Proliferation Of Renal Epithelial Cells Research Articles

Expression of Augmenter of Liver Regeneration in Rats with Gentamicin-Induced Acute Renal Failure and Its Protective Effect on Kidney

Augmenter of liver regeneration (ALR) enhances the proliferation of hepatocytes and accelerates recovery from acute liver failure in animal models. ALR is expressed in both the liver and kidney; however, the specific location of ALR expression and its biological effects in the kidney remain unknown. We aimed to investigate the efficacy of ALR in rats with gentamicin (GM)-induced acute renal failure (ARF). Rats were randomized into the normal group, GM+saline group, GM+vehicle group, and GM+rrALR group. Blood urea nitrogen, serum creatinine, and urine beta-N-acetyl-D-glucosaminidase were measured, and histological analyses of the kidneys were performed. The expression of ALR protein was determined by immunohistochemistry and Western blotting. In vitro incorporation of tritiated thymidine was used to measure the proliferation of renal tubular epithelial cells. In normal rats, the expression of ALR protein was faint in the medulla and absent in the cortex. However, in ARF rats, ALR expression increased significantly in both the renal cortex and medulla. Histological analyses revealed that treatment with recombinant rat ALR (rrALR) reduced the extent of injury of tubular cells in the renal cortex. Serum/urine biochemical parameters also showed that renal dysfunction was improved by the administration of rrALR. Intraperitoneal injection of rrALR enhanced the proliferation of tubular cells in vivo. We also confirmed that rrALR could promote the proliferation of renal tubular cells in vitro. These results indicate that rrALR effectively accelerates kidney recovery after ARF induced by gentamicin, and that the protective effect is associated with enhanced proliferation of renal tubular cells.

Netrin-1 Overexpression Protects Kidney from Ischemia Reperfusion Injury by Suppressing Apoptosis

Netrin-1, a diffusible laminin-related protein, is highly expressed in the kidney. However, the pathophysiological roles of netrin-1 in the kidney are unknown. To address this question directly, we used transgenic mice that overexpress chicken netrin-1 in the kidney. Netrin-1 overexpression was confirmed by real-time RT-PCR and Western blot analysis. Eight-week-old wild-type and transgenic mice were subjected to 26 minutes of renal ischemia followed by reperfusion for 72 hours. Wild-type mice developed more severe renal dysfunction by 24 hours than netrin-1 transgenic mice. Functional improvement was associated with better preservation of morphology, reduced cytokine expression, and reduced oxidative stress in the kidney of transgenic mice as compared with wild-type mice. In addition, both basal and reperfusion-induced cell proliferation were dramatically increased in transgenic kidneys as determined by Ki-67 staining. Interestingly, ischemia reperfusion induced a large increase in apoptosis in wild-type mice but not in netrin-1 transgenic mice that was associated with reduced caspase-3 activation in the transgenic kidney. These results suggest that netrin-1 protects renal tubular epithelial cells against ischemia reperfusion-induced injury by increasing proliferation and suppressing apoptosis.

High prevalence of simple kidney cysts in patients with primary hyperparathyroidism

PTH has been demonstrated to promote renal epithelial cell proliferation and cysts development. The study aimed to evaluate the prevalence of kidney cysts in patients with primary hyperparathyroidism (PHPT). The prevalence of renal cysts diagnosed at abdominal ultrasound examinations in 172 PHPT patients (59.4+/-15.1 yr, mean age+/-SD; female/male 2.8) with preserved renal function was compared with that observed in 210 age- and sex-matched healthy controls. All patients underwent clinical, serum, and urine evaluations, and bone mineralization assessment by dual X-ray absorptiometry. Simple kidney cysts occurred with a higher prevalence in both male and female PHPT patients in comparison with healthy controls (34.9% vs 16.2% p<0.001). Kidney cysts were absent in patients younger than 39 yr, whereas they were present in one third of PHPT patients in their 4th, 5th, and 6th decades, increasing up to 45% after the age of 70. Multiple renal cysts were larger and more frequent than single cysts. PHPT patients with renal cysts were affected by a more active PTH secretion than patients without renal cysts as indicated by significant higher hypercalcemia and lower tubular maximal phosphate (TmP) reabsorption, while renal function, the occurrence of kidney stones, and osteoporosis were similar in both groups. Reduced TmP values were associated with about 3-fold increase in the risk of kidney cysts. In conclusion, simple renal cysts might be considered as a benign kidney complication of PHPT and might be related to the action of the chronic elevated PTH levels on tubular epithelial cells.

Netrin-1 increases proliferation and migration of renal proximal tubular epithelial cells via the UNC5B receptor

The cellular hallmark of kidney repair is a rapid proliferation of renal tubular epithelial cells ultimately leading to the restoration of nephron structure and function. Netrin-1 was discovered as a neural guidance cue and found to be expressed outside the nervous system, including in kidney. Previous work showed that netrin-1 is upregulated in response to ischemic injury and ameliorates ischemic injury. The objectives of this study were to determine the role of netrin-1 in renal tubular epithelial cell proliferation and migration in vitro. Real-time RT-PCR analysis showed that netrin-1 and its receptors UNC5B and neogenin are highly expressed in cultured mouse renal epithelial cells (TKPTS), whereas the expression of the Deleted in Colon Cancer (DCC), UNC5A, UNC5C, and UNC5D receptors is negligible or undetectable. Netrin-1 protein was induced in the edges of mechanical wounds in vitro. Netrin-1 increased TKPTS cell proliferation in a dose-dependent manner. The netrin-1-induced increase in TKPTS cell proliferation was completely prevented by small interfering RNA (siRNA) inhibition of UNC5B receptor but not UNC5C receptor expression. Netrin-1 also increased TKPTS cell migration in vitro, and this was also mediated through the UNC5B receptor. Netrin-1 increased the phosphorylation of Akt and ERK. Inhibition of phosphatidylinositol 3-kinase and MEK1/2 completely inhibited netrin-1-induced cell proliferation but not migration. These results indicate that netrin-1 increases renal tubular epithelial cell proliferation and migration through the UNC5B receptor. Moreover, the increase in cell proliferation, but not migration, was mediated via activation of Akt and ERK pathways.

Transglutaminase-1 Regulates Renal Epithelial Cell Proliferation through Activation of Stat-3

Transglutaminase-1 (TGase-1) is a Ca(2+)-dependent enzyme capable of cross-linking a variety of proteins and promoting wound healing in the skin. In this study, we examined the role of TGase-1 in proliferation of renal proximal tubular cells (RPTC). TGase-1, but not TGase-2, -5, and -7, was expressed in RPTC. Treatment with monodansylcadarevine (MDC), a selective TGase inhibitor or down-regulation of TGase-1 with small interfering RNA (siRNA) decreased RPTC proliferation. Proliferation of RPTC was accompanied by activation of Akt and Stat-3 (signal transducer and activator of transcription-3). Treatment with MDC or TGase-1 siRNA decreased Stat-3 but not Akt phosphorylation. Further studies showed that the Janus-activated kinase 2 (JAK2) mediates phosphorylation of Stat-3, and knockdown of either JAK2 or Stat-3 by siRNA decreased RPTC proliferation. However, inhibition of TGase-1 decreased phosphorylation of Stat-3 but not JAK2. Overexpression of Stat-3, JAK2, and/or TGase-1 in RPTC revealed that JAK2 is indispensable for TGase-1 to induce Stat-3 phosphorylation and TGase-1 potentiates JAK2-induced Stat-3 phosphorylation. Consistent with these observations, we found that inhibition of TGase-1 and the JAK2-Stat-3 signaling pathway decreased the transcriptional activity of Stat-3 and expression of the Stat-3-targeted genes, cyclin D1 and cyclin E. Conversely, overexpresssion of TGase-1 enhanced the JAK2-dependent transcriptional activity of Stat-3. Finally, TGase-1 was found to interact with JAK2, and this interaction was inhibited by MDC. These results demonstrate that TGase-1 plays an important role in regulation of renal epithelial cell proliferation through the JAK2-Stat-3 signaling pathway.

Changes in Cell-Cycle Kinetics Responsible for Limiting Somatic Growth in Mice

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-H]thymidine and 5'-bromo-2'deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-wk-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.

20-HETE Mediates Proliferation of Renal Epithelial Cells in Polycystic Kidney Disease

Polycystic kidney diseases are characterized by abnormal proliferation of renal epithelial cells. In this study, the role of 20-hydroxyeicosatetraenoic acid (20-HETE), an endogenous cytochrome P450 metabolite of arachidonic acid with mitogenic properties, was evaluated in cystic renal disease. Daily administration of HET-0016, an inhibitor of 20-HETE synthesis, significantly reduced kidney size by half in the BPK mouse model of autosomal recessive polycystic kidney disease. In addition, compared with untreated BPK mice, this treatment significantly reduced collecting tubule cystic indices and approximately doubled survival. For evaluation of the role of 20-HETE as a mediator of epithelial cell proliferation, principal cells isolated from cystic BPK and noncystic Balb/c mice were genetically modified using lentiviral vectors. Noncystic Balb/c cells overproducing Cyp4a12 exhibited a four- to five-fold increase in cell proliferation compared with control Balb/c cells, and this increase was completely abolished when 20-HETE synthesis was inhibited; therefore, this study suggests that 20-HETE mediates proliferation of epithelial cells in the formation of renal cysts.

Src regulates cell cycle protein expression and renal epithelial cell proliferation via PI3K/Akt signaling-dependent and -independent mechanisms.

Our recent studies showed that Src family kinases (SFKs) are important mediators of proliferation in renal proximal tubular cells (RPTC). In this study, we elucidate the signaling mechanisms that mediate SFK regulation of cell proliferation and cycle protein expression, and identify the SFK member responsible for these responses in a mouse RPTC line. Akt, a target of phosphoinositide-3-kinase (PI3K), and ERK1/2 were constitutively phosphorylated in RPTC cultured in the presence of serum. While treatment of cells with PP1, a specific SFK inhibitor, completely blocked phosphorylation of ERK1/2 and Akt, only inhibition of PI3K/Akt resulted in decreased RPTC proliferation. Incubation of cells with PP1 decreased cyclin D1 expression, decreased p27 and p57 phosphorylation, and increased p27 and p57 expression, two cyclin-dependent kinase inhibitors. Inhibition of the PI3K pathway decreased expression of cyclin D1 without altering expression of p27 and p57. In contrast, PP1 and PI3K inhibition had no effect on cyclin E and p21. Although RPTC expressed Src, Fyn, and Lyn, only siRNA-mediated knockdown of Src decreased RPTC proliferation, decreased cyclin D1 expression, and increased p27 and p57 expression. These data reveal that Src is a crucial mediator of RPTC proliferation and Src-mediated proliferation is associated with PI3K-dependent upregulation of cyclin D1 and PI3K-independent downregulation of p27 and p57.

Heparin Binding‐Epidermal Growth Factor and Src Family Kinases in Proliferation of Renal Epithelial Cells

This study was to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate. RPTC expressed both pro‐ and cleaved heparin binding epidermal growth factor (HB‐EGF) forms and several metalloproteinases (MMP‐2, ‐3 ‐9, and ADAM10, ADAM17) that have been reported to cleave HB‐EGF. Treatment of RPTC with CRM 197, an inhibitor of HB‐EGF binding to EGF receptor, or down‐regulation of HB‐EGF with siRNA inhibited RPTC proliferation following plating. Furthermore, blocking metalloproteinases with GM6001, TAPI‐1, GW280264X, but not GI254023X, attenuated the proliferation following plating. Although EGF receptor activation is required for RPTC proliferation following oxidant injury, CRM197, GM6001, and TAPI‐1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation in oxidant‐injured RPTC. In addition, PP1 treatment attenuated HB‐EGF‐enhanced RPTC proliferation. We suggest that RPTC proliferation following plating is regulated by HB‐EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB‐EGF production.

Epidermal growth factor receptor ligands and renal epithelial cell proliferation

a critical issue in understanding how the mammalian kidney responds to pathological states and nephrotoxic chemicals involves the identification of modulating agents, growth factors, and signaling pathways that regulate responses. Although some of the modulating agents identified, including

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of gamma-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, gamma-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis.

Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1

The root cause for most cases of autosomal-dominant polycystic kidney disease (ADPKD) is mutations in the polycystin-1 (PC1) gene. While PC1 has been implicated in a perplexing variety of protein interactions and signaling pathways, what its normal function is and why its disruption leads to the proliferation of renal epithelial cells are unknown. Recent results suggest that PC1 is involved in mechanotransduction by primary cilia measuring the degree of luminal fluid flow. PC1 has also recently been shown to regulate the mTOR and signal transducers and activators of transcription (STAT) 6 pathways. These two pathways are normally dormant in the healthy kidney but are activated in response to injury and appear to drive a proliferative repair response. This review develops the idea that a critical function of PC1 and primary cilia in the adult kidney may be to sense renal injury by detecting changes in luminal fluid flow and to trigger proliferation. Constitutive activation of these pathways in ADPKD would lead to the futile attempt to repair a nonexisting injury, resulting in cyst growth. The existence of many known cellular and molecular similarities between renal repair and ADPKD supports this model.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

ERK1/2‐driven and MKP‐mediated inhibition of EGF‐induced ERK5 signaling in human proximal tubular cells

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.

Evidence of angiogenesis and microvascular regression in autosomal-dominant polycystic kidney disease kidneys: A corrosion cast study

Autosomal-dominant polycystic kidney disease (ADPKD) accounts for about 10% of all cases of chronic renal failure requiring dialysis. The disease is characterized by proliferation of renal epithelial cells and formation of cysts that expand over years and replace the normal parenchyma of the kidney. As the cysts grow, the volume of the kidney can increase by more than 10-fold, implying that remodeling and expansion of the vasculature must occur to provide oxygenation and nutrition to the cyst cells. Our previous studies support the notion that there is angiogenesis in ADPKD. We report here results from resin casting of ADPKD kidneys vasculature. In this study, the corrosion-casting method was used in conjunction with scanning electron microscopy to study the vascular architecture and the evidence for angiogenesis in ADPKD kidneys. We found a well-defined vascular network around the cysts forming a 'vascular capsule' somewhat similar to that described in avascular leiomyomata. We also found that the normal vascular architecture is lost and replaced by an assortment of capillaries of larger size than those in the normal kidney, mixed with flattened and spiral arterioles, damaged glomeruli, and atresic venules, indicative of regression of the microvasculature. In the same areas, there was capillary sprouting, considered the hallmark of angiogenesis. The present study documents regression changes of the vasculature and confirms the existence of angiogenesis in ADPKD kidneys, and suggests the use of inhibitors of angiogenesis as a possible avenue for the treatment of the disease.

Inhibition of polycystin-L channel by the Chinese herb Sparganum stoloniferum Buch.-Ham.

The Chinese herb Sparganum stoloniferum Buch.-Ham. (SBH) is frequently used to improve blood circulation and to rehabilitate vascular obstruction in traditional Chinese medicine. It was recently reported that SBH reduces the proliferation of renal epithelial cells stimulated by epidermal growth factor (EGF), and inhibits the phosphorylation of the EGF receptor. SBH has also been used as a trial drug to treat polycystic kidney disease (PKD) patients in China. The potential molecular actions of SBH on PKD remain unknown. Autosomal dominant PKD (ADPKD) is associated with mutations in polycystin-1 or polycystin-2 (PC2). PC2 and its homologue, polycystin-L (PCL), are nonselective cation channels permeable to potassium, sodium, and calcium. Here, we examine the effects of SBH on the human PCL channel expressed in Xenopus oocytes, using 2-microelectrode voltage-clamp electrophysiology and radiotracer uptake measurements. In PCL-expressing oocytes, with or without preincubation with SBH, the PCL channel was inhibited by SBH in a dose-dependent and reversible manner; a concentration of 2% SBH completely abolished the channel activation. The IC50 value for SBH was 0.48% +/- 0.03%, with a 10-min preincubation period. SBH was also found to inhibit the PCL-mediated 45Ca tracer uptake in oocytes. Our study suggests that SBH contains 1 or more yet-to-be determined components that are inhibitors of PCL channel. The therapeutic potential of SBH for ADPKD and its chemical composition remain to be investigated.

Sustained cell proliferation of renal epithelial cells in mice with inv mutation

A tubule system is an important component of the nephron, which is the structural and functional unit of the kidney. Expansion of renal tubules results in renal cysts. Hereditary forms of renal cystic diseases suggest that tubular size is determined genetically. The inv was discovered as a mutant with renal cysts and situs inversus. Inv/inv, inv deltaC::GFP (inv deltaC) mouse was created by the introduction of the inv gene lacking the C-terminus (inv deltaC) into inv/inv mice. The mouse develops multiple renal cysts without situs abnormality, giving us an opportunity to study inv function in renal tubular structure maintenance. In the present study, we showed that inv suppresses cyst progression in a dose-dependent manner and that the inv deltaC cystic kidneys showed increased cell proliferation and apoptosis. Cell cycle regulators for G1-S progression were activated in the cystic kidney. Furthermore, cDNA microarray and semiquantitative RT-PCR analysis showed that growth-related genes maintained a high level of expression in the cystic kidney at 4 weeks of age whereas they were decreased in control kidneys, suggesting that cells in inv deltaC kidney are still active in the cell cycle. One of the inv protein functions may provide a stop signal for renal epithelial cell proliferation.

Lixazinone Stimulates Mitogenesis of Madin-Darby Canine Kidney Cells

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.

Direct Effects of VEGF and VEGF Receptor Inhibitor on Proliferation of Human Renal Proximal Tubular Epithelial Cells (HRPTEC)

Clinical models link VEGF inhibition to hypertension. However, the role of VEGF in renal mechanisms of hypertension is poorly understood. We seek to determine if VEGF or VEGF receptor inhibitor (SU5416) directly affects proliferation of cultured HRPTEC, compared to human umbilical vein endothelial cells (HUVEC), and human aortic smooth muscle cells (HASMC). 3H-thymidine incorporation was determined in those cells following exposure to VEGF (10 ng/ml), VEGF plus SU5416 (10 μmol/L), SU5416 (1, 5, 10, & 20 μmol/L), vehicle control for 18 hrs. VEGF slightly but significantly increased proliferation of HRPTEC, compared to the control (10%, P=0.012, n=6). There was a significant decrease in proliferation of HRPTEC treated with VEGF plus SU5416, compared to the control (65%, P<0.001). VEGF caused a 2-fold increase in proliferation of HUVEC, compared to the control (P<0.001), but its action was completely abolished by SU5416. Neither VEGF nor SU5416 exerted any effect on proliferation of cultured HASMC. In addition, SU5416 caused a dose-related inhibition in proliferation of HRPTEC, but 20 μmol/L of SU5416 did not further blunt proliferation of HRPTEC. We first reported that VEGF receptor blocker inhibited proliferation of HRPTEC, and demonstrated that VEGF signaling system plays a major role in proliferation of HRPTEC though other signaling systems may be involved in regulating the proliferation. These findings also suggest autocrine and paracrine functions of VEGF on HRPTEC. Together, the results support the hypothesis that VEGF may play an important role in renal mechanisms of hypertension. (NHI/AA013821-01A2; NIH/HL51971).