The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38 α , SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X L and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo . Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis ( collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation ( PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate - dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

VEGF expression by proximal tubular epithelial cells may play a critical role in maintaining peritubular capillary endothelium in renal disease. Two major processes involved in renal injury include hypoxia (from vasoconstriction or vascular injury) and transforming growth factor (TGF)-beta-dependent fibrosis, both of which are known to stimulate VEGF. Because the TGF-beta/Smad pathway is activated in hypoxia, we tested the hypothesis that the induction of VEGF in hypoxia could be partially dependent on TGF-beta. Rat proximal tubular (NRK52E) cells treated with TGF-beta under normoxic conditions secreted VEGF at 24 h, and this was significantly reduced by blocking Smad activation by overexpressing the inhibitory Smad7 or by blocking p38 and ERK1/2 MAP kinase activation or protein kinase C activation with specific inhibitors. With acute hypoxia, rat proximal tubular cells also express VEGF mRNA and protein as well as TGF-beta. However, the induction of VEGF occurs before synthesis of TGF-beta and is not blocked by either a TGF-beta antagonist, by Smad7 overexpression, or by blockage of ERK1/2, whereas induction is blocked by PKC inhibition or partially blocked by a p38 inhibitor. Finally, the addition of TGF-beta with hypoxia results in significantly more VEGF expression than either stimulation alone. Thus TGF-beta and hypoxia act via additive/synergistic but distinct pathways to stimulate VEGF in proximal tubular cells, a finding that may be important in understanding how VEGF is stimulated in renal disease.

The vast majority of patients with essential hypertension have structural changes in their kidneys consisting of preglomerular vascular disease ('arteriolosclerosis') and tubulointerstitial injury. Most authorities have assumed that these structural changes occur secondary to hypertensive renal injury. However, Goldblatt proposed that primary renal microvascular disease might be the cause of some forms of hypertension. In this paper we present recent studies from our group that support a role for both preglomerular vascular disease as well as the tubulointerstitial inflammatory response in mediating salt-sensitivity. We propose that subtle acquired renal injury may underlie the etiology of some forms of salt-sensitive hypertension.

NephrologyVolume 7, Issue 1 p. A56-A56 BLOCKADE OF P38 ALPHA MAPK SIGNALING AMELIORATES RENAL INJURY IN ACUTE RAT ANTI-GBM GLOMERULONEPHRITIS C Stambe, C Stambe Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this authorRC Atkins, RC Atkins Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this authorGF Schreiner, GF Schreiner 2Search for more papers by this authorDJ Nikolic-Paterson, DJ Nikolic-Paterson Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this author C Stambe, C Stambe Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this authorRC Atkins, RC Atkins Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this authorGF Schreiner, GF Schreiner 2Search for more papers by this authorDJ Nikolic-Paterson, DJ Nikolic-Paterson Dept of Nephrology, Monash Medical Centre, Clatyon, Victoria; Scios Inc, CA, USASearch for more papers by this author First published: 09 October 2008 https://doi.org/10.1046/j.1440-1797.2002.00007-1-56.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume7, Issue1February 2002Pages A56-A56 RelatedInformation

Chronic exposure to high altitude is associated with the development of erythrocytosis, proteinuria, and, in some cases, hyperuricemia. We examined the relationship between high-altitude polycythemia and proteinuria and hyperuricemia in Cerro de Pasco, Peru (altitude, 4,300 m). We studied 25 adult men with hematocrits less than 65% and 27 subjects with excessive erythrocytosis (EE; hematocrit > 65%) living in Cerro de Pasco, Peru and compared them with 28 control subjects living in Lima, Peru (at sea level) and after 48 hours of exposure to high altitude. Serum urate levels were significantly elevated in patients with EE at altitude, and gout occurred in 4 of 27 of these subjects. Urate level strongly correlated with hematocrit (r = 0.71; P < 0.0001). Urate production (24-hour urine urate excretion and urine urate-creatinine ratio) was increased in this group compared with those at sea level. Fractional urate excretion was not increased, and fractional lithium excretion was reduced, in keeping with increased proximal reabsorption of filtrate. Significantly higher blood pressures and decreased renin levels in the EE group were in keeping with increased proximal sodium reabsorption. Serum urate levels correlated with mean blood pressure (r = 0.50; P < 0.0001). Significant proteinuria was more prevalent in the EE group despite normal renal function. Hyperuricemia is common in subjects living at high altitude and associated with EE, hypertension, and proteinuria. The increase in uric acid levels appears to be caused by increased urate generation secondary to systemic hypoxia, although a relative impairment in renal excretion also may contribute.

Salt sensitivity is present in about half of people with essential hypertension; decreasing salt intake ameliorates the hypertension. This review provides an explanation of how initially subtle renal injury promotes a tendency toward hypertension. The kidneys, initially normal in many persons with early primary hypertension, sustain subclinical injury over time, resulting in arteriolosclerosis and tubulointerstitial disease that lead to established hypertension.

Recent studies have demonstrated a role for microvascular and tubulointerstitial injury in some models of salt-sensitive hypertension. We utilized a model of post-cyclosporin A (CsA) nephropathy and hypertension to test the hypothesis that treatment with an angiogenic factor aimed at ameliorating the microvascular and renal injury would prevent the development of hypertension. CsA was administered with a low-salt diet for 45 days, resulting in a renal lesion characterized by afferent arteriolopathy, focal peritubular capillary loss, and tubulointerstitial fibrosis. Rats were then placed on a high-salt diet and randomized to receive either vascular endothelial growth factor (VEGF(121)) or vehicle for 14 days. Placement of rats with established CsA nephropathy on a high-salt diet results in the rapid development of salt-sensitive hypertension. VEGF(121) treatment resulted in lower blood pressure, and this persisted on discontinuing the VEGF. VEGF(121) treatment was also associated with a decrease in osteopontin expression, macrophage infiltration, and collagen III deposition and markedly stimulated resolution of the arteriolopathy (20.9 +/- 7.8 vs. 36.9 +/- 6.1%, VEGF vs. vehicle, P < 0.05). In conclusion, CsA-associated renal microvascular and tubulointerstitial injury results in the development of salt-sensitive hypertension. Treatment of animals with established CsA nephropathy with VEGF reduces the hypertensive response and accelerates histological recovery. The vascular protective effect of VEGF may be due to the improvement of arteriolopathy. Angiogenic growth factors may represent a novel strategy for treating CsA-associated hypertension and renal disease.

We investigated the relationship of changes in the microvasculature to age-related structural and functional changes in the kidney to determine whether there was evidence of impaired angiogenesis and whether the loss of microvasculature could be accounted for by changes in the local production of angiogenic or antiangiogenic factors. Glomerular and peritubular capillary number, density, and endothelial cell proliferation were determined in aging (24 months; n = 9) and young (3 months; n = 8) rat kidneys and correlated with renal functional and structural changes and alterations in renal expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aging rats showed a focal decrease in both peritubular capillary (peritubular capillary staining, 5.4% ± 1.8% versus 11.3% ± 2.0% per 100 tubules; rarefaction index, 10.6% ± 4.6% versus 0.6% ± 0.1%, aging versus young rats; P < 0.05 and P < 0.001, respectively) and glomerular capillary loops (27.3 ± 6.9 versus 50.7 ± 7.4/glomerulus, aging versus young rats; P < 0.001). The number of proliferating endothelial cells was decreased in aging rats compared with young rats (glomerular, 0.04 ± 0.01 versus 0.15 ± 0.03 positive cells/glomerular cross-section; peritubular, 0.7 ± 0.2 versus 4.3 ± 2.6 positive cells/mm2; P < 0.05). In the aging kidney, VEGF expression was focally increased in the cortex compared with young rats, whereas a profound decrease was observed in the outer and inner medulla (total area of VEGF expression, 19.2% ± 11.4% versus 39.3% ± 7.6%; P < 0.05). Tubular VEGF expression correlated with peritubular capillary density (r2 = 0.57; P < 0.01) and inversely correlated with tubular osteopontin (r2 = –0.55; P < 0.05) and macrophage infiltration (r2 = –0.64; P < 0.01). TSP-1 staining was increased in the glomeruli and tubulointerstitium of the aging rats. Glomerular TSP-1 score correlated inversely with glomerular capillary number (r2 = –0.89; P < 0.001). Tubulointerstitial TSP-1 also correlated with percentage of positive staining of peritubular capillary (r2 = –0.59; P < 0.001). Glomerular capillary number showed significant correlation with glomerulosclerosis score, as well as with 24-hour urinary protein excretion. Peritubular capillary density also inversely correlated with interstitial fibrosis score and urinary protein excretion. In conclusion, glomerular and peritubular capillary loss in the aging kidney correlate with alterations in VEGF and TSP-1 expression and also with the development of glomerulosclerosis and tubulointerstitial fibrosis. These findings suggest that impaired angiogenesis associated with progressive loss in renal microvasculature may have a pivotal role in age-related nephropathy.

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.

The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.