Glycogen synthase kinase 3beta: a novel marker and modulator of inflammatory injury in chronic renal allograft disease.

One key cell-signaling event central to inflammation in kidney diseases, including chronic renal allograft dysfunction or disease (CRAD), is the activation of NF-kappaB, which controls transcription of numerous proinflammatory mediators. Glycogen synthase kinase (GSK) 3beta is an indispensable element of NF-kappaB activation, however, the exact role of GSK3beta in the pathogenesis of inflammatory kidney diseases like CRAD is uncertain and was examined. Immunohistochemistry staining of GSK3beta was weak in normal kidneys, but was markedly induced in inflamed allograft kidneys, with prominent cytoplasmic staining of tubular cells in areas of inflammation. Net GSK3beta activity is regulated by inhibitory phosphorylation of its serine 9 residue, and this occurred in CRAD. Thus, the magnitude of GSK3beta inactivation was inversely correlated with the degree of injury as assessed by Banff criteria. In vitro in cultured human tubular epithelial cells, GSK3beta overexpression augmented, while GSK3beta silencing diminished proinflammatory cellular responses to TNF-alpha stimulation, including NF-kappaB activation and expression of chemokines MCP-1 and RANTES. These inflammatory responses were obliterated by GSK3beta inhibitors. Collectively, GSK3beta plays an important role in mediating proinflammatory NF-kappaB activation and renal inflammation. Suppression of GSK3beta activity might represent a novel therapeutic strategy to treat CRAD.

IgA nephropathy: markers of progression and clues to pathogenesis.

The unpredictable progression of IgA nephropathy hinders its treatment. The correlation of renal dysfunction with the immunophenotype of leukocytic infiltrates revealed interstitial CD20(+) cells and tubular NKG7(+)(GMP-17(+))/CD8(+) intraepithelial cells as predictive markers of progression in early-phase IgA nephropathy. This suggests that adaptive and innate immune responses mediate progression.

Excess risk of renal allograft loss and early mortality among elderly recipients is associated with poor exercise capacity.

BACKGROUND: Successful renal transplantation in the elderly offers substantial benefits in quality and life expectancy. However, in this group of patients there is an early increased risk of death compared with those remaining on dialysis. MATERIALS AND METHODS: Graft and patient outcomes in 64 older transplant recipients were compared with 338 patients aged 18 - 59 years. We identified potential risk factors that may predict clinical outcomes in older transplant recipients. A log-rank test and Cox regression analyses were performed to assess the impact of various patient characteristics on graft and patient survival. RESULTS: Among older patients, graft survival was 76.6% and 67% at 1 and 3 years, respectively. When graft survival was censored for death with functioning graft, the 1- and 3-year graft survival was 83% and 82%, respectively. Patient survival was 78% and 71% at 1 and 3 years, respectively. These survival rates were significantly lower than those of younger recipients. Pretransplant inactivity, delayed graft function, smoking history and longer waiting time predicted poor graft and patient survival. A history of chronic obstructive pulmonary disease, and peripheral vascular disease also predicted a higher mortality among older recipients. CONCLUSION: Older kidney transplant recipients are at high risk for allograft failure and early death. Poor functional capacity predicts a poor outcome for older patients undergoing renal transplantation. Therefore, careful patient selection is paramount, and every effort should be made to initiate timely interventions aimed at increasing physical activity in those with low fitness level.

Hepatocyte growth factor suppresses acute renal inflammation by inhibition of endothelial E-selectin.

Vascular endothelial activation, marked by de novo expression of E-selectin, is an early and essential event in the process of leukocyte extravasation and inflammation. Evidence suggests that hepatocyte growth factor (HGF) ameliorates inflammation in animal models of renal disease, implying that HGF might inhibit specific components of the inflammatory response. This study examined the effect of HGF on endothelial E-selectin expression in acute inflammation induced by tumor necrosis factor (TNF)-alpha. In vitro, HGF suppressed TNF-alpha-induced cell surface expression of E-selectin in human umbilical vein endothelial cells (HUVEC) and inhibited E-selectin mediated monocytic adhesion to endothelial monolayers. HGF activated phosphatidylinositol 3-kinase (PI3K)-Akt that in turn inhibited its downstream transducer glycogen synthase kinase (GSK)3. Blockade of the PI3K-Akt pathway with specific inhibitors abrogated HGF induced inhibitory phosphorylation of GSK3 and suppression of E-selectin. In addition, selective inhibition of GSK3 activity by lithium suppressed TNF-alpha-induced E-selectin expression and monocytic adhesion, reminiscent of the action of HGF. Moreover, ectopic expression of an uninhibitable mutant GSK3beta, in which the regulatory serine-9 is replaced by alanine, abolished HGF's suppressive effect on endothelial E-selectin. In vivo, administration of exogenous HGF reduced endothelial expression of E-selectin induced by bolus injection of TNF-alpha. This was associated with less sequestration of circulating fluorescence-labeled macrophages in the kidney. These findings suggest that HGF ameliorates acute renal inflammation in part by downregulating E-selectin mediated macrophage adhesion to the inflamed endothelium.

Cryptosporidium infection in renal transplant patients.

Cryptosporidium parvum, an intracellular protozoan parasite, is a significant cause of gastrointestinal disease worldwide. Transmission can occur from an infected person, animal or fecally contaminated environment. The clinical manifestations of cryptosporidiosis are dependent on the immunologic state of the host. Infection among immunocompetent hosts results in diarrhea that is typically self-limited. In immunocompromised hosts, however, the infection may be protracted and life-threatening with no reliable antimicrobial therapy. In transplant patients, a course of antimicrobial therapy along with concurrent reduction in immunosuppression optimize immunologic status and may potentially lead to resolution of the infection.

Residual renal function and mortality risk in hemodialysis patients.

Residual renal function, defined as the urinary clearance of urea and creatinine, is minimal in many patients treated with hemodialysis (HD) and tends to be ignored in most outcome studies involving HD patients. Recent studies showed that residual renal function, even at a low level, is influential in preventing mortality in the minority of patients with end-stage renal disease treated with peritoneal dialysis. This issue generally has not been examined in patients treated with HD. This prospective observational study of all 114 patients at a single community-based freestanding HD center is designed to examine the impact of residual renal function (defined as renal urea clearance and renal creatinine clearance derived from 24-hour urinary volumes) on mortality over a 2-year period. During that period, 50 deaths occurred in 114 patients. The presence of residual renal function was protective against mortality (odds ratio for death, 0.44; 95% confidence interval, 0.24 to 0.81; P = 0.008), even after adjustment for duration of dialysis treatment, age, smoking, presence of diabetes, presence of cardiovascular disease, serum albumin level, and urea reduction rate. In conclusion, the presence of residual renal function, even at a low level, is associated with a lower mortality risk in HD patients.

Genomic approaches to elucidating the pathophysiology of renal diseases.

The physiological and pathological processes of the kidney as a whole can now be analyzed with a molecular precision at a genomic-scale. Using massively parallel cDNA microarray technology, the mRNA expression of thousands of genes can be quantified simultaneously. The advantages of microarray analyses include the ability to examine the interaction of several genes or the entire genome in a single experiment. Bioinformatics approaches such as data mining through mathematical condensation of the massive gene expression profiles are essential for elucidating molecular and biological logic underlying gene expression programs. Genes that encode similar protein components are often coordinately regulated. Recent application of gene expression profiling to the normal human renal cortical tissue, experimental in vitro and in vivo models has shown that cellular activation is accompanied by changes of hundreds of genes in parallel. The databases of gene expression emerging from these studies will be used to interpret the pathological changes in gene expression that accompany a variety of human renal diseases.

Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways.

BACKGROUND: Hepatocyte growth factor (HGF) has been shown to promote tubule repair and renal regeneration following acute injury; however, whether HGF also modulates the development and progression of chronic renal diseases that are characterized by progressive tissue fibrosis is uncertain. To examine this question, this study investigated the functional consequence of blocking endogenous HGF signaling in vivo in a model of chronic renal disease. The effects of HGF on the processes of matrix synthesis and degradation in cultured renal epithelial cells were also examined. METHODS: The level of activity of the HGF/c-met axis was examined in rats following 5/6 nephrectomy at multiple time points. To determine the effects of HGF in modulating chronic renal injury, HGF action was blocked in remnant kidney rats using an anti-HGF antibody. The effects of HGF on extracellular matrix (ECM) synthesis and degradation were examined in renal epithelial cells by (35)S-methionine labeling, Western blotting, and zymographic analysis. RESULTS: An increase in renal and systemic production of HGF coupled with an increase in renal c-met was observed in rats with remnant kidneys. When HGF action was blocked by the administration of an anti-HGF antibody, rats experienced a rapid decrease in glomerular filtration rate and increased renal fibrosis. Kidney sections from the antibody-treated rats displayed a marked increase in ECM accumulation and in alpha-smooth muscle actin-positive cells in both the interstitium and tubular epithelium. In vitro studies revealed that HGF reduced net ECM accumulation by human proximal tubule cells (HKC), and this effect was abolished by incubating cells with an anti-HGF antibody. HGF did not alter the ECM synthetic rate in HKC cells. Rather, it markedly increased collagenase such as matrix metalloproteinase-9 (MMP-9) protein expression, as evidenced by Western blotting and zymographic analysis. HGF also decreased the expression of tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) and TIMP-2, the endogenous inhibitors of MMPs. CONCLUSION: These results suggest that HGF is a potent antifibrogenic factor both in vitro and in vivo. Endogenous activation of HGF tends to preserve kidney structure and function in rats with chronic renal disease by activating matrix degradation pathways.

Onset of glomerular hypertension with aging precedes injury in the spontaneously hypertensive rat.

The changes in renal hemodynamics that develop with aging in spontaneously hypertensive rats (SHR) were examined. Micropuncture studies revealed that glomerular capillary pressure was elevated in SHR at 9 mo of age compared with 3-mo-old SHR and 9-mo-old normotensive Wistar-Kyoto rats. Glomerular hypertension developed because of a small increase in systemic blood pressure and a decline in preglomerular vascular resistance, allowing transmission of elevated systemic pressure to the glomerular capillaries. The hemodynamic alterations were not a compensatory response to injury, inasmuch as vascular and glomerular morphology were normal in 9-mo-old SHR. To determine the mechanism of these changes, the activity of several vasoactive systems was examined. Similar changes in renal hemodynamics were observed in young and old SHR after blockade of nitric oxide production and after intravenous administration of endothelin. However, ANG II produced a proportionally greater reduction in glomerular filtration rate than renal blood flow in older SHR. These data suggest that reduced endogenous activity of the renin-angiotensin system leads to glomerular hypertension in aging SHR. Late development of glomerular hypertension may contribute to the subsequent appearance of glomerular sclerosis and progressive renal failure in these rats.

Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.

BACKGROUND: Hepatocyte growth factor (HGF) and its c-met receptor comprise a signaling system that has been implicated in tissue repair and regeneration. HGF action is specifically targeted to the damaged organ following injury; however, the mechanism underlying this important targeting process remains to be elucidated. We reasoned that induction of c-met expression might be a critical factor in determining the site specificity of this receptor-ligand system. To test this hypothesis, we examined changes in activity of the HGF/c-met system in the folic acid model of acute tubular injury and repair. METHODS: Tissue HGF and c-met mRNA levels were detected by RNase protection assay and Northern blot analysis following acute renal injury induced by a single injection of folic acid. HGF and c-met proteins were examined by a specific enzyme immunoassay and Western blotting, respectively. C-met expression and trans-activation were investigated by exposing renal epithelial mIMCD-3 cells to various cytokines in vitro. RESULTS: Extremely rapid induction of renal HGF and c-met mRNA was observed beginning one hour following injection of folic acid. Circulating plasma HGF protein level rose dramatically (approximately 16-fold), peaking first at two hours and again at 24 hours following injection. Despite elevated HGF mRNA in the kidney, total kidney HGF protein actually decreased significantly at 24 hours following injury. On the other hand, both c-met mRNA and c-met protein were markedly increased in the kidney, where active renal tubule repair and regeneration take place. In vitro studies suggested that increased levels of HGF, as well as other cytokines, might account for enhanced c-met expression in renal tubular epithelial cells. Pretreatment of the cells with actinomycin D totally blocked c-met induction, suggesting that induced c-met expression occurs primarily at the transcriptional level. Using a cloned region of the c-met promoter coupled to a reporter gene, we demonstrated that HGF directly stimulated c-met promoter transactivation in renal epithelial cells. CONCLUSION: These results suggest that local up-regulation of c-met transcription in the kidney is crucial to renal tubule repair and regeneration, not only because it increases overall activity of this receptor-ligand system, but also as a mechanism targeting HGF action specifically to renal epithelia.

Current strategies for management of hypertensive renal disease.

The incidence of hypertensive end-stage renal disease continues to increase annually. To reduce this incidence, it is necessary to control systolic and diastolic hypertension. Reversible causes should always be sought in any hypertensive patient who develops renal insufficiency. Blood pressure should be reduced to 130/85 mm Hg, and in African Americans with hypertensive renal failure, reducing the blood pressure to 120/75 mm Hg may be beneficial. Any antihypertensive treatment regimen that effectively lowers blood pressure will help slow progressive renal failure. Whenever possible, an angiotensin-converting enzyme inhibitor should be part of the treatment, since these drugs have been shown to be renoprotective beyond their antihypertensive effect in certain renal disease categories.

Antihypertensive therapy and progression of chronic renal disease.

This paper reviews findings on the relationship between blood pressure control and the progression of renal disease. Experimental studies demonstrate a correlation between systemic blood pressure and histologic glomerular injury and the delay in progression of renal disease with antihypertensive therapy, particularly with angiotensin-converting enzyme inhibitors. Recent clinical findings are reviewed, including epidemiologic data linking hypertension to subsequent renal disease, and clinical studies showing a beneficial effect on progression of renal disease with lower than usual blood pressure targets.

Expression of Na+/H+ exchanger isoforms in inner segment of inner medullary collecting duct.

Na+/H+ exchangers (NHE) play a critical role in many cellular and transport processes in the inner medullary collecting duct (IMCD). Morphologically, the IMCD is divided into the outer (IMCD1), middle (IMCD2), and inner (IMCD3) segments. The inner, IMCD3 segment contains only one cell type, the IMCD cell, which is distinct in ultrastructure and in function from the principal and intercalated cells that are present in other portions of the IMCD. NHEs constitute a gene family containing several isoforms (NHE1, NHE2, NHE3, NHE4 and NHE5) which possess distinct characteristics and serve specialized functions. To understand the molecular basis of NHE-related processes in the IMCD, it is critical to know the molecular identity of the NHEs in this tubule segment. The purpose of the present study was to identify the NHE isoforms present and their polar distribution in IMCD3. Applying the reverse transcription-polymerase chain reaction (RT-PCR) technique to IMCD3 (obtained from distal 50% of inner medulla) of mouse and rat kidneys, we found that NHE1, NHE2 and NHE4, but not NHE3 were expressed in both species. The polar localization of NHE in IMCD3 was examined in tubules isolated from rats and perfused in vitro with HEPES-buffered solutions under isotonic conditions. pHi was measured by BCECF fluorescence. Na+-dependent, amiloride-inhibitable pHi recovery from cell acidification (consistent with NHE) was detected in the basolateral, but not the apical, membrane of IMCD3. We conclude that NHE1, NHE2 and NHE4, but not NHE3, are present in both the mouse and rat IMCD3. Functionally, NHE is limited to the basolateral membrane. Additional studies are needed to determine the physiological roles and regulation of basolateral NHE isoforms in this tubule segment.

Constitutive expression of HGF modulates renal epithelial cell phenotype and induces c-met and fibronectin expression.

Hepatocyte growth factor (HGF) is a potent renotropic factor that has been shown to play important roles in kidney development and recovery from acute renal injury. To examine the effects of HGF on renal tubular epithelium, we generated HGF-producing renal epithelial cells by stably transfecting mIMCD-3 and OK cells with an expression plasmid containing human HGF cDNA. Expression of HGF in the transfected cells was confirmed by detection of HGF mRNA by Northern blot analysis and detection of HGF secretion into the conditioned medium by ELISA. HGF-transfected cells exhibited fibroblast-like scattered morphology and increased cell motility. They formed branching tubules when grown in 3-D collagen gel. In addition, HGF-producing cells grew faster than their parental cells, but failed to form colonies in soft agar. These phenotypic changes were inhibited by a specific, neutralizing anti-HGF antibody. Interestingly, both c-met transcript and c-met protein were increased in HGF-transfected cells, suggesting that HGF amplifies its own action via stimulation of c-met expression. Autocrine expression of HGF and c-met in renal epithelial cells also stimulated fibronectin gene expression, which was totally blocked by incubation with a neutralizing anti-HGF but not a pan-specific anti-TGF-beta antibody, suggesting that it is independent of TGF-beta production. Our data demonstrate that HGF as a single factor stimulates renal epithelial cell proliferation, migration, differentiation, and extracellular matrix remodeling, making it uniquely suited to promote renal tubulogenesis during development, as well as to reconstitute tubular integrity following acute injury.

Antihypertensive drugs, dietary salt, and renal protection: how low should you go and with which therapy?

Although it is clear that hypertension accelerates the rate of progression of most forms of chronic renal disease, many unanswered questions remain concerning how to optimally preserve kidney function in patients with hypertension and renal insufficiency. The mechanisms by which hypertension accelerates progression of renal disease have been extensively studied in experimental models. Glomerular capillary hypertension, consequent to an increase in systemic blood pressure combined with a reduction in preglomerular resistance and/or an increase in postglomerular resistance, results in increased hydraulic stress to the glomerular capillary wall. This and other mechanisms result in the release of growth-promoting cytokines and soluble mediators of fibrosis that stimulate cellular proliferation and matrix accumulation, ultimately leading to glomerular sclerosis and interstitial fibrosis. Almost without exception, studies in animals demonstrate that blood pressure reduction reduces the rate of progression of experimental renal disease. Angiotensin-converting enzyme inhibitors and, possibly, calcium antagonists may have a therapeutic advantage compared with other antihypertensive drugs in preventing kidney damage. This has been linked to both blood pressure-dependent and -independent actions. However, most experimental studies have failed to reduce blood pressure to a level sufficient to establish the clinical relevance of potential blood pressure-independent effects. Experimental studies comparing various types of antihypertensive drugs in which a mean arterial pressure (MAP) of approximately 92 mm Hg is achieved are necessary to determine whether clinically important differences in the effects of these drugs on the rate of progression of renal disease exist. Clinical experience with high blood pressure and kidney disease in humans suggests that the risk of developing hypertension-associated renal disease is a continuous variable across the entire range of systolic and diastolic blood pressures. Logically, optimal protection of kidney function may therefore be a continuous function of declining systemic blood pressure. Consistent with this view, recent clinical trials suggest that reducing MAP to 92 mm Hg, corresponding to a blood pressure reading of 125/75 mm Hg, provides more optimal stabilization of renal function in patients with nondiabetic proteinuric kidney disease (>1 g/d) compared with more conventional therapy with a blood pressure goal of 140/90 mm Hg (MAP 107 mm Hg). Clinical trials in patients with diabetes mellitus and renal insufficiency also demonstrate the benefits of reducing blood pressure to approximately 95 mm Hg MAP. Dietary salt consumption may be another important variable affecting the rate of progression of renal disease due to both direct, salt-dependent effects on renal growth and the action of decreased salt intake to augment the antihypertensive and antiproteinuric properties of many drugs. The precise role of alterations in dietary salt consumption on progression of renal disease directly as well as on the effectiveness of various antihypertensive drugs has yet to be examined in clinical trials.

Hepatocyte growth factor protects renal epithelial cells from apoptotic cell death.

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in renal tubular repair and regeneration following injury. Studies indicate that administration of exogenous HGF to animals stimulates renal epithelial cell DNA synthesis and accelerates recovery from acute renal failure (ARF). However, whether increased cell proliferation accounts for all of the beneficial effects of HGF in ARF is unknown. In this study, we demonstrate that HGF protects renal epithelial cells from undergoing apoptotic cell death. Treatment of renal epithelial mIMCD-3 cells with 25 microM cisplatin in the serum-free medium induced significant apoptosis, as assessed by fluorescent Dye H-33342 staining, TUNEL staining, light and electron microscopy, and DNA laddering analysis. However, constitutive expression of HGF by transfection in mIMCD-3 cells resulted in resistance to cisplatin-induced apoptotic death. The survival rate of HGF-producing C1 cells was more than 2-fold greater as compared to control, mIMCD-3 cells following treatment with 25 microM cisplatin for 2 days. These results suggest that HGF may not only activate tubular repair processes but also ameliorate the initial injury by protecting renal epithelial cells from undergoing apoptosis.

Clinical outcome in three patients with postpartum hemolytic uremic syndrome treated with frequent plasma exchange.

This study reviewed 3 cases of postpartum hemolytic uremic syndrome (HUS) at our hospital over a 3 year period. The 3 patients had clinical and laboratory abnormalities similar to those of 12 patients with other causes of thrombotic microangiopathy presenting during the same time span. Both groups were treated with 4-7 plasma exchange treatments/week and prednisone, 60 mg/day or its intravenous equivalent, until remission. The postpartum HUS patients had a more complicated, prolonged course; 3 of 3 required dialysis compared to 4 of 12 in the nonpostpartum group (p < 0.05), and they required more plasma exchange treatments (49 +/- 17 vs. 10 +/- 8, p = 0.0001) and a longer duration of therapy (70 +/- 31 vs. 19 +/- 17 days, p < 0.01) before remission. All postpartum HUS patients discontinued dialysis and survived whereas 4/12 nonpostpartum patients died before attaining remission. Compared to other variants of thrombotic microangiopathy, postpartum HUS requires a longer duration of therapy, but with aggressive therapy, renal and overall prognoses may be better.

Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb.

Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.