A mechanistic approach to inherited polycystic kidney disease

There are approximately six and a half million people, of the estimated world population of six billion, with inherited polycystic kidney disease. Polycystic kidney diseases have a broad spectrum of associated findings that distinguish and define them as specific disease states. The dysregulation of renal tubular epithelial cell biology, including cell polarity, cell signaling, proliferation and apoptosis, basement membrane and matrix abnormalities, and fluid transport, has been postulated to contribute to cystogenesis. Evidence is currently accumulating that supports an association of the primary cilium and basal body, as well as the focal adhesion assembly, with polycystic kidney diseases. Renal cystogenesis may be the result of a disruption of a critical feedback loop that regulates tissue morphology based on the epithelial cell environment.     

Pathways of apoptosis in human autosomal recessive and autosomal dominant polycystic kidney diseases

Autosomal dominant polycystic kidney disease (ADPKD) is a major cause of end-stage renal disease in adults. Autosomal recessive (AR) PKD affects approximately 1:20,000 live-born children with high perinatal mortality. Both diseases have abnormalities in epithelial proliferation, secretion, and cell-matrix interactions, leading to progressive cystic expansion and associated interstitial fibrosis. Cell number in a kidney reflects the balance between proliferation and apoptosis. Apoptosis results from extrinsic (ligand-induced, expression of caspase-8) and intrinsic (mitochondrial damage, expression of caspase-9) triggers. Previous studies have suggested a role for apoptosis in PKD cyst formation and parenchymal destruction. Mechanisms underlying apoptosis in human ADPKD and ARPKD were examined by quantitative immunohistochemistry and Western immunoblot analyses of age-matched normal and PKD tissues. Caspase-8 expression was significantly greater in small cysts and normal-appearing tubules than in larger cysts in ADPKD kidneys. Caspase-8 also appeared early in the disease process of ADPKD. In ARPKD, expression of caspase-8 was most pronounced in later stages of the disease and was not confined to a specific cyst size. In conclusion, apoptosis in human ADPKD is an early event, occurring predominantly in normal-appearing tubules and small cysts, and is triggered by an extrinsic factor, but it occurs later in ARPKD.     

肾缺血/再灌注时肾组织损伤及细胞凋亡的实验研究

目的：检测肾缺血／再灌注不同时问点肾组织、功能损伤及细胞凋亡的变化，探讨细胞凋亡在肾缺血／再灌注损伤中发生的机制。方法：应用苦味酸法和二乙酰一肟反应法测定大鼠肾缺血／再灌注不同时间点血肌酐和尿素氮值检测肾功能变化，用HE染色光镜下观察缺血／再灌注石蜡包埋切片肾组织损伤形态学改变，用酚／氯仿抽提小片断DNA，在琼脂糖电泳上测定不同时间点DNA Ladder及利用原位凋亡检到法观察各时间点TUNEL阳性细胞数检测细胞凋亡情况。结果：肾功能检测发现，缺血45min后血肌酐和尿素氮值明显增高和正方对照差异显(P＜0.05)，再灌注早期上升缓慢，3h后再次增高，组内对比差异显(P＜0．05)。HE染色光镜下观察肾组织细胞以近端肾小管变性为主、坏死轻微，缺血／再灌注各时间点组织损伤变化与肾功能变化规律相一致。琼脂糖电泳和TUNEL检测在缺血45min再灌注3h时可测到DNA Ladder和TUNEL阳性细胞增加，再灌注12h细胞凋亡最明显。结论：肾缺血／再灌注可引起肾组织轻、中度的损伤和明显的肾功能损害；肾缺血／再灌注后期肾组织及功能损伤加重可能与细胞凋亡的发生有关。     

Reduced postischemic macrophage infiltration and interstitial fibrosis in osteopontin knockout mice

BACKGROUND: Osteopontin (OPN) is a phosphoprotein that is up-regulated in several experimental models of renal disease, including ischemia/reperfusion injury. OPN has been described as a macrophage chemoattractant, may serve as a survival factor for tubular cells, and is implicated in the development of tubulointerstitial fibrosis. However, the precise role of this protein in renal pathophysiology remains unclear. METHODS: OPN knockout and wild-type mice were subjected to 30 minutes of warm renal ischemia combined with a contralateral nephrectomy, and sacrificed at six different time points, ranging from 12 hours to seven days after reperfusion. Besides functional and morphological parameters of postischemic acute renal failure (ARF), macrophage infiltration, apoptosis and expression of collagen types I and IV were investigated. RESULTS: Postischemic ARF in OPN knockouts and wild-types showed a similar course and severity, without significant differences in either functional or morphological disease parameters. However, macrophage infiltration was significantly diminished in OPN knockouts after five and seven days, in cortex as well as in the outer stripe of the outer medulla (OSOM). Furthermore, OPN knockout mice showed significantly enhanced apoptosis in the injury phase and significantly less collagen I and IV expression in the regeneration phase of postischemic ARF. CONCLUSIONS: There was no influence of OPN protein on the severity or course of functional impairment or morphological injury in the first seven days after an ischemic insult to the kidney. However, our results demonstrate that OPN favors macrophage recruitment to the postischemic kidney, inhibits apoptosis, and stimulates the development of renal fibrosis after an acute ischemic insult.     

Comparative profile of commercially available urinary biomarkers in preclinical drug-induced kidney injury and recovery in rats

We designed a study to provide reversibility and comparative injury data for several candidate urinary biomarkers of kidney injury in the United States Food and Drug Administration biomarker qualification process. The nephrotoxin gentamicin was given to rats once on each of three days and the animals were killed during dosing or over the following 42 days. Between days one and three, all biomarkers except albumin were elevated, peaked at day 7, and returned to control levels by day 10 (μ- and α-glutathione S-transferases, and renal papillary antigen-1) or day 15 (kidney injury molecule-1, lipocalin-2, osteopontin, and clusterin). All biomarkers performed better during injury than during recovery except osteopontin, which performed equally well in both time periods. During the evolution of injury, kidney injury molecule-1, renal papillary antigen-1, and clusterin best mirrored the histopathologic lesions. During injury resolution, kidney injury molecule-1, osteopontin, and blood urea nitrogen best reflected recovery. Based on histopathology, necrosis, or apoptosis scoring, kidney injury molecule-1 was the best biomarker of overall renal injury. Evaluation by regeneration score showed that renal papillary antigen-1 best reflected tubular and/or collecting duct regeneration, especially during recovery. Thus, these biomarkers performed with different effectiveness when evaluated by individual pathological processes such as necrosis, apoptosis, and regeneration.     

Adult stem cells in renal injury and repair

There has been considerable focus on the ability of bone marrow-derived cells to differentiate into non-haematopoietic cells of various tissue lineages, including cells of the kidney. This growing evidence has led to a reconsideration of the source of cells contributing to renal repair following injury. The kidney has an inherent ability for recovery and regeneration following acute damage. It is thought that dedifferentiation of glomerular and tubular cells to a more embryonic/mesenchymal phenotype represent key processes for recovery in response to damage. However, there has been much contention as to the source of regenerating renal cells. The present review focuses on new aspects of the plasticity of intrinsic renal cells and their role in renal remodelling and scarring. Growing support also suggests that bone marrow-derived cells have the ability to contribute to structural and functional repair following acute renal failure. Evidence for bone marrow cell engraftment in the repairing kidney leading to incorporation into a variety of tissue types is discussed. Because cell death and fibrosis is a common end-point in a variety of acute and chronic renal nephropathies, the paradigm of stem cell plasticity may have important implications in the cellular and pathological mechanisms of renal injury and repair. A better understanding of the processes controlling extra-renal cell engraftment and intrinsic renal cell differentiation may provide important clues for the development of new cell-based therapies in the field of renal reparative medicine.     

Endoplasmic reticulum stress and its effects on renal tubular cells apoptosis in ischemic acute kidney injury

Ischemia is the most frequent cause of acute kidney injury (AKI), which is characterized by apoptosis of renal tubular cell. A common result of ischemia in AKI is dysfunction of endoplasmic reticulum (ER), which causes the protein-folding capacity to lag behind the protein-folding load. The abundance of misfolded proteins stressed the ER and results in induction of the unfolded protein response (UPR). While the UPR is an adaptive response, over time it can result in apoptosis when cells are unable to recover quickly. Recent research suggests that ER stress is a major factor in renal tubular cell apoptosis resulting from ischemic AKI. Thus, ER stress may be an important new progression factor in the pathology of ischemic AKI. In this article, we review UPR signaling, describe pathology and pathophysiology mechanisms of ischemic AKI, and highlight the dual function of ER stress on renal tubular cell apoptosis.     

TWEAK, a multifunctional cytokine in kidney injury

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF superfamily that activates the Fn14 receptor. TWEAK may regulate cell proliferation, cell death, cell differentiation, and inflammation. TWEAK and Fn14 are constitutively present in the kidney. Sources of TWEAK and Fn14 include intrinsic renal cells and infiltrating leukocytes. Basal Fn14 expression is low, but Fn14 is greatly upregulated during kidney injury. TWEAK contributes to kidney inflammation promoting chemokine secretion by renal cells through canonical and non-canonical NFκB activation. TWEAK also promotes tubular cell proliferation. However, TWEAK induces mesangial and tubular cell apoptosis under proinflammatory conditions. These data indicate that TWEAK is a multifunctional cytokine in the kidney, the actions of which are modulated by the cell microenvironment. Confirmation of the role of TWEAK in kidney injury came from functional studies in experimental animal models. The TWEAK/Fn14 pathway contributed to cell death and interstitial inflammation during acute kidney injury, to glomerular injury in lupus nephritis, to hyperlipidemia-associated kidney injury, and to tubular cell hyperplasia following unilateral nephrectomy. Circulating soluble TWEAK (sTWEAK) levels are a potential biomarker of adverse outcomes in chronic kidney disease and urinary sTWEAK is a potential biomarker of lupus nephritis activity. The available evidence suggests that TWEAK may provide diagnostic information and be a therapeutic target in renal injury. Its role in human kidney disease should be further explored.     

Rarefaction of peritubular capillaries following ischemic acute renal failure: a potential factor predisposing to progressive nephropathy

PURPOSE OF REVIEW: Long-term renal complications of acute renal failure have generally not been expected in patients that recover from acute renal failure. However, as the incidence of acute renal failure is rising, the incidence of long-term complications is likely to increase. As a corollary to ischemic acute renal failure, ischemic injury in the setting of transplant is a leading cause of delayed graft function. Unlike acute renal failure in native kidneys, delayed graft function is highly predictive of chronic nephropathy and organ failure. It is generally well accepted that acute reversible injuries mediated by ischemia render grafts susceptible toward future demise. The nature of the susceptibility that is conveyed to grafts following ischemic injury is not well understood. RECENT FINDINGS: Evidence from animal models suggests that acute injury results in microvascular damage and vessel loss in the kidney, which, as opposed to tubular damage, is largely persistent. In addition, various studies of biopsies of renal transplants suggest that ischemia imposes an early and sustained loss in peritubular capillaries in the transplanted graft. The loss of peritubular capillaries has been associated with nephropathies of diverse etiologies and may represent a single, common pathway towards progressive damage. SUMMARY: It is hypothesized that rarefaction of peritubular capillaries represents a critical event, following ischemic injury, that permanently alters renal function and predisposes patients to the development of chronic renal insufficiency. Factors that affect vascular reactivity or the structural dynamics of the kidney vascular system following injury may represent future treatment modalities following renal injury.     

Role of apoptosis in the pathogenesis of acute renal failure

Renal tubular cells die by apoptosis as well as necrosis in experimental models of ischemic and toxic acute renal failure as well as in humans with acute tubular necrosis. It is not yet possible, however, to determine the relative contribution of these two forms of cell death to loss of renal tubular cells in acute tubular necrosis. The beneficial effect of administering growth factors to animals with acute tubular necrosis is probably related to the potent antiapoptotic (survival) effects of growth factors as well as to their proliferative effects. Rapamycin inhibits both of these effects of growth factors and delays the recovery of renal function after acute tubular necrosis by inhibiting renal tubular cell regeneration and by increasing renal tubular cell loss by apoptosis. The administration of caspase inhibitors ameliorates ischemia-reperfusion injury in multiple organs including the kidney. However, the extent to which this protective effect of caspase inhibition is caused by reduced intrarenal inflammation, or by amelioration of renal tubular cell loss due to apoptosis, remains uncertain. In addition to caspase inhibition, the apoptotic pathway offers many potential targets for therapeutic interventions to prevent renal tubular cell apoptosis.     

Leukemia inhibitory factor is involved in tubular regeneration after experimental acute renal failure

Leukemia inhibitory factor (LIF) is known to play a crucial role in the conversion of mesenchyme into epithelium during nephrogenesis. This study was carried out to test the hypothesis that LIF and LIF receptor (LIFR) are involved in the renal epithelial regeneration after acute renal failure. First, the authors investigated the spatiotemporal expression of LIF and LIFR in fetal and adult rat kidney. In developing kidney, LIF was expressed in the ureteric buds and LIFR was located in nephrogenic mesenchyme and the ureteric buds; in adult kidney, LIF and LIFR expression was confined to the collecting ducts. Next, the authors examined the expression of LIF and LIFR during the recovery phase after ischemia-reperfusion injury. Real-time PCR analysis revealed that LIF mRNA expression was significantly increased from day 1 to day 7 after reperfusion and that LIFR mRNA was upregulated from day 4 to day 14. Histologic analysis demonstrated that the increased expression of LIF mRNA and protein was most marked in the outer medulla, especially in the S3 segment of the proximal tubules. To elucidate the mitogenic role of LIF in the regeneration process, cultured rat renal epithelial (NRK 52E) cells were subjected to ATP depletion (an in vitro model of acute renal failure), and LIF expression was found to be enhanced during recovery after ATP depletion. Blockade of endogenous LIF with a neutralizing antibody significantly reduced the cell number and DNA synthesis during the recovery period. These results suggest that LIF participates in the regeneration process after tubular injury.     

CDK/GSK-3 inhibitors as therapeutic agents for parenchymal renal diseases

Drug discovery to lessen the burden of chronic renal failure and end-stage renal disease remains a principle goal of translational research in nephrology. In this review, we provide an overview of the current development of small molecule cyclin-dependent kinase (CDK)/glycogen synthase kinase-3 (GSK-3) inhibitors as therapeutic agents for parenchymal renal diseases. The emergence of this drug family has resulted from the recognition that CDKs and GSK-3s play critical roles in the progression and regression of many kidney diseases. CDK/GSK-3 inhibitors suppress pathogenic proliferation, apoptosis, and inflammation, and promote regeneration of injured tissue. Preclinical efficacy has now been demonstrated in mesangial proliferative glomerulonephritis, crescentic glomerulonephritis, collapsing glomerulopathy, proliferative lupus nephritis, polycystic kidney diseases, diabetic nephropathy, and several forms of acute kidney injury. Novel biomarkers of therapy are aiding the process of drug development. This review will highlight these advancements in renal therapeutics.     

Matrilysin (MMP-7) expression in renal tubular damage: association with Wnt4

BACKGROUND: Matrilysin, a secreted matrix metalloproteinase and target gene of Wnt signaling, functions in epithelial repair and host defense, but no role in renal injury has been described. METHODS: Matrilysin expression was assessed in human kidney specimens by immunohistochemistry, and in experimental renal injury in mice by immunohistochemistry, Northern blotting, and RNase protection assays (RPA). A relationship to Wnt4, which is also induced in renal injury, was determined by RPA and in situ hybridization. RESULTS: Matrilysin was not detected in the normal human renal tubular epithelium by immunohistochemistry. However, prominent staining was detected in sections from autosomal-dominant polycystic kidney disease in the cyst lining epithelium, atrophic tubules, and cyst micropolyps, and from hydronephrosis in dilated and atrophic tubules. Matrilysin expression was also induced by acute folic acid nephropathy and unilateral ureteral obstruction (UUO) in the mouse, and expression increased as acute injury progressed to tubulointerstitial fibrosis. Matrilysin staining was primarily localized to epithelium of distal tubule/collecting duct origin in both human and murine renal disease. Wnt signaling can induce matrilysin expression, and we found that the pattern of matrilysin expression during progression of renal fibrosis in the mouse after UUO or folic acid nephropathy, and in the jck model of murine polycystic kidney disease, closely paralleled that of Wnt4. CONCLUSION: These observations suggest that matrilysin may have a role in renal tubular injury and progression of tubulointerstitial fibrosis, and that Wnt4 may regulate matrilysin expression in the kidney.     

Multiple caspases mediate acute renal cell apoptosis induced by bacterial cell wall components

The stimulus for caspase-mediated renal cell apoptosis in septic acute renal failure (ARF) is unclear. To demonstrate the nephrotoxic effects of bacterial cell wall components, the anti-cellular activity of bacterial muropeptides (muramyl dipeptides), peptidoglycans, and lipopolysaccharides was investigated in rabbit kidney cells. Changes in the cell membrane (APOPercentage? dye uptake), caspase activities, and DNA degradation were quantified colorimetrically and using densitometric assays and their inhibition by caspase-specific and pan-caspase inhibitors was determined. The onset and levels of APOPercentage? dye-positive rabbit kidney cells, caspase activities, and DNA degradation were closely associated. Specific caspase-1, -2, -3, -4, -8, -10, and -12 inhibitors reduced caspase-3 activity by ≥40%, but only caspase-3 and -8-specific inhibitors reduced apoptotic DNA levels. Pan-caspase inhibitor Q-VD-OPh was 10-fold more effective at inhibiting rabbit kidney cell death, caspase activation, and DNA degradation than caspase-family inhibitor Z-VAD-FMK. Apoptosis was inhibited effectively by both pan-caspase inhibitors when applied early during the stimulus-to-response period. Multiple initiator and effector caspases were activated suggesting extrinsic, intrinsic, and endoplasmic reticulum/stress apoptotic pathway stimulation in rabbit kidney cells treated with bacterial cell wall components. The results provide in vitro support for bacterial cell wall-induced apoptosis as a pathogenic mechanism of renal cell death in septic ARF and support the potential prophylactic use of pan-caspase inhibitors to suppress septic ARF.     

Altered expression pattern of polycystin-2 in acute and chronic renal tubular diseases

Polycystin-2 represents one of so far two proteins found to be mutated in patients with autosomal-dominant polycystic kidney disease. Evidence obtained from experiments carried out in cell lines and with native kidney tissue strongly suggests that polycystin-2 is located in the endoplasmic reticulum. In the kidney, polycystin-2 is highly expressed in cells of the distal and connecting tubules, where it is located in the basal compartment. It is not known whether the expression of polycystin-2 in the kidney changes or whether it can be manipulated under certain instances. Therefore, the distribution of polycystin-2 under conditions leading to acute and chronic renal failure was analyzed. During ischemic acute renal failure, which affects primarily the S3 segment of the proximal tubule, a pronounced upregulation of polycystin-2 and a predominantly combined homogeneous and punctate cytoplasmic distribution in damaged cells was observed. After thallium-induced acute injury to thick ascending limb cells, polycystin-2 staining assumed a chicken wire-like pattern in damaged cells. In the (cy/+) rat, a model for autosomal-dominant polycystic kidney disease in which cysts originate predominantly from the proximal tubule, polycystin-2 immunoreactivity was lost in some distal tubules. In kidneys from (pcy/pcy) mice, a model for autosomal-recessive polycystic kidney disease in which cyst formation primarily affects distal tubules and collecting ducts, a minor portion of cyst-lining cells cease to express polycystin-2, whereas in the remaining cells, polycystin-2 is retained in their basal compartment. Data show that the expression and cellular distribution of polycystin-2 in different kinds of renal injuries depends on the type of damage and on the nephron-specific response to the injury. After ischemia, polycystin-2 may be upregulated by the injured cells to protect themselves. It is unlikely that polycystin-2 plays a role in cyst formation in the (cy/+) rat and in the (pcy/pcy) mouse.     

A mouse model for polycystic kidney disease through a somatic in-frame deletion in the 5' end of Pkd1

Autosomal dominant polycystic kidney disease, a leading cause of end-stage renal disease in adults, is characterized by progressive focal cyst formation in the kidney. Embryonic lethality of Pkd1-targeted mice limits the use of these mice. Here we developed a floxed allele of Pkd1 exons 2-6. Global deletion mutants developed polyhydramnios, hydrops fetalis, polycystic kidney and pancreatic disease. Somatic Pkd1 inactivation in the kidney was achieved by crossing Pkd1(flox) mice with transgenic mice expressing Cre controlled by a gamma-glutamyltranspeptidase promoter. These mutants developed cysts in both proximal and distal nephron segments and survived for about 4 weeks. Somatic loss of heterozygosity was shown in a reporter mouse strain to cause cystogenesis. Some cysts in young mice are positive for multiple tubular markers and a mesenchymal marker, suggesting a delay in tubular epithelial differentiation. A higher cell proliferation rate was observed in distal nephron segments probably accounting for the faster growth rate of distal cysts. Although we observed an overall increase in apoptosis in cystic kidneys, there was no difference between proximal or distal nephron segments. We also found increased cyclic AMP, aquaporin 2 and vasopressin type 2 receptor mRNA levels, and apical membrane translocation of aquaporin 2 in cystic kidneys, all of which may contribute to the differential cyst growth rate observed. The accelerated polycystic kidney phenotype of these mice provides an excellent model for studying molecular pathways of cystogenesis and to test therapeutic strategies.