Bryan R. Conway

Predicting mortality and uptake of renal replacement therapy in patients with stage 4 chronic kidney disease

BACKGROUND Novel strategies are required to efficiently manage the increasing number of patients diagnosed with chronic kidney disease (CKD). We sought to identify factors predicting outcome in patients with stage 4 CKD and to determine whether low-risk patients could be managed in primary care. METHODS We performed a two-centre, retrospective cohort study including 396 patients with stage 4 CKD referred to nephrology clinics from 1998 to 2002. We utilized electronic databases to determine the incidence of renal replacement therapy (RRT) and mortality and the rate of deterioration in estimated glomerular filtration rate (eGFR) to the year end 2005. RESULTS This was an elderly cohort, with 71.7% of patients aged > or =65 years. The risk of surviving to require dialysis fell with increasing age (HR 0.44; 95% CI: 0.23-0.84 for those >74 years verses those <65 years), in part due to the slower rate of decline in renal function in older patients (median fall in eGFR was -2.25, -1.38 and -0.86 ml/ min/1.73 m(2)/year in those aged <65 years, 65-74 years and >74 years, respectively, P < 0.0001). Additional independent risk factors predicting RRT included: high baseline proteinuria (HR 6.26; 95% CI: 2.74-14.23 for >3 g/24 h versus <0.3 g/24 h), greater early decline in renal function (HR 3.86; 95% CI: 2.34-6.38 for > or =4 ml/min/1.73 m(2)/year versus <4 ml/min/1.73 m(2)/year), low baseline eGFR (HR 2.92; 95% CI: 1.61-5.30 for 15-19 versus 25-29 ml/min/1.73 m(2)) and low haemoglobin (HR 3.16; 95% CI: 1.64-6.08 for <10 versus >12 g/dl). The 98 (24.7%) patients discharged to primary care had more stable renal function than those remaining under nephrology care (median change in eGFR of +0.20 versus -1.88 ml/ min/1.73 m(2)/year, P = 0.0001). CONCLUSIONS Most patients with stage 4 CKD, in particular the elderly, die without commencing RRT. Patients at low risk of progression can be identified and discharged safely to primary care with an active management plan.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up‐regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF‐β function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP‐2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C‐ζ to the leading edge of migrating cells. Inhibition of CTGF‐induced protein kinase C‐ζ activity with a myristolated PKC‐ζ inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC‐ζ kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF‐induced migration compared with wild‐type. Furthermore, CTGF stimulated phosphorylation and activation of GSK‐3β. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC‐ζ and GSK‐3β. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

MicroRNA-214 Antagonism Protects against Renal Fibrosis

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-β signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-β blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-β signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.

Recent Advances in Animal Models of Diabetic Nephropathy

Diabetic nephropathy (DN) is the single most common cause of end-stage kidney disease. Therefore, it is imperative that novel therapies are developed. Progress has been hindered, however, by the lack of robust animal models. In the current review we describe recent advances in the field, including the impact of background strain, hypertension and transcriptomic profiling. While the C57BL/6J strain is relatively resistant to DN, the FVB strain appears more susceptible and Ove26 and db/db mice on this background may be useful in modelling types 1 and 2 DN, respectively. Black and tan, brachyury (BTBR) mice deficient for the leptin receptor (ob/ob) develop many of the pathological features of human DN and, remarkably, treatment with exogenous leptin ameliorates hyperglycaemia, albuminuria and glomerulosclerosis. Hypertension plays a key role in the progression of human DN and exacerbates nephropathy in diabetic rodents. Endothelial nitric oxide synthase deficiency (eNOS-/-) results in moderate hypertension and the development of nodular glomerulosclerosis and hyaline arteriosclerosis in streptozotocin-induced diabetic C57BL/6J mice. In Cyp1a1mRen2 rats, renin-dependent hypertension synergises with streptozotocin-induced hyperglycaemia to produce a 500-fold increase in albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. Renal transcriptional profiling suggests that many of the gene expression changes observed in human DN are replicated in eNOS-/- mice and Cyp1a1mRen2 rats. Despite these advances, no model faithfully recapitulates all the features of human DN and further refinements are required. In the interim, it is likely that researchers may use publically available transcriptomic data to select the most appropriate model to study their molecule or pathway of interest.

Measuring urinary tubular biomarkers in type 2 diabetes does not add prognostic value beyond established risk factors

Tubulointerstitial disease plays an important role in the pathophysiology of diabetic kidney disease. To determine whether biomarkers of tubular injury could predict renal outcome and mortality in patients with type 2 diabetes, we measured urinary levels of kidney injury molecule-1 (KIM-1) and glycoprotein non-metastatic melanoma B (Gpnmb), both normalized to the urinary creatinine, in 978 individuals from the Edinburgh Type 2 Diabetes Study. At baseline, 238 patients had an estimated glomerular filtration rate (eGFR) below 60 ml/min/1.73 m2 while 147 and 15 patients had microalbuminuria or overt proteinuria, respectively. Both the urine KIM-1 and Gpnmb to creatinine ratios correlated with the urinary albumin to creatinine ratio, the duration of diabetes, and the stringency of glycemic control but not with blood pressure or baseline eGFR. Higher ratios of each marker were associated with a faster decline in kidney function during 4 years of follow-up; however, this was not independent of the urinary albumin to creatinine ratio. Higher KIM-1, but not Gpnmb ratios were associated with an increased risk of mortality, but this association was no longer significant after adjustment for other risk factors, in particular albuminuria. Thus, tubular injury in persons with type 2 diabetes may contribute to the decline in kidney function; however, measuring the urinary concentration of these two tubular biomarkers does not confer additional prognostic information beyond established risk factors.

Hyperglycemia and Renin-Dependent Hypertension Synergize to Model Diabetic Nephropathy

Rodent models exhibit only the earliest features of human diabetic nephropathy, which limits our ability to investigate new therapies. Hypertension is a prerequisite for advanced diabetic nephropathy in humans, so its rarity in typical rodent models may partly explain their resistance to nephropathy. Here, we used the Cyp1a1mRen2 rat, in which the murine renin-2 gene is incorporated under the Cytochrome P4501a1 promoter. In this transgenic strain, administration of low-dose dietary indole-3-carbinol induces moderate hypertension. In the absence of hypertension, streptozotocin-induced diabetes resulted in a 14-fold increase in albuminuria but only mild changes in histology and gene expression despite 28 weeks of marked hyperglycemia. In the presence of induced hypertension, hyperglycemia resulted in a 500-fold increase in albuminuria, marked glomerulosclerosis and tubulointerstitial fibrosis, and induction of many of the same pathways that are upregulated in the tubulointerstitium in human diabetic nephropathy. In conclusion, although induction of diabetes alone in rodents has limited utility to model human diabetic nephropathy, renin-dependent hypertension and hyperglycemia synergize to recapitulate many of the clinical, histological, and gene expression changes observed in humans.

Opiate toxicity in patients with renal failure

### Case 1 A 68 year old woman with type 2 diabetes, angina, and obesity had an uncomplicated below knee amputation. Baseline creatinine was 133 μmol/l (estimated glomerular filtration rate 36 ml/min).2 In the first 36 hours after surgery she received 50 mg of morphine and 76 mg of codeine. On the second day she developed oliguria despite intravenous fluid resuscitation, and her serum creatinine rose to 213 μmol/l. She became increasingly drowsy and her respiratory rate fell to 8 breaths/min. Opiate toxicity was suspected after consultation with the on-call nephrologist. She did not respond to 400 μg intravenous naloxone, however, and therefore she was transferred to the regional renal unit. On admission she was drowsy and uncommunicative. Observations showed oxygen saturation 91% on 28% inspired oxygen; respiratory rate 6 breaths/min; pulse 50 beats/min, and blood pressure 132/68 mm Hg. Pupils were pinpoint. Initial investigations detected sodium 130 mmol/l, potassium 7.6 mmol/l, urea 28.4 mmol/l, creatinine 320 μmol/l, pH 7.39, Po2 7.9 kPa, and Pco2 5.9 kPa. Electrocardiogram showed no hyperkalaemic changes. We noticed that the cannula previously used to administer naloxone had tissued. We gave 400 μg of naloxone via a new cannula, …

An Update on the Use of Animal Models in Diabetic Nephropathy Research

In the current review, we discuss limitations and recent advances in animal models of diabetic nephropathy (DN). As in human disease, genetic factors may determine disease severity with the murine FVB and DBA/2J strains being more susceptible to DN than C57BL/6J mice. On the black and tan, brachyuric (BTBR) background, leptin deficient (ob/ob) mice develop many of the pathological features of human DN. Hypertension synergises with hyperglycemia to promote nephropathy in rodents. Moderately hypertensive endothelial nitric oxide synthase (eNOS−/−) deficient diabetic mice develop hyaline arteriosclerosis and nodular glomerulosclerosis and induction of renin-dependent hypertension in diabetic Cyp1a1mRen2 rats mimics moderately severe human DN. In addition, diabetic eNOS−/− mice and Cyp1a1mRen2 rats recapitulate many of the molecular pathways activated in the human diabetic kidney. However, no model exhibits all the features of human DN; therefore, researchers should consider biochemical, pathological, and transcriptomic data in selecting the most appropriate model to study their molecules and pathways of interest.

Cellular orchestrators of renal fibrosis

Renal fibrosis is a key determinant of the progression of many renal diseases and represents a final common pathway that adversely affects outcome. The evolution of renal fibrosis is complicated and involves many cellular and molecular mediators. In this review we will outline the key players in the fibrotic response of the injured kidney and discuss emerging research in the field.

Association between haptoglobin gene variants and diabetic nephropathy : Haptoglobin polymorphism in nephropathy susceptibility

Background/Aims: The Hp1/Hp2 DNA polymorphism has previously been implicated in susceptibility to diabetic nephropathy in some but not all studies. In an attempt to clarify these conflicting findings, we conducted a case-control association study in a Caucasian population. Methods: We recruited 224 and 285 type 1 diabetic patients with (cases) and without (controls) nephropathy, respectively, from 2 centres based in Northern Ireland and the Republic of Ireland. Hp1/Hp2 genotyping was performed using a combination of long-range and multiplex PCR. Allele and genotype frequencies in cases and controls were compared using the χ2 test. Results: There was a statistically significant increase in the frequency of the Hp2 allele in cases compared to controls (65.6 vs. 58.6%, OR = 1.35, 95% CI: 1.04–1.76, p = 0.03). The distributions of genotypes were in Hardy-Weinberg equilibrium for both cases and controls, and the overall frequency of the Hp1 allele was 38.3%, which is similar to that found in other Western European populations. Conclusions: The results suggest that the Hp2 allele may confer susceptibility to nephropathy in patients with type 1 diabetes.