Hematopoietic growth factor inducible neurokinin-1 (Gpnmb/Osteoactivin) is a biomarker of progressive renal injury across species

We sought to find a urinary biomarker for chronic kidney disease and tested hematopoietic growth factor inducible neurokinin-1 (HGFIN, also known as Gpnmb/Osteoactivin) as it was found to be a kidney injury biomarker in microarray studies. Here, we studied whether HGFIN is a marker of kidney disease progression. Its increase in kidney disease was confirmed by real-time PCR after 5/6 nephrectomy, in streptozotocin-induced diabetes, and in patients with chronic kidney disease. In the remnant kidney, HGFIN mRNA increased over time reflecting lesion chronicity. HGFIN was identified in the infarct portion of the remnant kidney in infiltrating hematopoietic interstitial cells, and in distal nephron tubules of the viable remnant kidney expressed de novo with increasing time. In vitro, it localized to cytoplasmic vesicles and cell membranes. Epithelial cells lining distal tubules and sloughed luminal tubule cells of patients expressed HGFIN protein. The urine HGFIN-to-creatinine ratio increased over time after 5/6 nephrectomy; increased in patients with proteinuric and polycystic kidney disease; and remained detectable in urine after prolonged freezer storage. The urine HGFIN-to-creatinine ratio compared favorably with the urine neutrophil gelatinase-associated lipocalin (NGAL)-to-creatinine ratio (both measured by commercial enzyme-linked immunosorbent assays (ELISAs)), and correlated strongly with proteinuria, but weakly with estimated glomerular filtration rate and serum creatinine. Thus, HGFIN may be a biomarker of progressive kidney disease

The Role of Epithelial Sodium Channel ENaC and the Apical Cl-/HCO3- Exchanger Pendrin in Compensatory Salt Reabsorption in the Setting of Na-Cl Cotransporter (NCC) Inactivation.

The absence of NCC does not cause significant salt wasting in NCC deficient mice under basal conditions. We hypothesized that ENaC and pendrin play important roles in compensatory salt absorption in the setting of NCC inactivation, and their inhibition and/or downregulation can cause significant salt wasting in NCC KO mice.WT and NCC KO mice were treated with a daily injection of either amiloride, an inhibitor of ENaC, or acetazolamide (ACTZ), a blocker of salt and bicarbonate reabsorption in the proximal tubule and an inhibitor of carbonic anhydrases in proximal tubule and intercalated cells, or a combination of acetazolamide plus amiloride for defined durations. Animals were subjected to daily balance studies. At the end of treatment, kidneys were harvested and examined. Blood samples were collected for electrolytes and acid base analysis.Amiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p0.05). The increase in UO in NCC KO mice was associated with a significant increase in sodium excretion (from 0.25 mmol/24 hrs at baseline to 0.35 mmol/24 hrs after amiloride injection, p80% reduction of kidney pendrin expression in both WT and NCC KO mice. However, ACTZ treatment noticeably increased urine output and salt excretion only in NCC KO mice (with urine output increasing from a baseline of 1.1 ml/day to 2.3 ml/day and sodium excretion increasing from 0.22 mmole/day before to 0.31 mmole/day after ACTZ) in NCC KO mice; both parameters were significantly higher than in WT mice. Western blot analysis demonstrated significant enhancement in ENaC expression in medulla and cortex of NCC KO and WT mice in response to ACTZ injection for 6 days, and treatment with amiloride in ACTZ-pretreated mice caused a robust increase in salt excretion in both NCC KO and WT mice. Pendrin KO mice did not display a significant increase in urine output or salt excretion after treatment with amiloride or ACTZ.1. ENaC plays an important role in salt reabsorption in NCC KO mice. 2. NCC contributes to compensatory salt reabsorption in the setting of carbonic anhydrase inhibition, which is associated with increased delivery of salt from the proximal tubule and the down regulation of pendrin. 3. ENaC is upregulated by ACTZ treatment and its inhibition by amiloride causes significant diuresis in NCC KO and WT mice. Despite being considered mild agents individually, we propose that the combination of acetazolamide and amiloride in the setting of NCC inhibition (i.e., hydrochlorothiazide) will be a powerful diuretic regimen

Effect of Amiloride and Acetazolamide on water and salt excretion in WT and pendrin KO mice.

<p>Fig 7a depicts urine output in pendrin KO mice compared to WT mice, before and after amiloride injection. Fig 7b shows water intake in the two genotypes at baseline and after amiloride injection. Fig 7c and 7d compares urine output and water intake in WT vs pendrin KO mice post ACTZ injection.</p

Effect of amiloride on urine output and water intake in WT and NCC KO mice.

<p>After acclimation in metabolic cages, wild type and NCC KO mice were treated with daily intraperitoneal (IP) injection of amiloride. Water intake and urine output were collected daily for 5 days. Water intake (a) and urine output (b) on day 3 and beyond were comparable.</p

Effect of Acetazolamide on pendrin expression, urine output and water intake in WT and NCC KO mice.

<p>Fig 5a shows the expression of pendrin in WT and NCC KO mice treated with acetazolamide for 2 or 6 days (left and right panels). Fig 5b and 5c depicts the urine output (i) and water intake (ii) in WT and NCC KO mice treated with ACTZ. The increase in urine output by ACTZ in NCC KO mice correlated with a reduction in pendrin expression. Fig 5d demonstrates enhanced sodium excretion by ACTZ in NCC KO mice vs. WT animals.</p

Expression of ENaC in kidneys of WT and NCC KO mice.

<p>Expression of ENaC subunits by (a) Northern hybridization and (b). Western blot in kidneys of WT and NCC KO mice. (a) Expression levels of all 3 subunits increased by Northern hybridization, with α, β and γ subunits increasing by 110, 80 and 70%, respectively, vs. wt littermates. Western blots show significant increase in the cleaved ENaC γ subunit in NCC KO mice, consistent with published reports (29). (b) The abundance of cleaved ENaC γ subunit form increased by ~420% in NCC KO mice. The full length bands for α, β and γ subunits are shown, which did not show significant changes vs. WT mice. c. Expression of NKCC2 isotypes in WT and NCC KO mice.</p

Effect of Acetazolamide on ENaC expression: Impact of amiloride on salt excretion in WT and NCC KO mice.

<p>Fig 6a shows the expression of ENaC in kidney medulla and cortex of WT and NCC KO mice treated with acetazolamide for 6 days (left and right panels). Fig 6b depicts the effect of amiloride treatment on salt excretion in WT and NCC KO mice pretreated with ACTZ for 6 days.</p

Expression of pendrin in kidneys of WT and NCC KO mice.

<p>Expression of pendrin by (a) Northern hybridization and (b) Immunofluorescence labeling in kidneys of WT and NCC KO mice. Northern hybridizations showed significant increase in pendrin expression in NCC KO mice vs. WT littermates (with expression of pendrin increasing by 130% vs. wt littermates, p<0.03). Immunofluorescence labeling showed significant increase in the number of pendrin positive cells in kidneys of NCC KO mice vs. WT littermates (p<0.01 vs. WT).</p

Effect of amiloride on sodium excretion in WT and NCC KO mice.

<p>Daily sodium excretion before and after treatment with amiloride is shown in WT and NCC KO mice. Sodium excretion on day 3 and beyond were comparable.</p