Journal of the American Society of Nephrology Pathophysiology of Renal Disease and Progression

PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

2007-05-25

Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease. Studies in mouse models suggest that the vasopressin (AVP) V2 receptor (V2R) pathway is involved in renal cyst progression, but potential changes before cystogenesis are unknown. This study used a noncystic mouse model to investigate the effect of Pkd1 haploinsufficiency on water handling and AVP signaling in the collecting duct (CD). In comparison with wild-type littermates, Pkd1^+/– mice showed inappropriate antidiuresis with higher urine osmolality and lower plasma osmolality at baseline, despite similar renal function and water intake. The Pkd1^+/– mice had a decreased aquaretic response to both a water load and a selective V2R antagonist, despite similar V2R distribution and affinity. They showed an inappropriate expression of AVP in brain, irrespective of the hypo-osmolality. The cAMP levels in kidney and urine were unchanged, as were the mRNA levels of aquaporin-2 (AQP2), V2R, and cAMP-dependent mediators in kidney. However, the (Ser256) phosphorylated AQP2 was upregulated in Pkd1^+/– kidneys, with AQP2 recruitment to the apical plasma membrane of CD principal cells. The basal intracellular Ca²⁺ concentration was significantly lower in isolated Pkd1^+/– CD, with downregulated phosphorylated extracellular signal–regulated kinase 1/2 and decreased RhoA activity. Thus, in absence of cystic changes, reduced Pkd1 gene dosage is associated with a syndrome of inappropriate antidiuresis (positive water balance) reflecting decreased intracellular Ca²⁺ concentration, decreased activity of RhoA, recruitment of AQP2 in the CD, and inappropriate expression of AVP in the brain. These data give new insights in the potential roles of polycystin-1 in the AVP and Ca²⁺ signaling and the trafficking of AQP2 in the CD.

Mesenchymal Stem Cells Prevent Progressive Experimental Renal Failure but Maldifferentiate into Glomerular Adipocytes

2007-05-25

Glomerulonephritis (GN) is a major cause of renal failure. This study sought to determine whether intrarenal injection of rat mesenchymal stem cells (MSC) can preserve renal function in a progressive rat model of GN. Early in GN (day 10), fluorescently labeled rat MSC localized to more than 70% of glomeruli, ameliorated acute renal failure, and reduced glomerular adhesions. Fifty days later, proteinuria had progressed in controls to 40 ± 25 mg/d but stayed low in MSC-treated rats (13 ± 4 mg/d; P < 0.01). Renal function on day 60 in the MSC group was better than in medium controls. Kidneys of the MSC group as compared with controls on day 60 contained 11% more glomeruli per 1-mm² section of cortex but also significantly more collagen types I, III, and IV and -smooth muscle actin. Approximately 20% of the glomeruli of MSC-treated rats contained single or clusters of large adipocytes with pronounced surrounding fibrosis. Adipocytes exhibited fluorescence in their cytoplasm and/or intracellular lipid droplets. Lipid composition in these adipocytes in vivo mirrored that of MSC that underwent adipogenic differentiation in vitro. Thus, in this GN model, the early beneficial effect of MSC of preserving damaged glomeruli and maintaining renal function was offset by a long-term partial maldifferentiation of intraglomerular MSC into adipocytes accompanied by glomerular sclerosis. These data suggest that MSC treatment can be a valuable therapeutic approach only if adipogenic maldifferentiation is prevented.

Interstitial Vascular Rarefaction and Reduced VEGF-A Expression in Human Diabetic Nephropathy

2007-05-25

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes. Although the majority of DN models and human studies have focused on glomeruli, tubulointerstitial damage is a major feature of DN and an important predictor of renal dysfunction. This study sought to investigate molecular markers of pathogenic pathways in the renal interstitium of patients with DN. Microdissected tubulointerstitial compartments from biopsies with established DN and control kidneys were subjected to expression profiling. Analysis of candidate genes, potentially involved in DN on the basis of common hypotheses, identified 49 genes with significantly altered expression levels in established DN in comparison with controls. In contrast to some rodent models, the growth factors vascular endothelial growth factor A (VEGF-A) and epidermal growth factor (EGF) showed a decrease in mRNA expression in DN. This was validated on an independent cohort of patients with DN by real-time reverse transcriptase–PCR. Immunohistochemical staining for VEGF-A and EGF also showed a reduced expression in DN. The decrease of renal VEGF-A expression was associated with a reduction in peritubular capillary densities shown by platelet-endothelial cell adhesion molecule-1/CD31 staining. Furthermore, a significant inverse correlation between VEGF-A and proteinuria, as well as EGF and proteinuria, and a positive correlation between VEGF-A and hypoxia-inducible factor-1 mRNA was found. Thus, in human DN, a decrease of VEGF-A, rather than the reported increase as described in some rodent models, may contribute to the progressive disease. These findings and the questions about rodent models in DN raise a note of caution regarding the proposal to inhibit VEGF-A to prevent progression of DN.

Smad7 Gene Therapy Ameliorates an Autoimmune Crescentic Glomerulonephritis in Mice

2007-05-25

Autoimmune crescentic glomerulonephritis is characterized by severe immune response with glomerular crescentic formation and fibrosis in the kidney. Recent studies indicate that overexpression of renal Smad7 attenuates both renal fibrosis and inflammation in rat remnant kidney. However, little attention has been paid to the potential role of TGF-ß/Smad signaling in autoimmune kidney disease. This study tested the hypothesis that blocking TGF-ß signaling by overexpression of Smad7 may have a therapeutic effect in a mouse model of autoimmune crescentic glomerulonephritis that was induced in C57BL/6 x DBA/2J F1 hybrid mice by giving DBA/2J donor lymphocytes. Smad7 gene was transfected into the kidney using the ultrasound-microbubble–mediated system. Results showed that overexpression of Smad7 blocked both renal fibrosis and inflammatory pathways in terms of Smad2/3 and NF-B activation (P < 0.01), thereby inhibiting -smooth muscle actin; collagen I, III, and IV accumulation; and expression of inflammatory cytokines (IL-1ß and IL-6), adhesion molecule/chemokine (intercellular adhesion molecule-1, monocyte chemoattractant protein-1), and inducible nitric oxide synthase (all P < 0.01). Leukocyte infiltration (CD4⁺ cells and macrophages) was also suppressed (P < 0.005). Severe histologic damage (glomerular crescent formation and tubulointerstitial injury) and functional injury including proteinuria were significantly improved (all P < 0.05). This study provides important evidence that overexpression of Smad7 may have therapeutic potential for autoimmune kidney disease.

Slowly Progressive, Angiotensin II-Independent Glomerulosclerosis in Human (Pro)renin Receptor-Transgenic Rats

2007-05-25

For defining the pathogenic effects of the (pro)renin receptor–transgenic rat, strains that overexpressed the human receptor were generated. Although transgenic rats were normotensive and euglycemic and had a renal angiotensin II (AngII) level that was comparable to that of wild-type rats, transgenic rats developed proteinuria with aging and significant glomerulosclerosis at 28 wk of age. In kidneys of 28-wk-old transgenic rats, mitogen-activated protein kinases (MAPK) were activated without recognizable tyrosine phosphorylation of the EGF receptor, and expression of TGF-ß1 was enhanced. In vivo infusion of the (pro)renin receptor blocker peptide (formerly handle region decoy peptide) significantly inhibited the development of glomerulosclerosis, proteinuria, MAPK activation, and TGF-ß1 expression in the kidneys, but the angiotensin-converting enzyme inhibitor did not attenuate these changes despite a significant decrease in the renal AngII level. In addition, recombinant rat prorenin stimulated MAPK activation in the human receptor–expressed cultured cells, but human receptor was unable to evoke the enzyme activity of rat prorenin. Thus, human (pro)renin receptor elicits slowly progressive nephropathy by AngII-independent MAPK activation in rats. This study clearly provided in vivo evidence for the AngII-independent MAPK activation by human (pro)renin receptor and induction of glomerulosclerosis with increased TGF-ß1 expression.

Combination Therapy with an Angiotensin-Converting Enzyme Inhibitor and a Vitamin D Analog Suppresses the Progression of Renal Insufficiency in Uremic Rats

2007-05-25

Monotherapy with angiotensin-converting enzyme inhibitors has been shown to be beneficial in suppressing the progression of experimentally induced kidney diseases. Whether such therapy provides additional benefits when combined with vitamin D or an analog of vitamin D has not been established. Rats were made uremic by 5/6 nephrectomy and treated as follows: Uremic + vehicle (UC), uremic + enalapril (30 mg/L in drinking water; E), uremic + paricalcitol (19-nor; 0.8 µg/kg, three times a week), and uremic + enalapril + paricalcitol (E + 19-nor). A group of normal rats served as control (NC). BP was significantly elevated in the UC and 19-nor groups compared with the NC group but was indistinguishable from normal in the E and E + 19-nor groups. The decrease in creatinine clearance and the increase in the excretion of urinary protein that were observed in the UC group were ameliorated by the use of E alone or by E + 19-nor (P < 0.05 versus UC). The glomerulosclerotic index was significantly decreased in both the 19-nor (P < 0.01) and E + 19-nor groups (P < 0.01) compared with the UC group. Tubulointerstitial volume was significantly decreased in both the E (P < 0.05) and E + 19-nor groups (P < 0.01) compared with the UC group. Both macrophage infiltration (ED-1–positive cells) and production of the chemokine monocyte chemoattractant protein-1 were significantly blunted in E + 19-nor compared with E group. TGF-ß1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E + 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor–treated rats (5.5-fold versus NC). When rats were treated with E + 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-ß signaling pathway, and this effect is amplified when BP is controlled via renin-angiotensin system blockade.

Evidence for the Role of Reactive Nitrogen Species in Polymicrobial Sepsis-Induced Renal Peritubular Capillary Dysfunction and Tubular Injury

Wu, L., Gokden, N., Mayeux, P. R. — 2007-05-25

Acute kidney injury (AKI) remains a frequent and serious complication of human sepsis that contributes significantly to mortality. For better understanding of the development of AKI during sepsis, the cecal ligation and puncture (CLP) murine model of sepsis was studied using intravital video microscopy (IVVM) of the kidney. IVVM with FITC-dextran was used to determine the percentage of capillaries with continuous, intermittent or no flow at 0 (sham), 10, 16, and 22 h after CLP. There was a dramatic fall in capillary perfusion as early as 10 h after CLP that persisted through 22 h. The percentage of vessels with continuous flow at 16 h decreased from 73 ± 2% in shams to 16 ± 2% (P < 0.05), whereas the percentage of vessels with no flow increased from 4 ± 1% in shams to 42 ± 2% (P < 0.05). The capillary perfusion defect preceded the rise in serum creatinine. IVVM with dihydrorhodamine-123 was used to quantify in real time reactive nitrogen species (RNS) generation by renal tubules, and the inducible nitric oxide synthase inhibitor l-iminoethyl-lysine (mg/kg) was used to examine the role of inducible nitric oxide synthase inhibitor on capillary dysfunction and RNS generation. Tubular generation of RNS was significantly elevated at 10 h after CLP and was associated with tubules that were bordered by capillaries with reduced perfusion. l-Iminoethyl-lysine significantly reversed the capillary perfusion defect, blocked RNS generation, and reduced AKI. These data show that capillary dysfunction and RNS generation contribute to tubular injury and suggest that RNS should be considered a potential therapeutic target in the treatment of sepsis-induced AKI.

Genes Expressed by Both Mesangial Cells and Bone Marrow-Derived Cells Underlie Genetic Susceptibility to Crescentic Glomerulonephritis in the Rat

2007-05-25

The Wistar-Kyoto (WKY) rat shows marked susceptibility to crescentic glomerulonephritis. In the model of nephrotoxic nephritis (NTN) that is induced by a small dose of nephrotoxic globulin, WKY rats developed crescents in 80 ± 2% of glomeruli at day 10, whereas no crescents were seen in Lewis rats. This was associated with marked increase in monocyte chemoattractant protein-1 synthesis in WKY glomeruli. It was posited whether susceptibility depended on circulating cells or intrinsic renal cells. Bone marrow (BM) isografts from WKY to WKY or Lewis to Lewis did not affect susceptibility to NTN. When BM was transferred from WKY to Lewis rats, crescents developed in 35 ± 9% of glomeruli 10 d after induction of NTN, indicating that susceptibility could be transferred by BM cells. However, crescents were also seen in WKY rats that were given Lewis marrow. For assessment of the contribution of intrinsic renal cells, kidneys from WKY or Lewis rats were transplanted into F1 animals. In NTN, the ratio of crescents in the transplanted kidney to the native kidney was significantly higher for WKY-to-F1 than for Lewis-to-F1 transplants, demonstrating that the kidney itself also influences susceptibility. Mesangial cell responses were then examined in the two strains. Mesangial cells that were derived from WKY rats synthesized significantly more monocyte chemoattractant protein-1 basally and after stimulation with heat-aggregated rabbit IgG or TNF-. These results show that susceptibility to NTN in the WKY rat depends on both circulating and intrinsic renal cells and that there are genetic differences between the strains in mesangial responses to inflammatory stimuli.