Acidic organelles mediate TGF-ß1-induced cellular fibrosis via (pro)renin receptor and vacuolar ATPase trafficking in human peritoneal mesothelial cells.

TGF-ß1, which can cause renal tubular injury through a vacuolar-type H+-ATPase (V-ATPase)-mediated pathway, is induced by the glucose degradation product methylglyoxal to yield peritoneal injury and fibrosis. The present study investigated the roles of V-ATPase and its accessory protein, the (pro)renin receptor, in peritoneal fibrosis during peritoneal dialysis. Rats daily administered 20 mM methylglyoxal intraperitoneally developed significant peritoneal fibrosis after 7 days with increased expression of TGF-ß and V-ATPase, which was reduced by the inhibition of V-ATPase with co-administration of 100 mM bafilomycin A1. The (pro)renin receptor and V-ATPase were expressed in acidic organelles and cell membranes of human peritoneal mesothelial cells. TGF-ß1 upregulated the expression of collagens, α-SMA, and EDA-fibronectin, together with ERK1/2 phosphorylation, which was reduced by inhibition of V-ATPase, (pro)renin receptor, or the MAPK pathway. Fibronectin and the soluble (pro)renin receptor were excreted from cells by acidic organelle trafficking in response to TGF-ß1; this excretion was also suppressed by inhibition of V-ATPase. Soluble (pro)renin receptor concentrations in effluents of patients undergoing peritoneal dialysis were associated with the dialysate-to-plasma ratio of creatinine. Together, these results demonstrate a novel fibrosis mechanism through the (pro)renin receptor and V-ATPase in the acidic organelles of peritoneal mesothelial cells.

Rituximab is an efficient and safe treatment in adults with steroid-dependent minimal change disease.

Development of steroid dependency in patients with nephrotic syndrome may require a long-term multi-drug therapy at risk of drug toxicity and renal failure. Rituximab treatment reduces the steroid dosage and the need for immunosuppressive therapy in pediatric patients. Here we retrospectively analyze the efficacy and safety of rituximab in adult patients with steroid-dependent minimal change disease. To do this, we analyzed the outcome of all adult patients treated with rituximab for steroid-dependent minimal change nephrotic syndrome over a mean follow-up of 29.5 months (range 5.1-82 months). Seventeen patients with steroid-dependent or frequently relapsing minimal change nephrotic syndrome, unresponsive to several immunosuppressive medications, were treated with rituximab. Eleven patients had no relapses after rituximab infusion (mean follow-up 26.7 months, range 5.1-82 months) and nine of them were able to come off all other immunosuppressive drugs and steroids during follow-up. Six patients relapsed at least once after a mean time of 11.9 months (mean follow-up 34.5 months, range 16.9-50.1 months), but their immunosuppressive drug treatment could be stopped or markedly reduced during this time. No adverse events were recorded. Thus, rituximab is efficient and safe in adult patients suffering from severe steroid-dependent minimal change disease. Prospective randomized trials are needed to confirm this study.

TGF-beta1 mediates glucose-evoked up-regulation of connexin-43 cell-to-cell communication in HCD-cells.

BACKGROUND/AIMS: In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD). METHODS: RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry. RESULTS: At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells. CONCLUSIONS: High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

High glucose up-regulates ENaC and SGK1 expression in HCD-cells.

BACKGROUND/AIM: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. METHODS: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the alpha conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry. RESULTS: Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-beta1 were all found to evoke significant and time-dependent increases in both SGK1 and alphaENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level. CONCLUSION: Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy.

Glucose-evoked alterations in connexin43-mediated cell-to-cell communication in human collecting duct: a possible role in diabetic nephropathy.

Aberrant sodium absorption has been linked to the development of hypertension in both renal disease and diabetes. Efficient absorption depends on coordination of cellular activity across the entire epithelium via cell-to-cell coupling. In the current study we have utilized a model human collecting duct cell line (HCD) to assess the role of connexin43 (Cx43)-mediated gap junctions in the transfer of intracellular Ca(2+) transients within coupled cell clusters. HCD cells express Cx43 mRNA and protein, as well as that for the mechanosensitive transient receptor potential receptor (TRPV4). Mechanical stimulation of individual cells within a cluster evoked a transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) that propagated between cells via a heptanol-sensitive mechanism. The rise in [Ca(2+)](i) was dependent on both store release and Ca(2+)-influx pathways. Lucifer yellow dye transfer and Cx43 knockdown experiments confirmed direct cell-to-cell communication. Application of the Ca(2+) ionophore ionomycin, or an increase in glucose (5 to 25 mM), produced a time-dependent (48 h) increase in Cx43 protein expression. The transmission rate of touch-evoked Ca(2+) transients between coupled cells was accelerated after exposure to high glucose, providing a functional correlate to increased Cx43 expression. These data suggest a pivotal role for Cx43-mediated gap junctions in the synchronization of activity between HCD cells in response to stimuli that mimic osmotic and physical changes. Cx43 expression and cell-to-cell communication increased in response to high glucose and may protect the collecting duct from renal damage associated with more established diabetic nephropathy.

Urokinase (u-PA) is produced by collecting duct principal cells and is post-transcriptionally regulated by SV40 large-T, arginine vasopressin, and epidermal growth factor.

We have analyzed the expression and regulation of plasminogen activators (PA) in principal cells of the renal collecting duct. We used a rabbit principal cell line (RC.SVtsA58) infected with the temperature-sensitive SV40 strain tsA58. Transformed cells cultured at permissive temperature (33 degrees C) produced only tissue-type plasminogen activator (t-PA). Shifting the cells to nonpermissive temperature (39.5 degrees C) induced their differentiation and a marked increase in total fibrinolytic activity due to the induction of urokinase-type plasminogen activator (u-PA) synthesis and secretion. The effect on u-PA was post-transcriptional and it could be attributed to large-T inactivation at 39.5 degrees C since it was abolished by re-infecting the cells with wild-type SV40. Run-on assay and real-time RT-PCR of u-PA transcripts indicated that large-T altered post-transcriptional regulation. u-PA was also produced by primary cultures of collecting duct cells and was present in the rabbit urine. In the kidney, u-PA and its receptor (u-PAR) were almost exclusively expressed at the apex of collecting duct cells. We then analyzed the regulation of u-PA by arginine vasopressin (AVP) and epidermal growth factor (EGF), two key regulators of principal cell functions. We found that AVP and EGF, which have opposite hydro-osmotic effects in the collecting duct, also exhibited contrasted effects on u-PA synthesis in differentiated RC.SVtsA58 cells. EGF increased but AVP suppressed u-PA activity and protein, and these regulations occurred at post-transcriptional level. These results point to a physiological role of u-PA in principal cells of the renal collecting duct.

Klf6 is a zinc finger protein expressed in a cell-specific manner during kidney development.

Molecular mechanisms that are responsible for the development of the renal collecting duct system during embryogenesis are still poorly understood. A mouse cDNA encoding a zinc finger protein, called Klf6, which is a member of the Krüppel-like family of transcription factors, has been cloned. Northern blot analyses showed that Klf6 was already expressed in 11.5-d postconception mouse embryos and that its expression persisted after birth. They also disclosed that Klf6 had a restricted pattern of expression. In situ hybridization experiments using mouse embryos showed that during kidney development, Klf6 was expressed selectively in the Wolffian duct and in its derivatives. During mesonephros development, it was expressed in the Wolffian duct but not in the mesonephric mesenchyme. Thereafter, Klf6 was expressed in the ureteric bud and its branches and in the collecting ducts, whereas it was not expressed in tubular structures that derive from the metanephric mesenchyme. Glomeruli were not labeled during early stages of differentiation, and it is only at the capillary stage that a staining of the mesangial area was observed, which persisted after birth. This pattern of expression is strikingly similar to the one of GATA-3, which is another zinc finger protein. It suggests that Klf6 may play a role during kidney development and in particular during the development of the renal collecting duct system, possibly in association with GATA-3.