling by S1P depletion inhibited proliferation and expression of connective tissue growth factor in mesangial cells, proliferation, migration and VEGF expression in carcinoma cells, and proliferation and migration of endothelial cells. Upon intravenous injection of StSPL in mice, plasma S1P levels rapidly declined by 70% within 1 h and then recovered to normal 6 h after injection. Using the chicken chorioallantoic membrane model we further demonstrate that also under in vivo conditions StSPL, but not the inactive mutant, inhibited tumor cell-induced angiogenesis as an S1P-dependent process. Our data demonstrate that recombinant StSPL is active under extracellular conditions and holds promise as a new enzyme therapeutic for diseases associated with increased levels of S1P and S1P receptor signaling

Recombinant Expression, Purification, and Functional Characterisation of Connective Tissue Growth Factor and Nephroblastoma-Overexpressed Protein

The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated

The Interaction of LFA-1 on Mononuclear Cells and ICAM-1 on Tubular Epithelial Cells Accelerates TGF-β1-Induced Renal Epithelial-Mesenchymal Transition

The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-β1 on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-β1 stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-β1 (0.1 ng/ml) (HK-2-TGF-β1 (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-β1 (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-β1 (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-β1 (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-β1 (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-β1 (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72–96 hrs) TGF-β1 stimulation increased, that of HK-2-TGF-β1 (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-β1 induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-β1

Tubulointerstitial injury and the progression of chronic kidney disease

In chronic kidney disease (CKD), once injury from any number of disease processes reaches a threshold, there follows an apparently irreversible course toward decline in kidney function. The tubulointerstitium may play a key role in this common progression pathway. Direct injury, high metabolic demands, or stimuli from various other forms of renal dysfunction activate tubular cells. These, in turn, interact with interstitial tissue elements and inflammatory cells, causing further pathologic changes in the renal parenchyma. The tissue response to these changes thus generates a feed-forward loop of kidney injury and progressive loss of function. This article reviews the mechanisms of this negative cycle mediating CKD

Pathogenesis of progressive scarring trachoma in Ethiopia and Tanzania and its implications for disease control: two cohort studies.

BACKGROUND: Trachoma causes blindness through a conjunctival scarring process initiated by ocular Chlamydia trachomatis infection; however, the rates, drivers and pathophysiological determinants are poorly understood. We investigated progressive scarring and its relationship to conjunctival infection, inflammation and transcript levels of cytokines and fibrogenic factors. METHODOLOGY/PRINCIPAL FINDINGS: We recruited two cohorts, one each in Ethiopia and Tanzania, of individuals with established trachomatous conjunctival scarring. They were followed six-monthly for two years, with clinical examinations and conjunctival swab sample collection. Progressive scarring cases were identified by comparing baseline and two-year photographs, and compared to individuals without progression. Samples were tested for C. trachomatis by PCR and transcript levels of S100A7, IL1B, IL13, IL17A, CXCL5, CTGF, SPARCL1, CEACAM5, MMP7, MMP9 and CD83 were estimated by quantitative RT-PCR. Progressive scarring was found in 135/585 (23.1%) of Ethiopian participants and 173/577 (30.0%) of Tanzanian participants. There was a strong relationship between progressive scarring and increasing inflammatory episodes (Ethiopia: OR 5.93, 95%CI 3.31-10.6, p<0.0001. Tanzania: OR 5.76, 95%CI 2.60-12.7, p<0.0001). No episodes of C. trachomatis infection were detected in the Ethiopian cohort and only 5 episodes in the Tanzanian cohort. Clinical inflammation, but not scarring progression, was associated with increased expression of S100A7, IL1B, IL17A, CXCL5, CTGF, CEACAM5, MMP7, CD83 and reduced SPARCL1. CONCLUSIONS/SIGNIFICANCE: Scarring progressed in the absence of detectable C. trachomatis, which raises uncertainty about the primary drivers of late-stage trachoma. Chronic conjunctival inflammation appears to be central and is associated with enriched expression of pro-inflammatory factors and altered expression of extracellular matrix regulators. Host determinants of scarring progression appear more complex and subtle than the features of inflammation. Overall this indicates a potential role for anti-inflammatory interventions to interrupt progression and the need for trichiasis disease surveillance and surgery long after chlamydial infection has been controlled at community level

Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed