Immunological risk stratification and tailored minimisation of immunosuppression in renal transplant recipients.

BACKGROUND: The efficacy and safety of minimisation of immunosuppression including early steroid withdrawal in kidney transplant recipients treated with Basiliximab induction remains unclear. METHODS: This retrospective cohort study reports the outcomes from 298 consecutive renal transplants performed since 1st July 2010-June 2013 treated with Basiliximab induction and early steroid withdrawal in low immunological risk patients using a simple immunological risk stratification and 3-month protocol biopsy to optimise therapy. The cohort comprised 225 low-risk patients (first transplant or HLA antibody calculated reaction frequency (CRF ≤50% with no donor specific HLA antibodies) who underwent basiliximab induction, steroid withdrawal on day 7 and maintenance with tacrolimus and mycophenolate mofetil (MMF), and 73 high-risk patients who received tacrolimus, MMF and prednisolone for the first 3 months followed by long term maintenance immunosuppression with tacrolimus and prednisolone. High-risk patients not undergoing 3-month protocol biopsy were continued on triple immunosuppression. RESULTS: Steroid withdrawal could be safely achieved in low immunological risk recipients with IL2 receptor antibody induction. The incidence of biopsy-proven acute rejection was 15.1% in the low-risk and 13.9% in the high-risk group (including sub-clinical rejection detected at protocol biopsy). One- year graft survival was 93.3% and patient survival 98.5% in the low-risk group, and 97.3 and 100% respectively in the high-risk group. Graft function was similar in each group at 1 year (mean eGFR 61.2 ± 23.4 mL/min low-risk and 64.6 ± 19.2 mL/min high-risk). CONCLUSIONS: Immunosuppression regimen comprising basiliximab induction, tacrolimus, MMF and prednisolone with early steroid withdrawal in low risk patients and MMF withdrawal in high risk patients following a normal 3-month protocol biopsy is effective in limiting acute rejection episodes and produces excellent rates of patient survival, graft function and complications.

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-ß1 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-ß1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. RESULTS: Only the combination of high D-glucose and TGF-ß1 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-ß1 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-ß1; this rise was significantly reduced by both SGLT2 and MEK inhibition. CONCLUSIONS: We show that high D-glucose and TGF-ß1 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-ß1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

Evaluation of Urinary Biomarkers of Proximal Tubular Injury, Inflammation, and Fibrosis in Patients With Albuminuric and Nonalbuminuric Diabetic Kidney Disease.

INTRODUCTION: Albuminuric and nonalbuminuric pathways contribute to diabetic kidney disease. Proximal tubule and inflammation play important roles in these processes. Urinary biomarker(s) to detect early kidney damage and predict progression are needed. METHODS: Nine urinary biomarkers were measured at baseline in 400 patients with diabetes. Correlation and multivariate logistic and linear regression analyses were performed to assess the association of biomarkers with chronic kidney disease and progression. RESULTS: In the albumin/creatinine ratio (ACR) <3 cohort, the only biomarker significantly associated with estimated glomerular filtration rate < 60 ml/min was N-acetyl-ß-d-glucosaminidase. A combination of ACR and monocyte chemoattractant protein 1 (MCP1) were significantly associated with stage 2 chronic kidney disease in this cohort. Logistic models showed that in patients with all levels of albuminuria, ACR, retinol binding protein (RBP), and MCP1 were associated with progression. A model including MCP1, interleukin 6, and neutrophil gelatinase-associated lipocalin showed significant association with progression to chronic kidney disease 3/4 in the ACR <3 cohort. Linear mixed-model regression analyses demonstrated MCP1, RBP, and ACR as significant proteins associated with progression to stage 3 or worse, whereas MCP1 was the only significant biomarker in the ACR <3 cohort. Time-to-event and Cox proportional hazard models confirmed significant hazard ratios for progression for ACR, RBP, and MCP1, with significant differences noted between quantiles of biomarkers for ACR, RBP, and MCP1. CONCLUSION: In this study of diabetic patients with single baseline measurements of urinary biomarkers, albumin, RBP, and MCP1 were significantly associated with chronic kidney disease progression at all levels of albuminuria. Inflammatory cytokines, neutrophil gelatinase-associated lipocalin, and MCP1 were associated with progression in patients without albuminuria. N-acetyl-ß-d-glucosaminidase demonstrated a significant association with an estimated glomerular filtration rate < 60 ml/min in the ACR <3 cohort.

Connective tissue growth factor-(CTGF, CCN2)--a marker, mediator and therapeutic target for renal fibrosis.

Connective tissue growth factor (CTGF, CCN2) is a key mediator of tissue fibrosis. CCN2 plays an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. In this review, we discuss the biology of CCN2 with a focus on the regulation of CCN2 gene, cellular mechanisms of profibrotic CCN2 effects and the current in vivo and in vitro evidence for the role of CCN2 in the development of renal fibrosis. We also discuss the therapeutic potential of targeting CCN2 for the treatment of renal fibrosis.

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.

Tgf-beta auto-induction and connective tissue growth factor expression in human renal tubule epithelial cells requires N-ras.

BACKGROUND/AIMS: Transforming growth factor (TGF) beta is strongly implicated in the progression of renal fibrosis. TGFbeta1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFbeta production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFbeta1 inhibition of proliferation, TGFbeta auto-induction and TGFbeta1-induced CTGF expression in HKC human renal tubule epithelial cells. METHODS AND RESULTS: TGFbeta1 (0-25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5-10 min of incubation, TGFbeta1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFbeta3 caused an increase in extracellular TGFbeta1, which was significantly reduced in the presence of PD 98059. TGFbeta1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFbeta1 secretion and TGFbeta1-induced cellular and secreted CTGF. CONCLUSION: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFbeta1 auto-induction and TGFbeta1-induced CTGF expression in human renal tubule epithelial cells.

The TGFbeta1-induced fibronectin in human renal proximal tubular epithelial cells is p38 MAP kinase dependent and Smad independent.

BACKGROUND/AIM: Transforming growth factor beta 1 (TGFbeta1) is a fibrokine implicated in the progression of renal fibrosis. Following TGFbeta1 receptor activation, a number of signalling pathways are stimulated. This study investigates the role of p38 mitogen-activated protein (MAP) kinase and Smad pathways in the TGFbeta1-induced fibronectin (FN) production. METHODS: Transformed human proximal tubular epithelial cells of the line HKC were used. Secreted FN was analyzed by enzyme-linked immunosorbent assay and Smad proteins by Western blotting. Chemical inhibitors were used to study the role of p38 MAP kinase and the TGFbeta receptor ALK5. The Smad pathway was studied using a cell line overexpressing Smad7 and small interfering RNAs (siRNA). The FN mRNA expression was assessed by reverse transcription-polymerase chain reaction. RESULTS: TGFbeta1 produced a significant increase in FN secretion in both HKC and Smad7-HKC cells, and the p38 MAP kinase inhibitor SB202190 markedly reduced this (n = 3, p < 0.05 and p < 0.01). ALK5 inhibition also reduced the TGFbeta1-induced FN (n = 3, p < 0.05). Smad knockdown using the siRNA did not reduce the TGFbeta1-induced FN secretion. TGFbeta1 induced FN mRNA expression in HKC cells, and SB202190 decreased this induction (n = 5, p < 0.05). CONCLUSIONS: These results suggest that TGFbeta1-induced FN production in HKC cells is p38 MAP kinase dependent and Smad independent. Targeting p38 MAP kinase may be of therapeutic value in renal fibrosis.

The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFbeta1 responses in human proximal-tubule epithelial cells.

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGFbeta1-Smad (transforming growth factor-beta1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGFbeta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGFbeta1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, alpha-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGFbeta1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGFbeta1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGFbeta1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulo-interstitial fibrosis.

TGF-beta1-induced connective tissue growth factor (CCN2) expression in human renal proximal tubule epithelial cells requires Ras/MEK/ERK and Smad signalling.

BACKGROUND: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-beta. CCN2 induction by TGF-beta in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-beta1 and the possible mechanisms of this induction in human PTECs. METHODS: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-beta1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-beta1. RESULTS: TGF-beta1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-beta1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-beta1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-beta1 in Smad7 over-expressing HKCs. CONCLUSIONS: TGF-beta1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-beta induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

Intradialytic exercise as anabolic therapy in haemodialysis patients -- a pilot study.

Haemodialysis (HD) patients are characterized by muscle wasting and consequently decreased physical functioning and poor outcome. This pilot study investigated if a novel intradialytic exercise programme could increase lean mass via up-regulation of the insulin-like growth factor (IGF) system. Nine HD patients were assessed before (w-12) and after a 3-month control phase (w0), after a three-month intradialytic interval training programme using high intensity cycle exercise (w12), and after a withdrawal of treatment phase (w24). Body composition was determined by dual energy X-ray absorptiometry (DEXA) and bioelectrical impedance spectroscopy (BIS); physical functioning by knee extensor strength (KES) and 30-s sit stand test (SST); and IGF-I and IGFBP-3 in serum and muscle by radioimmunoassay. Despite significant increases in training load (+274%, P<0.001), peak power output (+71%, P<0.001) and physical function (KES: +19%, P<0.05; SST: +20%, P<0.05) following the intervention phase, lean masses by DEXA, intra cellular water by BIS (a surrogate measure of body cell mass) and serum and muscle IGFs remained unchanged following training. Although this novel exercise programme, utilizing high intensity interval training, was safe, clinically feasible and beneficial in terms of physical functioning, the 12 weeks of intradialytic cycle exercise failed to reverse the muscle atrophy characteristic of this population. Future studies, using primary outcome measures similar to those employed in the present study, should investigate other anabolic interventions to determine potential treatments for the muscle wasting associated with end stage renal disease.

Muscle insulin-like growth factor status, body composition, and functional capacity in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients typically have reduced muscle mass and diminished functional capacity. The role of the muscle insulin-like growth factors (IGFs), a principal anabolic system that is involved in protein synthesis and that has downregulation that is implicated in muscle loss in animal models of uremia, has previously not been assessed in vivo in HD patients. METHODS: Seventeen HD patients were compared cross-sectionally with 17 age-, sex-, and body mass index-matched healthy controls. Body composition was assessed by dual energy x-ray absorptiometry and bioelectrical impedance spectrometry; functional capacity by hand grip strength, quadriceps strength, and 30-second sit-to-stand test; systemic inflammation by tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1); serum and muscle IGF-I and IGFBP-3 by radioimmunoassay; and fragmentation of serum IGFBP-3 by Western immunoblotting. RESULTS: Appendicular lean mass was significantly decreased in HD patients compared with controls (17.6 +/- 0.9 versus 21.5 +/- 1.5 kg, P < .05), as were all measures of functional capacity (P < .01 to .001), and highly significant positive correlations between appendicular lean mass and functional capacity were evident (appendicular lean mass and hand-grip strength, quadriceps strength, 30-second sit-to-stand test, all P < .001). TNF-alpha and TNFR1 were elevated in patients (P < .001). Although serum IGF-I and IGFBP-3 levels did not differ between the groups (P = .295 and .379 respectively), fragmented IGFBP-3 levels were increased (53.1 +/- 16.0 versus 29.81 +/- 15.3%, P < .005). In contrast, muscle IGF-I was substantially diminished in the patient group (n = 7) relative to control (n = 5) levels (0.84 +/- 0.06 versus 2.78 +/- 1.80 pg/microg, P < .05). CONCLUSIONS: We provide evidence of reduced IGF-I in HD patients' skeletal muscle that may be a causal factor in the muscle wasting characteristic of this population. Future research should determine the exact consequences and causes of alterations to the muscle IGF system in HD patients.

Confusion after starting citalopram in a renal transplant patient.

The UK NICE (National Institute for Clinical Excellence) guidelines on management of depression in adults recommends use of selective serotonin re-uptake inhibitor (SSRI) as a first choice agent. Citalopram, an SSRI antidepressant, is a preferred antidepressant in those patients with concomitant chronic physical health problems. Hyponatraemia because of syndrome of inappropriate antiduretic hormone secrection has been reported with SSRI antidepressants but is usually mild and responds to fluid restriction and cessation of the drug. We report a case of severe hyponatraemia because of citalopram in a renal transplant patient who required treatment with hypertonic (3%) saline, with a good outcome avoiding any neurological complications.

Hyperparathyroidism in a renal transplant patient: an unusual cause of resistance to therapy.

A long-term renal transplant patient who was regularly followed up in the transplant clinic with stable renal allograft function was found to have elevated parathyroid hormone (PTH) levels on a biointact PTH assay. The elevated PTH levels were resistant to suppression on increasing doses of 1-alfacalcidol. Detailed history taking and clinical examination revealed the reason for apparent resistance to therapy.