Renal Cell Survival Research Articles

Generation and characterization of sodium-dicarboxylate cotransporter-deficient mice

The sodium-dependent dicarboxylate cotransporter (NaDC1) has a proposed function of reabsorbing various Krebs cycle intermediates in the kidney and the small intestine. Since Krebs cycle intermediates have been suggested to be important for renal cell survival and recovery after hypoxia and reoxygenation, the transporter may play a role in the recovery of the kidney. Additionally, mutations in the transporter homolog in Drosophila led to fly longevity which was thought to be similar to that induced by caloric restriction (CR). To clarify the role of the sodium dicarboxylate cotransporter in vivo we generated cotransporter-deficient mice. These knockout mice excreted significantly higher amounts of various Krebs cycle intermediates in their urine; thus confirming the proposed function to reabsorb these metabolic intermediates in the kidney. No other phenotypic change was identified in these mice, however. Transporter deficiency did not affect renal function under normal physiological conditions, nor did it have an effect on renal damage and recovery from ischemic injury. Additionally, the absence of the transporter did not lead to metabolic or physiological changes associated with CR. Our results suggest that although the sodium dicarboxylate cotransporter is involved in regulating levels of various Krebs cycle intermediates in the kidney, impaired uptake of these intermediates does not significantly affect renal function under normal or ischemic stress.

Renal tubule necrosis and apoptosis modulation by A1 adenosine receptor expression

We have shown that A1 adenosine receptors (A1ARs) are cytoprotective against renal tubular necrosis and apoptosis both in vivo and in vitro. To study the role of A1AR numbers on renal epithelial cell survival, we stably overexpressed the human A1 receptor in a porcine renal tubule cell line and utilized primary cultures of proximal tubules obtained from A1AR knockout mice. Receptor-overexpressing cells were protected against peroxide-induced necrosis and tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Conversely, cultured proximal tubule cells from receptor knockout mice showed more necrotic and apoptotic cell loss than corresponding cells from wild-type mice. Overexpression of the receptor resulted in a significantly higher baseline expression of both total and phosphorylated heat-shock protein (HSP)27; the latter due to A1 receptor enhancement of p38 and AP2 mitogen-activated protein kinase activities. The resistance to cell death in the porcine cells was reversed by selective A1 receptor antagonism and by a selective inhibitor of HSP synthesis. Receptor activation in wild-type mice in vivo led to increased total and phosphorylated HSP27, whereas receptor knockout mice showed decreased baseline and adenosine-mediated HSP phosphorylation. These studies show that endogenous A1AR activation produces cytoprotective effects in renal proximal tubules by modulating HSP27 signaling pathways.

STAT3 attenuates EGFR-mediated ERK activation and cell survival during oxidant stress in mouse proximal tubular cells

We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)-Ras-MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H(2)O(2))-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H(2)O(2) dose. On the other hand, both I/R and severe oxidative stress - but not moderate stress - increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury.

Lethal activity of FADD death domain in renal tubular epithelial cells

Fas-associated death domain (FADD) is an adaptor protein that is required for the transmission of the death signal from lethal receptors of the tumor necrosis factor superfamily. FADD contains a death domain (DD) and a death effector domain (DED). As death receptors contribute to renal tubular injury and tubular cell FADD increases in acute renal failure, we have studied the function of FADD in tubular epithelium. FADD expression was studied in kidney samples from mice. In order to study the contribution of FADD to renal tubular cell survival, FADD or FADD-DD were overexpressed in murine tubular epithelium. FADD is expressed in renal tubules of the healthy kidney. Both FADD and FADD-DD induce apoptosis in primary cultures of murine tubular epithelium and in the murine cortical tubular cell line. Death induced by FADD-DD has apoptotic morphology, but differs from death receptor-induced apoptosis in that it is not blocked by inhibitors of caspases. Neither an inhibitor of serine proteases nor overexpression of antiapoptotic BclxL prevented cell death. However, the combination of caspase and serine protease inhibition was protective. FADD and FADD-DD overexpression decreased nuclear factor kappa B activity. These data suggest that FADD has a death regulatory function in renal tubular cells that is independent of death receptors. FADD-DD is sufficient to induce apoptosis in these cells. This information is relevant to understanding the role of FADD in tubular injury.

Modulation of Renal Tubular Cell Survival: Where is the Evidence?

Tubular cell loss is prominent both in acute and chronic renal failure. Apoptosis and its regulatory mechanisms contribute to cell number regulation in the kidney. The potential role of apoptosis ranges from induction and progression to repair of renal injury. However, therapeutic interest has focused in preventing the apoptotic loss of tubular cells that leads to acute and chronic renal failure. Death ligands and receptors, such as tumor necrosis factor and Fas ligand, proapoptotic and antiapoptotic Bcl2 family members and caspases have all been shown to participate in apoptosis regulation in the course of renal cell injury. Nevertheless, the precise role of these proteins is unclear, and the participation of most known apoptosis regulatory proteins has not been studied. We now review the role of apoptosis in renal injury, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis, with emphasis in acute renal failure.

Genetic restoration of aldose reductase to the collecting tubules restores maturation of the urine concentrating mechanism

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenic line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kgH2O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism.

Caveolins in the repair phase of acute renal failure after oxidative stress

Ischaemia-reperfusion and toxic injury are leading causes of acute renal failure (ARF). Both of these injury initiators use secondary mediators of damage in oxygen-derived free radicals. Several recent publications about ischaemia-reperfusion and toxin-induced ARF have indicated that plasma membrane structures called caveolae, and their proteins, the caveolins, are potential participants in protecting or repairing renal tissues. Caveolae and caveolins have previously been ascribed many functions, a number of which may mediate cell death or survival of injured renal cells. This review proposes possible pathophysiological mechanisms by which altered caveolin-1 expression and localization may affect renal cell survival following oxidative stress.

Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors

Mouse proximal tubular cells (BUMPT), when cultured in the absence of growth factors, activate a default apoptotic pathway. Although Wnt signaling antagonizes the effect of proapoptotic triggers, its role in regulating the default pathway of apoptosis is less well defined. The present study examines the hypothesis that lithium (Li(+)) and (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), two glycogen synthase kinase-3beta (GSK3beta) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway. These studies demonstrate that Li(+) and BIO activate Wnt signaling as indicated by the following changes: phosphorylation (inhibition) of GSK3beta; decreased phosphorylation of beta-catenin (a GSK3beta substrate); nuclear translocation of beta-catenin; specific transcriptional activation of Tcf/catenin-responsive pTopflash constructs; and an increase in the expression of cyclin D1 (indicative of a promitogenic cell response). In addition, Li(+) or BIO significantly increases the phosphorylation (activation) of Akt, an anti-apoptotic protein, and inhibits apoptosis (decreases both annexin-V staining and caspase-3 activation), during serum deprivation. Inhibition of phosphatidylinositol 3-kinase (responsible for Akt activation) either by wortmanin or LY-294002 prevented Li(+)- or BIO-induced Akt phosphorylation and reduces cell survival without altering the phosphorylation state of GSK3beta. Li(+) or BIO also increases the expression of insulin-like growth factor-II (IGF-II), a potent proliferative signaling protein. Li(+) or BIO-free conditioned medium harvested from Li(+)- or BIO-exposed cells also induced Akt phosphorylation, mimicking the protective effect of the two GSK3beta inhibitors on serum-starved cells. Furthermore, the effect of conditioned medium on Akt phosphorylation could be inhibited by either LY-294002 or IGF-binding protein. BIO, a specific GSK3beta inhibitor, replicated the protective effect of Li(+) on cell viability, suggesting that GSK3beta activation is important for initiating the apoptotic pathway. Taken together, these data suggest that Li(+) or BIO promotes renal epithelial cell survival by inhibiting apoptosis through GSK3beta-dependent activation of the Wnt pathway and subsequent release of IGF-II. Extracellular IGF-II serves as an autocrine survival factor that is responsible, in part, for activating the anti-apoptotic phosphatidylinositol-3-kinase-Akt pathway during serum deprivation.

Carbonic Anhydrase IX (CAIX) expression predicts for renal cell cancer (RCC) patient response and survival to IL-2 therapy

4512 Background: RCC response to IL-2 Rx and patient (pt) survival have been correlated to histology including the presence of clear cell and alveolar and the absence of papillary and granular features (Upton et al Proc ASCO, 2003) and CAIX expression (Bui et al Clin Ca Res 2003;9:802). In an effort to confirm and expand these findings we undertook a nested case-control study within the Upton cohort. Methods: Paraffin-embedded tissue sections of RCC were immunostained with an Ab to CAIX and expression levels were correlated with pathology findings, clinical outcome and the Upton model relating them. The percentage of CAIX positive tumor cells was utilized to separate high (> 85%) vs low (≤ 85%) expressers. Results: Tissue specimens were obtained from 66 pts (51M, 15F). 27 of the selected pts (41%) had responded (10CR/17PR) to IL-2. 24 (36%), 31 (47%) and 11 (17%) were classified as good, intermediate, and poor prognosis according to the Upton model. 41 specimens (62%) had high CAIX expression. 21 of 27 (78%) responding pts had high CAIX expression compared to 20/39 (51%) non-responders (OR=3.3; p=0.04); a similar survival benefit was seen (p=0.04). Survival >5 yrs was seen only in high CAIX expressors. High CAIX staining was associated with better pathology prognosis, and when stratified by group, the ORs of CAIX expression were unequal across groups (p=0.08) . The stongest association was in the intermediate group where 9/9 responders has high CAIX expression vs.11/22 non-responders, suggesting a possible refinement of the Upton model. A resultant group with good pathology or intermediate pathology and high CAIX contained 26 of 27 (96%) responders compared to only 18/39 (46%) non-responders (OR=30; p<0.01). Survival benefit was also seen (p< 0.01). Conclusions: We conclude that CAIX expression is an important predictor of outcome in RCC pts receiving IL-2-based Rx and can enhance prognostic information obtained from pathology specimens. While this refinement to the pathology model requires validation, data suggest that IL-2 Rx might ultimately be reserved for pts with Upton model good pathology alone or intermediate pathology and high CAIX staining. No significant financial relationships to disclose.

Carbonic Anhydrase IX (CAIX) expression predicts for renal cell cancer (RCC) patient response and survival to IL-2 therapy

4512 Background: RCC response to IL-2 Rx and patient (pt) survival have been correlated to histology including the presence of clear cell and alveolar and the absence of papillary and granular features (Upton et al Proc ASCO, 2003) and CAIX expression (Bui et al Clin Ca Res 2003;9:802). In an effort to confirm and expand these findings we undertook a nested case-control study within the Upton cohort. Methods: Paraffin-embedded tissue sections of RCC were immunostained with an Ab to CAIX and expression levels were correlated with pathology findings, clinical outcome and the Upton model relating them. The percentage of CAIX positive tumor cells was utilized to separate high (> 85%) vs low (≤ 85%) expressers. Results: Tissue specimens were obtained from 66 pts (51M, 15F). 27 of the selected pts (41%) had responded (10CR/17PR) to IL-2. 24 (36%), 31 (47%) and 11 (17%) were classified as good, intermediate, and poor prognosis according to the Upton model. 41 specimens (62%) had high CAIX expression. 21 of 27 (7...

COX2 Activity Promotes Organic Osmolyte Accumulation and Adaptation of Renal Medullary Interstitial Cells to Hypertonic Stress

The mechanism by which COX2 inhibition decreases renal cell survival is poorly understood. In the present study we examined the effect of COX2 activity on organic osmolyte accumulation in renal medulla and in cultured mouse renal medullary interstitial cells (MMICs) and its role in facilitating cell survival. Hypertonicity increased accumulation of the organic osmolytes inositol, sorbitol, and betaine in cultured mouse medullary interstitial cells. Pretreatment of MMICs with a COX2-specific inhibitor (SC58236, 10 micromol/liter) dramatically reduced osmolyte accumulation (by 79 +/- 9, 57 +/- 12, and 96 +/- 10% for inositol, sorbitol, and betaine respectively, p < 0.05). Similarly, 24 h of dehydration increased inner medullary inositol, sorbitol, and betaine concentrations in vivo by 85 +/- 10, 197 +/- 28, and 190 +/- 24 pmol/microg of protein, respectively, but this increase was also blunted (by 100 +/- 5, 66 +/- 15, and 81 +/- 9% for inositol, sorbitol, and betaine, respectively, p < 0.05) by pretreatment with an oral COX2 inhibitor. Dehydrated COX2-/- mice also exhibited an impressive defect in sorbitol accumulation (88 +/- 9% less than wild type, p < 0.05) after dehydration. COX2 inhibition (COX2 inhibitor-treated or COX2-/- MMICs) dramatically reduced the expression of organic osmolyte uptake mechanisms including betaine (BGT1) and sodium-myo-inositol transporter and aldose reductase mRNA expression under hypertonic conditions. Importantly, preincubation of COX2 inhibitor-treated MMICs with organic osmolytes restored their ability to survive hypertonic stress. In conclusion, osmolyte accumulation in the kidney inner medulla is dependent on COX2 activity, and providing exogenous osmolytes reverses COX2-induced cell death. These findings may have implications for the pathogenesis of analgesic nephropathy.

Renal cell loss through cell suicide

Renal cell loss through cell suicide

Hepatocyte growth factor promotes renal epithelial cell survival by dual mechanisms.

Hepatocyte growth factor (HGF) has been shown to protect renal epithelial cells against apoptosis. To define the mechanism by which HGF inhibits apoptosis, we investigated the effect of HGF on the phosphorylation and expression of the Bcl-2 family proteins. Using a human proximal tubular epithelial cell (HKC) line as a model, we demonstrated that constitutive expression of HGF conveyed marked resistance to apoptotic death induced by serum withdrawal. HGF induced rapid phosphorylation of Akt in HKC cells, which was immediately followed by phosphorylation and resultant inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. Pretreatment of the HKC cells with 10 nM wortmannin completely abolished HGF-induced phosphorylation of Akt and Bad, suggesting that this pathway is dependent on phosphoinositide (PI) 3-kinase. Overexpression of Bad increased apoptotic death in wild-type HKC cells but not in HGF-producing H4 cells. Immunoblotting confirmed that the Bad protein over-expressed in H4 cells was fully phosphorylated at both Ser(112) and Ser(136) sites. Prolonged incubation of HKC cells with HGF also dramatically induced expression of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. These results suggest that the anti-apoptotic effect of HGF in renal epithelial cells is mediated by dual mechanisms involving two distinct Bcl-2 family proteins. HGF triggers Bad phosphorylation via the PI 3-kinase/Akt pathway, thereby inactivating this pro-apoptotic protein, while simultaneously inducing expression of anti-apoptotic Bcl-xL.

MAPK activation determines renal epithelial cell survival during oxidative injury.

Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.

Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability

Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHB-EGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent. We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRKCD9) or together with proHB-EGF (NRKboth). Juxtacrine mitogenic activity of NRKCD9 was no different than in cells transfected with vector alone (NRKvector), but was increased by NRKboth; juxtacrine mitogenic activity by NRKboth was twofold greater than when proHB-EGF was transfected alone (NRKproHB-EGF). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRKproHB-EGF grew 50% faster than NRKvector or NRKCD9, and NRKboth grew 20% to 50% faster than NRKproHB-EGF at one, two, and three days of culture. NRKproHB-EGF attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRKvector, and NRKboth was 20% to 30% greater than that of NRKproHB-EGF. Coating plates with either poly 2-hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell-extracellular matrix attachment, and NRKvector or NRKCD9 failed to attach or form cell-cell attachments. NRKproHB-EGF exhibited 300% and NRKboth exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRKproHB-EGF and NRKboth, but the expression of beta1 integrin was up-regulated only in NRKboth. Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.

The Membrane-bound Form of Heparin-binding Epidermal Growth Factor-like Growth Factor Promotes Survival of Cultured Renal Epithelial Cells

To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvector exhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10(-8) M) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGF attachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 x 10(3) cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.

Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction

Objectives. Acute unilateral ureteral obstruction (UUO) results in ipsilateral hydronephrosis characterized by a decrease in epidermal growth factor (EGF) mRNA expression and EGF protein levels in the distal renal tubules. UUO results in programmed cell death with increases in the characteristic markers of apoptosis. To suppress the apoptotic response during UUO, recombinant EGF was administered during renal obstruction and the ensuing molecular and histologic changes were studied. Methods. Mature Sprague-Dawley rats underwent left ureteral obstruction and the kidneys were harvested at 24, 48, and 72 hours. Markers of apoptosis included DNA laddering pattern on agarose gel electrophoresis, in situ gap labeling of fragmented DNA for quantitative apoptotic body determination, polyadenylated mRNA expression of SGP-2, and in situ hybridization for sulfated glycoprotein-2 (SGP-2) mRNA. Studies were repeated in rats following administration of 10, 20, and 40 μg of subcutaneous recombinant EGF on a daily basis after UUO. Results. Subcutaneous injection of EGF into unilaterally obstructed rats promotes renal tubular epithelial cell regeneration, as demonstrated by increased cortical mitotic activity. Systemic EGF supplementation in these unilaterally obstructed rats also resulted in a decrease in the intensity of the DNA laddering pattern associated with renal tubular apoptosis. An in situ labeling procedure to identify apoptotic nuclei in the ureterally obstructed kidneys revealed a 50% reduction in apoptosis after EGF administration. Northern blot analysis and in situ hybridization for SGP-2 mRNA or clusterin gene product also revealed a decreased expression in the obstructed and EGF-treated renal parenchyma. Conclusions. These data suggest that EGF, apart from its known role as a mitogenic substance for renal tubular epithelial cells, is also a critical in vivo renal cell survival factor for the developmentally mature kidney.

Molecular basis for osmoregulation of organic osmolytes in renal medullary cells.

Renal medullary cells are naturally exposed to extremely high and variable interstitial concentrations of NaCl and urea, consequent to operation of the urinary concentrating mechanism. They respond by accumulating large and variable amounts of sorbitol, glycerophosphocholine (GPC), glycine betaine (betaine), myo-inositol (inositol), and taurine both in vivo and in cell cultures. Sorbitol is synthesized from glucose, catalyzed by aldose reductase. Hypertonicity increases aldose reductase activity by raising this enzyme's transcription, mRNA level, and translation, and thereby increases production of sorbitol. GPC is synthesized from choline via phosphatidylcholine. A combination of high NaCl plus urea does not increase GPC synthesis, but does reduce its degradation by inhibiting GPC:choline phosphodiesterase. Betaine, inositol and taurine are taken up into the cells, each by a different sodium-dependent transporter. Hypertonicity increases mRNAs of all three transporters. This is due to increased transcription (at least of the inositol and betaine transporters). The eventual result is greater betaine, inositol and taurine uptake and accumulation. Osmoregulation of net sorbitol and GPC synthesis and of betaine, inositol and taurine transport is slow, requiring hours to days. However, following an acute fall in tonicity, these organic osmolytes exit from the cells within minutes, via specialized efflux mechanisms. As demonstrated by cloning efficiency studies, renal cell survival and growth following hypertonicity depend on the sum of all organic osmolytes that are accumulated; altering one experimentally changes the others to maintain a nearly constant total. Methylamine accumulation protects these cells against high urea; the methylamine that is preferentially accumulated in response to high urea is GPC.

Intracellular betaine substitutes for sorbitol in protecting renal medullary cells from hypertonicity

Renal medullary cells are normally exposed to a variably high extracellular NaCl concentration. They compensate by accumulating large amounts of organic osmolytes, including sorbitol and betaine. The sorbitol is synthesized from glucose, catalyzed by aldose reductase. Previously, inhibition of aldose reductase activity was noted to greatly reduce renal medullary cell survival and growth (measured by cloning efficiency) in tissue cultures of renal medullary cells in hypertonic medium. In contrast, inhibition of aldose reductase and renal medullary sorbitol accumulation is not associated with kidney damage in vivo. In the present experiments we find that addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by aldose reductase inhibitors and restores the cloning efficiency. We presume that in vivo uptake of betaine by renal medullary cells similarly protects them from harm when aldose reductase inhibitors lower sorbitol. The results also demonstrate that one organic osmolyte can substitute for another in protecting cells from hypertonicity, consistent with the compatible osmolytes hypothesis.