Richard J. Baines

Ligand-independent Activation of Peroxisome Proliferator-activated Receptor-γ by Insulin and C-peptide in Kidney Proximal Tubular Cells DEPENDENT ON PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY

Peroxisome proliferator-activated receptor γ (PPARγ) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARγ transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARγ transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARγ transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARγ. GW9662, a PPARγ antagonist, blocked PPARγ activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARγ transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARγ transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARγ transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARγ was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARγ transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARγ-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARγ activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPARγ-related cell functions.

Tubular toxicity of proteinuria.

Proteinuria is a prognostic indicator of progressive kidney disease and poor cardiovascular outcomes. Abnormally filtered bioactive macromolecules interact with proximal tubular epithelial cells (PTECs), which results in the development of proteinuric nephropathy. This condition is characterized by alterations in PTEC growth, apoptosis, gene transcription and inflammatory cytokine production as a consequence of dysregulated signaling pathways that are stimulated by proteinuric tubular fluid. The megalin–cubilin complex mediates the uptake of several proteins, including albumin, into PTECs. Megalin might also possess intrinsic signaling properties and the ability to regulate cell signaling pathways and gene transcription after processing regulated intramembrane proteolysis. Megalin could, therefore, link abnormal PTEC albumin exposure with altered growth factor receptor activation, proinflammatory and profibrotic signaling, and gene transcription. Evidence now suggests that other PTEC pathways for protein reabsorption of (patho)physiological importance might be mediated by the neonatal Fc receptor and CD36.

The molecular interactions between filtered proteins and proximal tubular cells in proteinuria.

Proteinuria is associated with progressive chronic kidney disease and poor cardiovascular outcomes. Exposure of proximal tubular epithelial cells to excess proteins leads to the development of proteinuric nephropathy with tubular atrophy, interstitial inflammation and scarring. Numerous signalling pathways are activated in proximal tubular epithelial cells under proteinuric conditions resulting in gene transcription, altered growth and the secretion of inflammatory and profibrotic mediators. Megalin, the proximal tubular scavenger receptor for filtered macromolecules, has intrinsic signalling functions and may also link albumin to growth factor receptor signalling via regulated intramembrane proteolysis. It now seems that endocytosis is not always a prerequisite for albumin-evoked alterations in proximal tubular cell phenotype. Recent evidence shows the presence of other potential receptors for proteins, such as the neonatal Fc receptor and CD36, in the proximal tubular epithelium.

CD36 mediates proximal tubular binding and uptake of albumin and is upregulated in proteinuric nephropathies

Dysregulation of renal tubular protein handling in proteinuria contributes to the development of chronic kidney disease. We investigated the role of CD36 as a novel candidate mediator of albumin binding and endocytosis in the kidney proximal tubule using both in vitro and in vivo approaches, and in nephrotic patient renal biopsy samples. In CD36-transfected opossum kidney proximal tubular cells, both binding and uptake of albumin were substantially enhanced. A specific CD36 inhibitor abrogated this effect, but receptor-associated protein, which blocks megalin-mediated endocytosis of albumin, did not. Mouse proximal tubular cells expressed CD36 and this was absent in CD36 null animals, whereas expression of megalin was equal in these animals. Compared with wild-type mice, CD36 null mice demonstrated a significantly increased urinary protein-to-creatinine ratio and albumin-to-creatinine ratio. Proximal tubular cells expressed increased CD36 when exposed to elevated albumin concentrations in culture medium. Expression of CD36 was studied in renal biopsy tissue obtained from adult patients with heavy proteinuria due to minimal change disease, membranous nephropathy, or focal segmental glomerulosclerosis. Proximal tubular CD36 expression was markedly increased in proteinuric individuals. We conclude that CD36 is a novel mediator influencing binding and uptake of albumin in the proximal tubule that is upregulated in proteinuric renal diseases. CD36 may represent a potential therapeutic target in proteinuric nephropathy.

Angiotensin II–Stimulated Phospholipase C Responses of Two Vascular Smooth Muscle–Derived Cell Lines: Role of Cyclic GMP

Vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) are known to show increased responsiveness to angiotensin II (Ang II) compared with cells of normotensive control Wistar-Kyoto rats (WKY). We investigated the hypothesis that differential levels of cGMP lead to the different responsiveness of the cells, using vascular smooth muscle cells in culture. cGMP levels in extracts of SHR-derived cells were lower than those of WKY-derived cells. This was true for both unstimulated cells and cells treated with equal concentrations of either sodium nitroprusside or S-nitroso-N-acetylpenicillamine. Stimulation of cells with Ang II did not affect levels of cGMP but increased levels of inositol 1, 4, 5-trisphosphate (IP3) and Ca2+, which were greater in SHR- than in WKY-derived cells. When SHR and WKY cells were preincubated with different concentrations of S-nitroso-N-acetylpenicillamine to generate similar cGMP levels in each cell type, the subsequent IP3 response to Ang II was the same in the two cell types. To reduce any influence of cGMP on responses, we permeabilized the cells with alpha-toxin. Stimulation of alpha-toxin-permeabilized the cells with high Ca2+ revealed an IP3 response in SHR- but not WKY-derived cells. Similarly, permeabilized SHR cells responded to Ang II but WKY cells did not. However, GTP and GTP gamma S elevated IP3 in both cell types. Taken together, these results indicate that the low response of WKY cells can be accounted for by the inhibitory influence of cGMP. However, when this inhibition is removed by permeabilization, further differences between the cells are revealed that will contribute to the elevated SHR response.

Stimulation of two vascular smooth muscle‐derived cell lines by angiotensin II: differential second messenger responses leading to mitogenesis

1 We show here that angiotensin II (All) and endothelin‐l (ET‐1) stimulate [3H]‐thymidine incorporation in a smooth muscle cell line derived from aortae of spontaneously hypertensive rats (SHR), but not in cells derived from normotensive controls (WKY). We have used the differential response of the two cell lines to investigate the relationship between second messenger systems and the mitogenic response. 2 All produced an increase in accumulation of inositol 1,4,5‐triphosphate which was greater in the SHR‐derived cell line than in the WKY cells. 3 All gave an increase in cytosolic Ca2+ in each of the cell lines, with both a larger peak (15–30 s) and plateau response (2min) in the SHR cells. ET‐1 gave an enhanced response in the SHR‐derived cells with respect to the peak but not the plateau of cytosolic Ca2+. 4 Phospholipase D activity was studied by monitoring the formation of [3P]‐phosphatidylbutanol in 32Pi prelabelled cells. All stimulation gave a larger phospholipase D response in SHR‐derived cells, while ET‐1 gave a larger response in WKY‐derived cells. 5 Stimulation of SHR‐derived cells with 100 nM All for 1 h, followed by 19 h in the absence of agonist, stimulated [3H]‐thymidine incorporation over the next 4 h. When the 1 h stimulation with All was in the presence of increasing concentrations of butanol, which diverts the product of the phospholipase D pathway, there was a loss of stimulated [3H]‐thymidine incorporation which was significant at 10 mM butanol and at 30‐50mM reached a maximum loss of 40%. 6 Contrasting with this there was no apparent loss of ET‐1‐stimulated thymidine incorporation when butanol was present at concentrations up to 40 mM. 7 These results suggest that phospholipase D is one of several pathways in the mitogenic response of SHR‐derived vascular smooth muscle cells to All.

An educational approach to improve outcomes in acute kidney injury (AKI): report of a quality improvement project.

Objective To assess the impact of a quality improvement project that used a multifaceted educational intervention on how to improve clinicians knowledge, confidence and awareness of acute kidney injury (AKI). Setting 2 large acute teaching hospitals in England, serving a combined population of over 1.5 million people. Participants All secondary care clinicians working in the clinical areas were targeted, with a specific focus on clinicians working in acute admission areas. Interventions A multifaceted educational intervention consisting of traditional didactic lectures, case-based teaching in small groups and an interactive web-based learning resource. Outcome measures We assessed clinicians’ knowledge of AKI and their self-reported clinical behaviour using an interactive questionnaire before and after the educational intervention. Secondary outcome measures included clinical audit of patient notes before and after the intervention. Results 26% of clinicians reported that they were aware of local AKI guidelines in the preintervention questionnaire compared to 64% in the follow-up questionnaire (χ²=60.2, p<0.001). There was an improvement in the number of clinicians reporting satisfactory practice when diagnosing AKI, 50% vs 68% (χ²=12.1, p<0.001) and investigating patients with AKI, 48% vs 64% (χ²=9.5, p=0.002). Clinical audit makers showed a trend towards better clinical practice. Conclusions This quality improvement project utilising a multifaceted educational intervention improved awareness of AKI as demonstrated by changes in the clinicians self-reported management of patients with AKI. Elements of the project have been sustained beyond the project period, and demonstrate the power of quality improvement projects to help initiate changes in practice. Our findings are limited by confounding factors and highlight the need to carry out formal randomised studies to determine the impact of educational initiatives in the clinical setting.

A 4-month programme of in-centre nocturnal haemodialysis was associated with improvements in patient outcomes.

Background Extended periods of haemodialysis (HD) can improve patient outcomes. In-centre nocturnal haemodialysis (INHD) should be explored as a method of offering extended periods of HD to patients unsuitable for or unable to perform home therapy. Methods Ten self-selecting, prevalent HD patients started an INHD programme to assess feasibility and patient satisfaction. Quality-of-life (QOL) measures were evaluated at enrolment and after 4 months of INHD using the EQ-5D, the Hospital Anxiety and Depression Scale (HADS) and the SF-12 questionnaires. Demographic, biochemical and haematological data and data on dialysis adequacy were collected before starting INHD and after 4 months. Results Three of the 10 patients failed to complete the 2-week run-in period. Seven patients completed the 4-month programme, with mean dialysis time of 355 ± 43.92 min throughout the period. The EQ-5D visual analogue score improved from 48 ± 16.89 to 72 ± 13.2 (P = 0.003) and the HADS anxiety score decreased from 9 ± 5.83 to 3.57 ± 3.04 (P = 0.029). The urea reduction ratio improved from 71.57 ± 2.29% to 80.43 ± 3.101% (P < 0.001), with improvements in phosphate control, reducing to within the target range from 1.73 ± 0.6 to 1.2 ± 0.2 (P = 0.08). Ultrafiltration (UF) volumes increased during the study from 2000 ± 510 to 2606 ± 343 mL (P = 0.015); there was a significant reduction in mean UF rate adjusted for body weight from 6.47 ± 1.71 to 4.61 ± 1.59 mL/kg/h (P = 0.032). Sensitivity analyses confirmed the significance of these results. Conclusions This single-centre study showed a 4-month programme of extended hours INHD is safe and associated with improvements in QOL measures, decreased UF rates and measures of dialysis adequacy. These data have been used to expand our service and inform the design of future randomized controlled trials to examine medical endpoints.

Induction treatment of previously undiagnosed ANCA-associated vasculitis in a renal transplant patient with Rituximab.

We report the case of a 40-year-old female transplant patient with undiagnosed ANCA-associated vasculitis (AAV) and renal allograft dysfunction who achieved disease remission with restoration of transplant function following induction therapy with rituximab. There are currently no trial data looking at the use of rituximab for induction of remission of renal transplant patients with AAV. Although recurrence of AAV following renal transplantation is rare, such patients have invariably had multiple previous exposures to induction and maintenance immunosuppressive regimens, often limiting treatment options post-transplantation. In this case, rituximab was well tolerated with no side effects, and was successful in salvaging transplant function. Optimal treatment regimens for relapsed AAV in the transplant population are not known, and clinical trials are needed to evaluate the efficacy and safety of rituximab at inducing and maintaining disease remission in relapsed AAV following transplantation.

The role of IgA in proximal tubular cell activation and tubulointerstitial scarring in IgA nephropathy

Abstract Background IgA nephropathy (IgAN) is the commonest form of glomerulonephritis worldwide, and around 30% of patients progress to end-stage renal failure within 20 years. One of the most powerful prognostic factors for progression in IgAN is the development of tubulointerstitial inflammation and fibrosis. The extent of tubulointerstitial damage in IgAN does not however correlate with the degree of mesangial IgA deposition. Changes in glomerular permeability in IgAN result in exposure of the proximal tubular epithelium to IgA. It is therefore possible that a pathogenic interaction between filtered IgA and proximal tubular cells drives tubulointerstitial scarring in IgAN, independent of the effect of albuminuria. We aimed to assess the interaction between IgA and proximal tubular cells to establish whether IgA could independently generate a proinflammatory and profibrotic phenotypic transformation in proximal tubular cells thereby favouring the development of renal fibrosis. Methods IgA1 was purified from serum from patients with IgAN and healthy individuals by jacalin-agarose affinity chromatography. Human proximal tubular epithelial HK2 cells (PTEC) were exposed for 24 h to IgA1 (100 μg/mL) from patients with IgAN or healthy individuals, or to control conditions IgM (100 μg/mL), IgG (100 μg/mL), albumin (100 μg/mL or 5 mg/mL), or medium alone, and then subjected to protein extraction, with supernatants assayed by sandwich ELISA. Findings IgA1 from patients with IgAN activated extracellular signal-regulated protein kinase in PTEC to a greater extent than did IgM or IgA1 from healthy individuals. IgA1 from patients with IgAN also stimulated PPAR-response-element-driven luciferase expression, indicating PPAR activation. PTEC synthesis of transforming growth factor β, fibronectin, and interleukin 6 was substantially increased by IgA1 (100 μg/mL) compared with IgM, IgG, and albumin at concentrations up to 50 times higher (5 mg/mL). PTEC production of complement component C3 and activation of the complement cascade with generation of the chemotactic component C5a were induced to a greater extent by IgA1 compared with the other conditions. Finally, IgA1 induced phosphorylation of a megalin cytoplasmic tail plus glutathione-s-transferase fusion protein in HK2 cells suggesting that IgA1 binding to PTEC can trigger the same intracellular signalling pathways as those activated after exposure to albumin. Interpretation We provide evidence that IgA1 in IgAN triggers a proinflammatory and profibrotic phenotypic transformation of PTECs. Interaction between filtered IgA1 and PTEC might be a key factor in driving tubulointerstitial damage and progression of renal fibrosis in IgAN. Further elucidation of the mechanism of this interaction might reveal novel therapeutic targets relevant in slowing progression of IgA nephropathy. Funding UK Medical Research Council.