Vincent H. Gattone

Developmental expression of urine concentration‐associated genes and their altered expression in murine infantile‐type polycystic kidney disease

Currently, there is little understanding of what factors regulate the development of urine concentrating capability in normal or polycystic kidney. The present study examined the developmental expression of genes associated with urine concentration in developing mice, including C57BL/6J-cpk/cpk mice with autosomal recessive-infantile (AR) polycystic kidney disease (PKD). Concentration of urine requires: 1) medullary collecting ducts (CD) located within a hypertonic interstitium, 2) CD cell expression of functional arginine vasopressin V2 receptors (AVP-V2R), and 3) the presence of appropriate CD water channels (aquaporins, AQP 2 and 3). An increase in urine osmolarity, normally seen between 1 and 3 weeks of age, was absent in cpk cystic mice. Aldose reductase mRNA expression (a gene upregulated by medullary hyperosmolarity) increased in normal mice, but remained low in the cystic kidney, suggesting the absence of a hypertonic medullary interstitium. AVP-V2R, AQP2, and AQP3 mRNA expression normally increase between 7 and 14 days. However, all were dramatically overexpressed even at 7 days of age in the cpk kidney in vivo, but decreased in vitro. Activation of the AVP-V2 receptor stimulates the production of cAMP, a substance known to promote cyst enlargement. To determine if CD cAMP, generated from increased AVP-V2Rs, was accelerating the PKD, cystic mice and their normal littermates were treated with OPC31260, a relatively specific AVP-V2R antagonist. OPC31260 treatment of cystic mice led to an amelioration of the cystic enlargement and azotemia. Treatment also decreased renal AQP2 mRNA but increased AVP-V2R and AQP3 mRNA expression in vivo. AVP upregulates the expression of AVP-V2R, AQP2, and AQP3 mRNAs in vitro. Renal EGF, known to inhibit AVP-V2R activity, downregulates AVP-V2R mRNA in vitro. Brief in vivo EGF treatment, known to decrease PKD in cpk mice, led to increased expression of AVP-V2R, AQP2, and AQP3 mRNAs at 2 weeks in both normal and cystic mice but no change was evident at 3 weeks of age. In conclusion, the development of urinary concentration ability correlates with the development of an increased medullary osmotic gradient which is diminished in murine ARPKD. However, CD genes associated with this process are overexpressed in vivo but underexpressed in vitro in the cystic kidney. The overexpression and/or overactivity of the AVP-V2R appears to contribute to the progression of PKD since an AVP-V2R antagonist inhibits cystic renal enlargement in the cpk mouse.

The SGP-2 gene is developmentally regulated in the mouse kidney and abnormally expressed in collecting duct cysts in polycystic kidney disease☆

Sulfated glycoprotein-2 (SGP-2) is a secreted, dimeric, glycosylated protein synthesized by a number of different epithelial cell types. Although its function is not yet understood, SGP-2 has been hypothesized to be involved in such diverse processes as the promotion of cell-cell interactions, spermatogenesis, modulation of the complement system, and programmed cell death. We have now found that the SGP-2 gene is developmentally regulated in the mouse kidney. SGP-2 gene expression is first detected in the condensing nephrogenic mesenchyme and is subsequently down-regulated during the maturation of the glomerular epithelia, proximal tubules, and collecting ducts. SGP-2 continues to be expressed in the mature kidney in distal tubules and in the urothelial lining of the calyx and papilla. We have also examined the expression of the SGP-2 gene in polycystic kidneys of the C57BL/6J-cpk mouse, a model of autosomal recessive polycystic kidney disease in which there is development of epithelial-lined cysts arising primarily from the collecting duct system. Abnormally high levels of SGP-2 mRNA were found in the cyst wall epithelium of polycystic kidneys. The expression of the SGP-2 gene in normal development suggests that it plays a role in differentiating epithelial structures; and the abnormally high levels of SGP-2 gene expression in polycystic kidneys suggests that the cells lining cysts are not fully differentiated. It is possible, therefore, that polycystic kidney disease is caused by a defective developmental process in which there is a delay in terminal differentiation.

Defective epidermal growth factor gene expression in mice with polycystic kidney disease

The C57BL/6J-cpk mouse has an inheritable form of polycystic kidney disease similar to the autosomal recessive disorder seen in humans. Between approximately 1 and 3 weeks of age, affected cpk mice develop numerous large cysts in the collecting tubule segment of kidney nephrons. The present study examined the ontogeny of renal and submandibular gland prepro-epidermal growth factor (preproEGF) gene expression in the cpk mouse using Northern blot hybridization and immunohistochemistry. There was a virtual absence of renal preproEGF gene expression in cystic kidneys over the 3-week postnatal period, during which time renal preproEGF mRNA and proEGF/EGF protein normally reach significant levels. PreproEGF mRNA was expressed in salivary glands of cystic mice; however, this mRNA could not be further elevated with testosterone suggesting that there are abnormalities in the regulation of the preproEGF gene in the submandibular gland, as well as in the kidney. Since renal preproEGF expression during the early postnatal period occurs when collecting duct cysts form, it is possible that a deficiency in renal proEGF or EGF contributes to the rapid development of collecting duct cysts and the concomitant renal failure in the C57BL/6J-cpk cystic mouse.

Effect of lovastatin on the development of polycystic kidney disease in the Han:SPRD rat.

Proliferation of tubular epithelial cells is a major element leading to cyst formation in Han:SPRD rats with autosomal dominant polycystic kidney disease (PKD). ras proteins are important in the control of renal cell proliferation, and ras gene expression is increased in PKD. Farnesyl pyrophosphate, an intermediate in the conversion of acetyl-CoA to cholesterol, is required for the activation of ras guanosine triphosphate (GTP)-binding proteins that are important in the execution of several cellular functions, including cell proliferation. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, such as lovastatin, reduce farnesyl production in responsive cells and thereby have potential for ameliorating the accelerated epithelial cell proliferation of PKD. We administered lovastatin to heterozygous (Cy/+) Han:SPRD rats (4 mg/kg/d subcutaneously) from age 4 to 10 weeks, a period of rapid cystic disease progression in these animals. Untreated male Cy/+ rats developed larger cystic kidneys and had more severe renal functional impairment than females, as reported previously. In males, lovastatin significantly decreased cystic kidney size (referenced to body weight), the volume density of cysts, and the serum urea nitrogen level 14.5%, 24.4%, and 25.6/%, respectively. The corresponding changes in females were insignificant, and lovastatin had no effect on kidney weight or serum urea nitrogen in homozygous (+/+) normal male animals. On the basis of these results we conclude that lovastatin diminishes the severity of PKD in heterozygous male Han:SPRD rats.

Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubules—A light microscopic autoradiography study with 109CdMT☆

Chronic, but not acute, exposure to inorganic Cd produces renal damage. However, a single injection of cadmium bound to metallothionein (CdMT) produces renal injury. It is hypothesized that an interorgan redistribution of Cd as CdMT is responsible for the chronic nephrotoxic effect of Cd. To better understand the mechanism(s) of CdMT-induced nephrotoxicity, the intrarenal distribution of 109CdMT was examined. 109CdMT isolated from rat liver was injected into mice at a nonnephrotoxic dose (0.1 mg Cd/kg, iv). The radioactivity in the kidney reached a maximum level (85% of the dose) as early as 30 min following administration and remained essentially constant for up to 7 days after injection. Within the kidney, 109Cd distributed almost entirely to the cortex. Light microscopic autoradiography of the kidney showed that, within the cortex, 109Cd distributed preferentially to the S1 and S2 segments of the proximal convoluted tubules. Within the S1 and S2 segments, the concentration of 109Cd in the basal and apical parts of the cells was similar to that after the nonnephrotoxic dose of CdMT, but after a nephrotoxic dose (0.3 mg Cd/kg) the radioactivity distributed preferentially to the apical portion of the cells. In contrast, light microscopic autoradiography studies with 109CdCl2 revealed that 109Cd was more evenly distributed throughout the proximal tubules. Moreover, after administration of a large dose of inorganic Cd (3 mg Cd/kg), a similar concentration of Cd was found in the convoluted and straight proximal tubules. These data support the hypothesis that CdMT-induced nephrotoxicity might be due, at least in part, to its preferential uptake of CdMT into the S1 and S2 segments of the proximal tubules, the site of Cd-induced nephrotoxicity.

Methylprednisolone retards the progression of inherited polycystic kidney disease in rodents.

Polycystic kidney disease in adult laboratory animals and humans is associated with enlarged kidneys and a progressive decline of renal function, resulting in death from uremia. Interstitial inflammation and fibrosis typically are observed in association with the development of renal insufficiency. To determine whether amelioration of interstitial inflammation and fibrosis may diminish cyst expansion/kidney enlargement and stabilize renal function, we administered methylprednisolone, an anti-inflammatory drug with antifibrogenic effects, to mice and rats with hereditary polycystic kidney disease. The experiment was repeated once for each species. Mice were studied both in America and in Japan. Weanling male and female mice (DBA/FG pcy/pcy [cystic] and +/+ [normal], n = 87 and 20, respectively) and rats (Han:SPRD Cy/+ and +/+, n = 70 and 33, respectively) were administered methylprednisolone (1 to 2 mg/kg/d) in the drinking water for 100 days (mice) or 42 days (rats). Control animals drank distilled water. In normal DBA +/+ mice, methylprednisolone had no effect on serum urea nitrogen (SUN) levels, kidney weight, or kidney/body weight. Untreated male and female mice developed cystic kidneys and azotemia to an equal extent. Methylprednisolone administered in America to mice with renal cystic disease decreased kidney weight, kidney/body weight, SUN levels, volume density of cysts, and severity of interstitial fibrosis. In Japan, methylprednisolone decreased kidney weight and SUN levels of animals with cystic disease, but the effect on kidney/body weight did not reach statistical significance. In contrast to mice, male rats developed more severe renal cystic changes and were more azotemic than female rats. Methylprednisolone administered to male rats with cystic disease decreased SUN levels, kidney weight, kidney/body weight, volume density of cysts, and severity of interstitial fibrosis. Methylprednisolone had no effect on kidney/body weight or SUN levels in female rats with renal cystic disease. In normal Han:SPRD (+/+) rats of both sexes, kidney and body weight were decreased by methylprednisolone, but kidney/body weight and SUN levels were unchanged. On the basis of this study, we conclude that methylprednisolone decreased the extent of renal enlargement, reduced renal interstitial fibrosis, and preserved kidney function in mice and rats with relatively severe forms of inherited polycystic kidney disease.

Gender and the effect of gonadal hormones on the progression of inherited polycystic kidney disease in rats

Human autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease and displays a gender dimorphism in renal disease progression. Han:SPRD-Cy rats manifest a form of ADPKD that is similar in many respects to that seen in humans. In Han:SPRD rats, male Cy/+ rats have more prominent renal changes and develop renal failure at an early age, whereas female Cy/+ rats exhibit less severe renal cystic change and have normal renal function until advanced age. To determine whether the male gonadal hormone, testosterone, contributes to this gender dimorphism, males were sham operated or castrated; some castrated rats were repleted with 5alpha-dihydrotestosterone. Female rats were sham operated or ovariectomized before sham operation or testosterone treatment. All treatments started at 4 weeks of age and ended at 10 weeks of age. Renal enlargement, cystic change, and renal function were assessed. In the males, castration reduced renal enlargement and cystic change; testosterone treatment abrogated these effects. Neither of these manipulations affected azotemia in male Cy/+ rats. In the females, testosterone was renotropic for both normal and cystic kidneys. In the Cy/+ females, testosterone treatment caused azotemia and an increase in the severity of the PKD. Ovariectomy blunted the effect of testosterone on cystic kidney enlargement. Testosterone treatment did not completely erase the gender-associated differences in azotemia in the Cy/+ rat. These data confirm the renotropic effects of testosterone and indicate that testosterone influences the progression of renal cystic change in male and female rats with ADPKD.

Modification of disease progression in rats with inherited polycystic kidney disease

The most common inherited form of human polycystic kidney disease (PKD), autosomal dominant PKD (ADPKD), is a leading cause of chronic renal failure, but has a variable clinical presentation, with end-stage renal disease occurring in only 25% to 75%. Several findings are consistent with the idea that factors in addition to the primary mutation can affect the progression of cystic change and chronic renal failure in PKD. Epithelial cell proliferation is a central element in the pathogenesis of renal cysts. We postulated that the superimposition of a growth-promoting stimulus might promote more intense proliferation of cystic epithelial cells in inherited cystic disease. To study this, we subjected Han:SPRD rats, with a form of ADPKD that resembles human ADPKD, from 4 until 10 weeks of age to diets designed to promote tubule cell growth. The diets included supplemental NH4Cl (280 mmol/L in drinking water), limited dietary K+ (0.016% of diet; control diet was 1.1% K+), and increased dietary protein (50%; control diet was 23% protein). Treatments designed to promote cell growth caused more aggressive PKD in males and females, worsened azotemia in males, and resulted in azotemia in females (which normally develop PKD but not azotemia at the ages studied). NH4Cl, K+ restriction, and increased dietary protein each caused greater kidney enlargement in males (kidney weight/body weight ratios increased by 35%, 78%, and 105%, respectively) and worsened azotemia in males (serum urea nitrogen values increased by 63%, 514%, and 224%, respectively); in contrast, decreased dietary protein (4%) caused less severe PKD in males (kidney weight/body weight ratios decreased by 43%) and lessened azotemia in males (serum urea nitrogen values decreased by 49%). Similarly, NH4Cl and K+ restriction caused greater kidney enlargement in females (kidney weight/body weight ratios increased by 206% and 203%, respectively) and caused azotemia in females (serum urea nitrogen values increased by 177% and 430%, respectively). On the basis of these results, we conclude that growth-promoting stimuli can alter the expression of hereditary renal cystic disease. These findings demonstrate that the progression of hereditary renal cystic disease can be altered by factors in addition to the primary genetic defect.

Accumulation and degradation of the protein moiety of cadmium-metallothionein (CdMT) in the mouse kidney.

Of major concern in Cd toxicity is its ability to produce renal damage after chronic exposure in humans and experimental animals. Renal injury affects predominantly the proximal tubules and more specifically the first segments of these tubules. Similar toxic effects to the kidneys are observed after administration of cadmium bound to metallothionein (CdMT). Therefore, CdMT was used in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has been recently demonstrated that Cd from CdMT was preferentially taken up by the proximal convoluted tubules. Therefore, the purpose of these studies was to determine if the organic portion of the complex was also accumulated in these tubules. [35S]CdMT prepared from rat liver was administered intravenously to mice at a nonnephrotoxic dose (0.1 mg Cd/kg). The radioactivity in the kidney showed maximum level (80% of the dose) 15 min after the injection. This preferential renal uptake was also observed after administration of various doses of [35S]CdMT. In contrast to the earlier observed persistency of 109Cd in the kidney after 109CdMT administration, 35S disappeared rapidly (with a half-life of approximately 2 hr), and 24 hr after injection of [35S]CdMT, there was very little 35S left in the kidneys. These observations indicate that the protein portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, 35S distributed mainly to the cortex. Light microscopic autoradiography showed that [35S]CdMT preferentially distributed to the proximal convoluted tubule (S1 and S2), which is the site of nephrotoxicity. Within the S1 and S2 segments, a greater distribution of 35S to the apical portion of the cells was observed after administration of both a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. 109Cd administered as 109CdMT also distributed to the apical portion of the S1 and S2 cells. Therefore, both the organic (35S) and inorganic (109Cd) portions of CdMT are rapidly and efficiently taken up by the S1 and S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily taken up by the proximal tubular cells as a complex, and then its protein portion is rapidly degraded to release Cd that binds permanently to intracellular sites and produces nephrotoxicity.

Renal expression of a transforming growth factor-α transgene accelerates the progression of inherited, slowly progressive polycystic kidney disease in the mouse

Polycystic kidney disease (PKD) is a prevalent inherited disease in human beings. The pathogenesis of PKD is as yet unclear. The epidermal growth factor family of proteins has been implicated in PKD based largely on in vitro data. To determine whether these growth factors contribute to the progression of inherited PKD in vivo, we crossed mice with a transgene for human transforming growth factor-alpha (TGF-alpha, a member of the epidermal growth factor (EGF) family) and mice with the pcy gene (which causes a slowly progressive form of PKD very similar to human autosomal dominant PKD). Renal expression of the TGF-alpha transgene in cystic mice (homozygous for the pcy gene) accelerated the development of PKD as shown by an increased kidney weight as a percent of body weight and an increased volume density of renal cysts at 8.5 weeks of age. However, renal expression of the TGF-alpha transgene did not appear to precociously initiate cyst development (at 6.5 weeks), nor did it cause an increase in the final degree of renal enlargement (at 29 weeks). Thus TGF-alpha accelerated the enlargement of cysts once initiated. At 8.5 weeks of age, renal expression of the TGF-alpha mRNA correlated positively with the amount of renal enlargement. At all time points studied, cystic kidneys exhibited increased expression of c-myc mRNA as compared with phenotypic normal kidneys, consistent with PKD being a hyperplastic disease of renal tubules. However, the renal expression of c-myc in 8.5 week cystic kidneys, with or without the transgene, did not correlate with the degree of renal enlargement. The results of this study suggest that EGF-like proteins may accelerate the progression of inherited renal cystic disease. However, the final degree of cystic change is dictated by the primary disease process rather than by the continued presence of growth factor.