Extracellular factors and immunosuppressive drugs influencing insulin secretion of murine islets.

Approximately 60% of transplanted islets undergo apoptosis within the first week post-transplantation into the liver attributed to poor engraftment, immune rejection and toxicity of immunosuppressive drugs. Understanding how extracellular matrix (ECM) components, immunosuppressive drugs and proinflammatory cytokines affect insulin secretion will contribute to an improved clinical outcome of islet transplantations. In this study, functional activity of isolated murine islets was measured by glucose-stimulated insulin secretion (GSIS) and by electrophysiological measurements using patch-clamp. Cultivating islets with soluble fibronectin or laminin, as opposed to with coated laminin, markedly increased GSIS. Addition of cyclosporin A reduced GSIS and suppressed glucose-induced spike activity. Tacrolimus affected neither GSIS nor spike activity, indicating a different mechanism. To evaluate the influence of proinflammatory cytokines, islets were incubated with interleukin (IL)-1ß, tumour necrosis factor (TNF)-α or with supernatants from cultured Kupffer cells, the main mediators of inflammation in the hepatic sinusoids. IL-1ß exerted a bimodal effect on insulin secretion, stimulating below 2 ng/ml and suppressing above 10 ng/ml. Soluble laminin in combination with a stimulatory IL-1ß concentration further increased insulin secretion by 20% compared to IL-1ß alone, while with high IL-1ß concentrations soluble laminin slightly attenuated GSIS inhibition. TNF-α alone did not affect GSIS, but with stimulatory IL-1ß concentrations completely abolished it. Similarly, supernatants derived from Kupffer cells exerted a bimodal effect on GSIS. Our data suggest that improved insulin secretion of transplanted islets could be achieved by including soluble laminin and low IL-1ß concentrations in the islet cultivation medium, and by a simultaneous inhibition of cytokine secretion from Kupffer cells.

Tubular atrophy, interstitial fibrosis, and inflammation in type 2 diabetic db/db mice. An accelerated model of advanced diabetic nephropathy.

OBJECTIVE: Advanced diabetic nephropathy (DN) is difficult to address experimentally in mice because available models of DN lack global glomerulosclerosis and major tubulointerstitial pathology. Accelerating the development of DN in mice would be desirable for feasible experimental validation of potential targets that mediate the progression to late stage DN. METHODS: 6 week old male db/db mice underwent uninephrectomy and the development of nephropathy was compared to wild-type mice and sham-operated db/db mice. RESULTS: Uninephrectomy at young age was associated with increased albuminuria and severe glomerulosclerosis in 37% of glomeruli at 24 weeks of age as compared to sham-operated db/db mice (8%). Uninephrectomy also increased the number of glomerular macrophages in db/db mice. The uninephrectomy-related acceleration of glomerular damage was associated with significant tubulointerstitial injury as indicated by an increase in indices of tubular cell damage, tubular dilatation, and expansion of interstitial volume. Uninephrectomy markedly increased the renal mRNA expression of Mcp-1/Ccl2, Tgf-beta, and collagen I. CONCLUSION: Early uninephrectomy can accelerate the development of advanced DN in db/db mice which may be instrumental in the design of interventional studies that intend to focus on the molecular pathology of the progression to late stage DN.

Isolation and functional characterization of pericytes derived from hamster skeletal muscle.

AIM: At the interface of tissue and capillaries, pericytes (PC) may generate electrical signals to be conducted along the skeletal muscle vascular network, but they are functionally not well characterized. We aimed to isolate and cultivate muscle PC allowing to analyse functional properties considered important for signal generation and conduction. METHODS: Pericytes were enzymatically isolated from hamster thigh muscles and further selected during a 16-30 days' cultivation period. PC markers were studied by fluorescence activated cell scanning (FACS) and immunocytochemistry. Electrical properties of the cultured PC were investigated by patch clamp technique as well as the membrane potential sensitive dye DiBAC(4) (3). RESULTS: The cultured cells showed typical PC morphology and were positive for NG2, alpha smooth muscle actin, PDGFR-ß and the gap junction protein Cx43. Expressions of at least one single or combinations of several markers were found in 80-90% of subpopulations. A subset of the patched cells expressed channel activities consistent with a Kv1.5 channel. In vivo presence of the channels was confirmed in sections of hamster thigh muscles. Interleukin-8, a myokine known to be released from exercising muscle, increased the expression but not the activity of this channel. Pharmacologic stimulation of the channel activity by flufenamic acid induced hyperpolarization of PC alone but not of endothelial cells [human umbilical vein endothelial cells (HUVEC)] alone. However, hyperpolarization was observed in HUVEC adjacent to PC when kept in co-culture. CONCLUSION: We established a culture method for PC from skeletal muscle. A first functional characterization revealed properties which potentially enable these cells to generate hyperpolarizing signals and to communicate them to endothelial cells.

Progression of kidney disease: blocking leukocyte recruitment with chemokine receptor CCR1 antagonists.

Chronic kidney disease (CKD) is usually associated with interstitial leukocytic cell infiltrates, which may contribute to disease progression by production of proinflammatory, proapoptotic, and profibrotic mediators. Recruiting leukocytes into the kidney involves local expression of chemotactic cytokines, that is, chemokines, that interact with respective chemokine receptors on the leukocyte's outer surface. Thus, specific chemokine receptor antagonists may represent an attractive therapeutic concept to interfere with renal leukocyte recruitment. Among the proinflammatory chemokine receptors, chemokine receptor (CCR)-1 has nonredundant roles for leukocyte adhesion to activated vascular endothelium and for transendothelial migration. In fact, blocking CCR-1 with specific small-molecule antagonists was shown to retard progression in various types of rodent CKD models. Here we discuss the perspective of CCR-1 as a new potential target for the treatment of CKD.

Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice.

Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.